cactus

Transcriptional Regulation

Transcription of cactus might be regulated by Dorsal (Kubola, 1995b).

The dorsoventral regulatory gene pathway, coded for by spatzle, Toll and cactus, controls the expression of several antimicrobial genes during the Drosophila immune response. This regulatory cascade shows striking similarities with the cytokine-induced activation cascade of NF-kappaB during the inflammatory response in mammals. The regulation of the IkappaB homolog Cactus has been studied in the fat body during the immune response. The cactus gene is up-regulated in response to immune challenge. Three hours after a bacterial challenge, cactus gene expression is markedly up-regulated in adults. A faint signal for Cact transcripts is present in unchallenged fat body and adult carcass and a remarkably rapid and strong up-regulation following bacterial challenge is observed. In both larvae and adults, peak values are observed after 2 or 3 h, after which the signals of Cact transcripts level off. These kinetics of induction/up-regulation, frequently referred to as acute phase kinetics, are similar to those of the cecropin A gene in these experiments. In contrast, the drosomycin and the diptericin genes reach their highest level of expression only 6-16 h postchallenge. Two Cact transcripts are observed during development; they are approximately 2.2 kb (referred to as maternal/zygotic) and 2.6 kb (zygotic) and encode proteins of 71 and 69 kDa, respectively, which differ in their C-terminal parts flanking the PEST sequence. Both transcripts are detectable in unchallenged tissues and are clearly up-regulated after bacterial challenge, the 2.2-kb transcript being predominant (Nicolas, 1998).

The immune response enhancer contains several sequence motifs homologous to insect and/or mammalian binding sites for Rel proteins: three sites are present upstream of a first intron P-transposon insertion site and two others are overlapping and located in intron 1. Intron 2 contains four sites, and intron 3 contains one site. These sites all contain the canonical three G residues in the 5' sequence, but differ in their 3' sequences; taken individually, some of these motifs are similar to counterparts in the various promoters of immune inducible genes encoding antimicrobial peptides (Nicolas, 1998).

Interestingly, the expression of the cactus gene is controlled by the spatzle/Toll/cactus gene pathway, indicating that the cactus gene is autoregulated. The two Cactus isoforms are expressed in the cytoplasm of fat body cells and they are rapidly degraded and resynthesized after immune challenge. This degradation is also dependent on the Toll signaling pathway. Altogether, these results underline the striking similarities between the regulation of IkappaB and cactus during the immune response (Nicolas, 1998).

The short gastrulation (sog) and decapentaplegic (dpp) genes function antagonistically in the early Drosophila zygote to pattern the dorsoventral (DV) axis of the embryo. This interplay between sog and dpp determines the extent of the neuroectoderm and subdivides the dorsal ectoderm into two territories. Evidence exists that sog and dpp also play opposing roles during oogenesis in patterning the DV axis of the embryo. Maternally produced Dpp increases levels of the IkappaB-related protein Cactus and reduces the magnitude of the nuclear concentration gradient of the NFkappaB-related Dorsal protein, and Sog limits this effect. Evidence is presented suggesting that Dpp signaling increases Cactus levels by reducing a signal-independent component of Cactus degradation. Epistasis experiments reveal that sog and dpp act downstream of, or in parallel to, the Toll receptor to reduce translocation of Dorsal protein into the nucleus. These results broaden the role previously defined for sog and dpp in establishing the embryonic DV axis and reveal a novel form of crossregulation between the NFkappaB and TGFbeta signaling pathways in pattern formation (Araujo, 2000).

In aggregate, the results support models in which Sog and Dpp proteins are produced by the follicle cells and then are delivered to the embryo. These proteins could be deposited in the vitelline membrane or in the oocyte plasma membrane, or might be sequestered in the perivitelline space and remain there protected until early embryogenesis. The fact that sog and dpp are expressed in follicle cells of stage 10 egg chambers, around the time that follicle cells are secreting major structural proteins of the vitelline envelope, is consistent with their products being delivered to the vitelline membrane or perivitelline space. Since sog and dpp are secreted proteins, they could be exported like components of the vitelline membrane to the extracellular compartment between the follicle cells and the oocyte. After stage 13, the vitelline membrane is thought to be an impermeant barrier separating the oocyte from follicle cells making it unlikely that sog and dpp products are transferred after this time. A similar model has been proposed to explain the functions of the dorsal group gene nudel and of the maternal terminal system gene torsolike (tsl). Both of these genes are expressed during midoogenesis, long before their activity is required during early embryogenesis. According to this model, the Sog and Dpp proteins would remain in the perivitelline space until early embryogenesis, when the Tl pathway is activated by Spatzle. In the early embryo, maternal Dpp would decrease the level of Tl-mediated nuclear translocation of Dorsal by decreasing Cactus signal-independent degradation through a pathway acting in parallel to Tl. Presumably, Sog antagonizes the action of Dpp, resulting in maximal nuclear Dorsal translocation (Araujo, 2000).

The Drosophila Toll-9, acting through Pelle and Cactus activates a constitutive antimicrobial defense

The Toll family of transmembrane proteins participates in signaling infection during the innate immune response. The nine Drosophila Toll proteins were analyzed and it was found that wild-type Toll-9 behaves similar to gain-of-function Toll-1. Toll-9 activates strongly the expression of Drosomycin, and utilizes similar signaling components to Toll-1 in activating the antifungal gene. The predicted protein sequence of Toll-9 contains a tyrosine residue in place of a conserved cysteine, and this residue switch is critical for the high activity of Toll-9. The Toll-9 gene is expressed in adult and larval stages prior to microbial challenge, and the expression correlates with the high constitutive level of drosomycin mRNA in the animals. The results suggest that Toll-9 is a constitutively active protein, and implies its novel function in protecting the host by maintaining a substantial level of antimicrobial gene products to ward off the continuous challenge of microorganisms (Ooi, 2002).

In both dorsal–ventral development and antifungal response, activated Toll-1 recruits Tube and Pelle to initiate signaling. Both Tube and Pelle contain death domains, and Pelle is a kinase. Recruitment of Pelle somehow leads to degradation of the inhibitor Cactus and release of the transcription factors, Dorsal and Dif. Whether Toll-9 employs the same signaling components to activate drosomycin expression was examined. A construct for Pelle containing only the death domain (Pelle^DD), but lacking the kinase domain, was generated. This mutated Pelle protein should function as dominant negative by binding to the death domain of Tube but cannot phosphorylate downstream substrates. Transfection of wild-type Pelle activated the reporter gene efficiently, consistent with an important role of the protein in antifungal response. As expected, Pelle^DD did not activate the reporter. In contrast, the Pelle^DD construct inhibited all the Toll-1-, Toll^10b- and Toll-9-mediated drosomycin reporter activities (Ooi, 2002).

Cactus uses its ankyrin repeats to bind to the Rel homology domains of Dif and Dorsal. The Cactus protein degradation is regulated both by signal dependent and signal independent mechanisms, through the N-terminal serine residues and C-terminal PEST sequence, respectively. Therefore, a construct CactusDelta¹²⁵Delta^PEST was used that contained only the ankyrin repeats. This mutant Cactus should stably bind to and inhibit Dif and Dorsal, even when the signaling pathway is stimulated. Co-transfection of wild-type Cactus did not lead to significant changes in the activation of drosomycin by Toll-1, Toll^10b and Toll-9. In contrast, the CactusDelta¹²⁵Delta^PEST construct abolished all these Toll signaling activities. Therefore, Cactus and Pelle, and probably the binding partners Dif and Tube, are likely signaling components that mediate the activation of drosomycin by Toll-9 (Ooi, 2002).

Protein Interactions

Cactus - Dorsal interaction

Transgenic flies containing the dorsal cDNA, driven by the constitutively active hsp83 promoter, exhibit rescue of the dorsal- mutant phenotype. Females with Dorsal levels roughly twice that of wild-type produced normal embryos, while a higher level of Dorsal protein resulted in phenotypes similar to those observed for loss-of-function cactus mutations. In contrast to a Dorsal/Cactus ratio of 2.5 resulting in fully penetrant weak ventralization, a Cactus/Dorsal ratio of 3.0 proved workable. By manipulating Dorsal levels in different cactus and dorsal group mutant backgrounds, it was found that the relative amount of ventral signal to that of the Dorsal-Cactus complex is important for the elaboration of the normal dorsoventral pattern. In a wild-type embryo, the activities of dorsal and cactus are not independently regulated; excess cactus activity is deployed only if a higher level of Dorsal protein is available (Govind, 1993).

A Cactus fusion protein, CACT-Bgl contains the six ankyrin repeat sequences which are necessary for specific binding to the Drosophila rel family transcription factor Dorsal. The purified CACT-Bgl protein can bind specifically to Dorsal and the protein adopts a largely alpha-helical secondary structure. A further analysis of the ankyrin repeat domains of Cactus, using an improved secondary structure prediction program, indicates that the N-terminal of the repeat will form into a loop structure and the C-terminal section into an interrupted, amphipathic alpha-helix. It is concluded that the ankyrin repeats of Cactus fold together into helical bundles interconnected by diverged loops (Gay, 1993).

By introducing intermolecular disulfide bonds in homogenates of embryos, three complexes of DL and/or CACT proteins were detected. Complex 1 (190 kDa) is a DL protein homodimer. Complex 2 (270 kDa) consists of one complex 1 and one CACT molecule. Complex 3 (200 kDa) is a CACT protein complex that does not contain DL protein. In wild-type embryos Complex 2 was detected as the major form of DL protein, and Complex 1 was minor. With this assay virtually no DL monomer is detected. Analysis of the DL protein complexes in ventralized and dorsalized mutant embryos indicates that Complex 2 is a cytoplasmic form, whereas the Dorsal homodimer is localized mainly in the nuclei. It seems that a small amount of Dorsal homodimer is also present in the cytoplasm (Isoda, 1994).

The Rel family of transcription factors show homology in an extended region spanning about 300 amino acids (the Rel homology domain [RHD]). The RHD mediates both DNA binding and interactions with a family of inhibitor proteins, including I kappa B alpha and Cactus. Previous studies have shown that an N-terminal region of the RHD (containing the sequence motif RXXRXRXXC) is important for DNA binding, while the C-terminal nuclear localization sequence is important for inhibitor interactions. Another sequence within the RHD (region I) that is essential for inhibitor interactions. There is a tight correlation between the conservation of region I sequences and the specificity of Rel-inhibitor interactions in both flies and mammals. Point mutations in the region I sequence can uncouple DNA binding and inhibitor interactions in vitro. Recent crystallographic studies suggest that the region I sequence and the nuclear localization sequence might form a composite surface which interacts with inhibitor proteins (Tatei, 1995).

Experiments in yeast with various Dorsal and Cactus derivatives showing that Cactus blocks the DNA binding and nuclear localization functions of Dorsal. In contrast, Dorsal's transcriptional activating region is functional in the Dorsal-Cactus complex. Two Dorsal mutants, Dorsal C233R and Dorsal S234P escape Cactus inhibition in vivo, and also fail to interact with Cactus in vitro. Thus the likely surface of Dorsal that binds Cactus can be identified. (Lehming, 1995).

Increased cytoplasmic calcium concentration and the expression of constitutively active Toll receptors can induce the relocalization of Dorsal. By contrast, activation of endogenous Protein kinase A and expression of wild-type Toll receptors, have only a marginal effect on the cellular distribution of the Dorsal protein. Treatment of cells with activators of Protein kinase C and radical oxygen intermediates, both of which activate nuclear factor kappa B, also has little effect on Dorsal protein localization. It is proposed that different threshold levels of Dorsal activation can be established by distinctly regulated signal transduction pathways (Kubota, 1993).

SLDL is a derivative of the SL2 cell line in which dorsal is expressed constitutively. Increased intra-cellular calcium levels induced by the ionophore ionomycin can activate Dorsal/Cactus complexes in the Drosophila cell line SL2. In a cell line (SLDL) in which dorsal is expressed constitutively ionomycin induces a rapid destruction of Cactus and dephosphorylation of Dorsal. These results suggest a role for the protein phosphatase Calcineurin in calcium mediated activation of Dorsal/Cactus complexes. They also indicate that in the resting cell constitutive phosphorylation of Dorsal is in equilibrium with calcium dependent dephosphorylation (Kubota, 1995a).

It has also been proposed that free cytoplasmic Dorsal protein is able to stimulate translation of the Cactus mRNA, either, directly or indirectly. Such an arrangement would enable the Dorsal protein to be buffered in the cytoplasm of the resting cell over a wide range of concentrations (Kubota, 1995b).

Dorsal is an embryonic phosphoprotein. Its phosphorylation state is regulated by an intracellular signaling pathway initiated by the transmembrane receptor Toll. The phosphorylation state of Dorsal is altered for the period during which Toll is activated. Moreover, mutations that constitutively activate Toll stimulated Dorsal phosphorylation, while mutations that block Toll activation reduced the level of Dorsal phosphorylation. Signal-dependent Dorsal phosphorylation is modulated by three intracellular proteins: Pelle, Tube, and Cactus. Free Dorsal is a substrate for a signal-independent kinase activity.These results imply that Dorsal is a substrate for a Toll-dependent kinase. (Gillespie, 1994).

Dorsal and Cactus are both phosphoproteins that form a stable cytoplasmic complex. The Cactus protein is stabilized by its interaction with Dorsal. The Dorsal-Cactus complex dissociates when Dorsal is targeted to the nucleus. While the phosphorylation of Cactus remains apparently unchanged during early embryogenesis, the phosphorylation state of Dorsal correlates with its release from Cactus and with its nuclear localization. This differential phosphorylation event is regulated by the dorsal group pathway (Whalen, 1993).

The Drosophila immune response uses many of the same components as the mammalian innate immune response, including signaling pathways that activate transcription factors of the Rel/NK-kappaB family. In response to infection, two Rel proteins, Dif and Dorsal, translocate from the cytoplasm to the nuclei of larval fat-body cells. The Toll signaling pathway, which controls dorsal-ventral patterning during Drosophila embryogenesis, regulates the nuclear import of Dorsal in the immune response, but the Toll pathway is not required for nuclear import of Dif. Dif is properly translocated from fat-body cytoplasm to nuclei in response to infection in Toll and pelle mutant larvae. Cytoplasmic retention of both Dorsal and Dif depends on Cactus protein; nuclear import of Dorsal and Dif is accompanied by degradation of Cactus. Therefore the two signaling pathways that target Cactus for degradation must discriminate between Cactus-Dorsal and Cactus-Dif complexes. New genes have been identified that are required for normal induction of transcription of antibacterial peptide diptericin during the immune response. Mutations in three of these genes prevent nuclear import of Dif in response to infection, and define new components of signalling pathways involving Rel. The 18-wheeler gene, which encodes transmembrane protein that is homologous to Toll, is important for the nuclear localization of Dif during the immune response, so two of these genes may encode products that are necessary for 18-wheeler activation or cytoplasmic components that act downstream of 18-wheeler. Mutations in three other genes, constituting a second class of mutations, cause constitutive nuclear localization of Dif; these mutations may block Rel protein activity by a novel mechanism. The gene immune deficiency (imd) belongs to this second class, as both Dif and Dorsal are constitutively nuclear in imd mutants. Cactus does not appear to be found in the nucleus in class II mutants. One hypothesis that fits these observations is that the class II genes are required to allow the formation of a nuclear complex of Dif with other proteins, and that this complex is required both for activation of diptericin transcription and for turnover of the nuclear Rel proteins (Wu, 1998).

Degradation of Cactus

Cactus, like its mammalian homolog I kappa B, inhibits nuclear translocation by binding Dorsal and retaining it in the cytoplasm. Like I kappa B, Cactus is rapidly degraded in response to signaling. More importantly, signal-dependent degradation of Cactus does not require the presence of Dorsal, indicating that Cactus degradation is a direct response to signaling, and that disruption of the Dorsal/Cactus complex is a secondary result of Cactus degradation. Cactus protein stability is regulated by two independent processes that rely on different regions within the protein: signal-dependent degradation requires sequences in the amino terminus or ankyrin repeats, whereas signal-independent degradation of free Cactus requires the carboxy-terminal region of the protein that includes a PEST sequence (Belvin, 1995).

A signal-dependent asymmetric distribution of Cactus in embryos has not previously been observed in whole mount stainings. Such an asymmetric distribution is expected if ventral degradation of CACT accounts for the nuclear uptake of Dorsal. The asymmetry might be reduced if CACT is constantly resynthesized from the large maternal CACT mRNA pool so that the total amount of CACT remains approximately the same. To observe CACT asymmetric distribution, protein synthesis was blocked with cyclohexamide in permeabilized wild-type embryos, and embryos were stained with anti-CACT antibody. CACT becomes diminished on the ventral side of embryos treated this way, probably as a result of active protein degradation. Nuclear localization of DL correlates with the reduction in CACT, suggesting a mechanistic link between nuclear translocation and cytoplasmic degradation. In nudel, snake, tube, and pelle mutants there is no detectable asymmetric distribution of CACT. Ventralized embryos from females carrying a dominant gain-of-function allele of Toll show a depletion of CACT staining in the apical but not the basal cytoplasm. This indicates the intracellular part of the dorsoventral signal transduction takes place predominantly in the narrow cytoplasmic space between the egg membrane, where the Toll receptor is located, and the nuclear layer. Dorsal controls the protein level of Cactus. Additional amounts of Dorsal lead to a corresponding increase in the amount of CACT. Thus, DL directly determines the amount of its inhibitor CACT. CACT in the DL/CACT complex is stabilized, whereas surplus CACT is unstable and gets degraded. This analysis distinguishes two different modes of CACT degradation: signal-induced degradation and single-independent degradation (Bergmann, 1996).

Due to signal-independent degradation, the level of CACT is down regulated in dl mutant embryos, but it is not completely absent. The ventral signal is present in dl mutants. There is a subtle difference in the abundance of CACT between the dorsal and the ventral side, suggesting that free, uncomplexed CACT is subject to signal-induced degradation on the ventral side. In double dl-pelle mutant flies in which the ventral signal is blocked, there is a significant increase in CACT levels. Deletion of the PEST sequence blocks signal independent degradation of CACT, while N-terminal deletions of CACT block the response of CACT to signal dependent degradation. Complete absence of CACT is not sufficient to translocate DL completely into the nucleus. It is concluded that in addition to signal-induced degradation of CACT, other mechanisms must operate for complete nuclear translocation of DL. Such mechanisms may include some signal input to DL such as phosphorylation of DL by Protein kinase A. Moreover, the existence of CACT/IkappaB homologs that can substitute for lacking CACT cannot be ruled out (Bergmann, 1996)

Signal-induced phosphorylation of IkappaBalpha targets this inhibitor of NF-kappaB for ubiquitination and subsequent degradation, thus allowing NF-kappaB to enter the nucleus to turn on its target genes. An IkappaB-ubiquitin (Ub) ligase complex has been identified that contains the F-box/WD40-repeat protein, beta-TrCP, a vertebrate homolog of Drosophila supernumerary limbs (Slimb). beta-TrCP binds to IkappaBalpha only when the latter is specifically phosphorylated by an IkappaB kinase complex. Moreover, immunopurified beta-TrCP ubiquitinates phosphorylated IkappaBalpha at specific lysines in the presence of Ub-activating (E1) and -conjugating (Ubch5) enzymes. A beta-TrCP mutant lacking the F-box inhibits the signal-induced degradation of IkappaBalpha and subsequent activation of NF-kappaB-dependent transcription. Furthermore, Drosophila embryos deficient in slimb fail to activate twist and snail, two genes known to be regulated by the NF-kappaB homolog, Dorsal. These biochemical and genetic data strongly suggest that Slimb/beta-TrCP is the specificity determinant for the signal-induced ubiquitination of IkappaBalpha (Spencer, 1999).

Dorsal-ventral polarity within the Drosophila syncytial blastoderm embryo is determined by the maternally encoded dorsal group signal transduction pathway that regulates nuclear localization of the transcription factor Dorsal. Nuclear uptake of Dorsal, a Rel/NFkappaB homolog, is controlled by the interaction with its cognate IkappaB inhibitor protein Cactus, which is degraded on the ventral side of the embryo in response to dorsal group signaling. Previous studies have suggested that an N-terminally located kinase target motif similar to that found in IkappaB proteins is involved in the spatially controlled degradation of Cactus. Studies of the in vivo function and distribution of fusion proteins comprising segments of Cactus attached to Escherichia coli ß-galactosidase (lacZ) are reported. Full-length Cactus-lacZ expressed in vivo normalizes the ventralized phenotype of embryos that lack Cactus and faithfully reconstitutes dorsal group-regulated degradation, while fusion protein constructs that lack the first 125 amino acids of Cactus escape dorsal group-dependent degradation. Furthermore, Cactus-lacZ constructs that lack only the putative IkappaB-dependent kinase target-like motif can nevertheless undergo spatially regulated dorsal group-dependent degradation and the regulatory determinant responsible for dorsal group-dependent degradation of Cactus in the absence of this motif has been identified. Taken together, these studies indicate the presence of two distinct redundantly acting determinants in the N terminus of Cactus that direct dorsal group-dependent degradation. Strikingly, the regulatory domain of human IkappaBalpha can also direct polarized degradation of Cactus-lacZ fusion protein (Fernandez, 2001).

Considerable study of the regulation of NFkappaB following activation of the IL-1R indicates that a specific kinase complex mediates the phosphorylation of IkappaBalpha at serines 32 and 36, followed by recruitment of SCF ß-TrCP complex, ubiquitination of IkappaBalpha at lysines 21 and 22 and degradation. Several experiments have demonstrated the key importance of the identified serines and lysines in the degradation of IkappaBalpha. Conversion of serines 32 and 36 to alanine or of lysines 21 and 22 to arginine results in a dominant gain-of-function effect, and in an inability to activate NFkappaB activity bound to the mutant IkappaBalpha. In contrast, conversion of serines 32 and 36 to glutamate results in constitutive degradation of IkappaBalpha and of constitutive NFkappaB activity (Fernandez, 2001).

The regulation of Dorsal nuclear localization by Cactus has remained less well understood. To some extent, this knowledge gap stems from a more complicated structure of Cactus, in comparison with other IkappaB proteins. Cactus, like IkappaBalpha and the other IkappaB proteins, carries six copies of the ankyrin repeat motif, which in all characterized IkappaB activities are involved in the physical interaction with their cognate Rel homologous proteins. The identification of the gain-of-function alleles cact^BQ and cact^E10 has directed attention at the region of Cactus N-terminal to the ankyrin repeat regions, as potentially containing determinants of dorsal group-mediated regulation. In IkappaBalpha, the 72 amino acids to the N-terminal side of the ankyrin repeat region contain three serine residues and four lysine residues, including the critical ones described above. In the case of Cactus, the 228 amino acids to the N terminus of the first ankyrin repeat contain 36 serine residues and 11 lysines, providing the possibility for a much greater level of complexity in the control of Cactus regulation (Fernandez, 2001).

In the N-terminal 100 amino acids of Cactus, a segment with strong homology to the putative IKK target motif has been identified and investigated via injection of RNA carrying mutations and deletions in this region. However no consensus was obtained as to whether the IKK target-like motif is essential for the regulation of Cactus. The phenotypes observed following expression of the mutant Cactus derivatives were a composite of the effects of the mutant together with the endogenous (wild-type or mutant) Cactus present in the embryos. Additional variability in the phenotypes is also likely to have arisen as a consequence of differences in the amount of RNA injected from embryo to embryo. In neither case was the distribution or function of the introduced Cactus assessed directly (Fernandez, 2001).

In order to address these technical issues and as a way of assessing the intrinsic ability of mutant Cactus derivatives to undergo spatially regulated degradation, the strategy was adopted of assaying directly the distribution and functionality of introduced Cactus in the context of Cactus-lacZ fusions. Full-length Cactus-lacZ reconstitutes function and binding to Dorsal. Strikingly, this protein also exhibits dorsal group-dependent regulation of subcellular distribution, as assessed by lacZ enzymatic activity, in spite of the fact that active ß-galactosidase acts as a tetramer. This suggests that tetramerization does not inhibit the ability of the protein to interact with Dorsal or the other cellular machinery mediating spatially regulated degradation (Fernandez, 2001).

By constructing specific mutant derivatives of Cactus in the context of a lacZ fusion protein, the redundant nature of the previously hypothesized IKK motif in Cactus has been demonstrated. Deletions mutants that lack this region and site-directed mutants in which the critical serines have been converted to alanine residues have illuminated a second putative regulatory target in the N terminus of Cactus, located at S116. Taken together, these results indicate that redundant determinants present in the N terminus have the potential to confer upon the protein the ability to be degraded in a graded fashion. Interestingly, serine 116 is contained within the context DSGIID. Substitution of the second aspartic acid residue to a serine would convert this to the IKK consensus target motif (DSGPsiXS). In view of the finding that conversion of the critical serines 32 and 36 in IkappaBalpha to glutamic acid results in a constitutively ubiquitinated protein, serine 116 may be present in the context of a second IKK-like target motif, one that is in fact predisposed towards an interaction with the degradation machinery, after a single phosphorylation event (Fernandez, 2001).

What is the basis for the presence of redundant regulatory determinants in the N terminus of Cactus? The formation of a gradient of nuclear Dorsal where high, intermediate, low and undetectable levels are required might necessitate a level of regulatory control in Cactus degradation that is not required of IkappaB in vertebrate cells where NFkappaB is simply active or inactive. On the ventral side of the Drosophila embryo, one might suppose that all regulatory serines in Cactus are phosphorylated, leading to complete degradation with partial phosphorylation in lateral regions, which leads to intermediate levels of degradation. While study of the distribution of Cactus-lacZ fusion that lacks either putative regulatory motif shows that both sites can act alone in the formation of a Cactus gradient, the possibility of subtle changes in degradation gradients formed by these mutant proteins, in comparison with the gradient exhibited by wild-type Cactus, cannot be ruled out. Interestingly, in support of the idea that multiple regulatory determinants are required for the formation of a normal Dorsal gradient, previous observations indicated that Cactus constructs lacking the previously identified putative IKK motif did not behave in an entirely wild-type manner, in comparison with introduced wild-type Cactus. Rather, this construct was weakly dorsalizing, leading to the suggestion that multiple determinants in Cactus are required for the formation of normal Cactus and Dorsal gradients (Fernandez, 2001).

Requirement for Cactus function in multiple processes in Drosophila provides a second possible reason for the presence of multiple regulatory determinants that mediate degradation. In addition to DV pattern formation, Cactus-mediated regulation of Dorsal/Rel-homologs plays a part in immune function in the fly as well as other processes. The presence of two identified regulatory domains may reflect a situation in which the N-terminal region of Cactus acts as an antenna to integrate several regulatory inputs, leading ultimately to the destabilization of Cactus. In this regard, it is interesting to speculate that additional members of the 36 serines present in the N-terminal domain of Cactus not identified in this study remain candidates for putative target sites for Cactus regulation in other processes. Whether either or both of the regulatory determinants identified in this study influence Cactus function in the Drosophila immune response remains an interesting topic for speculation (Fernandez, 2001).

In spite of the presence of IKK target-like motifs in the Cactus N terminus, there remains uncertainty about the identity of the kinase(s) that modifies these targets. Mutations have been identified that affect the Drosophila homologs of IKKß and IKKgamma, respectively, and while these mutations have striking effects on the bacterial arm of the Drosophila immune system, they have modest if any effects on the pathway affecting embryonic DV polarity. Furthermore, the Toll signaling pathway is not inhibited by RNAi-mediated inhibition of either DmIKKß or DmIKKgamma. It remains to be determined whether a Drosophila homolog of IKKepsilon, constitutes the Cactus kinase that acts in DV pattern formation (Fernandez, 2001).

How similar are the events of dorsal group dependent degradation of Cactus to the mechanism accomplishing degradation of IkappaBalpha? Previous studies have identified a role for the F-box protein B-TrCP, in the regulated degradation of phosphorylated IkappaBalpha. B-TrCP, a component of the IkappaB-Ubiquitin ligase (SCF), targets IkappaB for degradation in response to signals that induce NFkappaB activity, binding directly to phosphorylated IkappaBalpha and thereby providing specificity to the degradation process. By analogy to the situation for IkappaBalpha it is possible that phosphorylation of serines 74 and 78, or of serine 116 results in recruitment of an SCF complex containing an F-box specificity-conferring protein, followed by ubiquitination and degradation of Cactus. Drosophila embryos deficient for Slimb, a fly homolog of B-TrCP, show an impaired ability to activate the expression of Dorsal target genes. While a role for Slimb in targeting the degradation of Cactus during embryogenesis has been inferred, a direct test for an effect on the distribution of Cactus was not made. Cactus-lacZ expressed in slimb/slimb germline clones exhibits a weak though reproducibly asymmetric distribution, and the embryos produced develop polarized pattern elements along the DV axis (Fernandez, 2001).

Cactus-Pelle interaction

Pelle-mediated signaling induces the spatially graded degradation of Cactus. Using a tissue culture system that reconstitutes Pelle-dependent Cactus degradation, it can be shown that a motif in Cactus, resembling the sites of signal-dependent phosphorylation in the vertebrate Cactus homolog IkappaB is essential for Pelle-induced Cactus degradation. Substitution of four serines within this motif with nonphosphorylatable alanine residues generate a mutant Cactus that still functions as a Dorsal inhibitor but is resistant to degradation (Reach, 1996).

Although both Cactus and Dorsal are modified by phosphorylation in response to signaling, it is believed that signal-dependent modification of Cactus precedes modification of Dorsal. Signal-dependent modification of Dorsal presumably occurs downstream of Cactus degradation, since loss of Cactus function induces Dorsal modification in the absence of signaling. Phosphorylation of Dorsal upon release from Cactus could reflect unmasking of a phosphorylation site or an encounter with a protein kinase upon translocation into nuclei (Gillespie, 1994 and Reach, 1996).

Cactus-Casein kinase II interaction

Cactus is a phosphoprotein and has in its C-terminus a PEST protein stability domain. Like mammalian IkappaBalpha, the PEST domain of Drosophila Cactus is phosphorylated by Casein kinase II . The site of modification has been localized to a single residue, Ser468 in the PEST domain: no evidence for additional phosphorylation sites are found. The conservation of these sites in mammalian and invertebrate cytoplasmic anchor proteins suggests that phosphorylation by Casein kinase II may play a critical functional role, plausibly in the regulation of constitutive or inducible proteolysis (Packman, 1997).

Regulated proteolysis of Cactus, the cytoplasmic inhibitor of the Rel-related transcription factor Dorsal, is an essential step in the patterning of the Drosophila embryo. Signal-induced Cactus degradation frees Dorsal for nuclear translocation on the ventral and lateral sides of the embryo, establishing zones of gene expression along the dorsoventral axis. Cactus stability is regulated by amino-terminal serine residues necessary for signal responsiveness, as well as by a carboxy-terminal PEST domain. Drosophila casein kinase II (CKII) has been identified as a Cactus kinase: CKII specifically phosphorylates a set of serine residues within the Cactus PEST domain. These serines are phosphorylated in vivo and are required for wild-type Cactus activity. Conversion of these serines to alanine or glutamic acid residues differentially affects the levels and activity of Cactus in embryos, resulting in a longer half-life for endogenous Cactus. As a result, Cactus accumulates above wild-type levels, interfering with signal transduction. Mutation modification of the PEST domain does not inhibit the binding of Cactus to Dorsal. Taken together, these data indicate that wild-type axis formation requires CKII-catalyzed phosphorylation of the Cactus PEST domain. CKII phosphorylation may play a general role in PEST domain function (Liu, 1997).

Cactus-Cactin interaction

Rel transcription factors function in flies and vertebrates in immunity and development. Although Rel proteins regulate diverse processes, the control of their function is conserved. In a two-hybrid screen for additional components of the pathway using for bait the Drosophila I-kappaB protein Cactus, a novel coiled-coil protein with N-terminal Arg-Asp (RD)- like motifs was isolated and has been called Cactin (AAF66981; CG1676). Like the other components of this pathway, Cactin is evolutionarily conserved. Over-expression of cactin in a cactus^A2 heterozygous background results in the enhancement of the cactus phenotype. Both the embryonic lethality and ventralization are strongly increased, suggesting that cactin functions in the Rel pathway controlling the formation of dorsal-ventral embryonic polarity (Lin, 2000).

Cactin encodes a novel protein with a predicted molecular weight of 88 kDa. A striking feature is an N-terminal domain rich in charged residues, alternating between positively and negatively charged amino acids (RD-like repeats). The protein contains three potential bipartite nuclear localization signals and three coiled-coil domains, that may function in the self-association of the protein, or in the interaction of Cactin with proteins other than Cactus. Database searches have identified Cactin homologs in Arabidopsis, Dictyostelium, S. pombe, C. elegans, mouse, and human. In humans, several ESTs have been identified from such tissues as tonsillar cells enriched for germinal center B cells, kidney, testis, colon, T cells, and B cells. The human protein is predicted to be 758 aa in length and overall is 41% identical to the Drosophila protein. The Arabidopsis homolog is predicted to be 672 aa, and the S. pombe homolog is 517 aa in size: these two homologs show 32% and 24% identity, respectively, to the Drosophila protein. One mouse EST (161 aa) isolated from 13.5 to 14.5 day old embryos is also close to 50% identical to Drosophila Cactin. All Cactin homologs share a highly conserved region in the C-terminal half of the protein. In this domain there exists a block of 52 aa (amino acids 598-650 in Drosophila) in which only 2 aa are different between humans and flies. In the same domain 4 aa in Arabidopsis and 6 aa in C. elegans are different from the fly sequence. The most divergent sequence is that of the mouse. Since this sequence is relatively short and derived from an EST, these differences could be due to sequencing artifacts (Lin, 2000).

Over-expression of Cactin in a sensitized cactus background results in enhancement of embryonic lethality and of the ventralized phenotype, a result suggesting that Cactin may function in the dorsal-ventral pathway and that its normal function may be to positively regulate the nuclear targeting of Dorsal by regulating the stability of the Cactus protein. Cactus activity is regulated by both signal-independent and signal-dependent processes that target it for degradation. In dorsal protein-null backgrounds, no Cactus is present as a result of a signal-independent degradation process. The signal-dependent degradation of Cactus involves serine phosphorylation events in the N-terminal region. Phosphorylation at these sites triggers poly-ubiquitination and targets the IkappaB proteins for rapid degradation by the 26S proteasome. Cactin may function in either of the two degradation processes. The signal independent degradation of Cactus appears to proceed throughout early embryogenesis, and overexpression of Cactin could result in an excess of Cactus degradation resulting in an extended Dorsal nuclear gradient and a ventralized phenotype (Lin, 2000).

Alternatively, Cactin may function in the signal dependent degradation of Cactus. A 900 kDa IkappaB Kinase (IKK) complex that controls the phosphorylation of IkappaB has been identifed in vertebrates. Two IkappaB kinases, IKKalpha and IKKbeta, and IKKgama/NEMO (NF-kappaB essential modifier) as well as IKAP (IKK complex-associated protein) are components of this multiprotein complex. Studies on the ubiquitin-dependent proteolysis have identified an evolutionarily conserved SCF ubiquitin ligase complex in the regulation of the Rel/IkappaB signal transduction. Cactin could represent an additional component of either complex. The strong conservation of Cactin suggests that its function is conserved throughout evolution (Lin, 2000).

Cactus-Cactus kinase interaction

Eukaryotic organisms use a similar Rel/NF-kappaB signaling cascade for the induction of innate immune genes. In Drosophila, lipopolysaccharide (LPS) signal-induced activation of the Rel/NF-kappaB family transcription factors is an essential step in the transcriptional activation of inducible antimicrobial peptide genes. However, the mechanism by which the LPS-induced signaling pathway proceeds remains largely unknown. A novel Drosophila LPS-activated kinase (DLAK; Cactus kinase, abbreviated as IKK; identified as CG4201) has been cloned that is structurally related to mammalian IkappaB kinases. DLAK is expressed and transiently activated in LPS-responsive Drosophila cells following LPS stimulation. Furthermore, DLAK can interact with Cactus, a Drosophila IkappaB and phosphorylate recombinant Cactus, in vitro. Overexpression of dominant-negative mutant DLAK (DLAK^K50A) blocks LPS-induced Cactus degradation. DLAK-bound Cactus can be degraded in a LPS signal-dependent fashion, whereas the DLAK^K50A mutant-bound Cactus is completely resistant to degradation in the presence of LPS. The DLAK^K50A mutant also inhibits nuclear kappaB binding activity and kappaB-dependent diptericin reporter gene activity in a dose-dependent manner, but the kappaB-dependent diptericin reporter gene activity can be rescued by overexpression of wild type DLAK. Moreover, mRNA analysis of various kappaB-dependent antimicrobial peptide genes shows that LPS inducibility of these genes is greatly impaired in cells overexpressing DLAK^K50A. These results establish that DLAK is a novel LPS-activated kinase, which is an essential signaling component for the induction of antimicrobial peptide genes following LPS treatment in Drosophila cells (Kim, 2000).

Previous studies provided evidence that the expression of various kappaB-dependent Drosophila antimicrobial genes can be regulated independently by the Toll receptor-dependent pathway and/or the immune deficiency (imd) gene product-dependent pathway. Antibacterial diptericin gene appears to be regulated mainly by the imd gene product, whereas antifungal drosomycin gene appears to be controlled by the Toll pathway. Other antibacterial peptide genes, such as cecropin, defensin, and attacin, require both the Toll pathway and imd gene product for their immune signal-induced expression. An examination was performed to see whether the LPS inducibility of these antimicrobial genes is affected in the absence of DLAK activity using DLAK^K50A-overexpressed cells. Semi-quantitative RT-PCR analysis was carried out using specific primers for each gene following LPS challenge. In the absence of DLAK^K50A expression, all examined antimicrobial peptide genes are up-regulated 2.5-10 times (2.5 times for drosomycin expression, 4.6 times for diptericin expression, 6 times for defensin expression, 7.3 times for attacin expression, and 10 times for cecropin A expression) following LPS treatment. However, when DLAK^K50A is overexpressed, LPS inducibility of diptericin, as well as cecropin, defensin, attacin, and drosomycin, is severely impaired. defensin and attacin transcripts reach only 20% of control levels following infection, diptericin and cecropin transcripts reach 30% of control levels, and drosomycin transcripts reach 35% of control level. In control experiments, CuSO₄ treatment in untransfected control cells or a cell line stably expressing an unrelated Drosophila protein had no noticeable effect on the LPS inducibility of antimicrobial peptide genes. This result suggests that both Toll- and imd-dependent pathways require DLAK activity for full LPS inducibility of various antimicrobial peptide genes (Kim, 2000).

Until now, no signaling kinase has been found for LPS-induced Rel/NF-kappaB activation in Drosophila. During dorsoventral pattern formation, Pelle kinase is known to be essential for developmental signal-dependent Dorsal activation via Cactus degradation. However, in LPS signal transduction, Pelle is not required for the induction of diptericin, suggesting a kinase other than Pelle might be implicated in LPS signal-dependent Rel/NF-kappaB activation responsible for the induction of certain antibacterial peptides such as diptericin. The results presented here demonstrate that overexpression of a dominant-negative DLAK fully blocks LPS-induced Dipt-kappaB reporter gene (containing eight copies of the kappaB motif derived from the diptericin gene promoter) activity and also diminishes nuclear kappaB binding activity. Furthermore, in addition to diptericin, other antimicrobial peptide genes such as cecropin, attacin, defensin, and drosomycin also lose their LPS inducibility in dominant-negative DLAK-overexpressed cells. DLAK appears to be a good candidate for this LPS signaling process. To date, only three Rel/NF-kappaB family transcription factors, Dorsal, Dif, and Relish are known to be responsible for the induction of kappaB motif-containing antimicrobial genes. It is not known exactly which of these transcription factors is responsible for each antimicrobial peptide. Very recently, in the contrast to the induction of drosomycin, the induction of diptericin, cecropin, and attacin genes has been shown not to be affected in Dif and Dorsal double mutant flies. Involvement of an as yet unknown Rel/NF-kappaB transcription factor or Relish may participate in this process. It is not yet known how DLAK intervenes in the activation of the putative Rel/NF-kappaB, responsible for LPS-induced kappaB-responsive antimicrobial gene induction. In the case of mammalian cells, the activation of Rel/NF-kappaB transcription factors are thought to be regulated through the transient degradation of its cytoplasmic inhibitor protein (IkappaB), where signal-induced phosphorylation of IkappaB by specific IKKs initiates the inhibitor's conjugation to ubiquitin and subsequent degradation by the proteasome. Given that DLAK has the most extensive sequence similarity with IKK and that DLAK exists in the form of a complex with Cactus and LPS-induced Cactus degradation specifically blocked by dominant-negative DLAK, it can be speculated that the dominant-negative form of DLAK fails to transmit the Cactus degradation signal, essential for Rel/NF-kappaB activation and subsequently prevents LPS inducibility of at least certain kappaB-dependent antimicrobial peptide genes. The possible effect of DLAK activity on the stability of other proteins with IkappaB activity such as Relish is presently under investigation (Kim, 2000).

The essential involvement of DLAK for the induction of the kappaB-dependent antimicrobial peptide genes is yet another example of the striking similarities between the LPS-induced signal transduction in Drosophila and mammalian IKK-mediated innate immune signaling. In mammals, two kinases responsible for NF-kappaB activation (via IkappaB phosphorylation) have been identified, IKKalpha and IKKbeta. Both IKKalpha and IKKbeta contain three important functional domains as follows: a kinase domain for activity; a leucine zipper domain, necessary for dimerization, and a helix-loop-helix domain serving as a putative endogenous activator of IKK. In the case of DLAK, its kinase domain is most homologous with that of IKKbeta (34% identity and 57% similarity). Furthermore, the analysis of the predicted secondary structure shows that DLAK also contains the putative structural domains (leucine zipper and helix-loop-helix) at equivalent positions to those in the mammalian homologs. In addition to these structural motifs, it was recently demonstrated in the case of IKKbeta that a serine cluster, located between the helix-loop-helix motif and the COOH terminus, serves as a negative regulator of the kinase activity. A serine cluster (residues 673-705) containing 10 serine residues is found in the COOH terminus of DLAK corresponding to that of IKKbeta. Very recent studies using IKKalpha-/- and IKKbeta^-/- mice have further confirmed the importance of the structural motifs and further identified the roles of IKKalpha and IKKbeta in development and inflammation signal transduction, respectively. The strong structural resemblance between the mammalian IKKbeta and DLAK further corroborates functional results indicating that DLAK is a Drosophila functional homolog of IKKbeta. At present, it is unknown whether DLAK is also implicated in the regulation of kappaB-responsive genes involved in early Drosophila development. Further studies using DLAK-/- flies will elucidate the exact role(s) of DLAK in the Drosophila immune response and possibly in development (Kim, 2000).

Immune activation of NF-kappaB and JNK requires Drosophila TAK1, the Drosophila IkappaBkinase-activating kinase

Stimulation of the Drosophila immune response activates NF-kappaB and JNK signaling pathways. For example, infection by Gram-negative bacteria induces the Imd signaling pathway, leading to the activation of the NF-kappaB-like transcription factor Relish and the expression of a battery of genes encoding antimicrobial peptides. Bacterial infection also activates the JNK pathway, but the role of this pathway in the immune response has not yet been established. Genetic experiments suggest that the Drosophila homolog of the mammalian MAPK kinase kinase, TAK1 (transforming growth factor ß-activated kinase 1), activates both the JNK and NF-kappaB pathways following immune stimulation. Drosophila TAK1 functions as both the Drosophila IkappaB kinase-activating kinase and the JNK kinase-activating kinase. However, JNK signaling is not required for antimicrobial peptide gene expression but is required for the activation of other immune inducible genes, including Punch, Sulfated, and Malvolio (Mlv). Thus, JNK signaling appears to play an important role in the cellular immune response and the stress response (Silverman, 2003).

In the fly, the NF-kappaB homolog Relish is required for the induction of antimicrobial peptides in response to Gram negative bacterial infection, and relish mutants are hyper-susceptible to Gram-negative bacterial infections. Relish is a bipartite protein with an NF-kappaB (Rel homology) domain and an inhibitory IkappaB domain. In unstimulated cells, Relish is present in the cytoplasm as a full-length precursor protein and, upon infection, is endoproteolytically cleaved. The N-terminal Rel homology domain of Relish then translocates into the nucleus and activates antimicrobial gene expression, whereas the C-terminal IkappaB module remains in the cytoplasm. Signal-induced cleavage and activation of Relish requires a signaling pathway known as the Imd pathway, which includes the receptor PGRP-LC (peptidoglycan recognition protein LC), the intracellular signaling component Imd [a receptor interacting protein (RIP)-like death domain protein], TAK1 (a MAP3K), the Drosophila IKK complex (IKKß/Ird5 and IKKγ/Kenny), as well as the caspase Dredd and the adaptor known as the Fas-associated death domain (FADD). The detailed biochemical mechanisms required for this intracellular signaling pathway are not understood. Drosophila S2* cells have been used show that TAK1 is required for both LPS-induced JNK and IKK activation and for antimicrobial peptide gene induction. Surprisingly, the immune activation of the JNK pathway is not required for antimicrobial peptide gene induction. However, several other JNK-dependent, LPS-inducible genes were identified in microarray studies, suggesting a role for JNK signaling in the cellular immune response as well as protection from stress (Silverman, 2003).

To characterize the role of TAK1 in this pathway at a biochemical level, the Drosophila S2* cell line has been used. When these cells are treated with ecdysone, they differentiate and become responsive to LPS treatment, which leads to high levels of antimicrobial peptide gene expression. An additional advantage of this cell line is that RNAi can be used to target any gene of interest. Similar to the TAK1 mutant fly, targeting TAK1 with RNAi in S2* blocks LPS-mediated activation of antibacterial peptide gene expression, similar to that observed with IKKγ RNAi. TAK1 is unique among the Drosophila MAP3Ks tested because it is required for LPS signaling, whereas slpr and Drosophila MEKK4 are not (Silverman, 2003).

In mammals, TAK1 has been shown to play a critical role in IL-1-induced NF-kappaB activation. In vitro, TAK1 can directly phosphorylate and activate the human IKK complex. Thus, TAK1 has been proposed to function as the IKK activating kinase (IKK-K) in this signaling pathway. The immuno-compromised phenotype of TAK1 mutant flies and S2* cells suggest that TAK1 may function as the IKK-K in the insect immune response. To determine whether TAK1 function is required for IKK activation, an immunoprecipitation kinase assay was utilized. The endogenous Drosophila IKK complex was immunoprecipitated with anti-DmIKKgamma antisera from cell lysates prepared from LPS-treated or untreated cells. This immunoprecipitate was then tested for kinase activity in vitro using recombinant Relish as substrate. LPS treatment leads to a significant increase in Drosophila IKK activity. Treatment of cells with TAK1 RNAi inhibits the LPS-induced IKK activation as much as targeting DmIKKgamma itself. It is concluded that TAK1 is required for LPS-induced activation of the Drosophila IKK complex the subsequent expression of antimicrobial gene expression (Silverman, 2003).

The Drosophila TAK1 protein plays a critical role in the activation of the insect immune response. Genetic studies revealed that TAK1 mutant flies are unable to respond to Gram-negative infections and suggests that TAK1 functions upstream of the Drosophila IKK complex. Consistent with these results, TAK1 is shown to be required for activation of the LPS-induced immune signaling pathways in Drosophila cells in culture. In addition, TAK1 is required for activation of the Drosophila IKK complex in vitro. Thus, Drosophila TAK1 is likely to function as the IKK-K in the LPS signaling pathway, as has been proposed for human TAK1 (Silverman, 2003).

JNK signaling is also activated during the immune response in both flies and humans. However, the exact mechanism by which LPS leads to JNK activation in Drosophila is unclear, as is the role of JNK signaling during the immune response. Gene expression profiling has been used to infer that TAK1 is required for the activation of JNK signaling and that JNK signaling is important for wound healing. This study directly demonstrates that JNK activation requires TAK1. Thus, TAK1 appears to function both as a JNKK activating kinase and an IKK activating kinase, as proposed for mammalian TAK1. Furthermore, microarray results suggest that JNK signaling may have important functions in cellular immunity and the stress response (Silverman, 2003).

The gene expression profiling data presented in this study identifies a relatively small number of genes that specifically require the JNK signaling pathway for their LPS-induced expression. The expression of two genes (Punch and sulfated) identified in these experiments has been validated by real time RT-PCR. Punch is an immune inducible gene in cells in culture and in adult flies. However, although it has been shown that in adult flies the immune induction of Punch requires Relish (De Gregorio, 2001), the data presented in this study demonstrate that Punch induction in S2 cells requires JNK pathway components (hep, bsk, and TAK1) but not the Relish-activating kinase IKK. The experiments presented here were performed in an embryonic Drosophila cell line (that has macrophage-like qualities), whereas the data from De Gregorio (2001) was generated from entire adult flies. Thus, it is possible that the signaling pathways required for Punch induction vary depending on the developmental stage and cell type examined. In fact, Punch has at least two promoters that direct developmentally specific expression (Silverman, 2003).

Punch encodes the enzyme for GTP cyclohydrolase I, which is the first enzyme (and rate-determining step) in the formation of the cofactor tetrahydrobiopterin (BH4). This cofactor is required for the conversion of tyrosine to dopamine, which has at least two possible roles in immunity: (1) dopamine is one of two main Drosophila catecholamines, which are important for the stress response in both insects and mammals; (2) dopamine is the precursor of melanin, which is produced during wound healing and encapsulation processes in the fly. In fact, it has been proposed that increased Punch activity could lead to increased melanization (Silverman, 2003 and references therein).

The cofactor BH4 is also an essential cofactor for nitric oxide synthase (NOS). NO itself has at least two possible roles in the immune response: (1) NO is known to be a major microbicidal compound in mammalian phagocytic cells and is likely to function similarly in Drosophila macrophages; (2) NO has also been implicated in immune signaling in Drosophila. NO is required for transmitting a signal from the site of infection to the fat body, the major organ of immune responsive gene expression. Thus, Punch may contribute to the insect immune response in several ways, including protection against stress, melanization of wound sites, and activation of cellular and humoral immunity (Silverman, 2003 and references therein).

The potential role of sulfated in the immune response is less obvious. sulfated encodes an extracellular sulfatase that removes sulfate groups from heparin sulfate proteoglycans (HSPGs). In avian and Drosophila systems, it is thought that sulfated activity is crucial for the regulation of Wnt signaling, possibly by controlling the extracellular milieu in which the Wnt ligand travels (Silverman, 2003).

One of the most intriguing targets of both the JNK and IKK pathways is Mvl, the Drosophila NRAMP-1 homolog. Mvl mutants were first identified in the fly because they display gustatory behavioral defects caused by the inability to properly process sensory neuronal input. Mvl is expressed in both the nervous system and circulating hemocytes. In the mouse, NRAMP-1 is expressed in macrophages, and mutations in the NRAMP-1 gene are responsible for the sensitivity of some inbred mice strains to the Mycobacterium bovis bacille Calmette-Guérin (BCG) and other intracellular bacterial pathogens. NRAMP-1 is thought to control the levels of cations, possibly Fe²⁺ or Mn²⁺, in lysosomal compartments of mouse macrophages. A current model suggests that NRAMP-1 pumps cations out of the phagolysosome, thereby starving microbes of cations required by the enzymes (superoxide dismutase and catalase), which protect the bacteria from reactive oxygen intermediate (ROI)- and reactive nitrogen intermediate (RNI)-induced damage. In the fly, the role of Mvl in immunity is not yet characterized, but its induction during an immune response coupled with the activity of this protein in vertebrate macrophages suggests that it may play an important role in the cellular immune response (Silverman, 2003).

Microarray analysis has provided evidence that LPS-induced JNK activation is important for the stimulation of a gene expression program similar to that seen during dorsal closure. Thus, JNK may be important for wound healing. Expression of only a few of the JNK target genes (for example, Filamin) is reported in this study. The data argue that JNK signaling is required for the activation of cellular immunity and stress protection, whereas a connection to wound healing cannot be excluded by these data (Silverman, 2003).

Certain antimicrobial genes (e.g., diptericin) require a combination of transcription factors for their proper induction. It has been suggested, based on DNA footprinting and DNA sequence analysis, that Diptericin activation requires a kappaB binding site (now believed to be the site of Relish binding) as well as putative NF-IL6-like, and interferon regulatory factor (IRF)-like binding sites. However, of these only Relish is required for the immune inducible expression of diptericin. The data presented here show that the JNK signaling pathway and the AP-1-like factors activated by Drosophila JNK signaling are not involved in antimicrobial peptide gene induction in phagocytes. This would be quite different from immune activation of many mammalian cytokine genes, which require the coordination of several signaling pathways and the activity of several transcription factors for full immune induction. For example, IFN-ß induction requires the activation of three independent signaling cascades and the cooperative binding of three transcription factors, NF-kappaB, c-Jun/activating transcription factor 2 (ATF-2), and the interferon regulatory factor, to the enhancer region. Together, these transcription factors form a higher order complex known as the enhanceosome. Control of the insect antimicrobial genes may not require this complex enhancer architecture (Silverman, 2003).

These studies clearly demonstrate that activation of the innate immune response in Drosophila leads to the activation of JNK and NF-kappaB signaling pathways through a branched signal transduction cascade. The MAP3K TAK1 lies at the branch point of this cascade and likely functions as the JNKK activating kinase and the IKK activating kinase. These signaling pathways are highly conserved. TAK1 also serves similar functions in mammalian innate immune signaling. Furthermore, novel immune-induced targets of the JNK pathway have been identified, that may function in cellular immunity and stress protection (Silverman, 2003).

Graded maternal Short gastrulation protein contributes to embryonic dorsal-ventral patterning by delayed induction, probably by regulating degradation of the IkB homologue Cactus

Establishment of the dorsal–ventral (DV) axis of the Drosophila embryo depends on ventral activation of the maternal Toll pathway, which creates a gradient of the NFkappaB/c-rel-related transcription factor Dorsal. Signaling through the maternal BMP pathway also alters the dorsal gradient, probably by regulating degradation of the IkB homologue Cactus. The BMP4 homologue decapentaplegic (dpp) and the BMP antagonist short gastrulation (sog) are expressed by follicle cells during mid-oogenesis, but it is unknown how they affect embryonic patterning following fertilization. This study provides evidence that maternal Sog and Dpp proteins are secreted into the perivitelline space where they remain until early embryogenesis to modulate Cactus degradation, enabling their dual function in patterning the eggshell and embryo. Metalloproteases encoded by tolloid (tld) and tolkin (tok), which cleave Sog, are expressed by follicle cells and are required to generate DV asymmetry in the Dpp signal. Expression of tld and tok is ventrally restricted by the TGF-α ligand encoded by gurken, suggesting that signaling via the EGF receptor pathway may regulate embryonic patterning through two independent mechanisms: by restricting the expression of pipe and thereby activation of Toll signaling and by spatially regulating BMP activity (Carneiro, 2006).

This study has shown that sog, dpp, and tld act during oogenesis to promote the formation of dorsal anterior structures of the eggshell and to establish the embryonic DV axis. According to a proposed model, Sog is produced in follicle cells and is processed into different forms depending on DV location and stored in the perivitelline space. These forms of Sog then persist until early stages of embryogenesis at which time they act by a delayed induction mechanism to alter signaling mediated by maternally derived Dpp. It is proposed that an asymmetric distribution of Sog peptides is produced through the action of the ventrally localized Tld and Tok metalloproteases. Different forms of Sog act locally to inhibit Dpp signaling ventrally (e.g., N-Sog) or diffuse over considerable distances to concentrate Dpp dorsally (e.g., full-length Sog or C-Sog). According to this model, a dorsal-to-ventral gradient of Dpp activity is formed in the perivitelline space that counteracts and sharpens the inverse gradient of nuclear dorsal (Carneiro, 2006).

An important finding in this study is that Sog protein produced by follicle cells is secreted into the perivitelline space where it persists until the end of oogenesis and early embryogenesis, prior to initiation of zygotic sog expression. One way maternal Sog fragments might influence DV patterning in the embryo is to modify zygotic Dpp signaling. However, maternal Dpp signaling is involved in establishing the relative positions of the ventral mesoderm versus the lateral neuroectodermal territories, while zygotic Dpp activity determines the relative positions of dorsal and lateral domains. These distinct phenotypes suggest that maternal Sog acts by modulating the maternal rather than the zygotic component of Dpp signaling (Carneiro, 2006).

This analysis also suggests that the Dpp synthesized by follicle cells is secreted into the perivitelline space and stored there until advanced stages of oogenesis. These maternally synthesized Sog and Dpp proteins may act on the embryo following fertilization when signaling through the Toll pathway is initiated. Several lines of evidence support this hypothesis. (1) Through epistatic analysis, it was shown that maternal Dpp does not act upstream of the Toll receptor. Therefore, genes expressed in the follicle cell epithelium that regulate DV patterning exclusively via the Toll pathway should not be targets of maternal Dpp signaling. Alternatively, undescribed non-Toll mediators of DV patterning could potentially be targets of maternal Dpp in the follicular epithelium. (2) Blocking Tkv receptor function or reducing maternal Dpp activity (by 8xhssog, in follicle cells has no effect on the pattern of pip expression. It has been shown that maternal dpp does not alter grk expression. Thus, no evidence was found that the embryonic effects here described in this study are due to alterations in patterning of the follicular epithelium. (3) Maternal dpp signaling increases the levels of Cactus protein in the embryo by a mechanism that is independent of Toll. Finally, inhibition of Tkv with tkvDN expressed with an early maternal driver alters the embryonic expression domains of ventral and lateral genes such as vnd and snail, which are targets of dorsal activation but not of zygotic BMP signaling. tkvDN expression also alters expression of DV genes in lateralized embryos, which lack dorsal ectoderm and early zygotic dpp expression. In aggregate, these various data support the view that maternal dpp and sog act by delayed induction on the embryo itself. The possibility cannot be ruled out, however, that the embryonic DV phenotypes described in this study result from the combined effects of direct and indirect maternal dpp signaling with the predominant effect being direct (Carneiro, 2006).

Delayed inductive activities have been proposed for a variety of proteins synthesized during oogenesis. For example, activation of the terminal system relies on delayed inductive activity of the secreted product of the torsolike gene (tsl), which is expressed by follicle cells at the two poles of the oocyte and associates with the vitelline membrane. ndl has a dual action on chorion integrity and embryonic patterning. The embryonic patterning function of ndl is thought to be mediated by Nudel protein that is secreted into the perivitelline space where it associates with the embryonic plasma membrane and initiates a proteolytic cascade. It is proposed that Sog and Dpp secreted by follicle cells also serve two roles. First, they contribute to patterning the follicle cell epithelium and chorion, and secondly, they are transferred to and stored in the perivitelline space where it is proposed that they function after fertilization to modify Toll patterning in the embryo (Carneiro, 2006).

During embryogenesis, Sog protein diffuses dorsally from the neuroectoderm and may carry Dpp dorsally in a complex with Tld, Tsg, and Scw, resulting in the generation of peak Dpp activity in the dorsal midline. The spatial distribution of maternal Sog, Dpp, Tld and Tok during oogenesis could also create asymmetric BMP activity. Since tld and tok are expressed only in ventral follicle cells, a ventral-to-dorsal gradient of Sog fragments is likely to be produced. Because cleavage of Sog by Drosophila Tld and Tok is dependent on the amount of Dpp, cleavage of Sog by Tld and Tok should be increased near the source of Dpp, generating an oblique gradient of Sog fragments in the egg chamber. The existence of such a gradient is supported by the greater staining seen in anterior ventral cells with the anti-Sog 8A antiserum during stage 10B. However, greater asymmetry may exist as a result of differential distribution of an array of Sog fragments throughout the egg chamber. Unfortunately, visualization of such asymmetry would be hard to achieve due to limitations in the ability to recognize several fragments by existing Sog antisera (Carneiro, 2006).

The analysis of marked sog− and tld− follicle cell clones suggests that the mobility of Sog fragments in the extracellular compartment may contribute to creating a maternal Dpp activity gradient. Such clones resulted in different Sog staining patterns in the perivitelline space adjacent to the clones depending on where they were located along the DV axis. The staining pattern observed with the 8A antibody suggests that ventrally generated N-Sog cleavage products may be less diffusible than intact Sog or than C-Sog and remain restricted to their site of production. In contrast, full-length Sog and C-Sog fragments appear to diffuse more readily (Carneiro, 2006).

Diffusion of Dpp may also contribute to patterning the eggshell. The expression of dpp in anterior follicle cells is consistent with its role in the formation of dorsal anterior chorionic structures. An anterior-to-posterior gradient of Dpp activity in dorsal regions of the egg chamber is suggested by the Dpp-dependent activation of the A359 enhancer trap and graded repression of bunched along the AP axis. In addition, BR-C expression is lost in mad− clones away from the source of Dpp. sog is likely to contribute to establishing this BMP gradient since ventral sog−clones act non-cell-autonomously to decrease the size of the operculum. Since ventral tld− clones also alter the extent and angle of the operculum, Tld may process Sog to generate a fragment that diffuses and carries Dpp to a dorsal anterior location, concentrating and thus enhancing Dpp activity. Further evidence that a fragment with such activity exists derives from the observation that overexpression of a C-terminal Sog fragment generates chorionic phenotypes that strongly resemble dpp overexpression (Carneiro, 2006).

A dorsally produced form of Sog also appears to participate in patterning the eggshell since sog− clones located dorsally result in fusion of dorsal appendages along the dorsal midline. DV positioning of the dorsal appendages depends on several factors, most critically on EGFR signaling. In contrast, mild overexpression of dpp generates fusion of the dorsal appendages. Considering the well-established role of Sog in modulating Dpp activity, the fused appendage phenotype generated by dorsal sog− clones most likely reflects the loss of Dpp antagonism exerted by Sog (Carneiro, 2006).

In addition to the activities described above, N-Sog fragments which remain ventrally restricted could exert Supersog-like activity, antagonizing BMPs while acquiring resistance to further cleavage and degradation by Tld. This ventrally restricted activity most likely patterns the embryo but does not affect dorsal positioning of eggshell structures, which depends on the combined activity of Dpp/BMPR signaling and dorsally generated Grk/EGFR signals (Carneiro, 2006).

The assortment of Sog fragments in egg chambers is very similar to that in the embryo. Full-length and processed forms of Sog generated by Tld during oogenesis might remain asymmetrically distributed during embryogenesis and exert distinct activities. This hypothesis is in agreement with the effect of tld− and sog− follicle cell clones on the embryo. In the majority of cases, tld− follicle cell clones result in ventralized cuticles, indicating that Tld generates some activity that synergizes with Dpp. Reciprocally, the great majority of sog− follicle cell clones result in dorsalized cuticles and embryos, indicating that Sog primarily acts by antagonizing Dpp. Since only ventral sog− clones generate cuticle defects, ventrally produced Sog presumably generates a ventralizing activity that blocks Dpp locally. In contrast, since in a minority of cases ventral shifts are observed in embryonic gene expression domains resulting from sog− clones, as well as a minority of dorsalized cuticles from tld− clones, there may also be a form of Sog that can enhance Dpp signaling. This positive BMP promoting activity could be generated ventrally, as suggested above in the case of chorion patterning (Carneiro, 2006).

A model depicting the proposed effects of different Sog forms on formation of the chorion and embryonic patterning is presented. According to this model, ventrally restricted Tld cleaves Sog near the Dpp source in ventral anterior follicle cells generating N-Sog and C-Sog. It is suggested that N-Sog fragments remain restricted near ventral anterior cells to antagonize Dpp, while C-Sog fragments diffuse dorsally concentrating Dpp in dorsal anterior cells that direct formation of the operculum. This asymmetric production of Sog molecules would generate a dorsal-to-ventral gradient of Dpp, with the highest levels dorsally near the anterior Dpp source. Although direct visualization of the predicted resulting Dpp gradient in the embryo is hard to achieve with the tools available, it is proposed that such a similarly oriented gradient persists until early embryogenesis based on the asymmetric pattern of Dpp-GFP distribution during late oogenesis and the observed alterations in embryonic gene expression domains resulting from modifications in maternal Dpp signaling (Carneiro, 2006).

The slope of the Dl nuclear gradient ultimately defines the extent of the mesoderm (Mes), neuroectoderm (NE), and dorsal ectoderm (DE). A uniform increase or decrease in nuclear Dl along the DV axis can only alter the extent of the Mes and DE and positioning of the NE, while a change in the slope of the gradient will modify the extent of NE territories such as the vnd expression domain. Under all conditions that Dpp signaling was altered, modifications were observed in the width of the vnd domain. This suggests that graded maternal Dpp signaling helps determine the slope of the dorsal gradient. Earlier studies suggested that Dpp inhibits Cactus degradation and as a consequence decreases Dl translocation into the nucleus. Increased Dpp signaling should result in more Dl retained in the cytoplasm, with consequent narrowing of the mesoderm and ventral shift in lateral and dorsal expression domains. Conversely, inhibition of Dpp signaling would result in increased levels of Dl becoming available for nuclear translocation. Considering the proposal that maternal Dpp is highest dorsally, and that Cactus may also act to prevent Dl diffusion along the DV axis, decreasing Dpp should lower Cactus levels in dorsal–lateral regions of the embryo and result in the redistribution of free Dl from ventral to lateral regions. As a consequence of this redistribution of Dl, there would be a slight decrease in Dl levels ventrally and an increase laterally that would have the net effect of flattening the gradient. Such a mechanism would require a certain degree of mobility of dorsal dimers in the syncytial blastoderm. In future studies, it will be interesting to determine the relative mobilities of Dl/Cactus complexes in the cytoplasm (Carneiro, 2006).

Maternal BMP signaling may also increase the robustness of dorsal patterning. The prevailing view of DV patterning is that signaling through the Toll pathway is sufficient to generate threshold-dependent activation of several dorsal target genes along the entire DV axis. Activation of Toll triggered by the ON/OFF pip expression pattern must be transformed into a ventrally centered gradient of Toll signaling. Several mechanisms may contribute to generate this gradient, based on autoregulatory feedback mechanisms. Although the Toll system may be internally robust, regulatory inputs from other signaling pathways could also contribute further to its stability, such as suggested for the wntD pathway and for maternal Dpp. While a significant body of evidence supports the standard view that establishment of the dorsal gradient through the Toll pathway is central to DV axis specification, the maternal Dpp pathway may constitute an important secondary mechanism that sharpens and ensures robustness and stability of the dorsal gradient in response to a rapidly changing embryonic environment (Carneiro, 2006).

The initiating event in maternal DV patterning is localized activation of the Grk/EGFR pathway in dorsal cells. Grk functions by restricting the expression of both pip and tld/tok, providing two potentially independent means for spatially regulating the activity of Toll and Dpp. This dual action of the Grk/EGFR pathway is consistent with analysis in which it was found that embryonic cuticles from gd−; grk−; Tl[3] mothers displayed a phenotype distinct from those collected from gd−; Tl[3] mothers. While cuticles from both genotypes had denticle belts surrounding the entire circumference of the embryo, cuticles from gd−; grk−; Tl[3] mothers were more elongated than those from gd−; Tl[3] mothers and exhibited a more ventral character. This suggests that grk provides an additional signal for asymmetry downstream or in parallel to gd. It is suggested that the hypothetical system proposed acts downstream of grk/EGFR and in parallel to Toll may be the Dpp pathway (Carneiro, 2006).

Role for sumoylation in systemic inflammation and immune homeostasis in Drosophila larvae

To counter systemic risk of infection by parasitic wasps, Drosophila larvae activate humoral immunity in the fat body and mount a robust cellular response resulting in encapsulation of the wasp egg. Innate immune reactions are tightly regulated and are resolved within hours. To understand the mechanisms underlying activation and resolution of the egg encapsulation response and examine if failure of the latter develops into systemic inflammatory disease, parasitic wasp-induced changes in the Drosophila larva were correlated with systemic chronic conditions in sumoylation-deficient mutants. It has been reported that loss of either Cactus, the Drosophila (IkappaB) protein, or lesswright/Ubc9, the SUMO-conjugating enzyme, leads to constitutive activation of the humoral and cellular pathways, hematopoietic overproliferation and tumorogenesis. This study reports that parasite infection simultaneously activates NF-kappaB-dependent transcription of Spätzle processing enzyme (SPE) and cactus. Endogenous Spätzle protein (the Toll ligand) is expressed in immune cells and excessive SPE or Spätzle is pro-inflammatory. Consistent with this function, loss of Spz suppresses Ubc9- defects. In contrast to the pro-inflammatory roles of SPE and Spätzle, Cactus and Ubc9 exert an anti-inflammatory effect. Ubc9 maintains steady state levels of Cactus protein. In a series of immuno-genetic experiments, the existence of a robust bidirectional interaction between blood cells and the fat body was demonstrated, and it is proposed that wasp infection activates Toll signaling in both compartments via extracellular activation of Spätzle. Within each organ, the IkappaB/Ubc9-dependent inhibitory feedback resolves immune signaling and restores homeostasis. The loss of this feedback leads to chronic inflammation. These studies not only provide an integrated framework for understanding the molecular basis of the evolutionary arms race between insect hosts and their parasites, but also offer insights into developing novel strategies for medical and agricultural pest control (Paddibhatla, 2010).

Parasitic wasps are a large group of insects that typically attack other insects. Because of the absolute dependence on their insect hosts, parasitic wasps are of enormous commercial interest and can replace insecticides to control insect pests. The motivation of this study was to gain a clearer understanding of how insect larvae respond to attacks of these natural enemies. Using an immuno-genetic approach in Drosophila, this study found that the same Toll-dependent NF-kappaB mechanism that rids Drosophila of microbial infections also defends the host against metazoan parasites. However, because of critical differences in their size and mode of entry, the combination of immune responses summoned in the two cases is different. While phagocytosis and systemic humoral responses (the latter originating from the fat body and in the gut) are the principal mechanisms of host defense against bacteria and fungi, the development of parasitic wasp eggs is blocked primarily by encapsulation response (Paddibhatla, 2010).

Data is presented that demonstrate the critical requirement of the humoral arm in both the activation and resolution of egg encapsulation. The bi-directional interaction between the blood cells and the fat body occurs via cell non-autonomous effects of SPE/Spz, where these secreted proteins synthesized in one compartment can activate immune signaling in the other. Recent reports corroborate a signaling role for Spz derived from blood cells in the expression of antimicrobial peptides from the larval fat body in response to microbes. Because activation/deactivation of both immune arms is accomplished via the IkappaB/Ubc9-dependent feedback loop that has both, cell autonomous and cell non-autonomous effects, it is proposed that this shared mechanism allows efficient coordination between the immune organs and helps restore normal immune homeostasis within the infected host (Paddibhatla, 2010).

The mechanism that coordinates the activation and resolution of both immune arms after parasite infection involves a balance between the positive (SPE) and negative (Cactus) components. Infection induces nuclear localization of Dorsal and Dif, and the transcription of both SPE (which resolves over time) and cactus (transcription levels off). This Cactus-dependent regulation is essential for the downregulation of SPE transcription and the termination of the encapsulation response. The negative feedback loop of Cactus in flies is similar to the one identified for IkappaBα in mammalian cells (Paddibhatla, 2010).

In Ubc9 mutants, the stability of Cactus protein is compromised, and Toll signaling persists during the extended larval life. Accordingly, knockdown of Cactus in blood cells (Hml>cactus^RNAi) promotes inflammation, aggregation and melanization. It is proposed that loss of immune homeostasis leads to constitutive SPE expression and activation of Spätzle, which promotes the development of chronic inflammation. Thus, sumoylation serves an anti-inflammatory function in the fly larva (Paddibhatla, 2010).

This study has identified at least two distinct biological roles of sumoylation: first, an essential role in blood cells, where the post-translational modification curbs proliferation in the lymph gland in the absence of infection. This conclusion is also strongly supported by restoration of normal hematopoietic complement in mutants expressing wild type Ubc9 only within a limited lymph gland population. Second, sumoylation is essential to sustain significant, steady state levels of Cactus. In mammalian cells, sumoylation of IkappaBα protects it from antagonistic, ubiquitination-mediated degradation. The results are consistent with the mammalian model where Cactus sumoylation would be expected to modulate its half-life (Paddibhatla, 2010).

Cytokine activation and function are hallmarks of the normal inflammatory response in mammals. A key finding of this study is that active Spz serves a pro-inflammatory function in fly larvae. This first report of any pro-inflammatory molecule in the fly confirms that cytokines activate inflammation across phyla. As with mammalian cytokines that act as immuno-stimulants, Spz is expressed, and is therefore likely to activate the blood cells surrounding the parasite capsule. Active Spz promotes blood cell division, migration and infiltration much like high levels of Dorsal and Dif, suggesting that the cell biological changes triggered by SPE/Spz are mediated by target genes of Dorsal and Dif. It is intriguing that the integrity of the basement membrane (as visualized by Collagen IV expression pattern) appears to be important for orchestrating blood cells to the site of 'diseased self' (the mutant fat body in this study) in a manner that may be similar to recognition of the non-self parasitic egg, underscoring the parallel roles of basement membrane proteins in the origin and development of inflammation in both flies and mammals (Paddibhatla, 2010).

Although excessive (active) Spz is proinflammatory, its loss leads to reduction in the hematopoietic complement. For example mutants lacking spz (spz^rm7/spz^rm7) exhibit a 40% reduction in circulating blood cell concentration and these animals do not encapsulate wasp eggs as efficiently as their heterozygous siblings. These observations suggest that active Spz's normal proliferative/pro-survival functions, required for maintaining the normal hematopoietic complement, are fundamentally linked to its immune function for the activation and recruitment of blood cells to target sites. Thus, the autocrine and paracrine hematopoietic and inflammatory effects of Spz are amplified in the presence of hyperactive Toll receptor, excessive Dorsal/Dif, or the loss of Cactus/Ubc9 inhibition, resulting in production of hematopoietic tumors. It is possible that mutations in other, unrelated, genes that yield similar inflammatory tumors arise due to the loss of Toll-NF-kappaB dependent immune homeostasis (Paddibhatla, 2010).

These results highlight the central role of the Dorsal/Dif proteins not only in immune activation, but also in the resolution of these responses. Proteomic studies have confirmed that Dorsal is a bona fide SUMO target and its transcriptional activity is affected by sumoylation. Dorsal and Dif exhibit genetic redundancy in both the humoral and cellular responses. It is possible that this redundancy ensures that immune reactions against microbes and parasites are efficiently resolved to allow proper host development (Paddibhatla, 2010).

In nature, parasitic wasps are continually evolving to evade or suppress the immune responses of their hosts. To this end, they secrete factors or produce protein complexes with specific molecular activities to block encapsulation. These studies provide the biological context in which the effects of virulence factors produced by pathogens and parasites on primordial immune pathways can be more clearly interpreted. The molecular identity of wasp factors which actively suppress humoral and cellular responses (e.g., those in L. heterotoma remains largely unknown. Such virulence factors are likely to be 'anti-inflammatory' as they clearly interfere with host physiology that ultimately disrupts the central regulatory immune circuit defined in these studies (Paddibhatla, 2010).

Encapsulation reactions of non-self (wasp egg) or diseased self tissues (fat body) of the kind in the Drosophila larva are not only reported in other insects, but the reaction is likely to be similar to mammalian granulomas, which are characterized by different forms of localized nodular inflammation. Furthermore, the phenotypes arising from persistent signaling in mutants recapitulate the key features of mammalian inflammation: i.e., reliance on conserved signaling mechanism, the requirement for cytokines, and sensitivity to aspirin. These studies also reveal a clear link between innate immunity and the development and progression of hematopoietic cancer in flies, as has been hypothesized from work in mammalian systems. In the past, genetic approaches in Drosophila have served well to dissect signaling mechanisms governing developmental processes in animals. The fly model with hallmarks of acute and chronic mammalian inflammatory responses will provide deep insights into signaling networks and feedback regulatory mechanisms in human infections and disease. It can also be used to test the potency and mechanism of action of pesticides, anti-inflammatory and anti-cancer agents in vivo (Paddibhatla, 2010).

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