The Drosophila Jun N-terminal kinase (JNK) gene basket (bsk) promoter contains a DNA replication-related element (DRE) like sequence, raising the possibility of regulation by the DNA replication-related element-binding factor (DREF). Chromatin immunoprecipitation assays with anti-DREF IgG showed the bsk gene promoter region to be effectively amplified. Luciferase transient expression assays revealed the DRE like sequence to be important for bsk gene promoter activity, and knockdown of DREF decreased the bsk mRNA level and the bsk gene promoter activity. Furthermore, knockdown of DREF in the notum compartment of wing discs by pannier-GAL4 and UAS-DREFIR resulted in a split thorax phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered (puc)-LacZ enhancer trap line revealed the reduction in DREF knockdown clones. These findings indicate that DREF is involved in regulation of Drosophila thorax development via actions on the JNK pathway (Yoshioka, 2012).
Nutations in the apico-basal cell polarity regulators cooperate with oncogenic Ras (RasACT) to promote tumorigenesis in Drosophila melanogaster and mammalian cells. To identify novel genes that cooperate with RasACT in tumorigenesis, a genome-wide screen was carried out for genes that when overexpressed throughout the developing Drosophila eye enhance RasACT-driven hyperplasia. RasACT-cooperating genes identified were Rac1 Rho1, RhoGEF2, pbl, rib, and east, which encode cell morphology regulators. In a clonal setting, which reveals genes conferring a competitive advantage over wild-type cells, only Rac1, an activated allele of Rho1 (Rho1ACT), RhoGEF2, and pbl cooperated with RasACT, resulting in reduced differentiation and large invasive tumors. Expression of RhoGEF2 or >Rac1 with RasACT upregulated Jun kinase (JNK) activity, and JNK upregulation was essential for cooperation. However, in the whole-tissue system,
upregulation of JNK alone was not sufficient for cooperation with RasACT, while in the clonal setting, JNK upregulation was sufficient for RasACT-mediated tumorigenesis. JNK upregulation was also sufficient to confer invasive growth of RasV12-expressing mammalian MCF10A breast epithelial cells. Consistent with this, HER2+ human breast cancers (where human epidermal growth factor 2 is overexpressed and Ras signaling upregulated) show a significant correlation with a signature representing JNK pathway activation. Moreover, genetic analysis in Drosophila revealed that Rho1 and Rac are important for the cooperation of RhoGEF2 or Pbl overexpression and of mutants in polarity regulators, Dlg
and aPKC, with RasACT in the whole-tissue context. Collectively this analysis reveals the importance of the RhoGEF/Rho-family/JNK pathway in cooperative tumorigenesis with RasACT (Brumby, 2011).
Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. This study shows that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. It is proposed that in an immortalized ('undead') model of AiP, in which the activity of the effector caspases is blocked, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer (Fogarty, 2016).
The role of ROSs as a regulated form of redox signaling in damage detection and damage response is becoming increasingly clear. This study has shown that in Drosophila, extracellular ROSs generated by the NADPH oxidase Duox drive compensatory proliferation and overgrowth following hid-induced activation of the initiator caspase Dronc in developing epithelial tissues. At least one consequence of ROS production is the activation of hemocytes at undead epithelial disc tissue. Furthermore, the work implies that extracellular ROS and hemocytes are part of the feedback amplification loop between Hid, Dronc, and JNK that occurs during stress-induced apoptosis. Finally, hemocytes release the TNF ligand Eiger, which promotes JNK activation in epithelial disc cells (Fogarty, 2016).
This work helps to understand why JNK activation occurs mostly in apoptotic/undead cells but occasionally also in neighboring surviving cells. Because the data indicate that hemocytes trigger JNK activation in epithelial cells, the location of hemocytes on the imaginal discs determines which epithelial cells receive the signal for JNK activation. Nevertheless, the possibility is not excluded that there is also an autonomous manner of Dronc-induced JNK activation in undead/apoptotic cells (Fogarty, 2016).
In the context of apoptosis, hemocytes engulf and degrade dying cells. However, there is no evidence that hemocytes have this role in the undead AiP model. No Caspase-3 (CC3) material is observed in hemocytes attached to undead tissue. Therefore, the role of hemocytes in driving proliferation is less clear and likely context dependent. In Drosophila embryos, hemocytes are required for epidermal wound healing, but this is a nonproliferative process. With respect to tumor models in Drosophila, much of the research to date has focused on the tumor-suppressing role of hemocytes and the innate immune response. However, a few reports have implicated hemocytes as tumor promoters in a neoplastic tumor model. Consistently, in the undead model of AiP, this study found that hemocytes have an overgrowth- and tumor-promoting role. Therefore, the state of the damaged tissue and the signals produced by the epithelium may have differential effects on hemocyte response (Fogarty, 2016).
In a recent study, ROSs were found to be required for tissue repair of wing imaginal discs in a regenerative (p35-independent) model of AiP, consistent with the current work. Although a role of hemocytes was not investigated in this study, it should be noted that p35-independent AiP models do not cause overgrowth, whereas undead ones such as the ey>hid-p35 AiP model do. It is therefore possible that ROSs in p35-independent AiP models are necessary for tissue repair independent of hemocytes, whereas ROSs in conjunction with ROS-activated hemocytes in undead models mediate the overgrowth of the affected tissue. Future work will clarify the overgrowth-promoting function of hemocytes. These considerations are reminiscent of mammalian systems, where many solid tumors are known to host alternatively activated (M2) tumor-associated macrophages, which promote tumor growth and are associated with a poor prognosis (Fogarty, 2016).
Because tumors are considered 'wounds that do not heal', the undead model of AiP is seen as a tool to probe the dynamic interactions and intercellular signaling events that occur in the chronic wound microenvironment. Future studies will investigate the specific mechanisms of hemocyte-induced growth and the tumor-promoting role of inflammation in Drosophila as well as roles of additional tissue types, such as the fat body, on modulating tumorous growth (Fogarty, 2016).
The hindsight gene regulates cell morphology, cell fate specification, planar cell polarity and epithelial integrity during Drosophila retinal development. In the third instar larval eye imaginal disc, Hnt protein expression begins in the morphogenetic furrow and is refined to cells in the developing photoreceptor cell clusters just before their determination as neurons. In hnt mutant larval eye tissue, furrow markers persist abnormally, posterior to the furrow; there is a delay in specification of preclusters as cells exit the furrow; there are morphological defects in the preclusters, and recruitment of cells into specific R cell fates often does not occur. Additionally, genetically mosaic ommatidia with one or more hnt mutant outer photoreceptor cell, have planar polarity defects that include achirality, reversed chirality and misrotation. Mutants in the JNK pathway act as dominant suppressors of the hnt planar polarity phenotype, suggesting that Hnt functions to downregulate JUN kinase (JNK) signaling during the establishment of ommatidial planar polarity. Hnt expression continues in the photoreceptor cells of the pupal retina. When an ommatidium contains four or more hnt mutant photoreceptor cells, both genetically mutant and genetically wild-type photoreceptor cells fall out of the retinal epithelium, indicating a role for Hnt in maintenance of epithelial integrity. In the late pupal stages, Hnt regulates the morphogenesis of rhabdomeres within individual photoreceptor cells and the separation of the rhabdomeres of adjacent photoreceptor cells. Apical F-actin is depleted in hnt mutant photoreceptor cells before the observed defects in cellular morphogenesis and epithelial integrity. The analyses presented here, together with previous studies in the embryonic amnioserosa and tracheal system, show that during development Hnt has a general role in regulation of the F-actin-based cytoskeleton, JNK signaling, cell morphology and epithelial integrity (Pickup, 2002).
The fact that all of the symmetrical ommatidia along the borders of hnt clones are of the R3/R3 conformation suggests that Hnt function is necessary for correct R4 fate and orientation. It has been suggested that, owing to its closer proximity to the polarizing signal from the equator, a stronger activation of the JNK pathway occurs in the R3 precursor cell. Activated JUN would then be responsible for the upregulation of the target gene, Delta, in the R3 precursor cell relative to the R4 precursor cell. Since results in the eye and results in the embryo imply that Hnt is necessary for downregulating JNK function, it is proposed that the wild-type function of Hnt is to downregulate JNK activity in the R4 precursor cell. Such downregulation would enhance JNK signaling differences between the R3 and R4 cells. In the absence of the Hnt gene product, JNK signaling would be inappropriately elevated in the R4 precursor cell, thereby upregulating the transcription of JNK targets such as Delta, leading that cell to behave more like an R3 precursor cell. Consistent with this model, it has been found that Delta hypomorphs act as enhancers of the hntpeb rough eye phenotype. R3/R3 symmetric clusters are observed both when the R4 cell is mutant for hnt and the R3 precursor is hnt+, and when the R3 cell is mutant for hnt and the R4 precursor is Hnt+. In the latter case, the above model would lead one to expect normal R3/R4 clusters. Since only R3/R3 clusters are observed, it is speculated that Hnt can affect the R4 precursor cell when expressed only in the neighboring R3 precursor cell (i.e. that there may be some communication feedback between these cells leading to local non-autonomy of the hnt phenotype) (Pickup, 2002).
These results clearly implicate Hnt in regulation of several types of cellular events that are common to the different contexts in which Hnt functions. These include establishment or maintenance of the morphology of individual cells within an epithelium, as well as maintenance of the integrity of the epithelium per se. There are also shared molecular correlates of these Hnt functions. In particular, Hnt is required for establishment of localized F-actin- and phosphotyrosine-rich complexes in the leading edge epidermal cells, as well as in the photoreceptor cells. Hnt functions to regulate two JNK signaling dependent processes (planar polarity in the eye and dorsal closure of the embryo), possibly by downregulating JNK signaling in time and space. A fuller understanding of whether the functions of Hnt in different tissues and at different times during development derive from control of the same molecular pathway will await genetic and molecular analyses of the genes regulated by Hnt (Pickup, 2002).
Much of what is known about apoptosis in human cells stems from pioneering genetic studies in the nematode C. elegans. However, one important way in which the regulation of mammalian cell death appears to differ from that of its nematode counterpart is in the employment of TNF and TNF receptor superfamilies. No members of these families are present in C. elegans, yet TNF factors play prominent roles in mammalian development and disease. The cloning and characterization of Eiger, a unique TNF homolog in Drosophila, is described. Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution (Moreno, 2002).
Another pathway activated by mammalian TNF death factors is the JNK pathway, although its role in inducing apoptosis upon TNF signaling is less well defined. JNK signaling in Drosophila is reflected by the expression levels of puckered (puc), a gene encoding a dual-specificity phosphatase that forms a negative feedback loop by downregulating the activity of JNK. High levels of puc expression are induced by Eiger. Due to its function as a negative regulator of JNK, Puc can also be used as a powerful means to repress JNK activity if overexpressed by a constitutive promoter that is no longer dependent on this activity. Coexpression of Eiger and Puc completely blocks Eiger activity, strikingly reverting the eye and wing phenotypes to wild-type and blocking Eiger-induced elimination of cell clones. Forced expression of Puc does not prevent all forms of cell death. For example, when tested in the situation of polyglutamine repeat-induced neurodegeneration, which is also caused by apoptosis, coexpression of puc has no discernible protective effect. Taken together, these results are interpreted as firm evidence that Eiger induces the JNK signaling pathway and that Eiger-induced apoptosis is critically dependent on JNK activity (Moreno, 2002).
Consistent with this interpretation, several components of the Drosophila JNK pathway are rate limiting in mediating or preventing Eiger-induced apoptosis. The removal of one wild-type copy of either DTRAF1 (encoding the homolog of human TRAF2), misshapen (encoding a Ste20 kinase that binds to DTRAF1), or basket (encodes Drosophila JNK) suppresses Eiger-induced apoptosis. Conversely, animals heterozygous for a mutation in puc display an enhanced phenotype (Moreno, 2002).
The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).
Oncogenic mutations in Ras deregulate cell death and proliferation to cause cancer in a significant number of patients. Although normal Ras signaling during development has been well elucidated in multiple organisms, it is less clear how oncogenic Ras exerts its effects. Furthermore, cancers with oncogenic Ras mutations are aggressive and generally resistant to targeted therapies or chemotherapy. This study identified the exocytosis component Sec15 as a synthetic suppressor of oncogenic Ras in an in vivo Drosophila mosaic screen. Oncogenic Ras elevates exocytosis and promotes the export of the pro-apoptotic ligand Eiger (Drosophila TNF). This blocks tumor cell death and stimulates overgrowth by activating the JNK-JAK-STAT non-autonomous proliferation signal from the neighboring wild-type cells. Inhibition of Eiger/TNF exocytosis or interfering with the JNK-JAK-STAT non-autonomous proliferation signaling at various steps suppresses oncogenic Ras-mediated overgrowth. These findings highlight important cell-intrinsic and cell-extrinsic roles of exocytosis during oncogenic growth and provide a new class of synthetic suppressors for targeted therapy approaches (Chabu, 2014).
The Hippo and c-Jun N-terminal kinase (JNK) pathway both regulate growth and contribute to tumorigenesis when dysregulated. Whereas the Hippo pathway acts via the transcription coactivator Yki/YAP to regulate target gene expression, JNK signaling, triggered by various modulators including Rho GTPases, activates the transcription factors Jun and Fos. This study shows that impaired Hippo signaling induces JNK activation through Rho1. Blocking Rho1-JNK signaling suppressed Yki-induced overgrowth in the wing disk, whereas ectopic Rho1 expression promoted tissue growth when apoptosis was prohibited. Furthermore, Yki directly regulates Rho1 transcription via the transcription factor Sd. These results identify a novel molecular link between the Hippo and JNK pathways and implicate the essential role of the JNK pathway in Hippo signaling-related tumorigenesis (Ma, 2005).
Recent studies have revealed a complex interaction network between Hippo and other key signaling pathways, including TGF- β /SMAD and Wnt/β-catenin pathways, whereas its communication with JNK signaling remains elusive. This study provides genetic evidences that impaired Hippo signaling promotes overgrowth through Rho1-JNK signaling in Drosophila. First, loss of Hippo signaling induces JNK activation and its target gene expression. Second, Yki-induced overgrowth is suppressed by blocking Rho1-JNK signaling. Third, ectopic Rho1 expression phenocopies Yki-triggered overgrowth and proliferation when cell death is compromised (Ma, 2005).
Yki/YAP's ability in promoting tissue growth depends on transcription factors, including Sd/TEADs and SMADs. Consistent with this notion, this study found Sd, but not Mad, is essential for Yki-induced JNK activation, whereas ectopic Sd expression is sufficient to activate JNK signaling by itself. The Rho1 GTPase was further implicated as the critical factor that bridges the interaction between Hippo and JNK signaling. Rho1 not only mediates Yki-induced JNK activation and overgrowth, but also serves as a direct transcriptional target of Yki/Sd complex. Intriguingly, Rho1 activation was also found to promote nuclear translocation of Yki in wing discs, and reducing Yki activity significantly impeded Rho1 induced growth, implying the existence of a potential positive feedback loop to amplify Yki-induced overgrowth and to help maintain signaling in a steady state. Consistent with thi observation, recent studies
reported that GPCRs could activate YAP/TAZ through RhoA in mammals, whereas elevated JNK signaling in Drosophila could stimulate Yki nuclear translocation during regeneration and tissue growth. Thus, these results provide the other side of the story about a novel cross-talk between Hippo and JNK signaling (Ma, 2005).
Intriguingly, it was found that ectopic Yki expression driven by ptc-Gal4 induced MMP1 activation, puc-LacZ expression, rho1 transcription, and Yki target gene transcription predominantly in the proximal region of wing disk, but not that of the dorsal/ventral boundary. This is consistent with a recently published paper showing that tension in the center region of Drosophila wing disk is lower than that in the periphery, which correlates with lower Yki activity. It is also worth noting that despite the requirement of JNK signaling in Yki-induced wing overgrowth, JNK was not activated strictly in an autonomous manner upon Yki overexpression. This could be caused by supercompetitive activity of Yki expression clones, or, alternatively, through a propagation of JNK signal into neighboring cells, which would be very interesting to study further (Ma, 2005).
Apart from its role in growth control, the Hippo pathway also regulates tumor invasion and metastasis. Similarly, JNK signaling plays a major role in modulating metastasis in both flies and mammals. Rho1 was also reported to cooperate with oncogenic Ras to induce large invasive tumors. Hence, it is likely that Rho1 also acts as the molecular link between Yki and JNK signaling in modulating metastasis as well (Ma, 2005).
Reactive oxygen species (ROS) and mitochondrial defects in neurons are implicated in neurodegenerative disease. This study finds that a
key consequence of ROS and neuronal mitochondrial dysfunction is the
accumulation of lipid droplets (LD) in glia. In Drosophila, ROS triggers c-Jun-N-terminal Kinase (JNK) and Sterol Regulatory
Element Binding Protein (SREBP) activity in neurons leading to
LD accumulation in glia prior to or at the onset of neurodegeneration. The accumulated lipids were peroxidated in the presence of ROS. Reducing LD accumulation in glia and lipid peroxidation via targeted lipase overexpression and/or
lowering ROS significantly delayed the onset of neurodegeneration.
Furthermore, a similar pathway led to glial LD accumulation in Ndufs4
mutant mice with neuronal mitochondrial defects, suggesting that LD
accumulation following mitochondrial dysfunction is an
evolutionarily conserved phenomenon, and represents an early,
transient indicator and promoter of neurodegenerative disease (Liu, 2015).
This study shows that neuronal mitochondrial defects that lead to elevated levels of ROS, induce activation of JNK and SREBP, which in turn elevate lipid synthesis in neurons and formation of LD in glial cells. These LDs contribute to and promote ND through elevated levels of lipid peroxidation. LDs form in glia prior to or at the onset of the appearance of obvious degenerative histological features in Drosophila and mice. Reducing the number and size of LD pharmacologically or genetically delays ND in the fly. This is the first indication that SREBP, lipid droplet biogenesis, and lipid metabolism play a role in the pathogenesis of several neurodegenerative diseases (Liu, 2015).
A growing body of evidence points to the importance of glial health and function in nervous system energy metabolism and homeostasis. Nevertheless, given the number and prevalence of different types of neurodegenerative diseases, very few reports have documented the presence of LDs in either neuron or glia in patients and in animal models. LD accumulation in the brain has been reported in cells that line the ventricles in the globus pallidus and substantia nigra in mutant mice lacking both subunits of the liver X receptor, apolipoprotein E, or a peroxisomal biogenesis factor (Pex5) . In addition, in vitro studies using immortalized cell lines and explants show that LD may form and accumulate in glia under conditions of nutrient deprivation or lipopolysaccharide induced stress. However, LDs have not been shown to play an active role in neurodegenerative processes. Furthermore, LD accumulation has not been reported in patients with or animal models of Leigh syndrome (NDUFS4/Ndufs4, NDUFAF6/sicily), CMT-2A2 or HMSN6 (MFN2/Marf), and ARSAL (MARS2/Aats-met). The lack of neuropathological reports of LDs in animal models or in patients with ND may be attributed to the fact that LD accumulation is transient and mostly occur during presymptomatic stages of the disease (Liu, 2015).
Although these genes/mutants are implicated in very different mitochondrial processes, they exhibit a common phenotype of elevated levels of ROS, leading to LD accumulation. Similar morphological changes of glia have been reported under stress conditions. Interestingly, mid- and late-stage Ndufs4-/- mice exhibit CNS lesions in the same brain regions where the LD accumulate in early stage animals, showing a strong correlative relationship. Similarly, LD accumulation in Drosophila mutants occurs prior to or at the onset of physical signs of ND. Importantly, the delivery of AD4 is able to significantly ameliorate LD accumulation in Drosophila and delay the onset of ND in flies and mice. Hence, the molecular mechanisms underlying these phenotypes are likely to be conserved between these species and potentially also in higher organisms (Liu, 2015).
In the clinical setting, the prescription of antioxidants toward treatment of neurodegenerative diseases has been tested repeatedly on patients with neurodegenerative disorders, without compelling results. The LD accumulation phenotype in these mutants occurs prior to histopathological and physical signs of ND. A brief period of AD4 delivery prior to the onset of symptoms in mutant mice is effective in delaying onset of clinical signs. Thus, therapy with an effective antioxidant that penetrates the blood-brain barrier should be started early and sustained over long periods. In addition, pharmacological manipulation of JNK or lipid levels in the brain may serve as a potential therapy to delay the onset of ND. However, similar to antioxidant treatment, this may need to be administered at an early stage. Hence, early identification of potential ROS related neurological disease based on genetic/genomic diagnosis or by biomarkers may be critical. Since LD accumulation is one of the earliest presymptomatic changes that occurs in the nervous system, detection of LD itself or changes in neurometabolism may be a promising biomarker (Liu, 2015).
In summary, this study provides evidence for the role of altered lipid metabolism and a neuron-glia interplay that promotes ND. In some mitochondrial mutants, an upregulation of SREBP was observed, as well as lipid biogenesis and glial LD formation. The accumulation of LD is not sufficient to promote the ND process itself. However, in the presence of ROS the accumulated lipids are peroxidated and promote ND, possibly by promoting the release of lipids from LD, elevating the cytoplasmic load, and causing a progressive loss of LD. Hence, the synergistic effects of increased lipid synthesis and/or LD accumulation in combination with elevated ROS and lipid peroxidation promote ND. Finally, it was shown that LD accumulation occurs at the onset or precedes ND in flies and mice, suggesting that LD and changes in lipid metabolism in the nervous system may be a promising biomarker to identify brain regions susceptible to but not yet exhibiting symptoms of ND (Liu, 2015).
Although Jun amino-terminal kinase (JNK) is known to mediate a physiological stress signal that leads to cell death, the exact role of the JNK pathway in the mechanisms underlying intrinsic cell death is largely unknown. Through a genetic screen, it has been shown that a mutant of Drosophila tumor-necrosis factor receptor-associated factor 1 (DTRAF1) is a dominant suppressor of Reaper-induced cell death. Reaper modulates the JNK pathway through Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), which negatively regulates DTRAF1 by proteasome-mediated degradation. Reduction of JNK signals rescues the Reaper-induced small eye phenotype, and overexpression of DTRAF1 activates the Drosophila ASK1 (apoptosis signal-regulating kinase 1; a mitogen-activated protein kinase kinase kinase) and JNK pathway, thereby inducing cell death. Overexpresson of DIAP1 facilitates degradation of DTRAF1 in a ubiquitin-dependent manner and simultaneously inhibits activation of JNK. Expression of Reaper leads to a loss of DIAP1 inhibition of DTRAF1-mediated JNK activation in Drosophila cells. Taken together, these results indicate that DIAP1 may modulate cell death by regulating JNK activation through a ubiquitin-proteasome pathway (Kuranaga, 2002).
The molecular circuitry underlying innate immunity is constructed of multiple, evolutionarily conserved signaling modules with distinct regulatory targets. The MAP kinases and the IKK-NF-kappaB molecules play important roles in the initiation of immune effector responses. The Drosophila NF-kappaB protein Relish plays a crucial role in limiting the duration of JNK activation and output in response to Gram-negative infections. Relish activation is linked to proteasomal degradation of TAK1, the upstream MAP kinase kinase kinase required for JNK activation. Degradation of TAK1 leads to a rapid termination of JNK signaling, resulting in a transient JNK-dependent response that precedes the sustained induction of Relish-dependent innate immune loci. Because the IKK-NF-kappaB module also negatively regulates JNK activation in mammals, thereby controlling inflammation-induced apoptosis, the regulatory cross-talk between the JNK and NF-kappaB pathways appears to be broadly conserved (Park, 2004).
The JNK and IKK/Relish branches of the Imd pathway mediate distinct gene induction responses in Drosophila innate immunity. After diverging downstream from TAK1, these two signaling cascades regulate two separate groups of target genes that are distinct in their induction kinetics and function. The IKK/Relish targets have been extensively characterized and most encode products whose role in innate immunity is relatively well established. In contrast, the JNK-regulated, LPS-responsive genes represent a largely uncharacterized set of loci whose function in innate immunity is not clear. These genes exhibit transient induction kinetics, reaching a maximum ~1 h after induction. It was found that the transient kinetics of the JNK target genes is controlled by the transient kinetics of the JNK module of the Imd pathway and that the IKK/Relish branch plays an active role in turning off JNK activity. Hence, the two seemingly independent branches of the Imd pathway are wired in such a way as to coordinate the temporal order of individual responses (Park, 2004).
The evidence indicates that Relish-mediated JNK inhibition involves proteasomal degradation of TAK1, the MAPKKK responsible for JNK activation in response to LPS. Treatment with proteasomal inhibitors or RNAi against a component of the proteasome complex results in sustained JNK activation during the LPS response. Furthermore, in cells expressing constitutively active Relish, the stability of TAK1 is greatly decreased. Based on these findings, it is suggested that certain targets of Relish that are induced during immune responses facilitate destruction of TAK1 and switch off the JNK cascade. The fact that cycloheximide and actinomycin D also block the down-regulation of JNK activity indicates the involvement of targets of Relish rather than Relish itself. The Relish target involved in this cross-talk likely increases the susceptibility of TAK1 to proteasomal degradation by direct targeting of TAK1 or by antagonizing factors responsible for TAK1 stabilization (Park, 2004).
In considering this model, it should be noted that TAK1 is critically required for activating both IKK and JNK. Elimination of TAK1 during the LPS response thus turns off both of the downstream signaling cascades. Yet IKK/Relish target genes do not show a transient expression pattern, whereas JNK targets do. One possible explanation for this discrepancy lies in the nature of JNK and Relish activation. JNK is activated through its phosphorylation, a modification that is highly reversible. Thus, termination of the input that contributes to JNK phosphorylation is sufficient to result in its rapid inactivation, especially when one of the JNK targets encode a JNK phosphatase, Puckered. Relish, in contrast, is activated through proteolysis, an irreversible modification. Once activated Relish enters the nucleus, it may remain bound to its target genes for some time even after termination of the upstream signal (Park, 2004).
Interestingly, the antagonism between NF-kappaB and JNK signaling is evolutionarily conserved. In mice, inactivation of either IKKß or NF-kappaB RelA (p65) in cells leads to a sustained JNK activation in response to TNFalpha. The sustained JNK activation by TNFalpha has been associated with TNFalpha-induced apoptosis. Several independent studies have proposed different molecules as mediators for the NF-kappaB inhibition of JNK signaling in the TNFalpha pathway including XIAP, GADD45ß, and reactive oxygen species. Nevertheless, the underlying mechanism remains largely unknown. As the Drosophila Imd pathway and the signaling pathway downstream of mammalian TNF receptor share many conserved features, a similar mechanism may govern the antagonistic relationship between IKK/NF-kappaB and JNK in both flies and mammals. Thus, the mechanistic insights gained from studies in Drosophila should be relevant when elucidating the mechanism that connects NF-kappaB to JNK signaling in mammals (Park, 2004).
MAPK phosphatases (MKPs) are important negative regulators of MAPKs in vivo, but
ascertaining the role of specific MKPs is hindered by functional redundancy in
vertebrates. MKP function was characterized by examining the function of
Puckered (Puc), the sole Drosophila Jun N-terminal kinase (JNK)-specific MKP,
during embryonic and imaginal disc development. Puc is a key
anti-apoptotic factor that prevents apoptosis in epithelial cells by restraining
basal JNK signaling. Furthermore, JNK signaling plays an
important role in gamma-irradiation-induced apoptosis, and this study examined how JNK signaling
fits into the circuitry regulating this process. Radiation upregulates both JNK
activity and puc expression in a p53-dependent manner; apoptosis induced by
loss of Puc can be suppressed by p53 inactivation. JNK signaling acts upstream
of both Reaper and effector caspases. JNK signaling
directs normal developmentally regulated apoptotic events. However, if cell
death is prevented, JNK activation can trigger tissue overgrowth. Thus, MKPs are
key regulators of the delicate balance between proliferation, differentiation
and apoptosis during development (McEwen, 2005).
Mammalian JNK signaling promotes apoptosis in response to both extrinsic (e.g.
TNF) and intrinsic (e.g. DNA damage) cues. Drosophila JNK
signaling plays a similar role in apoptosis triggered by extrinsic cues,
but its role in response to intrinsic cues
remains to be established. The role of Puc in regulating responses to either
sort of signal has also been unclear (McEwen, 2005).
This study found that removal of Puc triggers apoptosis in epithelia, even in the
absence of stress. Thus, basal JNK signaling exists in the absence of stress in
embryonic and larval epithelia, and if Puc is not present to restrain this
intrinsic JNK activity, it exceeds a threshold and triggers apoptosis. This
suggests cells may regulate apoptosis without exogenous JNK stimulation, by
regulating MKP levels. Biochemical studies suggest a possible mechanism. Several
MKPs are labile proteins, whose half-lives can be shortened or lengthened by
post-translational modification.
Thus, cell signals may influence apoptosis by modulating MKP accumulation (McEwen, 2005).
Although Puc is crucial to restrain JNK activity and prevent apoptosis in most
epithelia, high-level JNK signaling does not always induce apoptosis. JNK
signaling is required for embryonic dorsal closure. High JNK activity is
normally restricted to the dorsal-most epithelial cells, though in puc mutants
it extends into adjacent cells. However, this JNK
signaling does not trigger apoptosis. By contrast, ectopic JNK
signaling in ventral and lateral epithelial cells in puc mutants does trigger
apoptosis. Thus, cells where JNK activity is normally high are somehow
refractory to JNK-induced apoptosis. JNK signaling normally activates Dpp in
dorsal epithelial cells, with ectopic Dpp activation in more lateral cells in
puc mutants -- perhaps this is anti-apoptotic, since Dpp is a survival factor in
wing discs. Interplay between death and survival
signals may also explain the strongly synergistic stimulation of apoptosis in
the embryonic epidermis observed when
zygotic Puc (lowering the threshold for JNK signaling) and the survival
signal Wg are simultaneously eliminated (McEwen, 2005).
Studies of JNK signaling in mammalian cells suggest that it plays a role in
radiation-induced apoptosis. This hypothesis was tested in Drosophila. When JNK
activation was blocked by expressing Myc-Puc using a JNK-independent promoter,
radiation-induced apoptosis is attenuated. Thus, JNK signaling plays a crucial
role in promoting apoptosis in response to radiation in Drosophila, paralleling
its essential role in mammalian UV-induced apoptosis (McEwen, 2005).
In mammalian cells, prolonged JNK activity correlates with apoptosis,
while transient activation induces cell proliferation.
Thus, differences in both signal amplitude and duration are key
determinants of the biological outcome. One mechanism modulating signal
amplitude is a negative feedback loop; JNK signaling induces expression of MKPs
such as Puc. These data provide an instance of how this may regulate the DNA
damage response. puc is upregulated by gamma-irradiation in a JNK-dependent manner,
and artificially prolonged Myc-Puc expression prevents radiation-induced
apoptosis. Thus, the initial increase in Puc expression following irradiation
may create a 'grace period' during which Puc elevates the threshold of JNK
activation required to induce apoptosis. During this time, cells could attempt
to repair radiation-induced damage. However, if damage persists and JNK
stimulation continues, the Puc-defined threshold may be exceeded. Indeed,
artificially prolonged p53 overexpression overcomes the anti-apoptotic effects
of Puc overexpression, perhaps overwhelming the ability of Puc to restrain JNK (McEwen, 2005).
p53 is a key regulator of decisions between life and death in response to DNA
damage. Thus, how JNK signaling and p53 are integrated was investigated.
Expression of p53 activates JNK, as revealed by its effect on a JNK enhancer trap reporter JNKREP,
while loss of p53 prevents radiation-induced JNK activation. These results suggest
that p53 acts upstream of JNK signaling in response to cellular stress. Several
mechanisms are possible; e.g., p53 might upregulate transcription of JNK pathway
components, whose overexpression can induce apoptosis (McEwen, 2005).
p53 normally monitors genome integrity. Loss of p53 significantly, although not
completely, suppresses apoptosis induced by Puc inactivation. One model to
explain this suggests that basal levels of DNA damage or replication errors act
through p53 to regulate basal JNK activity during normal development. This basal
activity is kept below the apoptotic threshold by Puc. In p53 mutants, basal JNK
activity would be lowered enough so that it could not exceed the apoptotic
threshold, even in the absence of Puc. Alternatively, p53 may also function
downstream of JNK. Consistent with this, p38 can activate p53 in response to UV,
and JNK can regulate p53
stability/activity via direct phosphorylation. This
would set up a positive-feedback loop: DNA damage-triggered activation of p53
would induce JNK activation, which would further elevate p53 activity.
Additional experiments are required to test these alternate hypotheses (McEwen, 2005).
Signal transduction pathways regulate apoptosis, at least in part, by regulating
transcription of rpr, hid and grim (the RHG proteins), the key developmental effectors of apoptosis in Drosophila. The
relationship between JNK signaling, RHG proteins and caspase activation are not
well understood. Caspase 3 cleavage of Mst1, an upstream regulator of JNK and
p38, has been suggested to amplify apoptotic responses,
while other data suggest that Mst1 activates caspases via a JNK-dependent
pathway. Likewise, in Drosophila, initiation of an apoptotic
response by inactivation of DIAP1 may lead to caspase-independent induction of a
JNKREP (McEwen, 2005).
Regulatory relationships between JNK activation, RHG
proteins and caspase activation were directly assessed. The data suggest that JNK signaling acts
through RHG proteins and caspases to induce apoptosis. JNK signaling is required
for rpr-reporter induction in response to radiation. Thus, RHG proteins may be
JNK-responsive target genes upregulated to elicit apoptosis. Consistent with
this hypothesis, mis-expression of eiger, a known JNK activator, has been shown
to promote hid expression and apoptosis in eye discs (McEwen, 2005).
To assess whether JNK signaling can be triggered by caspase activation, JNKREP activity
was examined in response to Rpr or Hid expression. Both induce
apoptosis without concomitant JNK activation. Furthermore, these data suggest
that, in at least some contexts, caspases act downstream of JNK: p35-mediated
caspase blockade allows cells with elevated JNK signaling to survive in imaginal
discs, but does not prevent JNK activation in response to irradiation. Thus, RHG
proteins elicit caspase-mediated apoptosis downstream of JNK activation (McEwen, 2005).
Work on JNK-induced apoptosis in
Drosophila has focused on its role in the complex processes shaping organ size.
Wing disc cells make decisions about whether to die or proliferate by
integrating levels of different developmental signals they receive, and
comparing their status with that of their neighbors. This occurs in part by
competition for survival signals like the TGFbeta family member Dpp. Complex crosstalk
among this and other signaling pathways precisely regulates the size and pattern
of the wing, in a process that is very resistant to tissue damage or
developmental errors. Discontinuities in smooth morphogen gradients, which may
arise from errors in patterning or tissue injury, are corrected in part by
JNK-dependent apoptosis (McEwen, 2005).
These data clarify roles for JNK signaling in adult development.
Failure to recover puc clones anywhere in the wing disc suggests that
basal JNK activity is sufficient to promote cell-autonomous apoptosis
independent of other signals activating JNK. However, JNK signaling does not
play a crucial role in wing patterning; JNK inactivation in the posterior
compartment (by Myc-Puc mis-expression) does not have drastic consequences.
Roles for JNK signaling can be identified in genitalia and thoracic bristles, where
it regulates developmentally programmed apoptosis (McEwen, 2005).
Can MKPs act as tumor suppressors? JNK signaling plays complex roles during
oncogenesis. It can prevent tumorigenesis by promoting apoptosis, and it can
promote tumorigenesis by supporting Ras-mediated or BCR-Abl-mediated
transformation. Since each tumor type has a unique set of mutations in oncogenes
and/or tumor suppressors, the phenotypic effects of JNK activation probably
differ depending on the activity of other pathways (McEwen, 2005).
Inhibition of apoptosis is one prerequisite for tumorigenesis. Thus
the consequences of JNK activation were examined when apoptosis was blocked. When cell death
was blocked in the posterior compartment of the wing and the restraints on JNK
activation were relaxed by puc heterozygosity, tissue overgrowth occurred.
Groups of posterior cells, presumably of clonal origin, exhibited elevated
levels of JNK activation, and formed small overgrowths both in developing
imaginal discs and in the resulting adult wings and legs. Thus, when apoptosis
is suppressed, JNK activation can lead to tissue over-growth (McEwen, 2005).
Related results have been reported with respect to
inducing apoptosis by diap inactivation, irradiation or Hid expression, while
simultaneously blocking caspase activation. This triggers non-autonomous
proliferation of neighboring cells, presumably to compensate for cell lost by
apoptosis. Furthermore, some of the 'undead' cells produced by these treatments
show JNK-dependent upregulation of Wg or Dpp. Wg signaling
has been shown to be required for compensatory proliferation. The current results extend previous studies,
since the current experiment did not actively induce
apoptosis, but simply blocked caspase activation in puc heterozygotes. Thus,
when apoptosis is inhibited and Puc repression is reduced, cells become
susceptible to runaway JNK activation. It is likely that analogous focal JNK
activation occurs in cells in which apoptosis is not blocked (e.g., anterior
compartment cells in these experiments), but JNK-induced apoptosis rapidly
eliminates them. At least a subset of the undead cells
activate expression of Wg, which may promote excess growth. Interestingly,
however, this ectopic Wg expression does not alter cell fates, at least in those
situations where overgrowths survive to be observed in the adult wing (McEwen, 2005).
Together, these data have interesting implications. In tumor cells in which
apoptosis is prevented, JNK signaling might switch from promoting apoptosis to
promoting proliferation by inducing Wnt or TGFbeta, suggesting how JNK signaling
can be both pro- and anti-tumorigenic. Future experiments should address
mechanisms by which JNK activation triggers Wnt and TGFβ signaling. These data
also suggest that runaway JNK activation can promote cell-autonomous
proliferation, since groups of cells with elevated JNK activity are associated with
overgrowths. Thus, JNK activation or loss of MKP-mediated JNK repression may
play multiple roles in tumors whose cells have lost the ability to die (McEwen, 2005).
Ectopic JNK activation occurred in small groups of cells in random positions
throughout the posterior compartment. The event(s) that initiate unrestrained
JNK activation in these cells remain to be defined. Perhaps there are stochastic
variations in JNK signaling that are normally below the threshold triggering
apoptosis. However, when Puc activity is reduced, some cells may exceed this
threshold, triggering runaway JNK activation in the initial cell and its
descendents. Variations in JNK activity may also be induced by spontaneous DNA
damage. Normally, such damage would trigger JNK-dependent apoptosis; however, if
apoptosis is blocked, the cells proliferate. Finally, loss-of-heterozygosity at
the puc locus could create cells lacking restraints on JNK signaling. Since mitotic
recombination in somatic tissue can occur, Puc might act in
a manner analogous to classic tumor suppressor genes. Additional studies will be
required to distinguish between these possibilities (McEwen, 2005).
In summary, this work establishes that Puc is a key negative regulator of
apoptosis throughout Drosophila development. In its absence, basal JNK activity
is poised to eliminate cells from the developing epithelium. The results
position JNK signaling in the hierarchy of events regulating radiation-induced
apoptosis. Finally, the data support the possibility previously suggested by
cytogenetics that MKPs may act as
tumor suppressor genes. These data prompt many new mechanistic questions
regarding the role of JNK signaling in apoptosis and oncogenesis (McEwen, 2005).
Tumours evolve several mechanisms to evade apoptosis, yet many resected carcinomas show significantly elevated caspase activity. Moreover, caspase activity is positively correlated with tumour aggression and adverse patient outcome. These observations indicate that caspases might have a functional role in promoting tumour invasion and metastasis. Using a Drosophila model of invasion, this study shows that precise effector caspase activity drives cell invasion without initiating apoptosis. Affected cells express the matrix metalloprotinase Mmp1 and invade by activating Jnk. These results link Jnk and effector caspase signalling during the invasive process and suggest that tumours under apoptotic stresses from treatment, immune surveillance or intrinsic signals might be induced further along the metastatic cascade (Rudrapatna, 2013).
Overall, these results indicate that effector caspase activity below levels sufficient to direct cell death might be optimal for migration of transformed cells. This signalling promotes migration through Jnk, consistent with previous studies showing that Jnk lies downstream of Dronc. Caspase activation of Jnk frequently leads to compensatory proliferation, a homeostatic programme of cell replacement after apoptosis. Compensatory proliferation studies of 'undead cells' have come to opposite conclusions concerning the role of Drice. The current work is consistent with the mammalian literature placing the JNK pathway as a caspase target (Rudrapatna, 2013).
Effector caspases are active in tumours in situ and are associated with metastasis; the current results indicate that cells with moderate caspase activity that are protected from apoptosis are prone to migration. In this view, therapeutic interventions proposed to increase tumour apoptosis might paradoxically exacerbate malignancy, as has been previously suggested. Tumour inflammation has also been suggested to promote metastasis and might do so via stimulation of the extrinsic apoptosis pathway. Tumour cells commonly contain high levels of XIAP, which blocks caspases' active site in a manner similar to P35. This might provide an important mechanism directing tumours to metastasize, though the experiments emphasize the importance of precise caspase activity. A better understanding of caspases' role in tumour progression might enhance ability to predict a tumour's progression and the impact of treatments designed to promote the apoptosis process (Rudrapatna, 2013).
Imd-mediated innate immunity is activated in response to infection by
Gram-negative bacteria and leads to the activation of Jun amino-terminal kinase
(JNK) and Relish, a NF-kappaB transcription factor responsible for the
expression of antimicrobial peptides. Plenty of SH3s (POSH) has been shown to
function as a scaffold protein for JNK activation, leading to apoptosis in
mammals. This study reports that POSH controls Imd-mediated immunity signalling in
Drosophila. In POSH-deficient flies, JNK activation and Relish induction were
delayed and sustained, which indicated that POSH is required for properly timed
activation and termination of the cascade. The RING finger of POSH, possessing
ubiquitin-ligase activity, is essential for termination of JNK activation.
POSH binds to and degrades TAK1, a crucial activator of both the JNK
and the Relish signalling pathways. These results establish a novel role for
POSH in the Drosophila immune system (Tsuda, 2005).
Among the components of the Imd pathway, TAK1 plays a crucial role as an
activator of both the JNK and the Relish pathways (Vidal 2001; Silverman, 2003; Park, 2004). Flies that are deficient in TAK1 do
not produce antibacterial peptides and are therefore highly susceptible to
Gram-negative bacterial infection (Vidal, 2001).
In Drosophila S2 cells, TAK1 has been shown to be required for the
peptidoglycan-induced activation of IkappaB
kinase (IKK) and JNK (Leulier, 2003; Silverman, 2003; Kaneko,
2004). TAK1 is also important for limiting the duration of JNK activation,
thereby resulting in a transient JNK-dependent response that precedes the
sustained induction of the Relish-dependent genes (Tsuda, 2005).
Although it has been shown that proteasomal degradation of
TAK1 leads to the rapid termination of JNK signalling (Park, 2004),
the degradation mechanism has yet to be entirely clarified,
and a ubiquitin ligase that promotes TAK1 degradation has not been reported.
This study identifies Plenty of SH3s (POSH) as a crucial component that controls the termination of immunity signalling in Drosophila. POSH, which was
initially isolated as a Rac1-interacting protein, consists of a RING-finger
domain and four SH3 domains, and has been shown to function as a scaffold
protein for JNK signalling components leading to apoptosis in mammals (Tapon, 1998; Xu, 2003). In Drosophila, overexpression of POSH in imaginal discs
activates JNK, which results in various defects in adult morphology (Seong, 2001). Based on flies that are deficient in
POSH, evidence was provided that POSH is required for the properly timed
activation and termination of Imd-mediated immune responses, and that the RING
finger of POSH, which shows ubiquitin-ligase activity, is essential for this
regulation (Tsuda, 2005).
To assess the role of POSH in vivo, a null allele of POSH
was created by the excision of a flanking P-element in EP(2)1026. A mutant was obtained
bearing a deletion that included the start codon and extended 1,282
nucleotide bases into the coding region of POSH. Northern blot analysis
indicated that POSH transcripts were not detectable in
POSH74 homozygous animals. Most
POSH74 homozygous individuals survived embryogenesis,
and the JNK cascade operating during dorsal closure remained intact. Furthermore,
POSH74 showed no genetic interaction with the JNK
pathway components in embryos. Taken together, these results
indicate that not every instance of JNK signalling requires POSH as a scaffold.
Although mutants showed no obvious developmental defects, adult
flies did not live as long as control flies, and older cultures often
contained dead larvae and pupae that were heavily melanized.
In addition, the viability of POSH-deficient flies
after injection with Escherichia coli was significantly lower than that
of wild type. A possible cause for such phenotypes is defective
immune function, and therefore a role for POSH in the immune
response was sought (Tsuda, 2005).
Whether the TAK1-mediated immune response was compromised by the
POSH74 mutation was tested. Infection with E. coli
activates TAK1, which consequently phosphorylates JNK and induces the expression
of Relish/NF-kappaB, resulting in the subsequent induction of a
battery of genes encoding antimicrobial peptides, such as Diptericin.
Northern blot analysis indicated that
the induction of relish and diptericin (dpt) messenger RNAs
after E. coli infection was significantly delayed in
POSH74 flies compared with that in controls.
Interestingly, it was also observed that expression of
relish was sustained for a longer period after infection in
POSH74 flies (Tsuda, 2005).
To investigate the effects of the POSH mutation more quantitatively,
real-time PCR analysis of immune response genes was performed.
Consistent with Northern blot analysis, the
induction of dpt was delayed, and relish induction was sustained,
with a maximum expression at 2 h after infection. The
expression pattern of attacin A (attA), which is dependent on
Relish, was also analyzed. Induction of attA was almost normal, but unlike
in the control flies, its expression level continued to increase up to 4 h after
infection. The maximum level of expression was twice that of control flies,
consistent with Relish activity being elevated in POSH mutant flies. The
expression profile of puckered (puc), a target
gene of the JNK pathway, was also analyzed. Induction of puc was slightly delayed, with the
highest level of expression at 1 h after infection, and expression was sustained
for a longer period in POSH74 flies compared with that
in controls. These results indicate that POSH is required for properly timed
activation and rapid termination of both JNK- and Relish-mediated immune
response pathways (Tsuda, 2005).
TAK1 has been shown to be required for the maintenance
of relish mRNA expression, and its
proteasomal degradation is responsible for the rapid termination of Relish and
JNK signalling (Park, 2004). POSH contains a
CH4C4-type RING-finger domain near its N terminus. It has been shown that the
RING-finger domain of mammalian POSH possesses E3 ubiquitin-ligase activity and
regulates the level of POSH protein through the proteasomal pathway (Xu, 2003).
Drosophila POSH also had E3 ubiquitin-ligase activity;
its auto-ubiquitination has been demonstrated. To assess the functional importance of the RING domain, POSHmR was constructed, bearing point mutations in the RING domain, and POSHDeltaR
lacking the RING-finger domain altogether. Neither of these
proteins was able to auto-ubiquitinate after expression in cultured
Drosophila cells (S2). It is conceivable, therefore, that POSH is
responsible for the feedback regulation of the immune response through TAK1
ubiquitination and its subsequent degradation. Observations indicate that
the RING-finger function of POSH is essential for the degradation of TAK1 (Tsuda, 2005).
To characterize further the role of
POSH in the regulation of JNK signalling, immunocompetent S2 cells were used in an
in vitro model system used to study the immune response triggered by
peptidoglycan in a commercial lipopolysaccharide preparation. Consistent with
puc expression results in POSH mutant flies, the reduction of POSH
by RNA interference (RNAi) significantly decreased the level of JNK activation,
but it was sustained for a longer period than in control cells.
In contrast, overexpression of POSH induced JNK
activation at a high level, but then inactivated it rapidly. When
POSHmR was transfected into S2 cells, phosphorylated JNK persisted
for a longer period than in control cells. The effects of POSH
overexpression and the role of the RING-finger function were examined
in vivo using
armadillo-GAL4 (arm-GAL4) as a ubiquitous expression driver.
Following bacterial infection, the expression level of
puc, a target of JNK signalling, rapidly declined in flies overexpressing
POSH (arm>POSH). In contrast, puc expression was sustained in
flies overexpressing POSHmR (arm>POSHmR),
which suggests that POSHmR has a dominant-negative effect on JNK
signalling. Together, these results indicate that POSH affects both activation
and inactivation of JNK signalling induced by microbial infection, and that the
RING-finger domain of POSH is essential for the negative feedback regulation of
JNK signalling. The profile of puc expression in
POSH74 flies is remarkably similar to that seen in
relish flies, which supports the idea that POSH mediates Relish-dependent inactivation of JNK in the immune system (Tsuda, 2005).
These results show that
POSH modulates the TAK1-mediated innate immune response. Following microbial
infection, POSH facilitates rapid JNK activation and the induction of
relish/NF-kappaB expression, both of which are mediated by TAK1
(Silverman, 2003). Conversely, POSH is also
required for the rapid termination of both JNK activation and relish
transcription through the E3 ubiquitin-ligase activity of the RING-finger
domain. The results indicate that POSH is involved in a mechanism of negative
feedback regulation of JNK and NF-kappaB
signalling. The Drosophila TAK1-mediated innate immune response is
similar to the tumour necrosis factor (TNF) signalling cascade in mammals.
Imd protein contains a death domain
with homology to that of mammalian TNF-receptor-interacting protein (RIP).
Most of the pathway
components (such as FADD, TAK1) and Drosophila homologues of
IKKgamma and IKKbeta are conserved in mammalian TNF signalling. The
activation of Relish involves its phosphorylation and cleavage by DREDD, the
Drosophila homologue of caspase 8. Eiger, a
Drosophila homologue of the TNF-alpha
superfamily ligand, has recently been identified. Overexpression of Eiger induces
cell death by activating the JNK signalling pathway, as in mammals. Although the
Eiger signalling pathway has not been fully elucidated in Drosophila, it
has been clearly shown that TAK1 is essential to transduce the cell-death
signal. It is possible that POSH is involved in Eiger signalling and modulates
TAK-mediated JNK signalling as in the immune system. It will be of interest to
determine whether POSH functions in mammalian TNF signalling (Tsuda, 2005).
Plenty of SH3s (POSH) functions as a scaffold protein for the Jun N-terminal kinase (JNK) signal transduction pathway, which leads to cell death in mammalian cultured cells and Drosophila. This study shows that POSH forms a complex with Apoptosis-linked gene-2 (ALG-2) and LKB1 modifiers, the JNK pathway was discovered as a novel component of LKB1 signaling. The JNK pathway is activated by LKB1 and mediates the LKB1-dependent apoptosis. Consistently, the LKB1-null mutant is defective in embryonic apoptosis and displayed a drastic hyperplasia in the central nervous system: these phenotypes are fully rescued by ectopic JNK activation as well as wild-type LKB1 expression. Furthermore, inhibition of LKB1 results in epithelial morphogenesis failure, which is associated with a decrease in JNK activity. Collectively, these studies unprecedentedly elucidate JNK as the downstream mediator of the LKB1-dependent apoptosis, and provide a new paradigm for understanding the diverse LKB1 functions in vivo (Lee, 2005).
Tumor suppressor LKB1 is a serine/threonine kinase that is heterozygotically mutated in the germ line of Peutz-Jegher syndrome (PJS) patients. PJS patients have a highly increased risk of malignant tumors, especially in the gastrointestinal tract, breast, uterine cervix, and ovary. Many of these tumors acquire somatic mutations in the remaining wild-type allele of LKB1. Moreover, various sporadic cancers are also associated with the loss of LKB1, implicating the general role of LKB1 in tumor suppression. Since point mutations of LKB1 found in PJS mostly reside in the highly conserved kinase domain, it is very likely that the kinase activity of LKB1 is essential for its tumor suppressive function (Lee, 2005 and references therein).
Since the discovery of LKB1 in 1998, various functions of LKB1 molecule have been suggested to be responsible for its tumor-suppressing activity, such as inhibition of cell cycle progression, cell growth retardation, apoptotic cell death, and cell polarity control. The LKB1 protein was originally characterized as a cell cycle inhibitor, and its putative downstream targets such as Brg1 and p21 were suggested to mediate LKB1-dependent cell cycle arrest. It has been recently proposed that LKB1 also regulates cellular growth by controlling another tumor suppressor, tuberous sclerosis complex (TSC), via the AMP kinase (AMPK)-dependent pathway. In addition, studies showing the absence of apoptosis in PJS polyps reveal that LKB1 is an inducer of apoptosis in vivo. Another tumor suppressor, p53, the first identified in vitro substrate of LKB1, was proposed as a mediator of this apoptosis. Finally, LKB1 was shown to be necessary for the polarization of intestinal epithelial cells and Drosophila ovary cells, demonstrating another important function of LKB1 in regulating cell structure (Lee, 2005).
Although considerable progress has been made to characterize the in vivo function of LKB1, only a limited amount of information concerning its molecular mechanisms has been found in detail; the major biological pathway responsible for the tumor suppressive function of LKB1 remains to be clarified (Lee, 2005).
The c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) family that mediates various cellular responses including programmed cell death, epithelial sheet movement, and planar polarity. The activity of JNK is tightly regulated by reversible phosphorylation, which is stimulated by a sequential cascade of protein kinases and inhibited by JNK-specific phosphatases. Activation of the JNK pathway is required for the release of cytochrome c from the mitochondria and the subsequent activation of the caspase cascade. Therefore, abrogation of the JNK signaling pathway causes various defects in developmental or stress-induced apoptosis. In addition to controlling apoptosis, regulation of epithelial morphogenesis is another well-known function of the JNK signaling pathway. Depletion of JNK activity results in various epithelial defects, including those of dorsal closure and planar polarity in Drosophila, and of the optic and neuronal system in mice (Lee, 2005 and references therein).
Since JNK is required for apoptosis and epithelial organization, disruption of the JNK signaling pathway is thought to be a prerequisite of tumorigenesis by enabling tumor cells to evade programmed cell death and to acquire a metastatic potential. Indeed, JNK1 and JNK2 are both required for the suppression of oncogenic transformation and tumorigenesis. Loss of JNK3, which is selectively expressed in neuronal cells, is also closely associated with human brain tumors. Furthermore, the Mkk4 gene, which encodes an upstream kinase of JNK, has been identified as a tumor suppressor gene and a metastasis suppressor gene (Lee, 2005 and references therein).
This paper shows a genetic connection between tumor suppressor LKB1 and the JNK signaling pathway. The results indicate that LKB1 activates the JNK pathway in vivo and that the JNK pathway mediates the LKB1-dependent apoptotic cell death (Lee, 2005 )
Using various Gal4 drivers, a high-level expression was obtained of both wild-type and kinase-dead LKB1 in the specific region of various tissues: the posterior region of the eye imaginal disc by the glass multiple reporter-Gal4 driver (gmr-Gal4), the dorsal region of the wing disc by the apterous-Gal4 driver (ap-Gal4), and the central pouch region of the wing disc by the ms1096-Gal4 driver). The expression levels of the LKB1 protein were determined by immunostaining using Drosophila LKB1-specific antisera. Eye-specific expression of wild-type LKB1 by the gmr-Gal4 driver induced a slight reduction in overall eye size, and this reduction became severer when the LKB1 expression level was elevated. However, the kinase-dead LKB1 overexpression did not induce this specific phenotype, showing that the kinase activity of LKB1 is important in inducing tissue-size reduction. The dorsal region-specific expression of wild-type and kinase-dead LKB1 by the ap-Gal4 driver displayed more dramatic phenotypes. Since Drosophila wing discs are composed of two layers of tissues, dorsal and ventral, alterations in size of only the dorsal layer give rise to bent-up or bent-down wing phenotypes. Although the dorsal tissue-specific expression of kinase-dead LKB1 did not induce any alterations, wild-type LKB1 induced a dramatic bent-up wing phenotype in a dose-dependent manner. Consistently, the ms1096-Gal4-driven expression of wild-type LKB1 dose-dependently reduced the overall wing size, although kinase-dead LKB1 did not. Therefore, it is concluded that Drosophila LKB1 negatively regulates the tissue and organ size in a kinase activity-dependent manner (Lee, 2005).
To examine the effect of LKB1 in individual cells, clonal analyses was performed by inducing LKB1 overexpression in a specific subset of cells within wing imaginal discs. Compared to the control clones expressing only GFP, the LKB1-expressing clones expanded very poorly. Similarly, overexpression of the TSC complex, another well-known tumor suppressor, also decreased the clone size, although with a milder severity than the overexpression of LKB1 (Lee, 2005).
Since the LKB1-dependent reduction of tissue size appropriately reflected the
tumor-suppressing activity of LKB1, whether this tissue-size reduction is caused by cell cycle arrest or apoptotic cell death was assessed. Both these phenomena have been proposed as major cellular processes underlying the tumor-suppressing function of LKB1. To check the G1-S cell cycle progression of LKB1-expressing tissues, the bromodeoxyuridine (BrdU) incorporation assay, which specifically labels S-phase cells, was performed. Although LKB1 is highly expressed in these tissues, no significant alterations in the S-phase cell number were detected with respect to the control tissues. Similarly, there was no change in the antiphosphospecific histone 3 (PH3) antibody staining pattern between the wild-type and the LKB1-expressing tissues, demonstrating that G2-M cell cycle transition was also unaffected. However, when the equivalent tissues were subjected to acridine orange (AO) staining, which specifically labels the dying cells with disrupted membrane integrity, they displayed prominent death signals, specifically in the region of LKB1 overexpression. These results strongly implied that the LKB1-induced organ size reduction is caused by apoptotic cell death, not by cell cycle arrest. Furthermore, TUNEL staining of the eye and wing discs also confirmed the existence of extensive apoptosis in the LKB1-expressing cells. Therefore, it was concluded that LKB1 is a potent apoptosis instigator in Drosophila (Lee, 2005).
Tumor suppressor LKB1 is a unique serine/threonine kinase with no other close relatives in the mammalian genome. Likewise, only a single LKB1 orthologue exists in the Drosophila genome, implying well-conserved functional characteristics between the mammalian and Drosophila LKB1. This study has discovered a novel functional connection between LKB1 and the JNK pathway: the JNK pathway is present downstream of the LKB1-dependent signaling pathway and controls apoptosis and organ size in response to LKB1 activities (Lee, 2005).
Previous studies on classical tumor suppressors in the Drosophila model system showed that hyperactivation of each tumor suppressor negatively regulates organ size by cell size reduction, cell cycle inhibition, or cell death promotion. LKB1 negatively regulates organ size in Drosophila in a kinase activity-dependent manner. Recent reports on the TSC tumor suppressor suggested that LKB1 suppresses TOR activity via the TSC complex, ultimately resulting in inhibition of cell growth and proliferation. Likewise, it was observed that TSC overexpression dramatically reduces cell size in Drosophila clonal overexpression analyses. However, unexpectedly, LKB1 overexpression did not induce any cell size reduction, in stark contrast to the case of TSC. Moreover, LKB1 overexpression in the clones of endoreplicating tissues altered neither cell size nor DNA content, confirming that the tumor-suppressing activity of LKB1 is not related to the negative regulation of cell growth. Therefore, it is concluded there is no physiological connection between LKB1 and TSC in the context of cell growth control in Drosophila (Lee, 2005).
However, histological analyses clearly demonstrate that the LKB1-overexpressing tissues suffer caspase-dependent programmed cell death, without apparent alteration in G1/S and G2/M cell cycle transition. The cell death induced by LKB1 overexpression displays the most typical phenotypes of apoptosis, such as membrane integrity disruption, DNA fragmentation, and caspase activation. Furthermore, endogenous LKB1 was physiologically required for normal embryonic apoptosis, and also essential for the size control of the central nervous system. Consistent with the results, LKB1 is reported to be highly expressed in apoptotic cells of the small intestine in humans, and PJS polyps that lack the functional LKB1 protein possessed fewer apoptotic cells than in adjacent normal tissues. Collectively, these results strongly suggested that instigation of apoptosis is a critical function of tumor suppressor LKB1 (Lee, 2005).
Surprisingly, in contradiction to previous research done with the mammalian system, the LKB1-induced apoptosis in Drosophila is a p53-independent event; it is not blocked by the expression of a dominant-negative version of p53. Moreover, apoptosis induced by DNA damage or p53 overexpression is also unaffected by downregulation of LKB1 activity. Furthermore, diagnostic symptoms and major tumor types of PJS patients are highly distinct from those of Li-Fraumeni syndrome patients Therefore, it is very likely that LKB1 and p53 are indirectly related since they both have caspase activation as a common downstream signaling event (Lee, 2005).
Genetic screening using the Drosophila model system enabled discovery or the JNK pathway as a downstream effector of the LKB1-dependent signaling pathway. Through simple genetic screening, more than 500 genes for interaction with LKB were found; this study covered various signal transduction pathways including the Ras/ERK, JNK, PI3K, TOR, Wnt, TGF-beta, and NF-kappaB pathways. Among them, some components of the JNK signaling pathway showed outstanding genetic interactions with LKB1, strongly enhancing the tissue-size reduction phenotype of LKB1. This result implied the specific involvement of the JNK pathway in LKB1-dependent signal transduction, prompting additional studies carried out concerning the relationship between LKB1 and the JNK signaling pathway (Lee, 2005).
These biochemical and histological analyses clearly demonstrated that JNK can be activated by LKB1 overexpression. Since the kinase-dead mutant of LKB1 did not induce this JNK activation, this phenomenon seems to be a direct consequence of LKB1 phosphotransferase activity. Moreover, blockage of the JNK signaling pathway strongly suppresses the LKB1-induced apoptosis, as well as the tissue-size reduction. Furthermore, the activation of JNK signaling is shown to be involved in the physiological apoptosis induced by endogenous LKB1. Therefore, it is concluded that the JNK pathway acts as a downstream effector of LKB1 by mediating the LKB1-induced apoptosis. Accordingly, any signals upregulating the kinase activity of LKB1 would also activate the JNK pathway, ultimately leading to apoptosis in the cell. In fact, some microtubule-disrupting agents that induce the LKB1-dependent cell death also activate the JNK pathway to promote apoptosis (Lee, 2005).
Another interesting point of this research is that inhibition of LKB1 results in defective epithelial morphogenesis, which is associated with low JNK activity. Several previous studies already demonstrated the requirement of LKB1 and JNK for epithelial integrity and polarity, both in the mammalian and Drosophila systems. Consistently, it was observed that inhibition of LKB1 activity during development causes various morphogenesis defects including malformation of adult thoraxes and failure in embryonic dorsal closure. Furthermore, LKB1 is shown to be required for maintaining adequate JNK activity during embryonic and larval development. Since loss of tissue integrity is a common feature of PJS polyps and cancer, the role of LKB1 in epithelial organization seems to be also important for mediating its tumor-suppressing activity (Lee, 2005).
This study has demonstrated that the JNK pathway is involved in the LKB1-dependent apoptosis and epithelial morphogenesis in Drosophila. Since deregulation of both LKB1 and the JNK pathways is highly implicated in cancer development, the novel connection between LKB1 and the JNK signaling pathway provides a new direction for future studies in better understanding the complex functions of tumor suppressor LKB1 (Lee, 2005).
Jun N-terminal kinase (JNK) signaling is a highly conserved pathway that controls both cytoskeletal remodeling and transcriptional regulation in response to a wide variety of signals. Despite the importance of JNK in the mammalian immune response, and various suggestions of its importance in Drosophila immunity, the actual contribution of JNK signaling in the Drosophila immune response has been unclear. Drosophila TAK1 has been implicated in the NF-kappaB/Relish-mediated activation of antimicrobial peptide genes. However, this study demonstrates that Relish activation is intact in dTAK1 mutant animals, and that the immune response in these mutant animals is rescued by overexpression of a downstream JNKK. The expression of a JNK inhibitor and induction of JNK loss-of-function clones in immune responsive tissue revealed a general requirement for JNK signaling in the expression of antimicrobial peptides. The data indicate that dTAK1 is not required for Relish activation, but instead is required in JNK signaling for antimicrobial peptide gene expression (Delaney, 2006).
Innate immune responses are critical for a rapid host defense against pathogens. The signaling pathways that control these responses are present in all multicellular organisms, ranging from humans to flies, and are remarkably well conserved. Although the innate response lacks the antigen recognition capacity of vertebrate adaptive immunity, it is nevertheless complex and crucial for host survival. Drosophila is a proven genetic model organism for the study of innate immunity and has provided invaluable insights into the control of responses to infection (Delaney, 2006).
Toll and Imd are the founding members of two principal innate immune response signaling pathways in Drosophila. Toll signals through two NF-kappaB/Rel family transcription factors, Dif and Dorsal, and is required for responses to fungal and Gram+ bacterial infections. Imd signaling controls primarily Gram- bacteria-specific responses through the cleavage and activation of a third Rel family transcription factor, Relish, by the Drosophila caspase Dredd. Relish activation also requires an IkappaB kinase (IKK) complex that is itself activated by Imd signaling. The transcriptional targets of Dif and Relish are not entirely distinct. For example, cecropinA expression requires either Relish or Dif, or both, depending on the type and strain of infecting microorganism. More than 20 Drosophila genes have been implicated in these signaling pathways and nearly all of them have mammalian homologues with conserved immune functions (Delaney, 2006 and references therein).
Jun N-terminal kinase (JNK) signaling has been linked to stress responses, cell migration, apoptosis, and immune responses in both insects and mammals. JNK activity can be induced by infection, lipopolysaccharide, and inflammatory cytokines such as tumor necrosis factor (TNF) in flies and mammals. Null mutations in JNK signaling components are typically embryonic lethal in flies and thus unlikely to appear as targets of mutagenesis screens designed to detect immune response genes in living animals. An exception to this rule is dTAK1. Overexpression and dominant-negative studies indicated that dTAK1 can act as a JNK kinase kinase (Delaney, 2006 and references therein).
Previously characterized dTAK1 mutations, however, showed no apparent JNK-like phenotype, but failed to express Relish-dependent antimicrobial peptides, suggesting a role in the Imd pathway (Vidal, 2001). Previous epistasis analysis using the UAS/GAL4 overexpression system to ectopically express dTAK1 placed dTAK1 downstream of imd and upstream of the IKK complex in the Relish signaling pathway (Vidal, 2001). In vitro experiments implicated dTAK1 in the IKK-dependent phosphorylation of Relish in S2 cells (Delaney, 2006).
Evidence has been uncovered for a Relish-independent function of dTAK1 in the control of antimicrobial peptide gene expression. Several aspects of Relish activation appeared normal in infected dTAK1 mutant animals, including cleavage, nuclear localization, and promoter binding. Therefore whether JNK pathway components mediate dTAK1 function in the immune response was examined. Several lines of evidence are reported for dTAK1 acting through the JNK cascade in the innate immune response. First, overexpression of Hemipterous, a JNKK, rescued attacin and diptericin expression in dTAK1 mutant animals, whereas overexpression of the downstream Imd component Dredd did not. Second, it was found that expression of the Puckered (Puc) phosphatase, an inhibitor of JNK activity, suppressed the expression of antimicrobial peptide genes. To directly test for a JNK requirement in immune signaling, JNK mutant clones were induced in the fat body of larvae. Strikingly, diptericin, attacin, Metchnikowin, and Drosomycin expression was lost in the mutant tissue (Delaney, 2006).
It is concluded that the JNK pathway is required to mediate dTAK1 signaling during the Drosophila immune response. Furthermore, a model is proposed where the JNK and NF-kappaB signaling are both required to activate antimicrobial peptide gene expression during the immune response in the Drosophila fat body (Delaney, 2006).
The function of TAK1 in vertebrates has remained enigmatic. It was originally identified as a TGFβ-activated kinase, hence the name, in mammalian cell culture assays. However, follow-up work in multicellular contexts and in vivo analyses in vertebrates, C. elegans, and Drosophila have shown no clear link to TGFβ signaling, but rather suggest a role for TAK family kinases in JNK activation or as upstream activators of Nemo-like kinases. In mammalian systems, TAK1 is one of a number of kinases that can activate IKK complexes and, consequently, NF-kappaB signaling in vitro. In vitro studies of human cells have shown that targeting of TAK1 by RNAi reduces NF-kappaB activation by TNFalpha and IL-1 stimulation. Recent studies using fibroblasts derived from TAK1 mutant mouse embryos and mice with a B-cell-specific deletion of TAK1 showed that JNK activation was impaired in response to all stimuli tested in TAK1 mutant cells. Although NF-kappaB activation was impaired in response to stimulation by IL-1β, TNF, and TLR3 and TLR4 ligands, NF-kappaB activation by B-cell receptor or LT-β stimulation remained intact, suggesting a specific role for TAK1 upstream of IKKβ and JNK, but not IKKalpha. Interestingly, IKKalpha activation leads to the phosphorylation and processing of NF-kappaB2 from the p100 to the active p52 form, reminiscent of Relish activation in Drosophila (Delaney, 2006 and references therein).
Biochemical analyses in mammalian systems have demonstrated that TAK1 functions in multimeric protein complexes that can include TAB1, TAB2, and different TRAF proteins. The exact composition of these complexes seems to determine TAK1 responsiveness and downstream effects. In the fly, genetic studies found an interaction between dTRAF1 and dTAK1 in the activation of JNK signaling and apoptosis. Gain- and loss-of-function analyses indicate that dTRAF2, but not dTRAF1, is necessary for the activation of Relish-dependent gene expression; however, no interaction between dTRAF2 and dTAK1 in the activation of antimicrobial peptides has been reported (Delaney, 2006).
Genome-wide analyses that examined in vivo responses in Drosophila identified dJun and puc as genes potentially regulated by Toll and Imd signaling, suggesting a cross-regulation between these pathways and the JNK signaling pathway. A study recently reported that RNAi knockdown of kayak, msn, hep, or aop blocked E. coli-induced attacin and drosomycin expression in S2 cells. Furthermore, in related studies, it was also observed that, although dTAK1 RNAi-treated S2 cells failed to express an attacin reporter gene, Relish cleavage and nuclear localization remain intact in these cells. Other RNAi analyses in S2 cells have concluded that JNK signaling does not have a significant role in antimicrobial peptide gene expression. However, RNAi against hep or bsk seemed to partially block antimicrobial peptide induction, especially of attacin and cecropinA and, accordingly, attacinD levels were lower in microarrays when the JNK pathway was blocked. The current results confirm a positive role for JNK signaling in the antimicrobial peptide response in vivo (Delaney, 2006).
The placement of dTAK1 function upstream of JNK, rather than IKK, requires a remodeling of the signaling pathways that activate the antimicrobial peptide genes. Earlier models were based on studies that showed that dTAK1 mutations blocked the constitutive activation of diptericin by Imd overexpression (Vidal, 2001). In turn, IKK mutations blocked dTAK1-induced diptericin expression. One interpretation of these data places IKK directly downstream of dTAK1. However, if the activation of both JNK and IKK signaling pathways is required, then a disruption in either branch would be sufficient to suppress any upstream activation (Delaney, 2006).
Overexpression of dTAK1 is sufficient to induce antimicrobial peptide expression (Vidal, 2001). However, dTAK1 is an extremely potent activator of JNK signaling and apoptosis, and overexpression of dTAK1 could activate proteins that are not normal phosphorylation targets. Based on RNAi studies in S2 cells, dTAK1 is required for dIKK complex-dependent phosphorylation of Relish in vitro. This could reflect a stringent requirement for dTAK1 in blood cell-derived S2 cells that is different in fat body tissue (Delaney, 2006).
The new model would predict that overexpression of the Dredd caspase would be insufficient to activate fully the antimicrobial peptides in dTAK1 mutant animals and this is indeed the case. Overexpression of Dredd may be sufficient to induce antimicrobial peptide gene expression in a wild-type background because of inadvertent JNK pathway activation by ectopic caspase activity or by the heat-shock protocol itself. Alternatively, an additional role for Dredd has been proposed in the ubiquitin-mediated activation of dTAK1 and the dIKK complex (Zhou, 2005). The suppression by dTAK1 mutants of ectopic Dredd expression is consistent with this model as well, and does not distinguish between the two potential functions of Dredd. The current data are consistent with a model that places dTAK1 activity in a pathway parallel to the functions of IKK and Relish and in which both these pathways are required for the activation of antibacterial peptide genes such as diptericin and attacin (Delaney, 2006).
Promoter analyses of most antimicrobial peptide genes have not revealed any obvious binding sites for activator protein-1 (AP-1) complexes, the Jun/Fos heterodimer, and transcriptional mediator of JNK signaling. However, AP-1 binding sites can be quite diverse and are not always predictable directly from DNA sequence. Nevertheless, a recent study identified a functional AP-1 binding site in the attacinA promoter. These data suggest that AP-1 binding represses attacinA transcription by recruiting histone deacetylase 1 (dHDAC1) to the promoter. In contrast, in mammalian studies, c-Jun function is itself repressed by association with HDAC3. This repression is relieved upon JNK signaling. A similar mechanism may be employed in the Drosophila fat body. Accordingly, the sustained expression of attacin and other antimicrobial peptide genes in vivo would require an activation (or de-repression) of AP-1 function at the onset of the immune response. Such positive cooperation between AP-1 and NF-kappaB transcription factors was also seen in molecular studies of the human β-defensin-2 promoter (Delaney, 2006).
AP-1-dependent gene expression is normally rapid. Thus, if AP-1 activity is not directly required for diptericin expression, it could act indirectly through the activation of other genes. Alternatively, JNK could phosphorylate some targets other than the AP-1 complex proteins Jun and Fos. In mammalian studies, it has been shown that JNK can phosphorylate, and thereby inhibit, Insulin Receptor Substrate-1. However, the recent finding that RNAi against kayak/dFos can block antimicrobial peptide expression and the current dJun loss-of-function studies in vivo suggest that JNK does indeed signal through AP-1 to control expression of these genes (Delaney, 2006).
It is intriguing that overexpressed Puc not only blocks Relish-dependent antimicrobial peptide gene expression, but it also strongly blocks drosomycin expression, which is not true in dTAK1 mutants. This suggests that JNK or JNK-related proteins, for example, p38a, p38b, and MPK2, may also be important for other aspects of the immune response, for example, the Toll/Dif-dependent antimicrobial genes. The clonal analysis of JNK mutant tissue confirms that JNK is required not only for the expression of Gram--specific peptides diptericin and attacin, but also for Metchnikowin (Gram+/fungal specific) and drosomycin (fungal specific). Mutations in dTAK1 had less of an impact on Metchnikowin or drosomycin expression than on attacin, for example. Furthermore, reduced dJun activation occurred in dTAK1 mutant animals, indicating that other upstream kinases may be involved in the control of these genes. JNK is a member of a large family of mitogen-activated protein kinases (MAPKs). In the fly, there are at least five MAPKKKs, four MAPKKs, and five MAPKs, and so the potential redundancies are many. If these other proteins contribute to the immune response, how they do so has yet to be tested in genetic loss-of-function in vivo studies in the fat body (Delaney, 2006).
How JNK and NF-kappaB signals integrate to positively control gene expression is a critical question. This study has demonstrated that both are required for the expression of a particular set of immune responsive genes in vivo. Through the use of Drosophila genetics, it should be possible to identify novel immune response genes that are controlled cooperatively by JNK and NF-kappaB signaling. From promoter analysis of these genes, it may be possible to predict additional genes that are important for other biological processes. Both the JNK and NF-kappaB signaling pathways have been implicated many times in many different contexts. Continued analysis in Drosophila may lead to a general understanding of their roles in normal biological processes and developmental malignancies (Delaney, 2006).
Apparent defects in cell polarity are often seen in human cancer. However, the underlying mechanisms of how cell polarity disruption contributes to tumor progression are unknown. Using a Drosophila genetic model for Ras-induced tumor progression, a molecular link has been shown between loss of cell polarity and tumor malignancy. Mutation of different apicobasal polarity genes activates c-Jun N-terminal kinase (JNK) signaling and downregulates the E-cadherin/β-catenin adhesion complex, both of which are necessary and sufficient to cause oncogenic RasV12-induced benign tumors in the developing eye to exhibit metastatic behavior. Furthermore, activated JNK and Ras signaling cooperate in promoting tumor growth cell autonomously, since JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras. The finding that such context-dependent alterations promote both tumor growth and metastatic behavior suggests that metastasis-promoting mutations may be selected for based primarily on their growth-promoting capabilities. Similar oncogenic cooperation mediated through these evolutionarily conserved signaling pathways could contribute to human cancer progression (Igaki, 2006).
Most human cancers originate from epithelial tissues. These epithelial tumors, except for those derived from squamous epithelial cells, normally exhibit pronounced apicobasal polarity. However, these tumors commonly show defects in cell polarity as they progress toward malignancy. Although the integrity of cell polarity is essential for normal development, how cell polarity disruption contributes to the signaling mechanisms essential for tumor progression and metastasis is unknown. To address this, a recently established Drosophila model of Ras-induced tumor progression triggered by loss of cell polarity has been used. This fly tumor model exhibits many aspects of metastatic behaviors observed in human malignant cancers, such as basement membrane degradation, loss of E-cadherin expression, migration, invasion, and metastatic spread to other organ sites (Pagliarini, 2003). In the developing eye tissues of these animals, loss of apicobasal polarity is induced by disruption of evolutionarily conserved cell polarity genes such as scribble (scrib), lethal giant larvae (lgl), or discs large (dlg), three polarity genes that function together in a common genetic pathway, as well as other cell polarity genes such as bazooka, stardust, or cdc42. Oncogenic Ras (RasV12), a common alteration in human cancers, causes noninvasive benign overgrowths in these eye tissues (Pagliarini, 2003). Loss of any one of the cell polarity genes somehow strongly cooperates with the effect of RasV12 to promote excess tumor growth and metastatic behavior. However, on their own, clones of scrib mutant cells are eliminated during development in a JNK-dependent manner; expression of RasV12 in these mutant cells prevents this cell death (Igaki, 2006).
To better quantify the metastatic behavior of tumors in different mutant animals, the analysis focused on invasion of the ventral nerve cord (VNC), a process in which tumor cells leave the eye-antennal discs and optic lobes (the areas where they were born) and migrate to and invade a different organ, the VNC. It was further confirmed that the genotypes associated with the invasion of the VNC in this study also resulted in the presence of secondary tumor foci at distant locations, although the number and size of these foci were highly variable (Pagliarini, 2003; Igaki, 2006).
In analyzing the global expression profiles of noninvasive and invasive tumors induced in Drosophila developing eye discs, it was observed that expression of the JNK phosphatase puckered (puc) was strongly upregulated in the invasive tumors. Upregulation of puc represents activation of the JNK pathway in Drosophila. Therefore an enhancer-trap allele, puc-LacZ, was used to monitor the activation of JNK signaling in invasive tumor cells. Strong ectopic JNK activation was present in invasive tumors, while only a slight expression of puc was seen in restricted regions of RasV12-induced noninvasive overgrowth. Intriguingly, more intense JNK activation was seen in tumor cells located in the marginal region of the eye-antennal disc and tumor cells invading the VNC. Analysis of clones of cells with a cell polarity mutation alone revealed that JNK signaling was activated by mutation of cell polarity genes. Notably, JNK signaling was not activated in a strictly cell-autonomous fashion. JNK activation in these cells was further confirmed by anti-phospho-JNK antibody staining that detects activated JNK (Igaki, 2006).
To examine the contribution of JNK activation to metastatic behavior, the JNK pathway was blocked by overexpressing a dominant-negative form of Drosophila JNK (BskDN). As previously reported (Pagliarini, 2003), clones of cells mutant for scrib, lgl, or dlg do not proliferate as well as wild-type clones, while combination of these mutations with RasV12 expression resulted in massive and metastatic tumors. Strikingly, inhibition of JNK activation by BskDN completely blocked the invasion of the VNC, as well as secondary tumor foci formation. Drosophila has two homologs of TRAF proteins (DTRAF1 and DTRAF2), which mediate signals from cell surface receptors to the JNK kinase cascade in mammalian systems. It was found that RNAi-mediated inactivation of DTRAF2, but not DTRAF1, in the tumors strongly suppressed their metastatic behavior. Inactivation of dTAK1, a Drosophila JNK kinase kinase (JNKKK), or Hep, a JNKK, also suppressed metastatic behavior. Drosophila has two known cell surface receptors that act as triggers for the JNK pathway, Wengen (TNF receptor) and PVR (PDGF/VEGF receptor). Intriguingly, it was found that RNAi-mediated inactivation of Wengen partially suppressed tumor invasion. Inactivation of PVR, in contrast, did not show any suppressive effect on metastatic behavior. It was also found that the metastatic behavior of RasV12-expressing tumors that were also mutated for one of three other cell polarity genes, bazooka, stardust, or cdc42, was also blocked by BskDN. These data indicate that loss of cell polarity contributes to metastatic behavior by activating the evolutionarily conserved JNK pathway (Igaki, 2006).
Next, whether JNK activation is sufficient to trigger metastatic behavior in RasV12-induced benign tumors was examined. Two genetic alterations can be used to activate JNK in Drosophila. First, JNK signaling can be activated by overexpression of Eiger, a Drosophila TNF ligand. While mammalian TNF superfamily proteins activate both the JNK and NFκB pathways, Eiger has been shown to specifically activate the JNK pathway through dTAK1 and Hep. Indeed, the eye phenotype caused by Eiger overexpression could be reversed by blocking JNK through Bsk-IR (Bsk-RNAi). Second, overexpression of a constitutively activated form of Hep (HepCA) can also activate JNK signaling. However, the eye phenotype caused by HepCA overexpression was only slightly suppressed by Bsk-IR, suggesting that HepCA overexpression may have additional effects other than JNK activation. Therefore Eiger overexpression was used to activate JNK in RasV12-induced benign tumors, and it was found that the RasV12+Eiger-expressing tumor cells did not result in the invasion of the VNC. This indicates that loss of cell polarity must induce an additional downstream effect(s) essential for metastatic behavior. A strong candidate for the missing event is downregulation of the E-cadherin/catenin adhesion complex, since this complex is frequently downregulated in malignant human cancer cells and is also downregulated by loss of cell polarity genes in Drosophila invasive tumors (Pagliarini, 2003). In addition, it has been recently reported that higher motility of mammalian scrib knockdown cells can be partially rescued by overexpression of E-cadherin-catenin fusion protein, suggesting a role of E-cadherin in preventing polarity-dependent invasion. Furthermore, overexpression of E-cadherin blocks metastatic behavior of RasV12/scrib−/− tumors (Pagliarini, 2003), indicating that loss of E-cadherin is essential for inducing tumor invasion in this model. It was found that loss of the Drosophila E-cadherin homolog shotgun (shg), combined with the expression of both RasV12 and Eiger, induced the invasion of the VNC. Intriguingly, loss of shg in RasV12-expressing clones also showed a weak invasive phenotype at lower penetrance. In agreement with the essential role of JNK in tumor invasion, clones of shg−/− cells weakly upregulated puc expression. It was further found that JNK activation in dlg−/− clones is not blocked by overexpression of E-cadherin, suggesting that mechanism(s) other than loss of E-cadherin also exist for inducing JNK activation downstream of cell polarity disruption. The metastatic behavior of RasV12+Eiger/shg−/− tumors was completely blocked by coexpression of BskDN, indicating a cell-autonomous requirement of JNK activation for this process. Furthermore, it was found that loss of the β-catenin homolog armadillo also induced metastatic behavior in RasV12-induced benign tumors. In contrast, overexpression of HepCA in RasV12/shg−/− cells resulted in neither enhanced tumor growth nor metastatic behavior. Together, these results suggest that, although the RasV12+Eiger/shg−/− does not completely phenocopy the effect of RasV12/scrib−/−, activation of JNK signaling and inactivation of the E-cadherin/catenin complex are the downstream components of cell polarity disruption that trigger metastatic behavior in RasV12-induced benign tumors (Igaki, 2006).
Aside from its evolutionarily conserved role in cell migration and invasion, JNK signaling is also a potent activator of cell death in Drosophila and mammals. Although RasV12-expressing tissues showed a weak and restricted activation of JNK at later stages of development, mutation of cell polarity genes in combination with RasV12 expression constitutively activated JNK signaling. Striking acceleration of tumor growth occurred during days 5 and 6, and these tumors outcompeted surrounding wild-type tissues, resulting in a loss of the unlabeled wild-type cells and a dramatic increase in the GFP-expressing mutant tissue. The activated JNK was correlated with this accelerated tumor growth, suggesting that JNK signaling may play a role in tumor growth. Indeed, in addition to blocking metastatic behavior, inactivation of JNK pathway components strongly suppressed the accelerated tumor growth caused by cell polarity disruption. These results reveal that JNK signaling activated by loss of cell polarity also stimulates tumor growth (Igaki, 2006).
Since JNK signaling is required for both tumor growth and invasion, it was next asked whether these two phenotypes are separable processes. To address this, different types of tumors caused by alterations in genes involved in cell proliferation, growth, and cell polarity were analyzed. Day 6 RasV12/scrib−/− tumors showed moderate tumor growth and VNC invasion phenotypes. Loss of the Akt gene, a component of insulin growth signaling, considerably reduced the tumor load of RasV12/scrib−/− animals but did not impair metastatic behavior. In contrast, overexpression of Akt, combined with mutations in both the scrib gene and the lats gene, a potent tumor suppressor, did not cause metastatic behavior despite accelerated tumor growth comparable to RasV12/scrib−/−. In addition, although RasV12/Tsc1−/− mutant cells resulted in extremely large tumors, these tumor cells never exhibited metastatic behavior. These data indicate that tumor growth and invasion are separable processes in this model system (Igaki, 2006).
It was found that JNK signaling is indeed activated in polarity-deficient cells, and acridine orange staining revealed that most of these cells die. Interestingly, ectopic cell death was mostly blocked within clones of polarity-deficient cells also expressing RasV12, despite strong JNK activation. In addition, coexpression of RasV12 and Eiger, a potent inducer of cell death, resulted in accelerated tumor growth, although neither RasV12 alone nor Eiger alone caused dramatic overgrowth. This massive overgrowth was completely blocked by coexpression of BskDN. Moreover, stimulation of JNK signaling by expressing Eiger dramatically enhanced tumor growth of RasV12/shg−/− tissues, although Eiger/shg−/− clones were very small, probably because of cell death of these mutant clones. The accelerated growth of the RasV12+Eiger/shg−/− tumors was again completely blocked by BskDN. Together, these data indicate that, in the context of oncogenic Ras, JNK activation is the primary mediator of tumor growth downstream of cell polarity disruption. These observations suggest that JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras, and that the dramatic tumor growth is caused by cooperation between oncogenic Ras and JNK signaling (Igaki, 2006).
This study provides a molecular link between loss of cell polarity and tumor malignancy, namely activation of JNK signaling and inactivation of the E-cadherin/catenin complex in the context of oncogenic Ras activation. Although RasV12 alone only induces noninvasive overgrowth, and loss of cell polarity alone results in JNK-mediated cell death, the combination of these two alterations promotes both tumor growth and invasion through oncogenic cooperation. Thus, the tumor-promoting alterations caused by loss of cell polarity do not function alone and rather act as oncogenic Ras modifiers or “oncomodifiers” (Igaki, 2006).
The JNK signaling is essential for a variety of biological processes such as morphogenesis, cell proliferation, migration, invasion, and cell death. Genetic studies in Drosophila have demonstrated that JNK signaling is essential for epithelial cell movements and invasive behavior during normal development. A genetic study in mice revealed that TNF-triggered JNK signaling stimulates epidermal proliferation. These studies suggest that JNK may play an important role in tumorigenesis, tumor growth, and metastasis. Indeed, a substantial body of evidence indicates that JNK activation and c-Jun phosphorylation play important roles in cancer development. In mammalian cell culture systems, Ras acts cooperatively with JNK or c-Jun to enhance cellular transformation. Furthermore, knockin mice expressing a mutant form of c-Jun (JunS63A,S73A) suppress development of skin tumors in response to Ras activation and also block development of intestinal epithelial cancers caused by APC mutation. Moreover, liver-specific inactivation of c-Jun impairs development of chemically induced hepatocellular carcinomas. Furthermore, JNK signaling is activated in many tumor types. On the contrary, however, it has been also shown that JNK functions as a negative regulator for tumor development in Ras/p53-transformed fibroblasts. Thus, the role of JNK signaling seems to be highly dependent on cellular context, and, this study provides the first evidence for a cell-autonomous oncogenic cooperation between JNK and Ras signaling that promotes tumor growth and malignancy (Igaki, 2006).
How is JNK signaling activated? Loss of cell polarity may directly influence activity of a JNK pathway component. Alternatively, cell polarity defects may activate a cell surface receptor that triggers JNK signaling. The genetic analysis of multiple JNK pathway components suggests that the pathway is activated through a cell surface receptor, Wengen. It would be interesting to further investigate whether mislocalization or disregulation of Wengen, which should be normally tightly regulated in polarized epithelial cells, results in stimulation of JNK pathway signaling (Igaki, 2006).
The discovery that metastasis-promoting alterations (i.e., JNK activation) also increase tumor growth may explain why tumor cells acquire such mutations; that is, they primarily provide a selective advantage in tumor growth. Given that cell polarity defects are frequently associated with human tumor malignancy, and that the pathways identified in Drosophila are evolutionarily conserved, similar molecular mechanisms could be involved in human tumor progression. It would be particularly interesting to study these processes in human tumors with high frequencies of Ras mutations. If such processes prove conserved, components of these pathways, especially JNK signaling, could serve as potential therapeutic targets against such cancers (Igaki, 2006).
Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, null and hypomorph alleles of the Drosophila Ral gene have been generated. Ral null animals are not viable. Reduced Ral expression in cells of the sensory organ lineage has no effect on cell division but leads to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggest that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst, a protein complex involved in vesicles trafficking, specifically the tethering and spatial targeting of post-Golgi vesicles to the plasma membrane prior to vesicle fusion, is a Ral effector. Epistasis between mutants of Ral and of misshapen (msn), the fly ortholog of HGK, suggests the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. It is conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. It is proposed that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK (Balakireva, 2006).
The Ral pathway is an essential component of physiological Ras signaling as well as Ras-driven oncogenesis. It can be instrumental in oncogenic transformation, and an activated form of a Ral exchange factor, Rlf, recapitulates the capacity of Ras to transform immortalized human cell cultures, either alone or together with other Ras effectors. Reciprocally, the lack of RalGDS, another Ral exchange factor, reduces tumorigenesis in a multistage skin carcinogenesis model and transformation by Ras in tissue culture. The molecular basis of the Ral contribution to oncogenesis remains to be elucidated (Balakireva, 2006).
None of the Ral effectors and their attributed cellular functions are obvious actors in oncogenesis. One of the two well-documented Ral effectors, RLIP76/RalBP1, is involved in endocytosis. The other, the exocyst complex, is involved in secretion, polarized exocytosis, and migration and can be found at the tip of filopods and at tight junctions. The exocyst complex is composed of eight proteins, which have been initially identified via mutants of secretion in the budding yeast. Exocyst complexes are bound to vesicles and are supposed to participate in vesicle trafficking and tethering to the plasma membrane. Globally, Ral appears to be a regulator of vesicle trafficking with consequences on cell proliferation, cell fate, and cell signaling (Balakireva, 2006).
In order to gain insight into Ral function, a genetic and cell biology approach was undertaken using Drosophila, which has a single Ral gene. Null and hypomorph alleles of Ral were generated, and Ral was shown to be an essential gene. Ral loss-of-function has dramatic effects on the differentiation of sensory organ precursor cells and leads to caspase-8-independent cell death by releasing ectopic tumor necrosis factor (TNF) receptor-associated factor 1-c-Jun N-terminal kinase (TRAF1-JNK) signaling. Sensory organ cell survival in Ral mutants is rescued by an activation of p38 mitogen-activated protein (MAP) kinase, revealing an antiapoptotic function of this latter. The influence of Ral on sensory organ cell fate is directly mediated by the exocyst complex together with a novel interaction partner, the MAP4K4 (also known as hepatocyte progenitor kinase-like/germinal center kinase-like kinase [HGK] in mammals and Misshapen [MSN] in flies). This suggests that a Ral/exocyst/JNK regulatory axis may represent a key component of developmental regulatory programs (Balakireva, 2006).
Hypomorph mutations of Ral displayed a loss-of-bristle phenotype with sockets without shafts, as do flies expressing dominant negative alleles of Ral). Whereas Ral is expressed in many if not all tissues, the only situation where a decreased level of Ral appears compatible with adult viability leads to a developmental phenotype in the bristle sensory organs. In Ral mutants, the pI precursor cells undergo the right number of divisions with a correct timing, but afterward shaft cells die by apoptosis, showing that death hits after cell division and determination has taken place, during the subsequent differentiation stage (Balakireva, 2006).
The various pathways that lead to apoptosis for their interactions with Ral have been explored. The caspase-8-mediated pathway did not contribute to the Ral phenotype, as opposed to a caspase-9-mediated pathway. The JNK pathway, a cascade of four kinases starting with MSN (MAP4K4 or HGK in human), which requires formation of a complex with TRAF1 for its full activity, and ending at the Jun N-terminal kinase, was tested. Puckered is a phosphatase that dephosphorylates and deactivates JNK (Balakireva, 2006).
Loss-of bristle and apoptosis phenotypes due to decrease of Ral signaling were suppressed by down-regulation of the JNK pathway and enhanced by its up-regulation. Symmetrically, a phenotype due to a hyperactivation of the Ral pathway by the overexpression of RalG20V was suppressed and enhanced by enhancing or decreasing JNK signaling, respectively (Balakireva, 2006).
The fact that the enhancement and suppression can be induced by genetic alterations of TRAF and MSN as well as of JNK proteins suggests that Ral is a general negative regulator of this cascade. Dominant negative alleles of transcriptional effectors of the JNK, Jun itself but also Fos, suppress the Ral phenotype, suggesting that Ral regulates transcriptional events involved positively or negatively in apoptosis (Balakireva, 2006).
Down-regulating the JNK pathway is not only suppresses apoptosis in Ral-defective cells but also rescues normal bristle development. Together with data in S2 cells, where Ral behaves also as a negative regulator of JNK in the absence of any cell death (Sawamoto, 1999), the results suggest a functional relationship between Ral and the JNK pathway wherein Ral activation keeps JNK down. Data using activated and dominant negative alleles of Ral in mammalian cell culture support a positive effect of Ral on JNK activation. The source of this discrepancy, which might be due to cell- and/or context-specific interactions of Ral with the JNK pathway, is not understood. However, the current data obtained by RNA interference in HeLa cells are consistent with the fly model (Balakireva, 2006).
Epistatic relationships between Ral and p38 MAP kinase mutants revealed another actor in Ral-dependent apoptosis: the p38 MAP kinase behaves as an antiapoptotic kinase, which could be positively regulated by Ral (Balakireva, 2006).
A control of the basic JNK activity might serve two purposes: (1) it minimizes JNK activity and avoids undesirable cell death in normal conditions; (2) a low level of basal JNK activity allows better differential in activation of JNK when this activation happens in response to stresses that lead eventually to apoptosis (Balakireva, 2006).
The molecular basis of Ral action on the JNK pathway was addressed genetically and biochemically. The model that emerges is that the exocyst complex is the matchmaker between Ral and the JNK pathway, and the simplest interpretation of genetic data is that the exocyst works like a negative regulator of HGK activity. Finally, the exocyst complex was found to bind in vivo to HGK, providing a biochemical basis for the functional effect of Ral on JNK (Balakireva, 2006).
Decreasing the JNK pathway seems to favor the oncogenic capacity of Ras in mouse primary fibroblasts. The current results can explain one of the contributions of the Ral pathway to oncogenesi: cancer cells have to sustain proliferative signals and relieve proapoptotic signals, and Ral via the exocyst complex might be in charge, at least, of this latter task in oncogenesis. Finally, it has been recently shown that the exocyst complex carries enzymatic activities working in the NF-kappaB pathway. These data together with the present report widen the role of the exocyst to functions other than directing vesicle traffic and contributing to exocytosis (Balakireva, 2006).
CYLD encodes a tumor suppressor that is mutated in familial cylindromatosis. Despite biochemical and cell culture studies, the physiological functions of CYLD in animal development and tumorigenesis remain poorly understood. To address these questions, Drosophila CYLD (dCYLD) mutant and transgenic flies were generated expressing wild-type and mutant dCYLD proteins. dCYLD is essential for JNK-dependent oxidative stress resistance and normal lifespan. Furthermore, dCYLD regulates TNF-induced JNK activation and cell death through dTRAF2, which acts downstream of the TNF receptor Wengen and upstream of the JNKK kinase dTAK1. dCYLD encodes a deubiquitinating enzyme that deubiquitinates dTRAF2 and prevents dTRAF2 from ubiquitin-mediated proteolytic degradation. These data provide a molecular mechanism for the tumor suppressor function of this evolutionary conserved molecule by indicating that dCYLD plays a critical role in modulating TNF-JNK-mediated cell death (Xue, 2007).
Shortened animal lifespan may result from compromised oxidative stress tolerance. To examine the oxidative stress resistance, 3-day-old flies were challenged with paraquat for a prolonged period of time and their survival rates were measured. It was found that dCYLD mutants exhibited a significant reduction in survival rate as compared with wild-type or heterozygous dCYLD flies after 24 hr or 36 hr of exposure to paraquat, suggesting dCYLD plays a pivotal role in regulating oxidative stress resistance (Xue, 2007).
JNK signaling has been reported to play an important role in regulating oxidative stress resistance and lifespan in Drosophila (Wang, 2003). Ubiquitous expression of Bsk, the Drosophila JNK ortholog, under the control of tubulin promoter, rescues both lifespan and oxidative stress resistance defects in dCYLD mutants, suggesting that dCYLD regulates these two physiological effects through the JNK signaling pathway (Xue, 2007).
This study was extended to other stress conditions and it was found that dCYLD mutants are less resistant to dry starvation (no food and water), a phenotype that has been associated with reduced JNK activity (Wang, 2003). In contrast, dCYLD mutants do not affect animal survival at high and low temperature conditions (Xue, 2007).
To further examine the role of dCYLD in regulating JNK signaling in animal development, the genetic interactions between dCYLD and Eiger (Egr), the Drosophila ortholog of TNF that triggers the JNK pathway, was tested. Ectopic expression of Egr, under the control of the GMR promoter (GMR > Egr) and using the Gal4/UAS binary system, induces JNK activation and cell death in the developing eye that results in vastly reduced adult eye size. The Egr-induced JNK activation and small-eye phenotype was suppressed modestly by deleting one copy of dCYLD and suppressed soundly by removing both copies. The strong suppression of the Egr eye phenotype in homozygous dCYLD mutants was partially reverted by adding one copy of dCYLDRes. These results indicate that dCYLD is required for Egr-triggered JNK activation and cell death (Xue, 2007).
dCYLD encodes a protein of 640 amino acids, containing in its N terminal portion a cytoskeleton-associated protein (CAP) domain that is present in proteins associated with microtubules and the cytoskeletal network, two ubiquitin carboxyl-terminal hydrolases (UCH) domains that are commonly associated with deubiquitinating enzyme activity, and three CXXC zinc-finger (ZF) motifs with potential protein-protein interaction ability. To functionally characterize these motifs, UAS transgenes were generated expressing the wild-type or three mutant versions of dCYLD that delete the CAP domain, the two UCH domains, or the three ZF motifs. When expressed under the control of the GMR promoter, neither the full-length nor the dCYLD mutants displayed any detectable phenotype. When introduced into the GMR > Egr; dCYLD−/− background, wild-type dCYLD released the suppression of the Egr eye phenotype, confirming that the suppressive effect was due to the loss of dCYLD functions. In contrast, dCYLDΔUCH had no effect on the suppression of the Egr eye phenotype, and dCYLDΔCAP could only partially relieve this suppression, implying that the UCH domains are necessary for dCYLD functions and that the CAP domain is essential for dCYLD to execute its full activity in vivo. Interestingly, expression of dCYLDΔZF completely abolished the suppression effect, suggesting that the ZF motifs are dispensable in dCYLD regulation of Egr-induced cell death (Xue, 2007).
Ubiquitous expression of the full-length dCYLD, but not dCYLDΔUCH, rescues both shortened lifespan and hypersensitivity to paraquat in dCYLD mutants, suggesting that the deubiquitinating activity is indispensable for dCYLD to regulate JNK-dependent oxidative stress resistance and lifespan (Xue, 2007).
TNF receptor-associated factors (TRAFs) are important adaptor proteins that bind to TNF receptors and relay TNF signals to the JNK and NF-κB pathways in mammals. In Drosophila, Egr signal is mediated exclusively by the JNK pathway. However, the role of Drosophila TRAF proteins in Egr-JNK signaling remains unclear. The Drosophila genome encodes two TRAFs: dTRAF1, the TRAF2 ortholog; and dTRAF2, the TRAF6 ortholog. To determine the role of dTRAF1 and dTRAF2 in Egr-JNK signaling, the effects were examined of loss-of-function mutations and RNAi-mediated downregulation of dTRAF1 or dTRAF2 on the Egr eye phenotype. The Egr-induced small-eye phenotype was not suppressed by either deletion of one copy of the dTRAF1 gene or coexpression of a dTRAF1 RNAi. In contrast, the Egr eye phenotype was suppressed strongly by removing half of the dosage of dTRAF2 and suppressed completely by deleting the dTRAF2 gene. Consistently, coexpression of a dTRAF2 RNAi significantly suppressed the Egr eye phenotype. In agreement with genetic data, dTRAF2 exhibited a much stronger physical interaction with Wgn and dTAK1 than did dTRAF1 . Together, the above results point to dTRAF2, but not dTRAF1, as the adaptor protein that mediates Egr signaling in Drosophila (Xue, 2007).
To investigate the physiological functions of dCYLD and dTRAF2 in JNK activation, the expression pattern of puckered (puc) was checked in dCYLD or dTRAF2 mutants. puc encodes a JNK phosphatase whose expression is positively regulated by the JNK pathway, and thus, the puc-LacZ expression of the pucE69 enhancer-trap allele can be used as a readout of JNK activity in vivo. puc is weakly expressed in wild-type third-instar eye discs, and can be detected by prolonged staining. It has been previously shown that puc expression posterior to the morphogenetic furrow (MF) depends on endogenous Egr signaling. This study found that such expression patterns are reduced dramatically in dCYLD mutants and dTRAF2 RNAi animals. In contrast, puc expression in the disc margin, which is independent of Egr signaling, was not affected. GMR > Egr strongly activated puc transcription posterior to the MF. This ectopic Egr-induced puc expression was largely blocked by loss of dCYLD or by expression of dTRAF2 RNAi. Taken together, these observations indicate that both dCYLD and dTRAF2 are physiologically required by the endogenous JNK pathway (Xue, 2007).
The role of CYLD in modulating JNK signaling in mammalian cells has remained controversial. Consistent with the current observation, it was reported that JNK activity diminished in Cyld−/− thymocytes, which implies that CYLD is physiologically required for JNK activation. However, CYLD was also reported to negatively regulate JNK signaling in culture cells and macrophages. Thus, CYLD could positively or negatively regulate JNK signaling in a cell-type-specific manner (Xue, 2007).
To genetically map the epistasis of dCYLD and dTRAF2 in the Egr-JNK pathway, the genetic interaction between dTAK1 (JNKKK) and dCYLD or dTRAF2 was examined in the developing eyes. Expression of dTAK1 under the control of the GMR promoter resulted in pupa lethality, while dTAK1 expression under the control of the sevenless (sev) promoter (sev > dTAK1) induced extensive cell death in larval eye discs and gave rise to rough eyes with a reduced size. Loss of dCYLD or dTRAF2, or coexpression of a dTRAF2 RNAi, had no effect on the sev > dTAK1 phenotype, while removal of one copy of hep (JNKK) or bsk (JNK) partially suppressed the sev > dTAK1 phenotype, suggesting that dCYLD and dTRAF2 operate upstream of dTAK1 in the Egr-JNK pathway (Xue, 2007).
Ectopic Egr expression in the dorsal thorax driven by the potent pannier-GAL4 driver resulted in pupa lethality. However, when reared at 18°C, these animals survived to adulthood, presumably due to lessened Egr expression caused by reduced Gal4 activity, and produced a small-scutellum phenotype. This phenotype could be suppressed by RNAi inactivation of JNK signaling components, e.g., wgn, dTRAF2, dTAK1, hep, or bsk, suggesting that the phenotype is caused by activation of JNK signaling. Ectopic expression of dCYLD driven by pannier-GAL4 produced a similar but weaker phenotype, which could be fully suppressed by the coexpression of an RNAi of dTRAF2 and dTAK1, but not that of wgn. These results indicate that dCYLD functions downstream of Wgn, but upstream of dTRAF2 and dTAK1, in modulating JNK signaling (Xue, 2007).
RNAi-mediated downregulation of dTRAF2, but not dTRAF1, resulted in compromised oxidative stress resistance and shortened lifespan, suggesting that the role of dTRAF2 in dCYLD-JNK signaling has been conserved in different physiological contexts (Xue, 2007).
Previous studies have reported that the Shark tyrosine kinase and Src42A regulate JNK signaling in epidermal closure during embryogenesis and metamorphosis. However, null mutants for egr and dCYLD are fully viable and do not display the epidermal closure defect, implying that Shark and Src42A act in parallel to Egr and dCYLD in modulating JNK signaling. Consistent with this interpretation, loss of shark or src42A failed to suppress the GMR > Egr or pnr > dCYLD phenotype. In addition, it was found that loss of the transcription factor dFOXO could suppress both GMR > Egr and pnr > dCYLD phenotypes, suggesting that dFOXO acts downstream of dCYLD in JNK signaling. Consistent with this observation, dFOXO is required downstream of JNK in modulating cell death, oxidative stress resistance, and lifespan (Xue, 2007).
Overexpression of CYLD in mammalian tissue culture cells negatively regulates NF-κB signaling by deubiquitinating TRAF2/6. dCYLD, like its mammalian counterpart, contains two UCH deubiquitinating domains. Indeed, genetic analysis revealed that the UCH deubiquitinating domains are crucial for the in vivo functions of dCYLD. Furthermore, genetic epistasis data show that dCYLD acts upstream of dTRAF2 in the JNK pathway. Thus, it was hypothesized that dCYLD might act in the JNK pathway to deubiquitinate and subsequently stabilize dTRAF2 by preventing its ubiquitination-mediated proteolytic degradation. To examine this hypothesis in vivo, a FLAG-tagged dTRAF2 transgene (GMR > FLAG-dTRAF2) was introduced into dCYLD mutants and transgenic flies. Proteins were extracted from the heads of these flies for biochemical analyses. It was found that loss of dCYLD resulted in a significant reduction in dTRAF2 protein level, while the ubiquitination of dTRAF2 was markedly enhanced. Both changes were suppressed by dCYLDRes. Consistently, overexpression of dCYLD, but not dCYLDΔUCH, increased dTRAF2 protein level and decreased its ubiquitination. These results show that dCYLD functions as a deubiquitinating enzyme that deubiquitinates dTRAF2 and promotes dTRAF2 accumulation in vivo (Xue, 2007).
Polyubiquitination chains are usually formed on two lysine residues, K48 and K63. It is generally believed that the K48-linked polyubiquitination mediates proteasome-dependent protein degradation, while the K63-linked polyubiquitination mediates endocytosis and signal transduction. Previous work in mammalian culture cells has implicated that CYLD encodes a deubiquitinating enzyme that preferentially cleaves K63-linked polyubiquitin chain from its target proteins for NF-κB signaling. However, a recent in vivo study in Cyld−/− mice has reported that CYLD could regulate the stability of its target protein by effectively removing K48-linked polyubiquitin chain in thymocytes. Interestingly, JNK activity also diminished in Cyld−/− thymocytes. Thus, the role of CYLD in regulating protein stability and positively modulating JNK signaling could be conserved in mammals (Xue, 2007).
CYLD mutations in human patients cause dramatic skin tumors. However, the physiological function of CYLD and the mechanism underlying CYLD deficiency-induced tumorigenesis remain largely unknown. By generating the null dCYLD mutation and dCYLD transgenic animals and performing genetic analysis, this study has shown that dCYLD is a critical regulatory component of the JNK signaling pathway. Genetic epistasis and biochemical analysis further reveal that dCYLD modulates JNK signaling by deubiquitinating dTRAF2 and thus preventing dTRAF2 from ubiquitination-mediated proteolytic degradation. Loss of dCYLD results in augmented ubiquitination and degradation of dTRAF2, which renders cells resistant to apoptosis triggered by JNK signaling. Deregulation of apoptosis has been implicated as a major cause of tumorigenesis. Consistently, mice deficient for both jnk1 and jnk2 were resistant to apoptosis induced by UV irradiation, anisomycin, and MMS, and jnk1−/− mice exhibited enhanced skin tumor development, a phenotype that is pathogenically similar to cylindromatosis in CYLD human patients. Together these data argue that modulation of JNK signaling could be a conserved mechanism underlying familial cylindromatosis in CYLD patients (Xue, 2007).
Cellular signaling networks have evolved to enable swift and accurate responses, even in the face of genetic or environmental perturbation. Thus, genetic screens may not identify all the genes that regulate different biological processes. Moreover, although classical screening approaches have succeeded in providing parts lists of the essential components of signaling networks, they typically do not provide much insight into the hierarchical and functional relations that exist among these components. This study describes a high-throughput screen in which RNA interference was used to systematically inhibit two genes simultaneously in 17,724 combinations to identify regulators of Drosophila JUN NH2-terminal kinase (JNK). Using both genetic and phosphoproteomics data, an integrative network algorithm was then implemented to construct a JNK phosphorylation network, which provides structural and mechanistic insights into the systems architecture of JNK signaling (Bakal, 2008).
Signaling networks, especially those maintaining cell viability and proliferation in response to environmental fluctuations and stress, may be more robust to perturbation than others. One signaling network dedicated to maintaining cell, tissue, and organism fidelity in the face of cellular stress involves stress-activated protein kinases (SAPKs), also known as JUN NH2-terminal kinases (JNKs). Classical in vivo genetic approaches in Drosophila have identified a highly conserved pathway consisting of a single JNK, a JNK-kinase (JNKK: Hemipterous), and a mixed-lineage kinase (MLK) that serves as a JNKK-kinase, but little is known as to how other signaling networks feed into this canonical cascade. To expand understanding of JNK regulation, cell-based RNA interference (RNAi) screens were conducted to systematically investigate JNK activity in various genetic backgrounds. Furthermore, to gain insight into the systems architecture of JNK signaling, a probabilistic computational framework was used to reconstruct a JNK phosphorylation network among components identified in the screen on the basis of phosphoproteomics data (Bakal, 2008).
To measure JNK activity in live migratory Drosophila cells, an RNAi screen was devised based on a dJUN-FRET sensor (fluorescence resonance energy transfer or FRET). dJUN-FRET is a single polypeptide composed of a modified Drosophila JUN phosphorylation domain and a FHA phosphothreonine-binding module separated by a flexible linker and flanked by a cyan fluorescent protein (CFP) donor and yellow fluorescent protein (YFP) acceptor modules. Drosophila BG-2 migratory cells were transfected with a plasmid that drives dJUN-FRET expression from an actin promoter and, 2 days later, were transfected with a set of 1565 double-stranded RNAs (dsRNAs) targeting all 251 known Drosophila kinases, 86 phosphatases (PPases), and predicted kinases and PPases, as well as regulatory subunits and adapters (the 'KP' set). JNK activity in single cells was determined by calculating the ratio of FRET signal (generated by FRET between YFP and CFP) to the level of CFP intensity (which provides the baseline level of dJUN-FRET expression in each cell regardless of JNK activity) within each cell boundary. A mean ratio is then derived for all cells treated with a particular dsRNA. The mean fold change in dJUN-FRET reporter activity for 16,404 control wells was 1.00 ± 0.04 (SD); however, in a screen of the KP set, multiple dsRNAs targeting JNK (Z = -2.06 and -2.05) and MLK (Z = -5.06, -2.60, and -2.13) produced significant decreases in dJUN-FRET reporter activity. Moreover, dsRNAs targeting the JNK PPase puckered (puc) resulted in significant increases in reporter activity, consistent with the role of Puc as a negative regulator of JNK. In the KP screen, 24 genes (5% of genes tested) were identified as putative JNK regulators, and the 6 out of 7 positive and negative JNK regulators previously identified in vivo were reidentified. Although the KP screen identified both previously known and novel JNK components and regulators, the results are notable in the genes that the screen failed to isolate. For example, the only Drosophila JNKK, encoded by the hemipterous gene, was not identified in the KP screen. Furthermore, although ERK emerged from the KP screen as a JNK suppressor because of ERK's potential positive effects on puc transcription, no dsRNAs targeting other components of the ERK pathway were seen. A high false-negative rate appears to be present in this genetic screen; therefore, a combinatorial strategy was developed to further enhance the sensitivity of the screen (Bakal, 2008).
Twelve different sensitized screens were developed in which cells were incubated with dsRNAs targeting a 'query' gene in combination with dsRNAs of the KP set. In choosing query genes, focus was placed primarily on components of Rho guanosine triphosphatase (GTPase) signaling, such as Rac1, Cdc42, the Rho guanine nucleotide exchange factor still-life (sif), and p190RhoGAP (GTPase-activating protein), because Rho activity couples JNK activation to a number of upstream signaling events. Cells were also sensitized by targeting canonical JNK components, such as JNK, puc, and MLK; other strong candidates from the KP screen, such as ERK; and genes, such as AKT, PTEN, hippo, and VHL, whose inhibition could result in the activation stress pathways even though they were themselves not identified in the KP screen. Genes were then identified as likely JNK regulators if two or more independent dsRNAs resulted in average increases or decreases in dJUN-FRET reporter activity in each screen, and a significance score was assigned based on how many total dsRNAs were tested for each gene across all screens. For example, a gene targeted by two to four dsRNAs was considered a JNK regulator if isolated in two or more screens, but a gene targeted by five to seven dsRNAs must be isolated in three or more screens to be included in the list of high-confidence regulators. No genes were isolated in the background of JNK inhibition, which showed that increases or decreases in dJUN-FRET reporter activity in both unmodified and modified backgrounds are JNK-dependent. Using this combinatorial approach, 55 new JNK suppressors and enhancers were identified in a test of 17,724 dsRNA combinations, which, together with results from the nonsensitized initial screen, provide a list of 79 likely JNK regulators (17% of the genes tested). Some of the hits identified in multiple screens were validated as bona fide JNK regulators by quantifying mRNA abundance of the JNK-specific transcriptional target MMP1 after dsRNA-mediated inhibition of candidate genes by quantitative real-time polymerase chain reaction (Bakal, 2008).
Why do depletion of certain kinases and PPases had effects in both unmodified and modified backgrounds, while others were isolated only in sensitized contexts. To answer this question the genetic screen was integrated with phosphoproteomics data and computational models of kinase specificity to derive networks on the basis of all of these experimental sources using the NetworKIN algorithm. NetworKIN was deployed on more than 10,000 unique high-confidence phosphorylation sites identified in a recent mass spectrometry study of Drosophila cells (Bodenmiller, 2007). This resulted in an initial network that was subsequently overlaid with the genetic hits in order to derive a model of the JNK phosphorylation network. Last, to determine which phosphorylation events make functional contributions to JNK signaling, data sets derived from combinatorial screens for epistatic interactions among kinases and substrates were examined and hierarchical clustering of mean Z scores for components of the JNK phosphorylation network were performed across several combinatorial RNAi screens to look for shared patterns of genetic interaction. Thus, through integrating genetic and phosphoproteomics data using a computational framework, a systems-level strategy was undertaken to describe the protein networks underlying genetic interactions (Bakal, 2008).
JNK regulators identified in all screens could be broadly grouped into different classes on the basis of previously described biological functions and/or structural similarity of protein products. Specifically, a number of protein and lipid kinases involved in axon guidance and cell migration were identified, such as FER, Ptp69d, otk, thickveins, RET, wunen2, GSK3, PDK1, and JAK. Genes encoding components of apicobasal polarity complexes were identifed, such as ZO-1, Caki, Magi, and discs large 1 (dlg1), largely as JNK suppressors, which is consistent with in vivo studies demonstrating unrestrained JNK activation associated with breakdown of polarity in backgrounds of hyperactivated Ras/ERK signaling. Furthermore, the results implicate the Warts-Hippo complex as a potential link between JNK activity and the remodeling of cytoskeletal structures. NetworKIN predicts that Hippo-mediated activation of JNK can occur through phosphorylation of MLK and that Hippo is also a direct target for JNK, which suggests that a feedback loop exists between JNK and Warts-Hippo signaling. Notably, it is also predicted that Dlg1 is extensively phosphorylated by a number of kinases in the JNK network, including JNK itself. This suggests that JNK, and other kinases such as ERK and CDK2, can act upstream of Dlg1 to remodel or dismantle polarized cell-cell adhesion complexes, which, in turn, promote the morphological changes required to complete division, migration, or extrusion from tissue during apoptosis. Compelling support of this idea is provided by the fact that mammalian Dlg1 is regulated by phosphorylation, is a substrate of JNKs, and becomes highly phosphorylated during mitosis. These findings highlight the ability of integrated genetic and computational approaches to provide systems-level insight into the complex regulation of JNK activity (Bakal, 2008).
In summary, this study demonstrates that combinatorial RNAi screening is a powerful strategy to reduce the false-negatives present in current screens and reveals functions for a large fraction of genes. Moreover, the data-integrative-powered approach unraveled both mechanistic and hierarchical associations of components in the JNK regulatory system and provides an invaluable starting point for understanding the genetic interactions and signaling networks that underpin various diseases (Bakal, 2008).
Post-translational modification by the small ubiquitin-related modifier (SUMO) is important for a variety of cellular and developmental processes. However, the precise mechanism(s) that connects sumoylation to specific developmental signaling pathways remains relatively less clear. This study shows that Smt3 (SUMO) knockdown in Drosophila wing discs causes phenotypes resembling JNK gain of function, including ectopic apoptosis and apoptosis-induced compensatory growth. Smt3 depletion leads to an increased expression of JNK target genes Mmp1 and puckered. Although knockdown of the homeodomain-interacting protein kinase (Hipk) suppresses Smt3 depletion-induced activation of JNK, Hipk overexpression synergistically enhances this type of JNK activation. This study further demonstrates that Hipk is sumolylated in vivo, and its nuclear localization is dependent on the sumoylation pathway. These results thus establish a mechanistic connection between the sumoylation pathway and the JNK pathway through the action of Hipk. It is proposed that the sumoylation-controlled balance between cytoplasmic and nuclear Hipk plays a crucial role in regulating JNK signaling (Huang, 2011).
Sumoylation is a post-translational modification that regulates multiple biological activities by modifying a variety of different substrates. This study shows that tissue-specific perturbation of the sumoylation pathway activates the JNK signaling pathway. In particular, knockdown of the Drosophila SUMO gene smt3 recapitulates several key gain-of-function features of the JNK pathway, including apoptosis and wg ectopic expression. These results suggest that sumoylation plays a crucial role in regulating JNK signaling. Further experiments demonstrate that Hipk is responsible for Smt3 depletion-induced JNK activation. These experiments show that Hipk itself is sumoylated and that its nuclear localization is dependent on the sumoylation pathway. Based on these findings, a model is proposed in which Hipk is normally kept in the nucleus, but a compromised sumoylation pathway (such as that produced by depletion of Smt3) allows some Hipk molecules to translocate to the cytoplasm and activate the JNK signaling pathway (Huang, 2011).
Sumoylation regulates the biological activities of its substrates through several distinct mechanisms. These mechanisms include altering subcellular localization of its substrate proteins and/or molecular shuttling between the nucleus and the cytoplasm, mediating protein-protein interactions, locking its substrates in a particular conformational state (i.e. active or inactive) or altering protein stability and clearance. This study highlights the importance of sumoylation-dependent subcellular localization of Hipk in regulating its biological activities. It is proposed that sumoylation normally restricts Hipk to the nucleus and facilitates the execution of its nuclear functions, such as interaction with and phosphorylation of transcriptional co-repressors. However, unsumoylated or desumoylated Hipk becomes accessible to the cytoplasm for executing its cytoplasmic function(s). As shown in this study, one such cytoplasmic function of Hipk is to modulate the JNK signaling pathway (Huang, 2011).
Hipk family members play roles in different biological processes, such as cell cycle progression, p53-dependent apoptosis, and transcriptional regulation. The mammalian cells contain four Hipk proteins that perform overlapping, but distinct functions. For example, Hipk1 and Hipk2 have functionally redundant roles in mediating cell proliferation and apoptosis during development. Hipk1 interacts with transcription factor c-Myb, while Hipk2 phosphorylates transcriptional co-repressor Groucho, suggesting their distinct roles in transcription regulation. Therefore, it would be interesting to elucidate whether Drosophila Hipk executes the functions of all the mammalian counterparts, although Drosophila Hipk shares most homology with Hipk2. This all-in-one mode of Hipk function requires different strategies to regulate its functions. Previous studies have shown that Drosophila Hipk promotes various signaling pathways such as the Wnt pathway through stabilizing Armadillo, and the Notch pathway through inhibiting the global co-repressor Groucho. This work reports that Drosophila Hipk potentiates JNK signaling through a sumoylation-dependent regulation of its subcellular localization. This work underscore the roles of Drosophila Hipk both inside and outside of the nucleus in fine-tuning signaling pathways. It remains to be determined precisely how Hipk regulates the JNK pathway and whether it involves a direct mechanism such as phosphorylating relevant components of this pathway (Huang, 2011).
The subcellular localization of Hipk represents an important mechanism in defining its functional specificity. In particular, Hipk controls the degradation of transcriptional co-repressor CtBP inside the nucleus, while the cytoplasmic Hipk interacts with the nonhistone chromosomal factor Hmga1 (high-mobility group A1) to inhibit cell growth. Hipk has also been shown to, within the speckled subnuclear structures, interact with p53 to promote its phosphorylation. The current results presented show that Hipk is normally sequestered in the nucleus but gains access to the cytoplasm, upon sumoylation perturbation, to activate the JNK signaling pathway. The idea that the subcellular localization of Hipk is crucial for its functional specificity also explains why overexpressing Hipk alone did not result in a robust activation of JNK. It is suggested that, without sumoylation perturbation, the majority of transgene-expressed Hipk is, like the endogenously expressed Hipk, sumoylated and kept in the nucleus, making it inaccessible to activating JNK (Huang, 2011).
The JNK signaling pathway is composed of stepwise actions of kinases. The canonical JNK pathway receives signals from death stimuli, such as tumor necrosis factor (TNF) and oxidative stresses. In addition to the JNK pathway, other factors such as Hipk proteins are also stimulated by a variety of stresses. For example, the human HIPK1 responds to the stimulation of TNFα to relocate itself from the nucleus to the cytoplasm. In addition, the mammalian Hipk2 phosphorylates p53 in response to UV irradiation and phosphorylates cyclic AMP response element-binding protein (CREB) to cope with genotoxic stress. It is proposed that stress signals such as TNF may activate not only the canonical JNK pathway but also the Hipk-dependent JNK activation mechanism(s). The idea that Hipk acts downstream of TNF is consistent with genetic evidence that RNAi ablation of Hipk partially rescues the Drosophila TNF (Egr)-induced phenotype in the eye. A major finding of the current study is that it establishes a cross-regulation between the sumoylation and the JNK pathways through the action of Hipk. It is currently unknown whether TNF or even JNK itself may regulate the sumoylation pathway, but it remains an interesting possibility that will require further investigation. It is noted that the relationship between sumoylation and JNK pathways is likely to be more complex than Hipk-mediated action described in this work. It has been shown that sumoylation is required for Axin-mediated JNK activation. Thus, it is possible that sumoylation may have different, even opposite, effects on the JNK pathway through distinct sumoylation targets. The robust increase of the JNK activity detected in Smt3-depleted cells in this study demonstrates an overall negative role of the sumoylation pathway in JNK signaling (Huang, 2011).
Increased expression of the histone deacetylase sir2 has been reported to extend the life span of diverse organisms including yeast, Caenorhabditis elegans, and Drosophila melanogaster. A small molecule activator of Sir2, resveratrol, has also been suggested to extend the fitness and survival of these simple model organisms as well as mice fed high calorie diets. However, other studies in yeast have shown that Sir2 itself may prevent life extension, and high expression levels of Sir2 can be toxic to yeast and mouse cells. This conflicting evidence highlights the importance of understanding the mechanisms by which Sir2 expression or activation affects survival of organisms. To investigate the downstream signaling pathways affected by Sir2 in Drosophila, transgenic flies were generated expressing sir2. Overexpression of sir2 in Drosophila promotes caspase-dependent but p53-independent apoptosis that is mediated by the JNK and FOXO signaling pathways. Furthermore, a loss-of-function sir2 mutant partially prevents apoptosis induced by UV irradiation in the eye. Together, these results suggest that Sir2 normally participates in the regulation of cell survival and death in Drosophila (Griswold, 2008).
Drosophila has five sir2-like genes, with sir2 being most homologous to the yeast sir2, C. elegans sir2.1, and human sirt1. Ubiquitous overexpression of sir2 in Drosophila by using EP lines has been reported to extend life span. To understand the consequences of Drosophila sir2 overexpression and to identify its downstream signaling pathways, transgenic flies were generated that can express sir2 in the Drosophila eye, a well established system for characterizing signaling pathways. The use of the gmr-Gal4 driver line to overexpress this gene in developing eyes causes a phenotype, specifically a lack of pigmentation and a rough, bristled appearance. The severity of this phenotype correlates well with dosage of sir2. Consistent with the known pattern of gmr-Gal4 expression, Sir2 expression was seen in the developing eye imaginal disc in cells posterior to the morphogenic furrow. The endogenous Sir2 is also found at a low level in whole heads of Drosophila as well as in photoreceptor cells posterior to the morphogenic furrow and regions of the antennal disc. Ubiquitous sir2 overexpression using the actin-5C-Gal4 or the pan-neuronal driver elav-gal4 resulted in premature death during development, suggesting that Sir2 affects survival in other cell types as well. Additionally,
it was found that the overexpressed Sir2 was enzymatically functional because it increased NAD+-dependent deacetylase activity in both larval eye imaginal discs and adult fly heads (Griswold, 2008).
To verify whether the observed phenotype is Sir2-specific, transgenic flies were generated to express a Sir2 paralog, CG5085, which shares 43% identity and 63% similarity in amino acid sequence in the deacetylase domain. CG5085 is indeed a functional Sir2 deacetylase family member because recombinant CG5085 demonstrates NAD+-dependent deacetylase activity. However, although structurally and enzymatically similar, when overexpressed by using the gmr-Gal4 driver, CG5085 did not alter the phenotype of the eye. Furthermore, overexpression of lacZ or the Drosophila G protein-coupled receptor methuselah had no effect on eye morphology. This indicates that the eye phenotype in the transgenic flies overexpressing sir2 is indeed Sir2-specific and not due to either general overexpression of proteins or the deacetylase activity itself (Griswold, 2008).
The finding that overexpression of sir2 results in a deleterious effect on various tissues of the fly contrasts with a previously reported effect of sir2 overexpression on longevity. To clarify this contradictory result, a sir2 overexpression line (EP2300) used in the previous report was crossed with the gmr-Gal4 driver but no defective eye phenotype was found, although it highly expressed sir2. It is plausible that the insertion of EP2300 could affect genes neighboring sir2, thus leading to modification of the eye phenotype. EP2300 is inserted in a 500-bp region upstream of a chaperone gene dnaJ-H, and an increase was observed in the transcription level of dnaJ-H in adult heads when EP2300 was crossed with the gmr-Gal4 driver. Because overexpression of dnaJ-H can suppress the effects of toxic proteins in the eye, transgenic flies were generated overexpressing UAS-sir2 and UAS-dnaJ-H together in the eye, and up-regulation of dnaJ-H was found to ameliorate the defective phenotype caused by sir2 overexpression. This result suggests an explanation for the lack of an eye phenotype in EP2300 line despite an increase in sir2 expression. Coexpression of sir2 and dnaJ-H by EP2300 raises the possibility that the reported effects of this line may give a misleading picture of the role of sir2 in Drosophila aging (Griswold, 2008).
Based on the defective eye phenotype, it was hypothesized that sir2 expression may cause cell death. Thus, the developing eye in third-instar larval imaginal discs was examined by using acridine orange staining, a vital dye that detects dying cells, and the TUNEL assay, which identifies cells undergoing programmed cell death. Staining with acridine orange showed an increase in dying cells in the posterior part of eye discs overexpressing sir2, suggesting that sir2 overexpression causes cell death. Numerous TUNEL-positive cells in the imaginal discs with sir2 overexpression were also found, whereas the control showed few positive cells, indicating that the phenotype is mediated by apoptotic cell death in the developing eye. In addition, in vitro caspase-3 activity in the eye imaginal disc overexpressing sir2 increased 1.5-fold when compared with the control. This increase in caspase-3 activity was also verified by immunostaining for active caspase-3 in the imaginal discs; overexpression
of sir2 showed an increase in the number of more intensely stained cells (Griswold, 2008).
A previous report has characterized mammalian Sir2 as an apoptosis inhibitor through its deacetylation of p53. In Drosophila, the role of p53 is still under investigation, although it has been shown to regulate cell death in response to stress, similar to its mammalian homolog. However, the relationship of p53 and Sir2 in Drosophila has not been explored. To determine whether p53 and Sir2 are in the same genetic pathway to cause apoptosis in the eye, p53 and sir2 were coexpressed. The eyes of the flies overexpressing both p53 and sir2 were more severely affected than either p53 or sir2 overexpressed alone. However, overexpression of dominant negative p53 constructs did not rescue the sir2 phenotype. This suggests that the sir2 overexpression effect is p53-independent and that p53 and sir2 overexpression work in parallel to induce cell death in Drosophila (Griswold, 2008).
Sir2 can alter the activity of mammalian FOXO3a by deacetylation. Additionally, to increase life span in C. elegans, overexpression of sir2.1 requires DAF-16, the FOXO3a transcription factor homolog. No such direct link between Sir2 and FOXO has been established in Drosophila; however, overexpression of foxo in the Drosophila eye exhibits a defective eye phenotype. Because the results show that sir2 overexpression induces apoptotic cell death in the Drosophila eye, whether FOXO activity might be involved in the induction of apoptosis as a result of sir2 overexpression in Drosophila was investigated. sir2 was overexpressed in a foxo null mutant background, and a less severe eye phenotype was found, suggesting a genetic interaction between Sir2 and FOXO in cell death pathways in Drosophila (Griswold, 2008).
Recently, it was shown that the foxo-induced defective eye phenotype can be modulated through the JNK signaling pathway. Furthermore, increased activation of JNK is associated with an apoptotic eye phenotype in Drosophila. Because JNK signaling interacts with FOXO and influences apoptotic pathways, whether the JNK pathway is also
involved in the Sir2-induced eye phenotype was examined. The transcription level of the JNK phosphatase puc, a downstream target of the JNK signaling pathway, is increased in the heads of flies overexpressing sir2, suggesting an increase in JNK-dependent transcription. Inhibition of this signaling pathway by overexpression of bskDN, a dominant negative form of Drosophila JNK, resulted in a major improvement of the eye phenotype caused by sir2 overexpression. Additionally, inhibition of JNK signaling by coexpression of puc with sir2 demonstrated a significant rescue in the eye, consistent with a report that constitutive overexpression of puc can rescue the eye phenotype caused by increased JNK activity. These rescue flies do not reduce the level of Sir2 below that expressed by coexpression of lacZ. Together, these results suggest that sir2 overexpression requires JNK signaling to induce cell death in the eye (Griswold, 2008).
In summary, the evidence that endogenous Sir2 in the Drosophila eye plays a role in apoptosis is consistent with the finding that sir2 overexpression induces apoptotic cell death in the eye imaginal discs. An important issue is thus the identification of signaling pathways that mediate these effects. JNK signaling is implicated by phenotypic alleviation via the expression of a dominant negative JNK or an inhibitor JNK signaling. Also, loss of function of foxo can ameliorate the eye phenotype induced by sir2 overexpression, suggesting that sir2 can activate a proapoptotic function of FOXO. These pathways may intersect at JNK to induce the proapoptotic function of FOXO. The result of these pathways is the observed increase in proapoptotic gene expression of reaper, grim, and hid. This in turn leads to increased caspase activity and ultimately cell death (Griswold, 2008).
The results show that sir2 overexpression in Drosophila does not necessarily promote longevity, and endogenous Sir2 plays a critical role in regulating cell survival and death in the animal. Hence, it will be of future interest to study the signaling pathways induced by sir2 expression that lead to JNK activation as well as the relationship between Sir2 and FOXO in modulating apoptotic and survival pathways in Drosophila. Together, these will determine pathways affected by sir2 expression and give insights as to how it can mediate both cell survival and cell death (Griswold, 2008).
Human tumours have a large degree of cellular and genetic heterogeneity. Complex cell interactions in the tumour and its microenvironment are thought to have an important role in tumorigenesis and cancer progression. Furthermore, cooperation between oncogenic genetic lesions is required for tumour development; however, it is not known how cell interactions contribute to oncogenic cooperation. The genetic techniques available in the fruitfly Drosophila melanogaster allow analysis of the behaviour of cells with distinct mutations, making this the ideal model organism with which to study cell interactions and oncogenic cooperation. In Drosophila eye-antennal discs, cooperation between the oncogenic protein RasV12 and loss-of-function mutations in the conserved tumour suppressor scribbled (scrib) gives rise to metastatic tumours that display many characteristics observed in human cancers. This study shows that clones of cells bearing different mutations can cooperate to promote tumour growth and invasion in Drosophila. The RasV12 and scrib- mutations can also cause tumours when they affect different adjacent epithelial cells. This interaction between RasV12 and scrib- clones involves JNK signalling propagation and JNK-induced upregulation of JAK/STAT-activating cytokines, a compensatory growth mechanism for tissue homeostasis. The development of RasV12 tumours can also be triggered by tissue damage, a stress condition that activates JNK signalling. Given the conservation of the pathways examined in this study, similar cooperative mechanisms could have a role in the development of human cancers (Wu, 2010).
Clones of mutant cells marked with green fluorescent protein (GFP) can be generated in the eye-antennal imaginal discs of Drosophila larvae by mitotic recombination. Clones expressing RasV12, an oncogenic form of the Drosophila Ras85D protein, moderately overgrow. Clones mutant for scrib lose apico-basal polarity and die. In contrast, scrib clones simultaneously expressing RasV12 grow into large metastatic tumours. To understand better the cooperation between these two mutations, animals were produced in which cell division after a mitotic recombination event creates two daughter cells: one expressing RasV12 and the other mutant for scrib. Discs containing adjacent RasV12 (GFP-positive) and scrib- clones developed into large tumours, capable of invading the ventral nerve cord. This shows that RasV12 and scrib also cooperate for tumour induction when they occur in different cells. These tumours are referred to as RasV12//scrib- tumours, to denote interclonal oncogenic cooperation and distinguish them from RasV12scrib- tumours, in which cooperation occurs in the same cells intraclonally (Wu, 2010).
This study has used Drosophila to investigate how oncogenic cooperation between different cells can promote tumour growth and invasion. These experiments, addressed to understanding interclonal cooperation in RasV12//scrib- tumours, uncovered a two-tier mechanism by which scrib- cells promote neoplastic development of RasV12 cells: (1) propagation of stress-induced JNK activity from scrib- cells to RasV12 cells; and (2) expression of the JAK/STAT-activating Unpaired cytokines downstream of JNK. These findings, therefore, highlight the importance of cell interactions in oncogenic cooperation and tumour development. It was also shown that stress-induced JNK signalling and epigenetic factors such as tissue damage can contribute to tumour development in flies. Notably, tissue damage caused by conditions such as chronic inflammation has been linked to tumorigenesis in humans. Furthermore, expression of the Unpaired cytokines promotes tumour growth as well as an antitumoural immune response, which parallels the situation in mice and humans. Future research into phenomena such as compensatory growth and interclonal cooperation in Drosophila will provide valuable insights into the biology of cancer (Wu, 2010).
The Abelson (Abl) family of non-receptor tyrosine kinases has an important role in cell morphogenesis, motility, and proliferation. Although the function of Abl has been extensively studied in leukemia, its role in epithelial cell invasion remains obscure. Using the Drosophila wing epithelium as an in vivo model system, this study shows that overexpression (activation) of Drosophila Abl (dAbl) causes loss of epithelial apical/basal cell polarity and secretion of matrix metalloproteinases, resulting in a cellular invasion and apoptosis. The in vivo data indicate that dAbl acts downstream of the Src kinases, which are known regulators of cell adhesion and invasion. Downstream of dAbl, Rac GTPases activate two distinct MAPK pathways: c-Jun N-terminal kinase signaling (required for cell invasion and apoptosis) and ERK signaling (inducing cell proliferation). Activated Abl also increases the activity of Src members through a positive feedback loop leading to signal amplification. Thus, targeting Src-Abl, using available dual inhibitors, could be of therapeutic importance in tumor cell metastasis (Singh, 2010).
This is the first study to provide in vivo evidence for the role of Abl in cell invasion. Cells expressing dAbl (in the dpp-domain) become invasive and migrate into the area of the posterior compartment, where they are located basally to the basement membrane. Although during this process many cells die, those that 'resist' cell death can be visualized by the presence of GFP at the base of the epithelium in either compartment. Furthermore, mechanistic evidence is provided for an Src-Abl signaling cascade and an Abl/Src signal amplification loop in epithelial cell invasion. Targeting both kinase types using dual Abl/Src inhibitors in cancer patients could thus be of clinical significance. It was also shown that increased cell proliferation associated with Abl can be separated from its cell invasion function by distinct downstream effectors. Different MAPKs are activated downstream of dAbl and Rac, and mediate the cell proliferation and cell invasion phenotypes, respectively (Singh, 2010).
Loss of cell polarity has been linked to tumor growth and cell invasion. The mechanism(s) by which dAbl downregulates cell adhesion/polarity genes like DE-Cadherin, β-Catenin/Armadillo and Dlg are not known. This could be a direct effect of dAbl on junctional complexes or a consequence of the cell invasive behavior. Downregulation of E-cadherin has been linked to several types of tumors. Furthermore, Src family members have been shown to increase the turnover of AJs, which in turn would cause an increase in cell mobility, a possible mechanism by which Abl can mediate loss of cell polarity. This hypothesis is further supported by the observation that overexpression of DE-cadherin suppresses the effects induced by Src upregulation (through Csk reduction, using UAS-dCsk-RNAi) in the retina. Consistent with this notion, overexpression of DE-Cadherin rescues the dAbl-induced cell invasion phenotype. Moreover, removing a genomic copy of mmp1 and mmp2 results in suppression of the dAbl cell invasion phenotype. On the basis of these data it is concluded that loss of cell polarity and MMP secretion are the key factors in contributing to cell invasive behavior of dAbl-expressing cells. However, the possibility of minor contributions of unknown factors in this process cannot be completely ruled out (Singh, 2010).
A complicated question is how dAbl causes cell proliferation in epithelial cells (dAbl lacks nuclear localization signals). The effect of dAbl expression results in cell-autonomous and non-autonomous cell proliferation. In Drosophila, cells destined to undergo apoptosis express specific growth factors (Wingless and Dpp; their upregulation is mediated by JNK activation), inducing non-autonomous compensatory proliferation in neighboring cells. This compensatory proliferation is important for maintaining proper tissue homeostasis and may also be relevant for the induction of tumor cell proliferation. As dAbl activation results in cell death in migrating cells, one argument could be that cell proliferation associated with dAbl activation is a consequence of compensatory proliferation. Interestingly, dAbl expression results in an increase in Wg expression, suggesting that compensatory proliferation takes place in response to dAbl. Taken together, these data suggest that at least some aspects of dAbl-mediated cell proliferation (mediated by activation of ERK) are cell-autonomous independent of such compensatory proliferation, as Bsk-DN co-expression in a dAbl overexpression background (which blocks JNK signaling and thus induction of Wg and Dpp expression), does not block excessive proliferation within the dAbl expression domain (Singh, 2010).
The cell invasion phenotype of dAbl overexpression is similar to Csk reduction (dCsk-RNAi) and the data indicate that dAbl acts downstream of dCsk. As Csk negatively regulates Abl/Src family kinases (SFKs), this suggested that Src mediates the effect of dCsk on dAbl. Previous studies have shown that Abl can act as a substrate of SFKs, though other studies have shown that the opposite can also be true. The data indicate that Src acts upstream of Abl and that Abl can feed back and amplify the signal through its positive effect on Src. How is the dAbl-Src feedback loop working mechanistically? From the in vivo experiments it is not possible to conclude whether dAbl acts directly on Src, dCsk, or unknown upstream components. dAbl does not co-immunoprecipitate either dCsk or the Src kinases in Drosophila S2 cells. Since binding between kinases can be of very transient nature, it is possible that even if dAbl would bind Src or dCsk in vivo, it may not be possible to detect it. However, in vivo data suggest that dCsk does not mediate the dAbl effect: if dAbl would act through dCsk (by inhibiting it), phospho-Src (pSrc) levels should be similar with dCsk-IR or dAbl expression, which is not the case. dAbl expression results in a much more robust activation Src with pSrc detected in all dAbl/GFP-positive cells, whereas dCsk-IR does not result in such strong activation. This observation suggests that dAbl does not act through dCsk in this process. Although the possibility cannot be excluded that dAbl could modulate an unknown component upstream of dCsk, the fact that co-expression of dCsk-IR and dAbl (dCsk-IR; UAS-Abl at 18°C) shows a synergistic effect (even at 18°C, where neither individual transgene has a phenotype on its own) suggests that dAbl and Csk act in parallel on Src. As Abl can phosphorylate Src kinases, a direct effect of dAbl on the Src kinases is favored (Singh, 2010).
JNK signaling is activated in response to environmental stress and by several classes of cell surface receptors, including cytokine receptors and receptor tyrosine kinases. In mammalian cells, JNK has been implicated in oncogenic transformation in fibroblasts and hematopoietic cells, and in cell invasion. In oncogenic transformation, JNK signaling can promote tumor growth, while it can also act as a tumor suppressor. It also functions in basement membrane remodeling during imaginal disc eversion and tumor invasion. This study provides evidence for a link between Src and JNK during cell invasion, mediated through dAbl. The cell invasion and apoptosis phenotypes induced by dAbl require JNK activity, whereas the cell proliferation function of dAbl appears to be mediated by ERK signaling. dAbl does not affect expression levels of JNK but instead causes an increase in active JNK (phospho-JNK). It is worth noting that removing a genomic copy of each of the Drosophila Rac genes suppresses all phenotypes associated with dAbl overexpression (cell invasion, death, and proliferation). These data are consistent with the study of BCR-Abl-mediated cell growth, which requires Rac function, suggesting a general relevance of Rac GTPases as Abl effectors (Singh, 2010).
It is not established how Abl mediates Rac activation. A possible link can be Crk, which primarily consists of SH2 and SH3 domains, serving as an adaptor. Crk-I can associate with and be phosphorylated by c-Abl. Furthermore, ectopic expression of Crk can result in JNK activation. As overexpression/activation of dAbl results in JNK activation, Crk may provide a missing link between dAbl and Rac for JNK activation. Another candidate to mediate an interaction between dAbl and Rac GTPases can be Trio, a guanine exchange factor. Trio has two putative Rac and Rho-binding domains. In Drosophila, Trio function has been studied extensively in the context of axon guidance where it has been shown to interact with dAbl. Interestingly, a recent report has identified Trio as one of the guanine exchange factors responsible for invasive behavior of glioblastoma. Thus, a potential role of Trio in the context of Abl-mediated cell invasion warrants further investigation (Singh, 2010).
In Drosophila, dorsal closure is a model of tissue morphogenesis leading to the dorsal migration and sealing of the embryonic ectoderm. The activation of the JNK signal transduction pathway, specifically in the leading edge cells, is essential to this process. In a genome-wide microarray screen, new JNK target genes during dorsal closure were identified. One of them is the gene scarface (scaf), which belongs to the large family of trypsin-like serine proteases. Some proteins of this family, like Scaf, bear an inactive catalytic site, representing a subgroup of serine protease homologues (SPH) whose functions are poorly understood. This study shows that scaf is a general transcriptional target of the JNK pathway coding for a secreted SPH. scaf loss-of-function induces defects in JNK-controlled morphogenetic events such as embryonic dorsal closure and adult male terminalia rotation. Live imaging of the latter process reveals that, like for dorsal closure, JNK directs the dorsal fusion of two epithelial layers in the pupal genital disc. Genetic data show that scaf loss-of-function mimics JNK over-activity. Moreover, scaf ectopic expression aggravates the effect of the JNK negative regulator puc on male genitalia rotation. scaf acts as an antagonist by negatively regulating JNK activity. Overall, these results identify the SPH-encoding gene scaf as a new transcriptional target of JNK signalling and reveal the first secreted regulator of the JNK pathway acting in a negative-feedback loop during epithelial morphogenesis (Rousset, 2010).
The trypsin- and chymotrypsin-like (S1 family) serine proteases (SPs) are mainly represented in animals and are implicated in food digestion, blood coagulation, immune response and development. They are almost exclusively localized outside the cytoplasm to perform their function and they are synthesized with a signal peptide for secretion. In addition, SPs are usually produced in an inactive form, which is cleaved upon a signal to form a functional protease. This mechanism favours a rapid and local response to a stimulus. For example, in mammals, cascades of localised SP activities control emergency blood clotting at a wound site. During Drosophila development, dorsoventral patterning of the embryo results from an extracellular cascade involving the SPs Nudel, Gastrulation defective, Snake and Easter and leading to the activation of the Toll pathway in the ventral region (Rousset, 2010).
SPs are endopeptidases that use a serine for their catalytic activity and are characterized by the presence of the residues histidine, aspartic acid and serine (HDS) in the active site. Serine protease homologues (SPHs) are also found in various animal genomes and are defined by the lack of at least one of the essential residues of the catalytic triad HDS. Only a few SPHs have been characterized. One well-known example in mammals is the Hepatocyte growth factor or Scatter factor (HGF, SF). In Drosophila, almost 30% of the 204 identified SP genes encode SPHs that are therefore most probably catalytically inactive. Only four of these SPH genes have been assigned a function. Mutations in masquerade (mas) lead to defects in somatic muscle attachment and in the formation of the nervous system during embryogenesis. The other three genes, spheroide, sphinx1 and sphinx2, were identified in an RNAi screen aimed at identifying genes involved in immunity (Rousset, 2010).
This study isolated the SPH encoding gene scarface (scaf) in a microarray screen designed to identify new genes transcriptionally regulated by the JNK (c-Jun N-terminal kinase) pathway during dorsal closure (DC) of the Drosophila embryo. DC is a major morphogenetic movement that takes place after germ-band retraction to close the eye-shaped opening of the dorsal embryonic epidermis, over the transient tissue called the amnioserosa (AS). Cell elongation is responsible for the dorsalward spreading of the lateral epithelial sheets. The AS, conversely, progressively reduces through cell constriction and engulfment accompanied by cell death. Precise activation of the JNK pathway, specifically in the dorsalmost row of epithelial cells in contact with the AS (the leading edge, LE), is a prerequisite for DC progression. Several transcriptional targets of the JNK pathway have already been described during DC, such as the profilin-coding gene chickadee, the transcription factor cabut, the integrin-coding genes scab and myospheroid or the trafficking gene Rab30. However, only two target genes are specifically expressed in the LE, decapentaplegic (dpp) and puckered (puc). Dpp, of the TGFβ family, participates in cell spreading of the lateral epithelium, cell constriction in the AS and connection of these two tissues. Dpp also regulates remodelling of the cytoskeleton in the segment border cells by activating Cdc42 and dPak. The gene puc encodes a phosphatase that sets up a negative-feedback loop at the level of the c-Jun kinase Basket (Bsk). In addition, other JNK target genes are involved in different biological processes. For instance, JNK-induced expression of matrix metalloproteinases is required for disc eversion and tumour invasion; JNK protection against oxidative stress is realized through expression of autophagy-related genes; and during immune response, JNK participates in the expression of antimicrobial peptide genes (Rousset, 2010).
This paper reports the characterization of scaf during Drosophila development. The catalytic triad of the SP domain of Scaf lacks two of the conserved HDS residues and Scaf was thus classified as a SPH. Although a previously isolated semi-lethal mutation led to adult escapers exhibiting a head scar under the proboscis, no function has been attributed to scaf. This study shows that the JNK signalling pathway regulates scaf expression in the embryo and in imaginal discs. In S2 cells and in vivo, the protein Scaf is secreted, indicating that it behaves as an extracellular SPH. Loss-of-function and gain-of-function in vivo studies uncovered an essential role of scaf during epithelial morphogenesis through antagonistic regulation of JNK signalling (Rousset, 2010).
This study shows that scaf is specifically induced by the JNK pathway in the LE cells that drive embryonic DC. This is the third gene, after dpp and puc, showing this very specific site of expression under the control of JNK. In addition, scaf expression has been demonstrated to be regulated by JNK in the A8 segment of the male genital disc. Scaf is also detected in the JNK-responsive cells (expressing puc-lacZ) of wing and leg discs, indicating that scaf can be considered as a general JNK target gene during epithelial morphogenesis. Like other proteins of the SP family, Scaf is a secreted SPH. Importantly, the results point to a negative role of scaf against JNK activity during DC in the embryo and during male terminalia formation in the pupa. Therefore, in addition to the inducible antagonist puc, the JNK pathway restrains its own activity during epithelial morphogenesis through the expression of the SPH gene scaf (Rousset, 2010).
The amount of trypsin- and chymotrypsin-like SP enzymes in metazoan genomes is highly variable, from a dozen in C. elegans to more than two hundred in dipteran species like Drosophila and about a hundred in mammals. Gene expansion that occurred in flies is remarkable and concerns both the active enzymes and their homologues, indicating specific adaptations to environment and life strategies. Catalytic domains of SP enzymes share high structural similarities and substrate specificity is insured by a binding cleft located near the active site, as well as surface loops present in the SP domain. SPH proteins have lost their catalytic activities and it is assumed that they have adopted new functions based on novel binding capacities. For instance, SPH could compete with an active SP for specific substrates (Rousset, 2010).
Classical genetics has revealed the implication of various genes in DC and other studies have identified several JNK target genes. However, scaf is the first member of the SP family with a role in this epithelial sealing movement. Like for the Drosophila SPH gene mas, mutations in scaf did not show complete expressivity and penetrance in the embryo. One straightforward explanation is the presence of a redundant protein among the various other SPH of the Drosophila genome. Compensatory mechanisms, which are known to be important during DC, could also hide the impact of scaf removal. Nevertheless, the results showed that scaf is important for morphogenesis by directly modulating the activity of the JNK pathway. How scaf negatively acts is still an open question, but it is probable that Scaf plays a role in the extracellular space, in contrast to the strong intracellular inhibitor Puc. The signal that activates JNK signalling (and therefore DC) has not yet been discovered, preventing a straightforward analysis of Scaf molecular action. SP proteins are well-described for their role in activating signal transduction pathways or in degrading the extracellular matrix (ECM), and defects in muscle attachment observed in mas mutations might be due to a decrease in cell-matrix adhesion. Accordingly, possible roles of Scaf could be to act locally on an unknown extracellular signal or at the level of the putative receptor of the JNK pathway, through interaction with the ECM. Functioning in a negative-feedback loop, scaf would allow a fine tuning of signalling in JNK-active tissues (Rousset, 2010).
The JNK signalling pathway is a major player of epithelial morphogenesis in many organs and developmental stages and its activity must be precisely controlled to coordinate tissue sealing. Two JNK target genes, puc and scaf, code for antagonists and therefore participate in this control. Whereas Puc acts intracellularly, Scaf is likely to operate in the extracellular space and represents the first secreted regulator of JNK-dependent epithelial sealing. Further studies of the role of scaf will highlight how a signal transduction pathway synchronizes collective cell behaviour in a three-dimensional environment, and how these cell rearrangements are controlled extracellularly to lead to a precise coordination of the movement (Rousset, 2010).
Cell-matrix interactions brought about by the activity of integrins and laminins maintain the polarized architecture of epithelia and mediate morphogenetic interactions between apposing tissues. Although the polarized localization of laminins at the basement membrane is a crucial step in these processes, little is known about how this polarized distribution is achieved. This study analysed the role of the secreted serine protease-like protein Scarface in germ-band retraction and dorsal closure -- morphogenetic processes that rely on the activity of integrins and laminins. Evidence that
scarface is regulated by c-Jun amino-terminal kinase and that scarface mutant embryos show defects in these morphogenetic processes. Anomalous accumulation of laminin A on the apical surface of epithelial cells was observed in these embryos before a loss of epithelial polarity was induced. It is proposed that Scarface has a key role in regulating the polarized localization of laminin A in this developmental context (Sorrosal, 2010).
During germ band retraction (GBR), the tail end of the germ band, or embryo proper, interacts with the amnioserosa (AS), an epithelium of large flat cells that does not contribute to the larva, and moves to its final posterior position. After GBR, the ectoderm has a gap on its dorsal side that is occupied by the amnioserosa (AS). The dorsal-most cells of the ectoderm, on both sides of the embryo, are in contact with the AS and are called leading edge (LE) cells. During DC, LE cells direct the movement of the epidermal sheets migrating dorsally over the apical side of the AS until they meet and fuse at the dorsal midline. Attention of this study centered on scarf on the basis of its embryonic expression pattern. Two piggyBac insertions, scarf PBss(GFP) and scarf M13.M2(lacZ), were expressed at a high level in LE cells during GBR and DC and at a low level in AS cells. The expression of
scarf was confirmed by in situ hybridization. It was also expressed in the head and tail regions, and in the ventral ectoderm in a segmental pattern (Sorrosal, 2010).
The JNK pathway is activated in LE cells and leads to the expression of the signalling molecule Dpp and the phosphatase Puckered (Puc). The
scarf gene was expressed in the same cells as puc and dpp at the LE. Reduced JNK - Basket (Bsk) in
Drosophila - or expression of a dominant-negative version of Bsk or Puc, which mediates a feedback loop repressing JNK activity, led to the loss of scarf expression in LE cells. Loss of puc activity, which leads to increased levels of JNK activity, or expression of an activated version of JNK -activating kinase (Hemipterous in Drosophila), led to the expansion of the scarf expression domain throughout the lateral ectoderm. Dorsal cells had a stronger response to increased JNK, suggesting that JNK requires the activity of another factor expressed in this region to induce scarf. As stated, the JNK cascade drives the expression of the secreted molecule Dpp. In embryos mutant for the Dpp receptor Thickveins, scarf expression at the LE was not lost and ectopic activation of the Dpp pathway did not induce the ectopic expression of scarf. These results indicate that scarf expression is induced by JNK in LE cells (Sorrosal, 2010).
Homozygosis or trans-heterozygosis for the piggyBac insertions
scarf PBss and scarf M13.M2 caused semi-lethality and survivors had scars on the head, phenotypes that were reverted by precise excision of the transposon elements. As embryos showed no obvious cuticle phenotype, stronger alleles of scarf were generated by imprecise excision of a P element located in the third intron of
scarf (scarf KG05129). One allele (scarf
Δ1.5) was isolated that led to the loss of scarf messenger RNA expression. This is an embryonic lethal allele and
trans-heterozygous combinations of scarf Δ1.5 or Df(2R)nap14, a deficiency that uncovers the scarf locus, with scarf PBss or scarf M13.M2 were semi-lethal and resulted in scars on the head. Homozygous animals for scarf Δ1.5 or Df(2R)nap14 showed similar phenotypes. Their larval cuticles were either dorsally wrinkled or presented a dorsal hole or a U-shaped phenotype, which is suggestive of failures in GBR and DC. A high proportion of embryos showed undifferentiated cuticles. Ubiquitous expression of
scarf largely rescued the scarf Δ1.5 phenotype (Sorrosal, 2010).
Time-lapse recordings indicated that these embryos were delayed in development and showed a failure in GBR. Frequently, the attachment of the AS to the tail end of the germ band was compromised and the germ band did not retract. In embryos that were able to retract, DC started and LE cells began to direct the movement of the epidermal sheets migrating dorsally over the apical
side of the AS. However, either the AS detached from the neighbouring epidermal sheet or the epidermal sheets met and fused at the dorsal midline but the dorsal epidermis reopened. The requirement of scarf in DC was analyzed in greater detail in fixed staged embryos. During the initial steps of DC, lateral ectodermal cells start to elongate dorsally and an actin cable at the LE is formed, which helps to generate a linear fence at the interface between LE and
AS cells. In scarf Δ1.5 embryos, elongation of the lateral ectoderm was compromised frequently, the actin cable was not formed properly, the interface between LE and AS cells became irregular, and eventually rips appeared at the AS-LE interface. The elongation of lateral ectodermal cells and adhesion between AS and LE cells are mediated by the activity of Dpp and JNK activity controls the polymerization of actin into a cable at the LE. In embryos lacking scarf, the activity of the Dpp and JNK signalling pathways was not affected. Thus, Scarf has a role in DC without affecting the activity levels of these pathways (Sorrosal, 2010).
Integrins and laminins have a fundamental role in GBR and DC by mediating interactions between AS cells and neighbouring cell populations. As defects were observed in these processes on scarf depletion and a reduction in scarf levels increased the frequency of cuticle phenotypes caused by depletion of the β-integrin position-specific (βPS) subunit, the contribution of Scarf to the regulation of the expression levels and subcellular localization of these proteins was examined. Cross-sectional views of properly staged wild-type and scarf Δ1.5 embryos stained for βPS integrin revealed similar levels and localization of integrins. The JNK signalling regulates the expression of the βPS integrin subunit during DC. Although ectopic activation of JNK induced increased expression of βPS integrin, ectopic expression of scarf did not exert this effect. The expression of LanA, one of the two existing fly
α-laminins. In wild-type embryos, high levels of LanA were localized at the BM of AS cells. Interestingly, scarf embryos revealed strong defects in the polarized distribution of LanA. It was localized on the apical side of the AS epithelium and its protein levels were largely reduced in the BM. Ubiquitous expression of scarf largely rescued the levels of LanA at the BM. Similar defects were observed in the polarized distribution of LanA in the lateral ectoderm of scarf mutant embryos, and these defects were also rescued largely by the ubiquitous expression of scarf. Apico-basal polarity of AS cells, visualized by the basal localization of βPS integrin and the localization of E-cadherin (E-cad) at the adherens junctions, was not
affected in hypomorphic scarf PBss and scarf
M13.M2 embryos and in some scarf Δ1.5 embryos even though LanA was localized on the apical side. However,
scarf depletion was frequently found to cause defects in the apico-basal polarity of AS cells and this was accompanied by multilayering of the AS epithelium. These observations suggest that before inducing a loss of epithelial integrity, the absence of Scarf caused aberrant localization of LanA on the apical side of the AS epithelium and reduced LanA in the BM, without affecting the distribution of apical and baso-lateral proteins. As integrins are involved in maintaining epithelial polarity, these results suggest that the observed loss of cell polarity is a consequence of reduced integrin-mediated adhesion to the BM. Consistent with this view, defects in the attachment of the AS cells to the
underlying yolk cell were observed frequently (Sorrosal, 2010).
Although the defects observed in scarf Δ1.5 embryos resemble those caused by βPS integrin depletion, only defects in the attachment of the AS with the underlying yolk cells are found in lanA mutant embryos. Mutations in wing blister (wb), the only other fly
α-laminin, cause defects in GBR and in the attachment between AS cells and the posterior germ band. These data suggest that LanA and Wb have a redundant function and that Scarf most probably
promotes BM localization of not only LanA but also Wb. Consistent with the proposed redundancy, halving the dose of
lanA or wb increased the frequency of the βPS integrin loss-of-function cuticle phenotype. The expression of Wb protein in AS cells using the available antibodies and the role of Scarf in the polarized distribution of other BM proteins, such as Perlecan or Collagen IV, were not addressed as these two proteins were not expressed in AS cells (Sorrosal, 2010).
These results indicate that Scarf depletion causes defects in the BM localization of LanA and in epithelial apico-basal polarity. The defects resemble those observed in follicle cells (that is, epithelial cells covering the fly oocyte) mutant for crag, a gene encoding for a protein localized in early and recycling endosomes and proposed to regulate protein transport and membrane deposition of BM proteins. Zygotic removal of
crag induced anomalous localization of LanA on the apical side of AS cells. Zygotic or both maternal and zygotic removal of crag produced cuticles with a weak dorsally wrinkled phenotype, suggesting that Crag has a redundant function with another protein in the fly embryo and that the low BM levels of LanA observed in these embryos are sufficient to exert their function. Interestingly, halving the dose of scarf expression gave rise to a large proportion of crag cuticles with phenotypes similar to those caused by
scarf depletion (Sorrosal, 2010).
Antibodies against Scarf were raised to analyse its subcellular localization. As the antibodies did not work properly at embryonic stages and did not detect endogenous levels of Scarf protein, the wing imaginal disc, a monolayered
epithelium, was used to analyse its subcellular localization. Scarf was not detected in wild-type wing cells. When
scarf and green fluorescent protein (GFP) were expressed in a restricted domain in the wing disc, Scarf was detected not only in the GFP-labelled
scarf-producing cells, but also on the apical side of the epithelium at long distances from the source. When atrial natriuretic factor-GFP (a fusion between the secreted rat atrial natriuretic peptide and GFP) was expressed,
the protein was also observed at long distances from the source; however, it did not accumulate on the apical side of the epithelium. Scarf was found to localize in small punctate structures throughout the cytoplasm of scarf non-producing cells. These structures corresponded either to Rab5-positive early endosomes, to Rab7-positive late endosomes, or to Rab11-positive recycling endosomes. Together, these results indicate that Scarf is secreted apically and is internalized through endocytosis by non-producing cells. To confirm that Scarf is a secreted protein,
Drosophila S2 cells were transfected with either a scarf-myc-tagged transgene or a transgene driving the expression of a myc-tagged membrane-tethered form of Scarf (Scarf-CD2). Scarf was isolated not only from the protein extract of the scarf-myc-expressing cells but also from the supernatant. Transfected Scarf-CD2 and endogenous actin were not observed in the supernatant (Sorrosal, 2010).
This study has characterized the role of scarf, a JNK-regulated gene, in GBR and DC, two morphogenetic processes that rely on cell-matrix interactions between the AS epithelium and neighbouring cell populations. Evidence that Scarf
is a secreted protein expressed in LE cells and involved in promoting the localized accumulation of LanA in the BM of AS cells. Although low levels of Scarf were detected in AS cells, the cuticle phenotype of
scarf mutant embryos and the defects observed in BM localization of LanA and epithelial integrity of AS cells were rescued largely by driving expression of a
scarf transgene only in ectodermal cells, indicating that Scarf is acting as a secreted protein. Three alternative hypotheses might explain the role of Scarf and Crag in the polarized localization of LanA to the BM. As Crag and Scarf are localized specifically on the apical side of the epithelium, they would have a repulsive role, inhibiting the targeting of LanA-containing vesicles with apical membranes. Alternatively, LanA might be secreted apically and basally and be stabilized preferentially on the basal side or degraded on the apical side. As Scarf encodes for a putative serine protease-like protein without catalytic activity, Scarf would function, in this scenario, to facilitate the effect of a cascade of serine proteases involved in the degradation of LanA
in the apical domain of epithelial cells. Finally, in scarf mutant cells, LanA might be transported from the basal to the apical side through transcytosis, a mechanism responsible for the formation of a small apical BM cap over pre-invasive epithelial cells during Drosophila oogenesis (Sorrosal, 2010).
Another secreted serine protease-like protein without catalytic activity, Masquerade, regulates cell-matrix interactions at the somatic-muscle attachment in the fly embryo, a process that also depends on integrin and laminin activity. It is speculated that a number of non-functional serine protease-like proteins, such as Masquerade and Scarf, are regulated temporally and spatially to promote the appropriate localization of BM proteins in a context-dependent manner to
facilitate cell-matrix interactions and to ensure the maintenance of epithelial integrity. Similarly, among the large class of vertebrate functional and non-functional serine protease-like proteins involved in remodelling the extracellular matrix, some of them might exert a similar action (Sorrosal, 2010).
Mitogen-activated protein kinase kinase kinase kinase-3 (MAP4K3) is a Ste20 kinase family member that modulates multiple signal transduction pathways. MAP4K3 has been identified as proapoptotic kinase using an RNA interference screening approach. In mammalian cells, MAP4K3 enhances the mitochondrial apoptosis pathway through the post-transcriptional modulation of selected proapoptotic Bcl-2 homology domain 3-only proteins. Recent data suggest that MAP4K3 mutations contribute to pancreatic cancer, which highlights the importance of studying the in vivo function of this kinase. To determine whether the cell death function is conserved in vivo and which downstream signalling pathways are involved, transgenic flies were generated expressing happyhour (hppy), the Drosophila MAP4K3 orthologue. This study shows that the overexpression of hppy promotes caspase-dependent apoptosis and that the hypothetical kinase domain is essential for inducing cell death. In addition, it was shown that hppy expression triggers the activation of both the c-Jun N-terminal kinase (JNK) and target of rapamycin (TOR) signalling pathways; however, only JNK signalling is required for apoptosis. Together, these results show that hppy has a JNK-dependent proapoptotic function in Drosophila, which reinforces the hypothesis that MAP4K3 might act as tumour suppressor by regulating apoptosis in higher eukaryotes (Lam, 2010).
The work reported here identifies Hppy, the Drosophila orthologue of human MAP4K3, as an in vivo modulator of apoptosis. In mammalian cells, MAP4K3 promotes apoptosis by inducing the post-transcriptional activation of a subset of BH3-only Bcl-2 family proteins. Expression of MAP4K3 leads to cell death through the mitochondrial (intrinsic) pathway; this action is suppressed by JNK and TOR inhibition. This study provides the first in vivo evidence that the fly orthologue of MAP4K3, Hppy, is a death-inducing kinase that promotes caspase-dependent apoptosis. Mitochondria have a crucial role in the intrinsic apoptosis pathway in vertebrates. In Drosophila, however, the role of mitochondria in apoptosis is unclear, as mutants of the Drosophila bcl-2 homologues debcl and buffy show no obvious defects in developmental apoptosis and this form of cell death is not blocked in cells with depleted Cyt-C (Lam, 2010).
The data suggest that Hppy-mediated cell death occurs independently of the mitochondrial pathway; buffy expression failed to block Hppy-dependent apoptosis, and negative modulation of the TOR pathway in flies failed to suppress Hppy-dependent cell death. Given that the human orthologue of Hppy employs the TOR pathway to stimulate the mitochondrial pathway of apoptosis, it is proposed that suppression of this pathway in flies fails to affect Hppy-dependent cell death because the mitochondrial apoptosis pathway may be of limited importance in flies. Genetic analysis suggests that the JNK pathway positively regulates Hppy-dependent cell death. It is unlikely that the JNK pathway induces cell death by activating the mitochondrial pathway in Drosophila. In flies, the genes reaper, grim and hid (RGH) promote caspase activation by antagonising the Drosophila inhibitor of apoptosis proteins. Although reaper and grim are only expressed in cells that are destined for death, hid expression is controlled at both the transcriptional and post-transcriptional levels. Hid has been shown to be transcriptionally activated in a JNK- and Foxo-dependent manner. Additionally, the proapoptotic activity of Foxo is opposed by the action of receptor tyrosine kinases (RTKs) such as EGFR and insulin-like growth factor receptor. Given the recent observation that Hppy is a potent negative modulator of EGFR signalling, it is tempting to propose that this kinase might promote apoptosis by modulating signalling pathways that affect the levels of RGH proteins. This idea is consistent with the observation that another Ste20 family member, Hippo, promotes hid expression (Lam, 2010).
A single amino-acid substitution in MAP4K3 has been associated with pancreatic cancer, suggesting that this kinase might be an important modulator of tumourigenesis (Lam, 2010).
It was also previously found that the levels of MAP4K3 are significantly reduced in pancreatic tumour samples (Lam, 2010).
Given that in Drosophila, Hppy acts to both stimulate JNK signalling it is tempting to propose that mammalian MAP4K3 might act as a tumour suppressor, not only by promoting JNK-dependent apoptosis but also by blocking the prosurvival effects of EGFR signalling mechanisms (Lam, 2010).
Metabolic homeostasis in metazoans is regulated by endocrine control of insulin/IGF signaling (IIS) activity. Stress and inflammatory signaling pathways -- such as Jun-N-terminal Kinase (JNK) signaling -- repress IIS, curtailing anabolic processes to promote stress tolerance and extend lifespan. While this interaction constitutes an adaptive response that allows managing energy resources under stress conditions, excessive JNK activity in adipose tissue of vertebrates has been found to cause insulin resistance, promoting type II diabetes. Thus, the interaction between JNK and IIS has to be tightly regulated to ensure proper metabolic adaptation to environmental challenges. This study identified a new regulatory mechanism by which JNK influences metabolism systemically. JNK signaling is required for metabolic homeostasis in flies and that this function is mediated by the Drosophila Lipocalin family member Neural Lazarillo (NLaz), a homologue of vertebrate Apolipoprotein D (ApoD) and Retinol Binding Protein 4 (RBP4). Lipocalins are emerging as central regulators of peripheral insulin sensitivity and have been implicated in metabolic diseases. NLaz is transcriptionally regulated by JNK signaling and is required for JNK-mediated stress and starvation tolerance. Loss of NLaz function reduces stress resistance and lifespan, while its over-expression represses growth, promotes stress tolerance and extends lifespan -- phenotypes that are consistent with reduced IIS activity. Accordingly, this study found that NLaz represses IIS activity in larvae and adult flies. The results show that JNK-NLaz signaling antagonizes IIS and is critical for metabolic adaptation of the organism to environmental challenges. The JNK pathway and Lipocalins are structurally and functionally conserved, suggesting that similar interactions represent an evolutionarily conserved system for the control of metabolic homeostasis (Hull-Thompson, 2009).
System-wide coordination of cellular energy consumption and storage is crucial to maintain metabolic homeostasis in multicellular organisms. It is becoming increasingly apparent that endocrine mechanisms that are required for this coordination impact the long-term health of adult animals and significantly influence lifespan and environmental stress tolerance. Insulin/IGF signaling (IIS) is central to this regulation, as loss of Insulin signaling activity impairs metabolic homeostasis, but induces stress tolerance and increases lifespan in a variety of model organisms. Interestingly, environmental stress and cellular damage can systemically repress IIS activity, suggesting the existence of adaptive response mechanisms by which metazoans manage energy resources in times of need. The mechanism(s) and mediators of this endocrine regulatory system are only beginning to be understood (Hull-Thompson, 2009).
Studies in flies and worms have recently identified the stress-responsive Jun-N-terminal Kinase (JNK) signaling pathway as an important component of such an adaptive metabolic response to stress. JNK activation, which can be induced by a variety of environmental stressors, including oxidative stress, represses IIS activity, extending lifespan but limiting growth. Interestingly, similar effects of JNK signaling are also observed in mammals, in which it represses Insulin signal transduction by various mechanisms, including an inhibitory phosphorylation of Ser-307 of the insulin receptor substrate, as well as activation of the transcription factor FoxO. This inhibition contributes to Insulin resistance and the metabolic syndrome in obese mice, suggesting that chronic inflammatory processes (which result in activation of JNK signaling) are central to the etiology of metabolic diseases in obese individuals (Hull-Thompson, 2009).
Endocrine interactions between Insulin-producing and various Insulin-responsive tissues are likely to coordinate the adaptive metabolic response described above. JNK-mediated activation of Foxo in Insulin Producing Cells (IPCs) of flies, for example, represses the expression of insulin-like peptide 2 (dilp2), regulating growth and longevity. At the same time, Foxo activation in the fatbody results in lifespan extension, presumably by an endocrine mechanism that feeds back to IPCs (Hull-Thompson, 2009).
Adipose tissue is increasingly being recognized as an important regulator of metabolic homeostasis. It secretes a variety of so-called adipokines, including the inflammatory cytokine TNF-alpha (Hotamisligil, 2006). TNF-alpha activates JNK signaling, contributing to JNK-mediated insulin resistance in mouse models for obesity. JNK activation in adipose tissue further induces expression of IL-6, which specifically induces Insulin resistance in the liver. While the chronic inhibition of insulin signaling by adipose-derived inflammatory cytokines thus has deleterious effects in obese individuals, it is likely that such endocrine interactions have evolved to govern metabolic homeostasis systemically in an adaptive manner. Supporting this view, adipose tissue is an important regulator of lifespan in worms, flies, and mice, and it is emerging that systemic inhibition of Insulin signaling by adipose-derived factors is involved in this effect (Hull-Thompson, 2009).
An endocrine role for adipose tissue in metabolic regulation has further been demonstrated in mice with adipose-specific deletion of the glucose transporter GLUT4, in which secretion of the Lipocalin family member RBP4 from fat cells induces insulin resistance throughout the organism. Such an endocrine system is expected to be adaptive, since it preserves glucose for only the most essential functions during starvation or environmental stress. At the same time, mis-regulation of this system is likely to contribute to metabolic diseases like type II diabetes. Accordingly, increased serum levels of RBP4 are found in obese and diabetic individuals, and polymorphisms in the rbp4 locus are associated with type II diabetes (Hull-Thompson, 2009 and references therein).
The Lipocalins are a large family of mostly secreted proteins that bind small hydrophobic ligands. Lipocalin family members are characterized by a low sequence similarity (reflecting diversification of biological functions), but a highly conserved tertiary protein structure and similar exon/intron structures of their genes. Recent studies implicate various Lipocalins in the regulation of systemic insulin action and of stress responses. Interestingly, the neuroprotective Lipocalin ApoD is strongly induced in aging mice, rhesus macaques and humans, suggesting evolutionarily conserved regulation of this gene, an it induces insulin resistance when overexpressed in the mouse brain (Hull-Thompson, 2009 and references therein).
The Drosophila genome contains three Lipocalin genes: NLaz, GLaz, and karl. Unlike the protein Lazarillo in more ancient insect lineages, which is GPI-anchored to the cell membrane of neurons, all Drosophila Lipocalins are secreted extracellular proteins, like ApoD and all other vertebrate Lipocalins. Recent studies have identified an important role for GLaz in stress resistance and lifespan control as well as in the regulation of lipid storage. While the function of NLaz remains unclear, in situ hybridization in Drosophila embryos shows that it is expressed in a subset of neuronal cells, and, interestingly, in the developing fat body, indicating a potential role in the systemic regulation of metabolism (Hull-Thompson, 2009).
This study shows that NLaz transcription is induced by oxidative stress and by JNK signaling in the fat body, influencing metabolic homeostasis in the fly. Importantly, NLaz induces stress and starvation tolerance downstream of JNK signaling, and negatively regulates Insulin signaling, disrupting glucose homeostasis, repressing growth, and extending lifespan. The results thus indicate that induction of NLaz mediates the antagonistic interaction between JNK and Insulin signaling in flies, acting as part of a stress response mechanism that adjusts metabolism and growth in response to environmental insults (Hull-Thompson, 2009).
The findings support a role for JNK-mediated NLaz induction in the fatbody as a central part of an adaptive endocrine system that coordinates metabolism in response to environmental stress by regulating insulin sensitivity of peripheral tissues. Recent studies have highlighted the role of adipose-derived endocrine factors in such adaptive responses. For example, reducing IIS activity or over-expressing Foxo specifically in adipose tissue leads to lifespan extension and stress tolerance in flies, mice and worms, presumably mediated by systemic repression of IIS. Furthermore, amino acid deprivation of Drosophila fat body cells leads to marked decreases in PI3K activity in wing imaginal discs and in the epidermis. In vertebrates, in contrast, excessive JNK activation in adipose tissue induces insulin resistance in the periphery, promoting Type II diabetes. The current results implicate NLaz as a mediator of such systemic repression of IIS activity by adipose tissue (Hull-Thompson, 2009).
JNK-mediated repression of IIS in flies is thus not only mediated by its
function in IPCs, where it represses dilp2 transcription, but also by adipose-specific induction of NLaz, which then inhibits IIS activity in insulin target tissues. This dual antagonism of IIS by JNK is intriguing, as it indicates that adaptive regulation of metabolism requires coordinated control of both insulin-like peptide production and peripheral insulin sensitivity. How the relative contribution of these effects regulates the organism's metabolic homeostasis, stress resistance and lifespan, is an interesting question that will require further investigation (Hull-Thompson, 2009).
Vertebrate Lipocalins have also been implicated in the modulation of insulin action, and recent studies suggest a protective role of these molecules under diverse stress conditions. This function of Lipocalins thus emerges as an evolutionarily conserved adaptive mechanism, and this work integrates this mechanism into the known antagonism between JNK and IIS. Based on the evolutionary conservation of this antagonism it is tempting to speculate that vertebrate Lipocalins also act as effectors of JNK in the regulation of systemic insulin sensitivity, with important implications for potential therapeutic targeting of these molecules (Hull-Thompson, 2009).
While generally promoting metabolic homeostasis and stress tolerance, functional specialization of different Lipocalin family members is expected due to their high sequence divergence. Accordingly, the data show that the Lipocalins present in Drosophila differ in regulation and function. While NLaz and GLaz both regulate stress sensitivity, only NLaz was found to be regulated by JNK signaling. Regulation of Karl, on the other hand, does not influence starvation tolerance (as NLaz does), but promotes resistance against infection by E. faecalis. Further investigation of this diversification of Lipocalin function promises to provide important insight into the systemic regulation of adaptation to diverse environmental challenges. Of particular interest will be to assess the role of Karl as a potential regulator of IIS during infection. Infection with Mycobacterium Marinum can result in significant repression of IIS activity, leading to phenotypes similar to wasting disease. It is intriguing to speculate that excessive JNK-induced Karl expression may cause this pathology (Hull-Thompson, 2009).
In humans, dysregulation of Lipocalins has been correlated with obesity, insulin resistance, and type II diabetes. The cause for this mis-regulation of Lipocalin expression remains unclear, however. The current results implicate JNK signaling, which is activated chronically in obese conditions, as a possible cause. The finding that mammalian lipocalin-2, which impairs insulin action, is induced by the JNK activator TNFalpha, is especially intriguing. Additional studies in vertebrates, as well as in the Drosophila model, will provide further insight into the physiological role of Lipocalins, their regulation by stress signaling, as well as their interaction with Insulin signaling. As Lipocalins are secreted molecules that bind hydrophobic ligands, it is further crucial to identify their physiological ligands in an effort to understand the mechanism(s) by which IIS activity is antagonized by Lipocalins. Such insight promises to provide a deeper understanding of the coordination of metabolic adaptation in metazoans as well as of the etiology of diabetes and other metabolic diseases (Hull-Thompson, 2009).
Epithelial homeostasis and the avoidance of diseases such as cancer require the elimination of defective cells by apoptosis. This study investigated how loss of apical determinants triggers apoptosis in the embryonic epidermis of Drosophila. Transcriptional profiling and in situ hybridisation show that JNK signalling is upregulated in mutants lacking Crumbs or other apical determinants. This leads to transcriptional activation of the pro-apoptotic gene reaper and to apoptosis. Suppression of JNK signalling by overexpression of Puckered, a feedback inhibitor of the pathway, prevents reaper upregulation and apoptosis. Moreover, removal of endogenous Puckered leads to ectopic reaper expression. Importantly, disruption of the basolateral domain in the embryonic epidermis does not trigger JNK signalling or apoptosis. It is suggested that apical, not basolateral, integrity could be intrinsically required for the survival of epithelial cells. In apically deficient embryos, JNK signalling is activated throughout the epidermis. Yet, in the dorsal region, reaper expression is not activated and cells survive. One characteristic of these surviving cells is that they retain discernible adherens junctions despite the apical deficit. It is suggested that junctional integrity could restrain the pro-apoptotic influence of JNK signalling (Kolahgar, 2011). This study has shown that apical, but not basolateral, disruption leads to apoptosis in a developing epithelium. It was also showed that JNK signalling is a key intermediate in the signal transduction mechanism that triggers apoptosis in response to the loss of apical determinants. Apical disruption leads to activation of JNK signalling, which in turn activates transcription of the pro-apoptotic gene rpr. Moreover, rpr expression is not activated in apically disrupted embryos that are prevented from activating JNK signalling. Interestingly, in bicoid-deficient embryos and other segmentation mutants, cell fate misspecification requires activation of a different pro-apoptotic gene, hid. Therefore, distinct quality control pathways might exist to ensure that different forms of defective cells are removed from developing epithelia. JNK signalling has been shown to mediate apoptosis in a variety of other situations, including after DNA damage. However, JNK signalling does not necessarily cause apoptosis. Indeed, this pathway modulates many other cell activities, such as proliferation, differentiation and morphogenesis. What conditions determine whether JNK triggers apoptosis or not is an important issue. Another obvious question raised by the current findings concerns the nature of the mechanism that triggers JNK signalling following the loss of apical determinants (Kolahgar, 2011). This study has shown that, in the embryonic epidermis, JNK signalling is activated by the loss of apical, not basolateral, determinants. In fact, reduction of lgl activity prevents JNK activation in crb mutant embryos. Similarly, Scrib knockdown prevents JNK activity in the mouse mammary epithelium, suggesting that the loss of the basolateral domain could have a general anti-JNK (and perhaps anti-apoptotic) activity. Although JNK activation has been documented in tissues that lack a basolateral determinant, it is suggested that this might be an indirect consequence of cell competition, which triggers JNK signalling, or of the specific experimental conditions (partial reduction of Puc activity). Overall, the results suggest the existence of an apical domain-dependent activity that modulates JNK signalling in the embryonic epidermis of Drosophila. This activity could be similar to that postulated to be at work in cultured MDCK cells, but is likely to be distinct from the process that leads to apoptosis in response to mosaic disruption of the basolateral domain in imaginal discs (Kolahgar, 2011). The mechanism underlying the activation of JNK signalling by loss of apical determinants remains unknown. For example, it is uncertain at this point whether there is an apically localised activity that directly modulates JNK signalling or whether a more indirect route is at work (paths 1 and 2 respectively; see Signalling upstream and downstream of JNK). Since apical organisation is required for the establishment of adherens junctions, it is conceivable that the effect of apical disruption on JNK signalling is mediated by junctional disruption. This possibility is compatible with the absence of ectopic JNK activation in basolateral mutants, in which E-cadherin remains localised to patches at the cell surfac. However, one would have to invoke that slight junctional disruption is sufficient to trigger JNK signalling, as this pathway is upregulated in the dorsal region of crb mutants, where the extent of junctional disruption is relatively mild. It has not been possible to discriminate between paths 1 and 2, partly because of the current difficulty in eliminating adherens junctions from early Drosophila embryos. Future work will require novel means of interfering specifically with adherens junctions. Considering the lack of involvement of Egr, it will also be necessary to identify the upstream components of JNK signalling that respond to epithelial disruption (Kolahgar, 2011). Overexpression of Puc, a feedback inhibitor of JNK signalling, prevents apoptosis in the ventral epidermis of crb embryos. This is clear evidence that JNK signalling is required for apical deficit to trigger apoptosis. However, it is well established that JNK signalling does not necessarily lead to apoptosis. This is particularly well illustrated by the situation at the dorsal edge of wild-type embryos, where JNK is highly active without triggering apoptosis. Moreover, in crb mutants, a 6- to 10-cell-wide band of dorsal tissue is refractory to the pro-apoptotic influence of JNK signalling. Therefore, additional conditions must be met for JNK signalling to activate rpr expression and trigger apoptosis. This study has identified two situations when refractory cells succumb to the pressure of JNK signalling (see Signalling upstream and downstream of JNK). One involves the reduction of Puc and the other the removal of zygotic E-cadherin activity. The first situation suggests that endogenous Puc can limit the ability of JNK to activate rpr expression and trigger apoptosis. The important role of Puc in preventing cell death is also highlighted by the extensive apoptosis seen in embryos lacking both maternal and zygotic Puc activity. Puc could act solely by limiting the extent of JNK signalling, thus preventing the very high level of signalling required for rpr expression. Alternatively, or in addition, Puc could have an activity that specifically prevents certain genes, such as rpr, from being spuriously activated. In any case, it is likely that the regulatory relationships between JNK, Puc and apoptosis are influenced by the cellular context (e.g., the state of adherens junctions (Kolahgar, 2011). JNK signalling triggers rpr expression (and apoptosis) more readily if adherens junctions are weakened or disrupted. Therefore, junctional integrity could also protect epithelial cells from the pro-apoptotic effects of JNK signalling. Dorsoventral differences in junctional integrity and remodelling have been noted in the embryonic epidermis of Drosophila and these might explain why these two regions respond differently to the loss of crb. The results suggest that residual junctional integrity in the dorsal epidermis prevents JNK signalling from activating rpr expression. It is conceivable that a protective signal emanates from adherens junctions. Alternatively, junctional disruption could interfere with the ability of Puc to rein in the effect of JNK signalling on rpr expression. Although differential junctional dynamics between the dorsal and ventral epidermis could determine the propensity to undergo apoptosis, the possibility cannot be excluded that other dorsoventral determinants are at work too (Kolahgar, 2011). This study has shown that loss of apical polarity leads to apoptosis by activating JNK signalling and causing junctional disruption. It is expected that this response, which is readily detectable in the crb mutant condition, might reflect a process that ensures the removal of abnormal and damaged cells during epithelial homeostasis. It is hoped that understanding the machinery that links epithelial disruption to JNK signalling and the transcription of pro-apoptotic genes will suggest means of reactivating this pathway in pathological situations (Kolahgar, 2011).
Homeostatic mechanisms can eliminate abnormal cells to prevent diseases such as cancer. However, the underlying mechanisms of this surveillance are poorly understood. This study investigated how clones of cells mutant for the neoplastic tumor suppressor gene scribble (scrib) are eliminated from Drosophila imaginal discs. When all cells in imaginal discs are mutant for scrib, they hyperactivate the Hippo pathway effector Yorkie (Yki), which drives growth of the discs into large neoplastic masses. Strikingly, when discs also contain normal cells, the scrib-
cells do not overproliferate and eventually undergo apoptosis through JNK-dependent mechanisms. However, induction of apoptosis does not explain how scrib-
cells are prevented from overproliferating. This study reports that cell competition between scrib- and wild-type cells prevents hyperproliferation by suppressing Yki activity in scrib- cells. Suppressing Yki activation is critical for scrib- clone elimination by cell competition, and experimental elevation of Yki activity in scrib- cells is sufficient to fuel their neoplastic growth. Thus, cell competition acts as a tumor-suppressing mechanism by regulating the Hippo pathway in scrib- cells (Chen, 2012).
This study shows that tumorigenic scrib- cells are removed
from Drosophila imaginal discs by a cell-cell signaling event that
suppresses elevated Yki activity in scrib- cells. Previous reports
implicated JNK as a mediator of cell competition of scrib-
clones, where it induces apoptosis and suppresses proliferation. However, it was not known how JNK prevents scrib- clones from hyperproliferating. This study now provides evidence that
JNK prevents scrib- clones from hyperproliferating by regulating
the activity of the Hippo pathway effector Yki. First, scrib-
clones that do not face cell competition up-regulate Yki activity,
which drives their hyperproliferation. Second, when scrib- clones
do face cell competition, then JNK signaling prevents the upregulation
of Yki activity. Third, experimental up-regulation of
Yki activity is sufficient to rescue scrib- clones from being
eliminated by cell competition. Fourth, experimental suppression
of Yki activity in scrib- clones not subjected to cell competition
is sufficient to suppress their hyperproliferation. Therefore, cell
competition suppresses up-regulation of Yki activity in scrib-
cells, and this suppression is important for the elimination of
scrib- clones by cell competition. Previous reports showed that
Hippo pathway reporters can be up-regulated in scrib- and lgl−
mutant discs and clones and that Yki is required for the overgrowth of scrib-+BskDN cells not subjected to cell competition. However, these studies did not analyze the
effects of cell competition on Yki activity in scrib- cells. This
analysis now shows that scrib- cells facing cell competition do not
up-regulate Yki activity and thereby identifies a mechanism that is critical for the elimination of scrib- cells. Although it was reported that scrib- and lgl− clones can upregulate ex-lacZ expression and Yki activity (Chen, 2012).
However, upon quantification it was found that the majority of
scrib- clones have normal or reduced levels of ex-lacZ expression,
and only a small percentage of scrib- clones have elevated
levels of ex-lacZ expression. Clones with elevated ex-lacZ expression
were observed mainly in the hinge region of wing discs,
which may provide an environment of reduced cell competition. Thus, outcompeted scrib- clones do not have elevated levels of Yki activity. In contrast, when scrib- clones are
rescued from cell competition, they show highly elevated levels
of ex-lacZ expression. Similarly, discs that are entirely mutant for scrib, thereby creating an environment that does not have competing normal cells, show hyperactivation of Yki. Cell competition thus prevents the hyperactivation of Yki in scrib- clones and turns a potential high-Yki 'supercompeting' scrib- cell into a
cell of lower fitness and less resistance to apoptosis. Importantly,
scrib- wts- and scrib-+Yki clones show greatly increased growth
and survival compared with scrib- clones. These results show that
elevated levels of Yki are sufficient to protect scrib- cells from
being outcompeted. Thus, if Yki activity already was high in
scrib- cells facing cell competition, those cells would not be
outcompeted, and overexpression of Yki or loss of wts would not
cause such dramatic effects on the survival and growth of scrib-
clones. Apparently, Yki levels in scrib- cells facing cell competition
are not high enough for these cells to evade cell competition.
Thus, the amount of Yki activity in scrib- cells is a critical
determinant of whether scrib- clones are eliminated or form
tumorous tissue, and the suppression of Yki activity in scrib-
clones is important for the elimination of scrib- clones by cell competition (Chen, 2012).
These studies show that JNK activity is required in scrib- cells for
the suppression of Yki activity by cell competition. In contrast,
JNK signaling can induce Yki activity during regeneration and
compensatory proliferation in imaginal discs. Therefore,
the effects of JNK signaling on Yki activity in scrib- cells are
different from those in normal cells: JNK signaling activates Yki
in normal cells promoted to regenerate but suppresses Yki in
scrib- cells induced to be eliminated. Interestingly, both these
effects are observed in discs with scrib- clones. In scrib- cells, JNK
activity suppresses the hyperactivation of Yki, but in neighboring
cells that are stimulated to proliferate and compensate for the loss
of scrib- cells, the activities of both JNK and Yki are elevated. However, non-cell-autonomous effects
on Yki reporters were still observed in egr−/− animals and in discs that ubiquitously
inhibited JNK signaling by BskDN. Therefore, JNK-independent
signals contribute to the non-cell-autonomous induction of Yki
activity around scrib- clones. The regulation of Yki by JNK signaling
thus is complex and context dependent and may involve several mechanisms (Chen, 2012).
The observation that wts- scrib- clones overgrow indicates that
JNK and Wts function in parallel to regulate Yki or that JNK
regulates the Hippo pathway upstream of Wts. JNK can phosphorylate
and activate Yap1 to regulate apoptosis in mammalian
cells. Notably, the JNK phosphorylation sites of Yap1
are different from the Lats phosphorylation sites, supporting
a model in which JNK functions in parallel with Wts to regulate
Yki activity. However, it is not known whether the same sites also
act to suppress the activity of Yki in other contexts (Chen, 2012).
Although several models have been proposed to explain how
cell-cell interactions between scrib- and normal cells lead to the
elimination of scrib- clones from epithelia, it was not clear what
properties normal cells must possess to perform this tumorsuppressive
role. The data demonstrate that for scrib-
cells to be eliminated they must be juxtaposed with cells that
have higher levels of competitive fitness, not just proper cellular
architecture. Overexpression of the Myc or RasV12 oncogenes in
scrib- clones increases their fitness. As a result, in scrib- clones
cell competition does not suppress Yki activity, which protects
these clones from being eliminated. Interestingly, Myc expression
also synergizes with loss of scrib to form tumors in mammals, and the data offer a model to explain this phenomenon. In addition to the cell-autonomous hyperproliferation, scrib-
cells that are not removed from imaginal discs have profound
non-cell-autonomous effects on the Hippo pathway. This non-cell-autonomous Hippo pathway-regulating signal may serve normally as a regenerative growth signal that facilitates the replacement
of eliminated or dying cells, such as outcompeted
scrib- cells. If scrib- clones are not eliminated efficiently,
however, this signal may persist longer than required to restore
the tissue, thereby causing overgrowth and deformation of neighboring
tissue. Thus, continued residence of tumorigenic cells can
stimulate growth beyond that needed for compensation, essentially
hijacking the proliferation and regeneration programs of their normal neighbors. Therefore, the non-cell-autonomous activation of Yki by scrib- cells may have important implications
for tumor-stromal interactions in human cancers (Chen, 2012).
In summary, it is concluded that cell competition is crucial in
suppressing the tumorigenic capacity of scrib- cells and does so
by regulating their Yki activity. Loss of this
regulation results in overproliferation of both tumorigenic cells
and neighboring wild-type cells. Efficient elimination
of scrib- clones by cell competition prevents Yki-fueled overgrowth
of mutant cells and prevents them from disrupting proliferation
control of their normal neighbors. Thus, this study identified
a tumor-suppression mechanism that depends on signaling between
normal and tumorigenic cells. These data identify evasion
of cell competition as a critical step toward malignancy and
illustrate a role for wild-type tissue in preventing the formation of cancers (Chen, 2012).
To form a gonad, germ cells (GCs) and somatic gonadal precursor cells (SGPs) must migrate to the correct location in the developing embryo and establish the cell-cell interactions necessary to create proper gonad architecture. During gonad morphogenesis, SGPs send out cellular extensions to ensheath the individual GCs and promote their development. Mutations have been identified in the raw gene that result in a failure of the SGPs to ensheath the GCs, leading to defects in GC development. Using genetic analysis and gene expression studies, it was found that Raw negatively regulates JNK signaling during gonad morphogenesis, and increased JNK signaling is sufficient to cause ensheathment defects. In particular, Raw functions upstream of the Drosophila Jun-related transcription factor to regulate its subcellular localization. Since JNK signaling regulates cell adhesion during the morphogenesis of many tissues, the relationship was examined between raw and the genes encoding Drosophila E-cadherin and beta-catenin, which function together in cell adhesion. It was found that loss of DE-cadherin strongly enhances the raw mutant gonad phenotype, while increasing DE-cadherin function rescues this phenotype. Further, loss of raw results in mislocalization of beta-catenin away from the cell surface. Therefore, cadherin-based cell adhesion, likely at the level of beta-catenin, is a primary mechanism by which Raw regulates germline-soma interaction (Jemc, 2012).
raw is an important regulator of embryonic gonad morphogenesis and the establishment of proper gonad architecture. raw mutants exhibit a failure of SGPs to ensheath GCs in the gonad, resulting in defects in GC development. It was also found that raw affects gonad morphogenesis primarily by acting as a negative regulator of the JNK signaling pathway. Finally, it was found that raw mutants exhibit defects in cadherin-based cell adhesion, and that this is the primary cause of the failure of gonad morphogenesis. These results have clear implications for understanding of how important cell signaling pathways are regulated to control normal organogenesis and may be misregulated to cause disease (Jemc, 2012).
Previously raw has been proposed to be a negative regulator of the JNK pathway during closure of the dorsal epidermis, based on changes in JNK-dependent expression of target genes such as dpp and puc. Indeed, an increase was seen in puc expression in the region of the embryonic gonad, and more broadly throughout the embryo. Further, upregulation of a dedicated AP-1 reporter construct was observed, indicating that the changes in target gene expression are directly due to changes in AP-1 transcriptional activity regulated by the JNK pathway. When the JNK pathway was upregulated via independent means, similar defects were observed in gonad morphogenesis, indicating that the changes in the JNK pathway were the primary mechanism by which raw mutants cause gonad defects. Therefore, the results support and extend the previous observations that raw acts as a negative regulator of JNK pathway, both in the gonad and in other tissues in which raw mutants exhibit defects in morphogenesis (Jemc, 2012).
How might raw be regulating the JNK pathway? The evidence indicates that raw regulates the JNK pathway at the level of transcription factor JRA. It was found that the nuclear localization of JRA, but not FOS, was altered in raw mutants. JRA was more strongly concentrated in the nucleus in a variety of cell types in raw mutants, whereas no changes were observed in the global levels of JRA protein. These observations are consistent with previous genetic epistasis experiments that indicated that raw acts at the level of JRA, rather than further upstream in the pathway. It has been proposed that raw acts as a general negative regulator of the JNK pathway to suppress basal activity and perhaps establish a threshold for pathway activation. The data are consistent with this hypothesis, as a general nuclear accumulation of JRA was seen in a variety of cell types in the embryo, along with generalized activation of the transcriptional reporter for AP-1 activity. Presumably, not all of these different cells are normally exposed to activators of the JNK pathway at this time, indicating that the pathway may be activated in cells in which the pathway would normally be turned off. Thus, rather than being just a modulator of the level of signal a cell might receive under conditions of JNK pathway activation, raw is likely also responsible for ensuring that the pathway remains inactive in cells that are not experiencing pathway activation (Jemc, 2012).
It is difficult to predict exactly how Raw may be regulating JNK signaling, as the Raw protein has no readily identifiable protein domains and exhibits only limited homology to proteins of other species. It may be the case that similar JNK pathway regulators are present in other species and have structural and/or functional conservation with Raw, but are difficult to identify based on primary sequence homology. Studies examining the subcellular localization of Raw in cultured mammalian or Drosophila cells indicate that it is primarily found in the cytoplasm. One attractive hypothesis is that Raw directly binds to JRA to block its nuclear translocation and sequester JRA in the cytoplasm. Unfortunately, efforts to identify a direct, physical interaction between Raw and JRA have so far been unsuccessful (Jemc, 2012).
The JNK pathway is subject to negative regulation at several levels. Most familiar are the MAP kinase phosphatases (MKPs, a subfamily of Dual-specificity phosphatases), like Drosophila Puckered, that provide negative feedback by dephosphorylating activated MAP kinases such as JNK. Additional modes of regulation include nuclear repressors of AP-1 target genes (e.g., Anterior open) and a secreted protease that acts in negative feedback on the JNK pathway (Scarface). Raw appears to represent a distinct mode of regulation, acting on the ability of JRA/JUN to translocate to the nucleus. Regulation of the subcellular localization of transcription factors and cofactors is a strategy that is commonly deployed to regulate signaling pathway activity, and many transcription factors are sequestered in the cytoplasm as a mechanism for negatively regulating their activity. It is proposed that JRA is subject to such regulation as a means to repress its activity in cells that are not experiencing sufficient levels of JNK pathway activity (Jemc, 2012).
Further studies of Raw are necessary to determine how Raw functions at a molecular level to regulate JRA subcellular localization (Jemc, 2012).
This study has found that raw mutants also exhibit defects in cadherin-based cell adhesion, which is known to be important for proper gonad morphogenesis and GC ensheathment by SGPs. Loss of DE-cad function exacerbates the gonad defects observed in raw mutants while increasing DE-cad function strikingly rescues these defects. It is likely that the increase in JNK pathway activity in raw mutants leads to defects in DE-cad-based adhesion and that this is the primary cause of the gonad morphogenesis defects. This is in contrast to the role of raw and the JNK pathway in the closure of the dorsal epidermis, which is largely thought to be due to regulation of dpp expression. Consistent with this, less up-regulation of dpp was observed in the region of the gonad, relative to the overall activation of the AP-1 transcriptional reporter (Jemc, 2012).
Previous studies in mammalian cells have implicated JNK signaling in negative regulation of cadherin-based cell adhesion, while in other contexts the JNK pathway has also been observed to upregulate DE-cad. The current results favor a repressive role for the JNK pathway on DE-cad in the gonad. It is also known that cadherins can act upstream of the JNK pathway, and that loss of cadherin can lead to an increase in c-Jun protein levels. However, the current results are consistent with DE-cad acting downstream of the JNK pathway, since DE-cadherin expression could rescue gonad morphogenesis independently of rescuing JRA localization. It is concluded that during gonad morphogenesis, raw acts as a negative regulator of the JNK pathway, and increased JNK pathway activity observed in raw mutants leads to a downregulation of DE-cadherin based cell adhesion and a failure of proper ensheathment of the GCs by the somatic gonad (Jemc, 2012).
While no change was observed in DE-cad localization in the gonad, the localization of ARM/ß-catenin was dramatically altered. Since ARM is essential for proper DE-cad function in cell adhesion, this indicates that DE-cadherin-based adhesion is strongly affected in raw mutants. It has been shown that JNK can directly phosphorylate ß-catenin and negatively regulate its activity. Consistent with this, a modest increase was observed in the relevant phospho-form of ARM/ß-catenin in raw mutants. Thus, this may represent one aspect of how the JNK pathway regulates DE-cad based adhesion in the gonad. However, the change in ARM/ß-catenin phosphorylation observed is unlikely to account for the more dramatic change in ARM/ß-catenin immunostaining observed in the gonad. Considering that a strong increase was also observed in transcriptional activation by AP-1 in raw mutants, and that mutations in the JRA transcription factor can partially suppress the gonad morphogenesis defects observed in raw mutants, it is concluded that at least some of the JNK pathway effect on DE-cad function and ß-catenin localization is likely to depend on changes in gene expression mediated by AP-1. Since no overall changes were observed in protein levels for DE-cad or ARM/ß-catenin in raw mutants, the changes in gene expression may reflect changes in other regulators of DE-cad based cell adhesion. Interestingly, previous work identified a zinc transporter, Fear of intimacy, that also affects gonad morphogenesis and GC ensheathment by regulating DE-cad. Regardless of whether there is an interesting connection between zinc transport and the JNK pathway, or these represent independent pathways, they highlight the importance of careful regulation of cadherin-based cell adhesion in controlling morphogenesis (Jemc, 2012).
Previous work has indicated that GC ensheathment requires preferential adhesion between SGPs and GCs, such that SGP-GC adhesion is favored over GC-GC or SGP-SGP adhesion. Indeed, just increasing the adhesion between GCs via DE-cadherin expression in these cells is sufficient to prevent ensheathment of the GCs by SGPs. In raw mutants, changes were primarily observed in the JNK pathway in SGPs and surrounding somatic cells. In addition, expression of DE-cad in the soma, but not the germline, is sufficient to rescue the ensheathment defects in raw mutants. Together, these data indicate that raw mutants likely affect gonad ensheathment by decreasing DE-cad function in the SGPs, which decreases SGP-GC adhesion relative to GC-GC adhesion. While it is possible that effects of raw on somatic cells outside of the gonad affect ensheathment within the gonad, it is less easy to imagine how decreasing DE-cad activity in these cells would influence ensheathment (Jemc, 2012).
The JNK pathway has been implicated in many diseases, including birth defects, neurodegeneration, inflammatory diseases, and cancer. Signaling pathways must be tightly regulated both positively, to ensure rapid and robust signaling responses, and negatively, to terminate signaling events and prevent inappropriate signaling. As a negative regulator of JNK pathway signaling, raw represents the type of gene that might be mutated or misregulated in diseases caused by altered JNK pathway activity. This idea is supported by the strong developmental phenotypes associated with mutations in negative regulators of the JNK pathway in Drosophila and mice (Jemc, 2012).
One disease where the JNK pathway has been particularly well studied is cancer. The JNK pathway's role in cancer is complex, however, and the pathway can act in tumor suppression or oncogenesis, depending on the context. In mouse and Drosophila models of cancer due to activated Ras, upregulation of the JNK pathway is required for tumor formation and disease progression. Interestingly, downregulation of E-cadherin is also associated with cancer progression, including in the models of activated Ras where the JNK pathway is involved. Thus, a similar link between the JNK pathway and cadherin regulation that was observed in morphogenesis of the gonad during development may play a role in oncogenesis. Since upregulation of the JNK pathway promotes cancer in these examples, negative regulators of the pathway such as the MAPK phosphatases or Raw would act as tumor suppressors whose mutation could contribute to disease progression. A better understanding of how the JNK pathway is regulated, and how Raw contributes to this regulation, is essential for understanding the normal roles of the JNK pathway in development and homeostasis, and how it is misregulated to cause disease (Jemc, 2012).
Neuronal signal transduction by the JNK MAP kinase pathway is altered by a broad array of stimuli including exposure to the widely abused drug ethanol, but the behavioral relevance and the regulation of JNK signaling is unclear. This study demonstrates that JNK signaling functions downstream of the Sterile20 kinase family gene tao/Taok3 to regulate the behavioral effects of acute ethanol exposure in both the fruit fly Drosophila and mice. In flies tao is required in neurons to promote sensitivity to the locomotor stimulant effects of acute ethanol exposure and to establish specific brain structures. Reduced expression of key JNK pathway genes substantially rescued the structural and behavioral phenotypes of tao mutants. Decreasing and increasing JNK pathway activity resulted in increased and decreased sensitivity to the locomotor stimulant properties of acute ethanol exposure, respectively. Further, JNK expression in a limited pattern of neurons that included brain regions implicated in ethanol responses was sufficient to restore normal behavior. Mice heterozygous for a disrupted allele of the homologous Taok3 gene (Taok3Gt) were resistant to the acute sedative effects of ethanol. JNK activity was constitutively increased in brains of Taok3Gt/+ mice, and acute induction of phospho-JNK in brain tissue by ethanol was occluded in Taok3Gt/+ mice. Finally, acute administration of a JNK inhibitor conferred resistance to the sedative effects of ethanol in wild-type but not Taok3Gt/+ mice. Taken together, these data support a role of a TAO/TAOK3-JNK neuronal signaling pathway in regulating sensitivity to acute ethanol exposure in flies and in mice (Kapfhamer, 2012).
Endocytosis plays an important role in the regulation of tumour growth and metastasis. In Drosophila, a number of endocytic neoplastic tumour suppressor genes have been identified that when mutated cause epithelial disruption and over-proliferation. This study characterises the Drosophila homologue of the Rab5 effector Rabaptin-5, and shows that it is a novel neoplastic tumour suppressor. Its ability to bind Rab5 and modulate early endosomal dynamics is conserved in Drosophila, as is its interaction with the Rab5 GEF Rabex5, for which neoplastic tumour suppressor characteristics were also demonstrated. Surprisingly, disruption of apico-basal polarity is not disrupted in Rabaptin-5 and Rabex-5 mutant tissues; instead the tumour phenotype is associated with upregulation of Jun N-terminal Kinase (JNK) and Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signalling (Thomas, 2013).
Endocytosis is increasingly recognised as an important mechanism for the growth and metastasis of tumours, both in the clinical setting and in cancer models. The categorisation of endocytic neoplastic tumour suppressor genes in Drosophila began less than ten years ago with the identification of Rab5, avl, Vps25 and Tsg101 and has since expanded with the discovery of at least 14 others, in particular multiple components of the ESCRT complex. Most mutations so far identified are zygotic lethal long before the third instar larval stage and so have been discovered through screens in which homozygous mutant clones were generated that spanned entire epithelial compartments in otherwise heterozygous animals. However, not all chromosome arms have been screened and screens have not been saturating, hence it is likely that many others remain to be found (Thomas, 2013).
This study identified two novel endocytic neoplastic tumour suppressor genes. Rbpn-5 has not been previously characterised in Drosophila, and this work is the first to show that not only does it act as a neoplastic tumour suppressor gene in Drosophila, but that its endocytic functions, initially investigated in mammalian cell culture and in vitro, are conserved in a multi-cellular organism during development. Unlike many of the Drosophila endocytic neoplastic tumour suppressor genes so far identified, Rbpn-5 has a human homologue that has been directly implicated in tumour growth and metastasis. Mammalian Rabaptin-5 controls integrin recycling during migration of invasive tumour cells, has been identified in patients suffering from myelomonocytic leukaemia, is involved in preventing hypoxia in primary kidney and breast tumours and has been shown to interact physically with the Tuberous sclerosis protein Tuberin. Although not all of these functions may be conserved in Drosophila, this work provides a basis for further investigating the mechanisms of Rbpn-5 dependent tumourigenesis in a whole animal system (Thomas, 2013).
Rabex-5 has been previously identified as a tumour suppressor gene in Drosophila (Yan, 2010), but its neoplastic characteristics were not described. Importantly, both Rbpn-5 and Rabex-5 mutants are homozygous viable until late larval stages, and thus may provide a more facile model than other early endocytic tumour suppressor genes for uncovering the mechanistic basis of neoplasia (Thomas, 2013).
This study initially set out to identify new regulators of planar polarity through an RNAi screen. As core planar polarity proteins are known to undergo internalisation and recycling, and Rbpn-5 and Rabex-5 are general endocytic regulators that are functional in epithelial tissues at the time of planar polarisation, it is highly likely that they are playing a role in trafficking of core proteins. Indeed, another Rab5 effector, Rbsn-5, which is also a neoplastic tumour suppressor protein, regulates Fmi localisation. Unlike Rbsn-5, cell-autonomous depletion of Rbpn-5 or Rabex-5 does not significantly alter planar polarity protein levels or localisation, suggesting that some level of endocytosis can occur in the absence of these proteins. One possibility is that Rbpn-5 and Rabex-5 proteins might be highly stable compared with Rbsn-5. Evidence in support of this comes from zygotic mutants, which survive to early pupal stages for Rbpn-5 and Rabex-5, presumably due to perdurance of maternal protein, but die before the second larval instar in the Rbsn-5 background. If this is the case, residual protein activity in clones or RNAi-treated tissue may allow a low level of general endocytosis to occur, which would be sufficient to correctly localise core planar polarity proteins. Another, non-mutually exclusive hypothesis is that a level of endocytosis can occur in the absence of either Rabex-5 or Rbpn-5. There are three other Rab5 GEF homologues in Drosophila, and these may be able to step in if Rabex-5 is absent (Thomas, 2013).
Whatever the reason may be, the lack of defects seen in clones or RNAi, combined with the severe pleiotropic effects on epithelial architecture observed in zygotic mutants, means that analysis of a specific role in planar polarity is extremely complex, and it was decided to focus attention on their tumour suppressor roles (Thomas, 2013).
In mammalian cells, it has been shown that Rabaptin-5 is an effector of Rab5 that promotes early endosome fusion through its interaction with Rabex-5. This study provides the first characterisation of a Rabaptin-5 homologue in a multicellular organism, and show that many of its functions are conserved. Rab5 effectors are defined by several criteria including GTP-dependent binding to Rab5 and the ability to modulate a Rab5-dependent process in response to Rab5 activity. This study shows that Drosophila Rbpn-5 fulfills the criteria of a Rab5 effector protein in the same way as its mammalian counterpart. Firstly this study has demonstrated that Rbpn-5 physically interacts specifically with GTP-bound Rab5, probably through its predicted C-terminal Rab5 binding domain. Secondly, that Rbpn-5 and Rab5 colocalise in Drosophila pupal wings. Thirdly, when Rbpn-5 was depleted using RNAi or in mutant clones, a strong reduction was seen in apical Rab5-positive vesicles indicating that Rbpn-5 is required for Rab5 recruitment. Lastly, if Rbpn-5 is over-expressed, subtle alterations is seen in early endosome structure, which are indicative of a role for Rbpn-5 in promoting early endosome fusion (Thomas, 2013).
Although the Rab5 effector function of Rabaptin-5 is conserved in Drosophila, its Rab4 effector function may not be. Mammalian Rabaptin-5 has been shown to bind Rab4 through a separate N-terminal domain, and the divalent Rab5-Rab4 binding ability allows Rbpn-5 to act as a bridge between the endocytic and fast recycling pathways. It was not possible to recapitulate Rab4 binding, and indeed the N-terminal Rab4 binding site is not highly conserved in Drosophila Rbpn-5. A similar result was found for the Drosophila homologue of another Rab5-Rab4 divalent effector, Rabenosyn-5 , suggesting that the mechanism of transfer of cargo from the endocytic to recycling routes might not be conserved across phyla (Thomas, 2013).
A large number of proteins have been shown to bind Rabaptin-5, but one of the best characterised is Rabex-5. The endogenous GEF activity of Rabex-5 is fairly low, and unless large quantities are overexpressed, the formation of a Rabaptin-5/Rabex5 complex is necessary to promote nucleotide exchange on Rab5. The effect of this is to enhance Rab5 activity, thus facilitating its recruitment of factors involved in tethering and fusion of early endocytic membranes (Thomas, 2013).
This study has provided evidence that the interaction between Rabex-5 and Rbpn-5, and their ability to alter early endosome dynamics is conserved in Drosophila. As with their mammalian counterparts, the two proteins bind in vitro: it is suspected that Rabex-5 binds to a central domain in Rbpn-5, as this is homologous to the Rabex-5 binding site in mammalian Rabaptin-5, and neither N- nor C-termini of Rbpn-5 can bind alone. As in mammalian cells, over-expression of Rabex-5 in Drosophila wings is sufficient to promote early endosome fusion even if Rbpn-5 levels are depleted by RNAi. However, co-expression of Rbpn-5 significantly enhances this, demonstrating that the complex acts synergistically. The dependency on Rabex-5 for Rabaptin-5 localisation to early endosomes is also conserved in Drosophila as this study showed that depletion of Rabex-5 levels using RNAi causes a reduction in Rbpn-5 puncta. This is surprising as not only does Rbpn-5 contain a Rab5 binding domain, but also a FYVE domain (unlike its mammalian homologue), which would be expected to promote its recruitment to early endosomes. It is possible that the FYVE domain is non-functional. Alternatively, its ability to bind PI3P may be somehow inhibited in the absence of Rabex-5, or the protein may be destabilised. Further work would be required to distinguish between these possibilities (Thomas, 2013).
This study found that over-expressing a mutant form of Rabex-5 lacking GEF activity prevents both Rbpn-5 and Rab5 from accumulating in apical puncta. It is surmiseed that the construct is acting in a dominant-negative manner to suppress Rbpn-5 recruitment and/or Rab5 activation via endogenous Rabex-5. In mammalian cells, localisation of all three proteins appears to be highly interdependent, suggesting that it is likely that Rabex-5 recruitment to early endosomes is also compromised in the absence of Rbpn-5 or Rab5. However, in the absence of reagents for visualising endogenous Rabex-5, this remains speculative (Thomas, 2013).
The mechanistic basis of neoplastic tumour formation is complex and much remains unclear. Drosophila models, in particular the classic baso-lateral polarity complex mutants scrib, dlg and lgl, which were discovered many years ago and which, unlike most of the endocytic mutants, are zygotically viable until late larval stages, have been extremely useful in describing the process of tumorigenesis. There are several different pathways and mechanisms that have been investigated. Given that Scrib, Dlg and Lgl regulate apico-basal polarity by inhibiting the apical Crb and Par-3/Bazooka (Baz) complexes, an expansion of the apical domain was proposed as one possible mechanism for tumourigenesis. This hypothesis was reinforced by the finding that over-expressing either Crb or atypical Protein kinase C (aPKC) is sufficient to promote tumourous discs, and that in the endocytic mutants, Crb accumulates massively and apico-basal polarity is disrupted. Crb accumulation is thought to promote the growth aspect of the neoplastic tumour phenotype through misregulation of the Hpo/Wts pathway. However, there is some evidence that Crb is not always strongly misregulated in neoplastic tumours, and it has been speculated that in scrib, dlg and lgl mutants it may be the concurrent disruption of endocytosis, rather than the polarity defect, which is the primary cause of their neoplastic phenotypes (Thomas, 2013).
The results show that neither Rabex-5 nor Rbpn-5 exhibit disruption in apico-basal polarity and there is no accumulation of Crb. It is suggested that these mutants are likely to represent a milder phenotype than the other endocytic mutants studied so far, possibly due to enhanced perdurance of maternal protein which allows the survival of zygotic mutants to early pupal stages. In any case, the lack of an apico-basal polarity defect in Rbpn-5 and Rabex-5 mutants indicates that loss of apico-basal polarity is not strictly required for neoplasia (Thomas, 2013).
Other pathways that have been found to be activated in Drosophila neoplastic tumours include N, JAK/STAT and JNK. Due to the differential ability of N to signal in different endocytic compartments, the pathway is upregulated in ESCRT mutant tumours, but not in early endocytic mutants, despite their accumulation of N protein on the cell surface. As with Crb, accumulation of N was not seen in Rbpn-5 or Rabex-5 mutant discs, suggesting that a certain level of endocytosis is still occurring, and unsurprisingly it was also found that the N pathway is not activated (Thomas, 2013).
In ESCRT mutant clones, activation of the N pathway leads to transcription of the JAK/STAT ligand Upd, which acts non-autonomously on wild-type tissue to induce JAK/STAT signalling and promote neoplasia. The massive upregulation of JAK/STAT signalling that was observed in this study must be activated via an alternate mechanism, as it is both cell autonomous and N-independent. Although autonomous JAK/STAT signalling in neoplastic discs has been documented and shown to contribute to excessive cell size and proliferation, it is unclear how it is activated in endocytic mutants. One explanation that was not possible to test, is that the endocytic block could directly disrupt the trafficking of the JAK/STAT receptor Domeless (Dome), as Dome localisation has been shown to be altered in ESCRT mutant neoplastic tumourous discs, and signalling ability is known to be influenced by the intracellular compartment in which the ligand-receptor complex finds itself. This study showed that a hypomorphic stat92E mutation is able to rescue many of the neoplastic defects, including over-proliferation, inability to differentiate and overall disc shape and structure, suggesting that ectopic activation of JAK/STAT signalling in endocytic mutants is indeed causative for these aspects of tumourigenesis (Thomas, 2013).
The upregulation of the JNK pathway that was observed in the Rbpn-5 and Rabex-5 mutants is likely to also contribute to the over-proliferation phenotype. The JNK pathway is unusual in having both pro-proliferation and pro-apoptotic roles, and was initially thought to be activated only where wild-type tissue abuts clones mutant for neoplastic tumour suppressor genes, promoting apoptosis within mutant cells and their elimination from the tissue. However, more recent work has shown that JNK signalling is also activated in tissues wholly mutant for endocytic neoplastic tumour suppressor genes where it promotes cell proliferation (Thomas, 2013).
It is thought that JNK pathway upregulation in neoplastic tumours is likely to act through the Eiger/TNF ligand receptor complex. Eiger and activated JNK have been shown to accumulate in early endosomes in scrib mutant clones to promote apoptosis, and there are several other pieces of evidence that implicate the endocytic pathway in Eiger regulation. Interestingly, the switch from JNK-mediated apoptosis to proliferation can be mediated by co-expression of the Ras oncogene. For example, expressing Ras in scrib mutant clones produces a highly invasive neoplastic phenotype such that clones that would usually be eliminated instead invade neighbouring wild-type tissue. Given that Ras is ubiquitinated by Rabex-5, and the Ras pathway has been shown to be upregulated in Rabex-5 mutant larvae, it is intriguing to speculate whether this may be contributing in some way to the neoplastic phenotype seen in mutant larval discs, not only in the Rabex-5 background but perhaps more generally in other endocytic neoplastic tumour suppressor mutants. Indeed, as Rabex-5-mediated ubiquitination targets cargo to early endosomes for degradation, it seems highly likely that Ras signalling is not only regulated by ubiquitination but also by endosomal dynamics. Further work will be needed to investigate a potential role for Ras in endocytic neoplastic tumours, to determine the mechanisms of JNK and JAK/STAT activation and to elucidate how differential activity of these pathways is controlled in clones versus whole mutant tissues (Thomas, 2013).
Autophagy is a highly conserved catabolic process that degrades and recycles intracellular components through the lysosomes. Atg9 is the only integral membrane protein among autophagy-related (Atg) proteins thought to carry the membrane source for forming autophagosomes. This study shows that Drosophila Atg9 interacts with Drosophila tumor necrosis factor receptor-associated factor 2 (dTRAF2: TNF-receptor-associated factor 6) to regulate the c-Jun N-terminal kinase (JNK) signaling pathway. Significantly, depletion of Atg9 and dTRAF2 compromised JNK-mediated intestinal stem cell proliferation and autophagy induction upon bacterial infection and oxidative stress stimulation. In mammalian cells, mAtg9 interacts with TRAF6, the homolog of dTRAF2, and plays an essential role in regulating oxidative stress-induced JNK activation. Moreover, it was found that ROS-induced autophagy acts as a negative feedback regulator of JNK activity by dissociating Atg9/mAtg9 from dTRAF2/TRAF6 in Drosophila and mammalian cells, respectively. These findings indicate a dual role for Atg9 in the regulation of JNK signaling and autophagy under oxidative stress conditions (Tang, 2013).
Macroautophagy (hereafter autophagy) is a conserved catabolic pathway in which double membrane vesicles called autophagosomes engulf macromolecules or organelles. Subsequently, autophagosomes fuse with lysosomes to form autolysosomes where degradation occurs. Autophagy is involved in cytoprotective responses to environmental stresses, stem cell maintenance and differentiation, tumorigenesis, and programmed cell death. There have been more than 30 autophagy-related (Atg) genes essential for autophagy process identified through genetic screens in yeast. Atg9 is the only one identified as a transmembrane protein, and it has been thought to promote lipid transport to the forming autophagosomes. Mammalian Atg9 (mAtg9) localizes on the trans-Golgi network and endosomes under nutrient-rich conditions, whereas it translocates to forming autophagosomes under starvation conditions. The recycling of mAtg9 during autophagy is regulated by several proteins including Ulk1, ZIPK, mAtg13, and p38IP. Interestingly, one recent study has reported that mAtg9 modulates innate immune response in an autophagy-independent manner . However, the physiological functions of Atg9 remain elusive (Tang, 2013).
Reactive oxygen species are highly reactive free radicals that can cause irreversible oxidative damage to proteins, lipids, or nucleotides in cells. Excessive production of ROS or depletion of antioxidants causes oxidative stress that often leads to cell dysfunction and diseases such as neurodegeneration, cancer, and aging. More importantly, ROS also plays critical roles in host defense and in the regulation of various cellular signaling pathways The ROS-induced signaling pathways include several mitogen-activated protein (MAP) kinase cascades involving the c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase. The JNK signaling pathway regulates diverse biological functions, including apoptosis, cytoprotection, metabolism, and epithelial homeostasis in response to several cytokines and environmental stresses. Depending on the duration and magnitude of exposure, ROS-induced JNK activation may lead to the promotion of either cell survival or apoptosis. In Drosophila, JNK signaling was found to protect cells from oxidative stress and extend lifespan of adult flies. It has been shown that the JNK pathway is required for intestinal epithelium renewal during bacterial infection-induced ROS/oxidative stress. One of the mechanisms that JNK meditates to protect flies against acute oxidative insults is the activation of autophagy. In response to oxidative stress, JNK signaling stimulates the expression of several ATG genes. Several recent studies have reported that overexpression of ATG genes and activation of autophagy are sufficient to extend lifespan and confer stress resistance in Drosophila (Tang, 2013).
How does ROS/oxidative stress trigger JNK activation? It has been shown that signaling molecules, including apoptosis signal-regulating kinase (Ask1), glutathione S-transferase Pi (GSTp), and Src kinase can function as molecular links between ROS and JNK. Ask1 is a MAPKKK that activates JNK by phosphorylating MKK4/7. Under normal physiological conditions, Ask1 is inhibited by forming a complex with the redox regulatory protein thioredoxin. Upon exposure to ROS/oxidative stress, the oxidized thioredoxin dissociates from Ask1 and results in the activation of Ask1 signaling pathway. GSTp has been identified as a JNK inhibitor. Under oxidative conditions, GSTp forms oligomers and dissociates from JNK, leading to JNK activation. A number of reports have also shown the involvement of Src and its downstream targets in H2O2-induced JNK activation, although the underlying molecular mechanism remains elusive. Recently, tumor necrosis factor receptor-associated factors (TRAFs) have been found to be involved in ROS-mediated JNK activation. In mammals, the TRAF family consists of seven members and functions as scaffold proteins that link cell surface receptors to the downstream effectors. Among them, TRAF2 and TRAF6 are found to associate with Ask1 and form the active Ask1 signalsome in response to ROS stimulation. Moreover, the involvement of TRAF4 in oxidative activation of JNK via its interaction with the NAD(P)H oxidase p47phox has been demonstrated. The Drosophila TRAF2 (dTRAF2), a homolog of human TRAF6, was found to mediate Eiger/Wegen (tumor necrosis factor/tumor necrosis factor receptor [TNF/TNFR])-induced JNK signaling. However, the role of dTRAF2 in ROS-mediated JNK activation remains unclear (Tang, 2013).
This study has identified a biological function of Atg9 in regulation of JNK signaling pathway. Drosophila Atg9 can activate JNK signaling through its interaction with dTRAF2. Depletion of Atg9 compromised oxidative stress-induced JNK activation, the JNK-mediated epithelium renewal, and autophagy induction. In mammalian cells, mAtg9 was found to be essential for JNK activation in response to ROS/oxidative stress, indicating a highly conserved role of Atg9 in regulating JNK activity. It was further found that ROS-induced autophagy negative feedback regulates JNK activity through the dissociation of Atg9/mAtg9 from dTRAF2/TRAF6 in Drosophila and mammalian cells, respectively. These findings provide insights into the crosstalk between autophagy and JNK signaling pathway in response to oxidative stress (Tang, 2013).
The Atg9 transmembrane protein has been shown to play an essential role in autophagy pathway in yeast and mammals. In this study, Drosophila Atg9 was also found to be required for autophagy induction upon nutrient deprivation or under oxidative stress conditions. More importantly, a role was uncovered for Atg9 in regulating the JNK signaling pathway. Upon bacterial infection, Atg9 interacts with dTRAF2 to activate JNK-mediated autophagy induction and epithelium renewal in Drosophila gut cells. The role of Atg9 in activating JNK signaling was also observed in mammalian cells. Moreover, this study found that ROS-induced autophagy in turn inhibits JNK signaling via a negative feedback mechanism by dissociation of Atg9 from dTRAF2 and TRAF6 in Drosophila and mammalian cells, respectively (Tang, 2013).
Atg9 is a highly conserved and the only multi-spanning transmembrane Atg protein essential for the formation of autophagosomes. In yeast, Atg9 cycles between the preautophagosomal structure (PAS) and peripheral cytoplasmic structures. Recently, using single particle tracking, Yeast Atg9 exists as highly motile vesicles that contribute to PAS formation. In mammalian cells, mAtg9 is localized mainly to the trans-Golgi network and endosomes. However, upon nutrient starvation, mAtg9 is enriched in endosomal pools and undergoes a dynamic interaction with forming autophagosomes. The current study found that Drosophila Atg9 not only distributed in cytoplasm, but also concentrated at cell-cell junctions, suggesting Atg9 may have additional roles besides its function in autophagy. For example, it has been reported that mAtg9 can function as a regulator for dsDNA-triggered innate immune response (Tang, 2013).
The involvement of Atg1/Ulk1 in Atg9 trafficking has been described in yeast and mammalian cells. Consistent with these findings, the current study found that Drosophila Atg9 redistributed from peripheral pools to forming autophagosomes in an Atg1-dependent manner. A previous reported that overexpression of Drosophila Atg1 induces cell death. Interestingly, this study found that overexpression of Atg1 did not induce JNK activation and the Atg1-induced cell death could not be rescued by inhibition of JNK signaling. The current findings highlight that, in addition to its role in autophagy, Atg9 plays a role in the regulation of JNK activation in response to oxidative stress (Tang, 2013).
The JNK signaling pathway is one of the mitogen-activated protein kinase (MAPK) cascades involved in stress responses. Activation of the JNK pathway has been implicated in a number of biological processes including cell proliferation, survival, apoptosis, and migration. The involvement of JNK in both proapoptotic and anti-apoptotic activities indicates a complex function of the JNK pathway, whereas the molecular mechanism that regulates JNK to mediate both processes remains elusive. This study study has shown that ectopic expression of Atg9 in the developing wing and eye leads to JNK activation and apoptotic cell death. Moreover, the results provided evidence that, upon ROS stimulation, Atg9, but not Atg12, is required for JNK-mediated intestinal stem cell proliferation and autophagy induction in Drosophila. These results indicate that Atg9 may play a critical role in regulating JNK-mediated cell survival and apoptosis. It was further shown that Atg9 regulates JNK signaling via its association with dTRAF2 and TRAF6 in Drosophila and mammals, respectively. GST-pull down assay revealed that the C terminus of Drosophila Atg9 can interact with dTRAF2. Surprisingly, Atg9 lacking the C-terminal region can still promote JNK activation and cell death. One possibility is that Atg9 may interact with dTRAF2 through multi-regions. On the other hand, yeast Atg9 has been shown to self-interact through the C terminus, and Atg9 self-association is critical for its function in autophagy. Sequence analysis revealed that Drosophila Atg9 also contains the conserved self-interacting motif (VGNVC) between amino acids 560 and 564. It is possible that Atg9ΔC may exert its function in regulating JNK activity by interacting with the endogenous Atg9 (Tang, 2013).
TRAF6 functions as a RING-domain containing ubiquitin ligase involved in a variety of biological processes including adaptive and innate immunity, bone metabolism and tissue development. TRAF6 is required for interleukin-1 (IL-1) and transforming growth factor-β-mediated JNK activation. In Drosophila, dTRAF2 plays a role in Eiger/Wegen (TNF/TNFR)-induced JNK signaling. How does Atg9 regulate TRAF-mediated JNK activation? One mechanism may be that Atg9 associate with TRAF6 to modulate its ubiquitin ligase activity. Indeed, a recent study indicates that Atg9 interacts and promotes TRAF6 ubiquitination. Alternatively, because Atg9 is a membrane protein with diverse subcellular localization, Atg9 may bind and target TRAF6 to peripheral membrane regions in response to bacterial infection and oxidative stress. These two mechanisms need not be mutually exclusive and can occur together (Tang, 2013).
Recent studies suggested there to be a complex relationship between the JNK pathway and autophagy. On the one hand, under nutrient starvation conditions, JNK has been found to phosphorylate Bcl-2, leading to the dissociation of Bcl-2 from beclin 1 and the activation of autophagy. JNK signaling also activates autophagy via the upregulation of ATG gene expression in response to oxidative stress and oncogenic transformation. On the other hand, JNK can act as a negative regulator of FoxO-dependent autophagy in neurons. It is interesting to note that, although Atg9 overexpression activates JNK, the current data showed that Atg9 overexpression could not induce autophagy in the larval fat body. Because Atg9 promotes JNK activation through its association with dTRAF2, dTRAF2 may not be expressed in the fat body. Indeed, RNA expression analysis reveals that dTRAF2 expresses in the fat body at a relatively low level . Alternatively, it has been reported that JNK overexpression activates autophagy independently of Atg1 and nutrient signal. However, the current results showed that Atg9 interacts with Atg1 and is required for starvation-induced autophagy. Overexpression of JNK may induce a noncanonical autophagy that is independent of 'core Atg proteins.' (Tang, 2013 and references therein).
This current study also demonstrates that autophagy can act as a negative feedback regulator for JNK activation upon oxidative stress. Inhibition of autophagy in flies fed with Ecc15 or paraquat resulted in a substantial increase in JNK activity, which led to increased ISC proliferation and cell death in adult Drosophila midgut. In mammalian cells, depletion of Atg5 led to prolonged JNK activation during hydrogen peroxide-induced oxidative stress. Moreover, activation of autophagy by rapamycin effectively blocked the interaction between Atg9 and TRAF6 and inhibits ROS-induced JNK activity. Considered together, these findings together indicate an important role of autophagy in restricting JNK activity by modulating the interaction between Atg9 and TRAF6 in response to oxidative stress. In conclusion, this work establishes a regulatory mechanism between Atg9, autophagy, and the JNK signaling pathway during oxidative stress conditions (Tang, 2013).
Parkinson's disease (PD) is one of the most common neurodegenerative disorders with limited clinical interventions. A number of epidemiological as well as case-control studies have revealed an association between pesticide exposure, especially of paraquat (PQ) and occurrence of PD. Hsp70, a molecular chaperone by function, has been shown as one of the modulators of neurological disorders. However, paucity of information regarding the protective role of Hsp70 on PQ-induced PD like symptoms led to a hypothesis that modulation of hsp70 expression in the dopaminergic neurons would improve the health of these cells. Advantage was taken of Drosophila, which is a well-established model for neurological research and also possesses genetic tools for easy manipulation of gene expression with limited ethical concern. Over-expression of hsp70 was found to reduce PQ-induced oxidative stress along with JNK and caspase-3 mediated dopaminergic neuronal cell death in the exposed organism. Further, anti-apoptotic effect of hsp70 was shown to confer better homeostasis in the dopaminergic neurons of the PQ-exposed organism, as evidenced by their improved locomotor performance and survival. The study has merit in the context of human concern since protection of dopaminergic neurons in PQ-exposed organism was observed by over-expressing a human homologue of hsp70, HSPA1L, in these cells. The effect was parallel to that observed with Drosophila hsp70. These findings reflect the potential therapeutic applicability of hsp70 against PQ-induced PD like symptoms in an organism (Shukla, 2014).
Cell number plasticity is coupled to circuitry in the nervous system, adjusting cell mass to
functional requirements. In mammals, this is achieved by neurotrophin (NT) ligands, which promote
cell survival via their Trk and p75NTR receptors and cell death via p75NTR and Sortilin. Drosophila NTs (DNTs; see NT1) bind Toll receptors (see Toll-6 & Toll-7) instead to promote neuronal survival, but
whether they can also regulate cell death is unknown. This study show that DNTs and Tolls can switch
from promoting cell survival to death in the central nervous system (CNS) via a three-tier
mechanism. First, DNT cleavage patterns result in alternative signaling outcomes. Second, different
Tolls can preferentially promote cell survival or death. Third, distinct adaptors downstream of
Tolls can drive either apoptosis or cell survival. Toll-6 promotes cell survival via MyD88-NF-κB and cell
death via Wek-Sarm-JNK. The distribution of adaptors changes in space and time and
may segregate to distinct neural circuits. This novel mechanism for CNS cell plasticity may operate
in wider contexts (Foldi, 2017).
Balancing cell death and cell survival enables structural plasticity and homeostasis, regeneration,
and repair and fails in cancer and neurodegeneration. In the nervous system, cell number plasticity
is linked to neural circuit formation, adjusting neuronal number to functional requirements. In mammals, the neurotrophin (NT) protein family [NGF, brain-derived
neurotrophic factor (BDNF), NT3, and NT4] regulates neuronal number through two mechanisms. First,
full-length pro-NTs, comprised of a disordered prodomain and a cystine-knot (CK) domain, induce cell
death; in contrast, mature NTs formed of CK dimers promote cell survival. Second,
pro-NTs bind p75NTR and Sortilin receptors, inducing apoptosis via JNK signaling, whereas mature NTs
bind p75NTR, promoting cell survival via NF-κB and TrkA, B, and C, promoting
cell survival via PI3K/AKT and MAPK/ERK. As the NTs also regulate connectivity and synaptic transmission, they couple the regulation of cell number to neural circuitry and function, enabling structural brain plasticity. There is abundant evidence that cell number plasticity occurs in Drosophila melanogaster central nervous system (CNS) development, with neurotrophic factors including NTs and mesencephalic astrocyte-derived neurotrophic factor (MANF), but fruit flies lack p75NTR and Trk receptors, raising the question of how this is achieved in the fly. Finding this out is important, as it could lead to novel mechanisms of structural plasticity for both flies and humans (Foldi, 2017).
The Drosophila NTs (DNTs) Spätzle (Spz), DNT1, and DNT2 share with mammalian NTs the characteristic
structure of a prodomain and a conserved CK of 13-15 kD, which forms a disulfide-linked dimer. Spz
resembles NGF biochemically and structurally, and the binding of its Toll-1 receptor resembles that
of NGF to p75NTR. DNT1 (also known as spz2) was discovered by homology to BDNF, and
DNT2 (also known as spz5) as a paralogue of spz and DNT1.
DNT1 and 2 promote neuronal survival, and DNT1 and 2, Spz, and Spz3 are required for connectivity
and synaptogenesis. Spz, DNT1, and DNT2 are ligands for Toll-1, -7, and -6, respectively, which function as NT receptors and promote
neuronal survival, circuit connectivity, and structural synaptic plasticity. Tolls belong to the Toll receptor superfamily, which underlies innate immunity. There are nine Toll paralogues in flies, of which only
Toll-1, -5, -7, and -9 are involved in immunity.
Tolls are also involved in morphogenesis, cell competition, and epidermal repair. Whether DNTs and Tolls can balance cell number plasticity is unknown (Foldi, 2017).
Like the p75NTR receptor, Toll-1 activates NF-κB (a potent neuronal prosurvival factor with
evolutionarily conserved functions also in structural and synaptic plasticity) signaling downstream. Toll-1
signaling involves the downstream adaptor MyD88, which forms a complex with Tube and Pelle. Activation of Toll-1
triggers the degradation of the NF-κB inhibitor Cactus, enabling the nuclear translocation of the
NF-κB homologues Dorsal and Dorsal-related immunity factor (Dif), which function as transcription
factors. Other Tolls have also been suggested to activate NF-κB. However, only Toll-1 has been shown to bind MyD88, raising
the question of how the other Tolls signal in flies (Foldi, 2017).
Whether Tolls regulate cell death is also obscure. Toll-1 activates JNK, causing apoptosis, but its
expression can also be activated by JNK to induce nonapoptotic cell death. Toll-2, -3, -8, and -9 can induce apoptosis via NF-κB and dSarm independently of
MyD88 and JNK. However, in the CNS, dSarm induces axonal degeneration, but
there is no evidence that it can promote apoptosis in flies. In other
animals, Sarm orthologues are inhibitors of Toll signaling and MyD88, but there is no evidence that dSarm is an inhibitor of MyD88 in Drosophila. Thus,
whether or how Tolls may regulate apoptosis in flies is unclear (Foldi, 2017).
In the mammalian brain, Toll-like receptors (TLRs) are expressed in neurons, where they regulate
neurogenesis, apoptosis, and neurite growth and collapse in the absence of any insult. However, their neuronal functions have been little explored, and their endogenous ligands in
neurons remain unknown (Foldi, 2017).
Because Toll-1 and p75NTR share common downstream signaling pathways and p75NTR can activate NF-κB
to promote cell survival and JNK to promote cell death, this study asked whether the DNTs and
their Toll receptors could have dual roles controlling cell survival and death in the Drosophila CNS (Foldi, 2017).
In the first regulatory tier, each DNT has unique features conducive to distinctive functions. Spz,
DNT1, and DNT2 share with the mammalian NTs the unequivocal structure of the CK domain unique to
this protein family. However, DNT1, DNT2, and Spz have distinct prodomain features and are processed
differently, leading to distinct cellular outcomes. Spz is only secreted full length and
cleaved by serine proteases. DNT1 and 2 are cleaved intracellularly
by conserved furins. In cell culture, DNT1 was predominantly secreted with a truncated prodomain
(pro-DNT1), whereas DNT2 was secreted mature. In vivo, both pro- and mature DNTs were produced from
neurons. Interestingly, DNT1 also has an isoform lacking the CK domain, and Spz
has multiple isoforms with truncated prodomains. Thus, in vivo, whether DNT1
and 2 are secreted full length or cleaved and whether Spz is activated will depend on the proteases
that each cell type may express. Pro-DNT1 activates apoptotic JNK signaling, whereas mature DNT1 and
2 activate the prosurvival NF-κB (Dorsal and Dif) and ERK signaling pathways. Mature Spz does not
activate ERK. This first tier is evolutionarily conserved, as mammalian pro-NTs can promote cell
death, whereas furin-cleaved mature NTs promote cell survival. NF-κB, JNK, and ERK
are downstream targets shared with the mammalian NTs, downstream of p75NTR (NF-κB and JNK) and Trks
(ERK), to regulate neuronal survival and death. Thus, whether a cell lives or dies will depend on the available proteases, the ligand type,
and the ligand cleavage product it receives (Foldi, 2017).
In a second regulatory tier, this study showed that the specific Toll family receptor activated by a DNT matters. Toll-6 and -7 could maintain neuronal survival, whereas Toll-1 had a predominant proapoptotic
effect. Because there are nine Tolls in Drosophila, some Tolls could have prosurvival functions,
whereas others could have proapoptotic functions. Different Tolls also lead to different cellular
outcomes in immunity and development. Thus, the life or death of a neuron will depend on the Toll
or combination of Tolls it expresses. Binding of Spz to Toll-1 is
most likely unique, but DNT1 and 2 bind Toll-6 and -7 promiscuously, and, additionally, DNT1 and 2 with Toll-6 and -7 activate NF-κB and ERK, whereas pro-DNT1 activates JNK. This
suggests that ligand prodomains might alter the affinity for Toll receptors and/or facilitate the
formation of heterodimers between different Tolls and/or with other coreceptors to induce cell
death. A 'DNT-Toll code' may regulate neuronal numbers (Foldi, 2017).
In a third tier, available downstream adaptors determine the outcome between cell survival and death. Toll-6 and -7 activate cell survival by binding MyD88 and activating NF-κB and ERK
(whether ERK activation depends on MyD88 is not known), and Toll-6 can activate cell death via Wek,
dSarm, and JNK signaling. Toll-6 was shown to bind MyD88 and Wek, which binds dSarm, and
dSarm binds MyD88 and promotes apoptosis by inhibiting MyD88 and activating JNK. Wek also binds
MyD88 and Toll-1. So, evidence suggests that Wek recruits MyD88 and dSarm
downstream of Tolls. Because Toll-6 binds both MyD88 and Wek and Wek binds both MyD88 and
dSarm, Wek functions like a hinge downstream of Toll-6 to facilitate signaling via MyD88 or dSarm,
resulting in alternative outcomes. Remarkably, adaptor expression profiles change over time,
switching the response to Toll-6 from cell survival to cell death. In the embryo, when both MyD88
and dSarm are abundant, there is virtually no Wek, and Toll-6 can only bind MyD88 to promote cell
survival. As Wek levels rise, Toll-6 signaling can also induce cell death. If the
Wek-Sarm-JNK route prevails, Toll-6 induces apoptosis; if the Wek-MyD88-NF-κB route prevails, Toll-6
signaling induces cell survival (Foldi, 2017).
Thus, the cellular outcome downstream of DNTs and Tolls is context and time dependent. Whether a
cell survives or dies downstream of DNTs and Tolls will depend on which proteases are expressed
nearby, which ligand it receives and in which form, which Toll or combination of Tolls it expresses,
and which adaptors are available for signaling (Foldi, 2017).
How adaptor profiles come about or change is not understood. A neuronal type may be born with a
specific adaptor gene expression profile, or Toll receptor activation may influence their
expression. In fact, MyD88 reinforces its own signaling pathway, as Toll-6 and -7 up-regulate
Dorsal, Dif, and Cactus protein levels and TLR activation increases Sarm
levels. This study showed that apoptosis caused by MyD88 excess depends on JNK
signaling. Because JNK functions downstream of Wek and dSarm, this suggests that MyD88, presumably
via NF-κB, can activate the expression of JNK, wek, or dsarm. By positively regulating wek expression, MyD88 and dSarm could establish positive feedback loops reinforcing their alternative
pathways. Because dSarm inhibits MyD88, mutual regulation between them could
drive negative feedback. Positive and negative feedback loops underlie pattern formation and
structural homeostasis and could regulate neuronal number in the CNS as well. Whether
cell-autonomous or -nonautonomous mechanisms result in the diversification of adaptor profiles,
either in time or cell type, remains to be investigated (Foldi, 2017).
Either way, over time the Toll adaptors segregate to distinct neural circuits, where they exert
further functions in the CNS. Toll-1, -6, and -8 regulate synaptogenesis and structural
synaptic plasticity. Sarm
regulates neurite degeneration, and in the worm, it functions at the synapse to determine neuronal
identity. The reporters used in this study revealed a
potential segregation of MyD88 to the motor circuit and dSarm to the sensory circuit, but this is
unlikely to reflect the endogenous complexity of Toll-signaling circuitry, as dsarmMIMIC- has a GFP
insertion into one of eight potential isoforms, and dsarm also functions in the motor system
(McLaughlin, 2016). Importantly, cell death in the normal CNS occurs mostly in late
embryogenesis and in pupae, coinciding with neural circuit formation and remodeling, when neuronal
number is actively regulated. Thus, the link by DNTs and Tolls from cell number to circuitry offers
a complex matrix of possible ways to regulate structural plasticity in the CNS (Foldi, 2017).
This study has uncovered remarkable similarities between Drosophila Toll-6 and mammalian TLR signaling
involving MyD88 and Sarm. All TLRs except TLR3 signal via MyD88 and activate NF-κB . Neuronal apoptosis downstream of TLRs is independent of NF-κB and
instead depends on TRIF and Sarm1. Sarm1 is a negative regulator of TLR signaling, an inhibitor of MyD88 and
TRIF. sarm1 is expressed in neurons, where it activates JNK and promotes
apoptosis. However, the endogenous
ligands for TLRs in the normal undamaged brains are not known. Preliminary analysis has revealed
the intriguing possibility that NTs either can bind TLRs or induce interactions between Trks,
p75NTR, and TLRs. It is compelling to find out whether TLRs regulate structural plasticity in the
mammalian brain in concert with NTs (Foldi, 2017).
To conclude, DNTs with Tolls constitute a novel molecular system for structural plasticity in the
Drosophila CNS. This could be a general mechanism to be found also in the mammalian brain and in
other contexts as well, such as epithelial cell competition and regeneration, and altered in cancer
and neurodegeneration (Foldi, 2017).
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