DJun
CBP is a Jun coactivator
p300 and CREB-binding protein are functional homologs and global transcriptional coactivators that are involved in the regulation of various DNA-binding transcription factors. p300/CBP interacts with nuclear receptors, CREB, c-Jun, C-Myb, c-Fos, and MyoD. DNA-binding factors recruit p300/CBP by not only direct but also indirect interactions through cofactors. p300/CBP is not only a transcriptional adaptor but also a histone acetyltransferase. The p300/CBP-histone acetyltransferase domain has no obvious sequence similarity to GCN5, another protein with known histone acetyltransferase activity, or to other previously described acetyltransferases. P300 acetylates all core histones in mononucleosomes and the four lysines in the Histone H4 N-terminal tail. These observations suggest that p300/CBP is not a simple adaptor between DNA binding factors and cellular p300/CBP associated factor (PCAF) or transcription factors; rather, p300/CBP per se may contribute directly to transcriptional regulation via targeted acetylation of chromatin (Ogryzko, 1996 and references).
p300 and CBP are functional homologs and global transcriptional coactivators that are involved in the regulation of various DNA-binding transcription factors. p300/CBP interacts with nuclear receptors, CREB, c-Jun, C-Myb, c-Fos, and MyoD. DNA-binding factors recruit p300/CBP by not only direct but also indirect interactions through cofactors. p300/CBP is not only a transcriptional adaptor but also a histone acetyltransferase. The p300/CBP-histone acetyltransferase domain has no obvious sequence similarity to GCN5, another protein with known histone acetyltransferase activity, or to other previously described acetyltransferases. P300 acetylates all core histones in mononuclesomes and the four lysines in the Histone H4 N-terminal tail. These observations suggest that p300/CBP is not a simple adaptor between DNA binding factors and cellular p300/CBP associated factor (PCAF) or transcription factors; rather, p300/CBP per se may contribute directly to transcriptional regulation via targeted acetylation of chromatin (Ogryzko, 1996 and references).
Calcium is the principal second messenger in the control of gene expression by electrical activity in neurons. Recruitment of the coactivator
CREB-binding protein, CBP, by the prototypical calcium-responsive transcription factor, CREB and stimulation of CBP activity by nuclear
calcium signals is one mechanism through which calcium influx into excitable cells activates gene expression. Another
CBP-interacting transcription factor, c-Jun, can mediate transcriptional activation upon activation of L-type voltage-gated calcium channels.
Calcium-activated transcription mediated by c-Jun functions in the absence of stimulation of the c-Jun N-terminal protein kinase
(JNK/SAPK1) signaling pathway and does not require c-Jun amino acid residues Ser63 and Ser73, the two major phosphorylation sites that
regulate c-Jun activity in response to stress signals. Similar to CREB-mediated transcription, activation of c-Jun-mediated transcription by
calcium signals requires calcium/calmodulin-dependent protein kinases and is dependent on CBP function. These results identify c-Jun as a
calcium-regulated transcriptional activator and suggest that control of coactivator function (i.e. recruitment of CBP and stimulation of CBP
activity) is a general mechanism for gene regulation by calcium signals (Cruzalegui, 1999).
Growth factors such as epidermal growth factor (EGF) and insulin regulate development and metabolism via genes containing both POU homeodomain (Pit-1) and phorbol ester (AP-1) response elements. Although
CREB binding protein (CBP) functions as a coactivator on these elements, the mechanism of transactivation has been unclear. CBP is recruited to these elements only after it is phosphorylated at serine 436 by growth factor-dependent signaling pathways. In contrast, p300, a protein closely related to
CBP that lacks this phosphorylation site, binds only weakly to the transcription complex and in a growth factor-independent manner. A small region of CBP (amino acids 312-440), which has been termed the GF box, contains a potent transactivation domain and mediates this effect. Direct phosphorylation represents a novel mechanism controlling coactivator recruitment to the transcription complex (Zanger, 2001).
Previous in vitro studies have suggested that CBP and p300 interact constitutively with both c-Fos and c-Jun and mediate activation on an AP-1 element. These constitutive interactions are confirmed in this study, where both CBP and p300 activate the AP-1 element 2- to 3-fold in the absence of growth factor stimulation. CBP is found to bind to c-Jun through its CREB binding domain. Although the constitutive interactions between p300/CBP and c-Jun may be functionally significant on the AP-1 element, the growth factor-dependent recruitment of CBP to the AP-1 complex may prove to be quantitatively more important (Zanger, 2001).
Jun functions upstream of cytoskeletal rearrangements Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth
factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to
invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express
low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by
sustained expression of the dominant negative mutant of c-Jun (TAM67), suggesting a role for the
transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, it has been established that
sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the
cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits
EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding.
Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431
cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac,
whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho.
Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67
(TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways
downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67
expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated
with its activation, is defective in TA cells. These data establish a novel link between AP-1 activity and
EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required
for cell motility and invasion (Malliri, 1998).
Jun photoperiod response Light-induced phase shifts of circadian rhythmic locomotor activity are associated with the expression
of c-Jun, JunB, c-Fos and FosB transcription factors in the rat suprachiasmatic nucleus, as shown in
the present study. In order to explore the importance of c-Fos and JunB, the predominantly expressed
AP-1 proteins for the phase-shifting effects of light, the expression of c-Fos and JunB were blocked in
the suprachiasmatic nucleus of male rats, housed under constant darkness, by intracerebroventricular
application of 2 microliters of 1 mM antisense phosphorothioate oligodeoxynucleotides (ASO)
specifically directed against c-fos and junB mRNA. A light pulse (300 lux for 1 h) at circadian time 15
induces a significant phase shift (by 125 +/- 15 min) of the circadian locomotor activity rhythm,
whereas application of ASO 6 h before the light pulse completely prevents this phase shift.
Application of control nonsense oligodeoxynucleotides has no effect. ASO strongly reduces the
light-induced expression of c-Fos and JunB proteins. In contrast, light pulses with or without the control
nonsense oligodeoxynucleotides evokes strong nuclear c-Fos and JunB immunoreactivity in the rat
suprachiasmatic nucleus. These results demonstrate for the first time that inducible transcription
factors such as c-Fos and JunB are an essential part of fundamental biological processes in the adult
mammalian nervous system, e.g. of light-induced phase shifts of the circadian pacemaker (Wollnik, 1995).
Jun, apoptosis and survival The c-Jun NH2-terminal kinase (JNK) can cause cell death by activating the mitochondrial apoptosis pathway. However, JNK is also capable of signaling cell survival. The mechanism that accounts for the dual role of JNK in apoptosis and survival signaling has not been established. JNK-stimulated survival signaling can be mediated by JunD. The JNK/JunD pathway can collaborate with NF-kappaB to increase antiapoptotic gene expression, including cIAP-2. This observation accounts for the ability of JNK to cause either survival or apoptosis in different cellular contexts. Furthermore, these data illustrate the general principal that signal transduction pathway integration is critical for the ability of cells to mount an appropriate biological response to a specific challenge (Lamb, 2003).
The survival signaling role of JNK in the response to TNF contrasts with the effects of JNK to mediate apoptosis in response to the exposure of cells to environmental stress. How can one signal transduction pathway mediate two very different responses? There are two general mechanisms that could account for these different roles of JNK in apoptosis signaling. These mechanisms are not mutually exclusive. One mechanism is represented by the time course of JNK activation. Studies of MAP kinases indicate that the time course of activation can determine the cellular response. This may also apply to the response of cells to JNK activation. Sustained JNK activation is required for apoptotic signaling and is sufficient for apoptosis. In contrast, TNF causes transient JNK activation. These considerations indicate that transient JNK activation may be important for mediating a survival response in TNF-treated cells and that chronic JNK activation may contribute to apoptotic responses. A second mechanism that may account for the different roles of JNK in apoptosis signaling is that the biological consequence of JNK function may depend upon the activation state of other signal transduction pathways. For example, increased AKT activation can suppress the apoptotic effects of activated JNK. A plausible hypothesis is that the JNK signaling pathway may cooperate with other signaling pathways to mediate cell survival (e.g., NF-kappaB and AKT). For example, target genes that are induced by the antiapoptotic NF-kappaB pathway may contain JNK-responsive elements in their promoters (e.g., AP-1 sites). The cIAP-2 gene represents an example of this class of gene. JNK increases the expression of such genes in cells with activated NF-kappaB and thus increases cell survival. In contrast, in the absence of a survival pathway that can cooperate with JNK, sustained JNK activation may lead to apoptosis (Lamb, 2003 and references therein).
This analysis demonstrates that JNK mediates a survival response in cells treated with TNF. The role of JNK is mediated by the transcription factor JunD, which can collaborate with NF-kappaB to increase the expression of prosurvival genes, including cIAP-2. In the absence of activated NF-kappaB, the JNK pathway mediates an apoptotic response. Together, these data provide a mechanism that can account for the dual ability of JNK to cause either survival or apoptosis in different cellular contexts (Lamb, 2003).
Cerebellar granule neurons die by apoptosis when deprived of survival signals. This death can be
blocked by inhibitors of transcription or protein synthesis, suggesting that new gene expression is
required. c-Jun mRNA and protein levels increase rapidly after survival signal
withdrawal and transfection of the neurons with an expression vector for a c-Jun dominant
negative mutant protects the neurons against apoptosis. Phosphorylation of serines 63 and 73 in the c-Jun
transactivation domain is known to increase c-Jun activity. By using an antibody specific for c-Jun
phosphorylated on serine 63, it has been shown that this site is phosphorylated soon after survival signal
withdrawal. To determine whether c-Jun phosphorylation is necessary for apoptosis, c-Jun phosphorylation site mutants were expressed in granule neurons. c-Junasp, a constitutively active c-Jun
mutant in which the known and potential serine and threonine phosphoacceptor sites in the
transactivation domain have been mutated to aspartic acid, induces apoptosis under all conditions
tested. In contrast, c-Junala, which cannot be phosphorylated because the same sites have been
mutated to alanine, blocks apoptosis caused by survival signal withdrawal. Cerebellar granule neurons contain high levels of Jun kinase activity and low levels of p38 kinase
activity, neither of which increases after survival signal withdrawal. Mitogen-activated protein kinase
activity decreases under the same conditions. These results suggest that c-Jun levels and c-Jun
phosphorylation may be regulated by novel mechanisms in cerebellar granule neurons (Watson, 1998).
c-Jun is a component of the transcription factor AP-1, which is activated by a wide variety of extracellular stimuli. The
regulation of c-Jun is complex and involves both increases in the levels of c-Jun protein as well as phosphorylation of
specific serines (63 and 73) by Jun N-terminal kinase (JNK). Fibroblasts derived from c-Jun null embryos were used
to define the role of c-Jun in two separate processes: cell growth and apoptosis. In fibroblasts, c-Jun is
required for progression through the G1 phase of the cell cycle; c-Jun-mediated G1 progression occurs by a mechanism
that involves direct transcriptional control of the cyclin D1 gene, establishing a molecular link between growth factor
signaling and cell cycle regulators. In addition, c-Jun protects cells from UV-induced cell death and cooperates with
NF-kappaB to prevent apoptosis induced by tumor necrosis factor alpha (TNFalpha). c-Jun mediated G1 progression is
independent of phosphorylation of serines 63/73; however, protection from apoptosis in response to UV, a potent
inducer of JNK/SAP kinase activity, requires serines 63/73. The results reveal critical roles for c-Jun in two different
cellular processes and show that different extracellular stimuli can target c-Jun by distinct biochemical mechanisms (Wisdom, 1999).
Although a number of studies have implicated c-Jun in neuronal death and axonal regeneration, it is unknown whether Jun function is essential for either response. One approach to resolve this issue is to analyze knock-out mice. However, c-jun-null mice die at midgestation, precluding critical investigation. Therefore, a xenograft paradigm was used in which retinas from embryonic day 12.5 (E12.5) c-jun nullizygous or wild-type mice were transplanted onto the superior colliculus of newborn rats. The rats were allowed to develop, and the grafts were assayed at various times for cell death and axon growth. Histologically, grafts of both genotypes develop in identical manners and have morphological characteristics of retinas. A functional c-jun allele is not essential for axogenesis, because ganglion cells in retinal grafts from c-jun nullizygous mice developed axons that projected into the colliculus. Programmed cell death (PCD) is also evident in the age-appropriate regions of the retina in both wild-type and c-jun-null grafts. Furthermore, there are no discernible differences in the number or location of dying cells in the two genotypes. That c-jun was not essential for PCD is supported by two additional findings: (1) a c-jun-lacZ reporter gene is expressed in many cells in developing and grafted retinas, although only a few of these cells are destined to die; (2) in E12.5 c-jun-null embryos there are normal levels of PCD in the trigeminal ganglion. Together, these data indicate that c-Jun is not essential for axon growth in the retina or for PCD in the retina and trigeminal ganglion (Herzog, 1999).
JunD is the most broadly expressed member of the Jun family and the AP-1 transcription factor complex. Primary fibroblasts lacking JunD display
p53-dependent growth arrest, upregulate p19Arf expression, and premature senescence. In contrast, immortalized cell lines lacking JunD show increased
proliferation and higher cyclinD1 levels. These properties are reminiscent of the effects of oncogenic Ras expression on primary and established cell cultures.
Furthermore, JunD-/- fibroblasts exhibit increased p53-dependent apoptosis upon ultraviolet irradiation and are sensitive to the cytotoxic effects of TNF-alpha. The antiapoptotic role of JunD has been confirmed using an in vivo model of TNF-mediated hepatitis. It is proposed that JunD protects cells from
senescence, or apoptotic responses to stress stimuli, by acting as a modulator of the signaling pathways that link Ras to p53 (Weitzman, 2000).
The mammalian UV response results in rapid and dramatic induction of c-jun. Induction of a protooncogene, normally involved in mitogenic responses, by a genotoxic agent that causes growth arrest seems paradoxical. An explanation for the role of c-Jun in the UV response of mouse fibroblasts is provided by this study. c-Jun is necessary for cell-cycle reentry of UV-irradiated cells, but does not participate in the response to ionizing radiation. Cells lacking c-Jun undergo prolonged cell-cycle arrest, but resist apoptosis, whereas cells that express c-Jun constitutively do not arrest and undergo apoptosis. This function of c-Jun is exerted through negative regulation of p53 association with the p21 promoter. Cells lacking c-Jun exhibit prolonged p21 induction, whereas constitutive c-Jun inhibits UV-mediated p21 induction (Shaulian, 2000).
Decreased Fas expression during tumor progression often results in a loss of Fas-ligand (FasL)-mediated apoptosis. Human and mouse melanoma exhibit an inverse correlation between the degree of Fas cell surface expression, tumorigenicity, and metastatic capacity. The expression of dominant negative Stat3 or c-Jun in
melanoma cells efficiently increases Fas expression and sensitizes cells to FasL-induced apoptosis. Stat3+/- as well as c-Jun-/- cells exhibit increased Fas cell surface expression and higher sensitivity to FasL-mediated apoptosis. Suppression of Fas expression by Stat3 and c-Jun is uncoupled from Stat3-mediated transcriptional activation. These findings indicate that Stat3 oncogenic activities could also be mediated through its cooperation with c-Jun, resulting in downregulation of Fas surface expression, which is implicated in the tumor's ability to resist therapy and metastasize (Ivanov, 2001).
Sympathetic neurons require nerve growth factor for survival and die by apoptosis in its absence. Key steps in the death pathway include c-Jun activation, mitochondrial cytochrome c release, and caspase activation. Neurons rescued from NGF withdrawal-induced apoptosis by expression of dominant-negative
c-Jun do not release cytochrome c from their mitochondria. Furthermore, mRNA for BIMEL, a proapoptotic BCL-2 family member, increases in level after NGF withdrawal and this is reduced by dominant-negative c-Jun. Overexpression of BIMEL in neurons induces cytochrome c redistribution and apoptosis in the presence of NGF, and neurons injected with Bim antisense oligonucleotides or isolated from Bim-/- knockout mice die more slowly after NGF withdrawal (Whitfield, 2001).
The transcription factor c-Jun mediates several cellular processes, including proliferation and survival, and is upregulated in many carcinomas. Liver-specific inactivation of c-Jun at different stages of tumor development was used to study its role in chemically induced hepatocellular carcinomas (HCCs) in mice. The requirement for c-jun is restricted to early stages of tumor development, and the number and size of hepatic tumors is dramatically reduced when c-jun is inactivated after the tumor has initiated. The impaired tumor development correlates with increased levels of p53 and BH3-only protein Noxa, which is a known target gene of p53; this response results in the induction of apoptosis without affecting cell proliferation. Primary hepatocytes lacking c-Jun shows increased sensitivity to TNF-alpha-induced apoptosis -- this sensitivity is abrogated in the absence of p53. These data indicate that c-Jun prevents apoptosis by antagonizing p53 activity, illustrating a mechanism that might contribute to the early stages of human HCC development (Efer, 2003).
Cardiac hypertrophic stimuli induce both adaptive and maladaptive growth
response pathways in the heart. Mice lacking junD develop
less adaptive hypertrophy in the heart after mechanical pressure overload, while
cardiomyocyte-specific expression of junD in mice results in spontaneous
ventricular dilation and decreased contractility. In contrast, fra-1
conditional knock-out mice have a normal hypertrophic response, whereas hearts
from fra-1 transgenic mice decompensate prematurely. Moreover,
fra-1 transgenic mice simultaneously lacking junD reveal a
spontaneous dilated cardiomyopathy associated with increased cardiomyocyte
apoptosis and a primary mitochondrial defect. These data suggest that
junD promotes both adaptive-protective and maladaptive hypertrophy in
heart, depending on its expression levels (Ricci, 2005).
Apoptosis is an important mechanism for sculpting morphology. However, the molecular cascades that control apoptosis in developing limb buds remain largely unclear. This study showed that bZip factor MafB was specifically expressed in apoptotic regions of chick limb buds, and MafB/cFos heterodimers repressed apoptosis, whereas MafB/cJun heterodimers promoted apoptosis for sculpting the shape of the limbs. Chromatin immunoprecipitation sequencing in chick limb buds identified potential target genes and regulatory elements controlled by Maf and Jun. Functional analyses revealed that expression of p63 and p73, key components known to arrest the cell cycle, was directly activated by MafB and cJun. The data suggest that dimeric combinations of MafB, cFos and cJun in developing chick limb buds control the number of apoptotic cells, and that MafB/cJun heterodimers lead to apoptosis via activation of p63 and p73 (Suda, 2014).
Developmental roles of Jun JunD is one of three mammalian Jun proteins that
contribute to the AP-1 transcription factor complex.
Distinct regulation and functions have been proposed for
each Jun member, but less is known about the biological
functions of each of these proteins in vivo. To investigate
the role of JunD, the murine gene was inactivated by
replacement with a bacterial lacZ reporter gene.
Embryonic JunD expression is initially detected in the
developing heart and cardiovascular system. Subsequent
broadening phases of JunD expression are observed
during embryonic development and expression in the adult
is widespread in many tissues and cell lineages. Mutant
animals lack JunD mRNA and protein and show no
evidence of upregulation of c-Jun and JunB mRNA levels.
In contrast to the other two Jun members, homozygous
JunD minus mutant animals are viable and appear healthy.
However, homozygous JunD minus animals show a reduced
postnatal growth. Furthermore, JunD minus males exhibit
multiple age-dependent defects in reproduction, hormone
imbalance and impaired spermatogenesis with
abnormalities in head and flagellum sperm structures. No
defects in fertility are observed in JunD minus female
animals. These results provide evidence of redundant
functions for members of the Jun family during
development and specific functions for JunD in male
reproductive function (Thépot, 2000).
Interactions between mesenchymal and epithelial cells are responsible for organogenesis and tissue homeostasis. This mutual cross-talk involves cell surface proteins
and soluble factors, which are mostly the result of regulated transcription. To elucidate dimer-specific functions of the AP-1 family of transcription factors, skin was reconstituted by combining primary human keratinocytes and mouse wild-type, c-jun-/-, and junB-/- fibroblasts. An antagonistic function of
these AP-1 subunits in the fibroblast-mediated paracrine control of keratinocyte proliferation and differentiation was discovered, and this effect was traced to the IL-1-dependent
regulation of KGF and GM-CSF. These data suggest that the relative activation state of these AP-1 subunits in a non-cell-autonomous, transregulatory fashion
directs regeneration of the epidermis and maintenance of tissue homeostasis in skin (Szabowski, 2000).
Two different lines of evidence have already suggested a critical role of AP-1 subunits in cytokine-dependent regulation of gene expression. (1) IL-1 and TNFalpha upregulate gene expression of jun, fos, and AP-1 target genes such as matrix metalloproteinases and Il-2. (2) IL-1 stimulates AP-1 and NFkappaB through activation of the MAP kinases JNK and p38 and the IkappaB kinase (IKK), respectively. c-Jun and ATF-2, which are responsible for transcriptional activation of c-jun expression in response to cytokines and genotoxic agents, are efficiently phosphorylated by JNK and p38, respectively. Interestingly, AP-1 sites have been found in the promoter region of the KGF and GM-CSF genes from mouse and human, suggesting that Jun proteins directly regulate expression by binding to the KGF and GM-CSF promoter. In vitro binding studies have confirmed direct binding of c-Jun and JunB to the AP-1 sites in the GM-CSF promoter. The heterodimeric partner of c-Jun involved in KGF and GM-CSF regulation is presently unknown. Since keratinocyte growth and differentiation was not disturbed in cocultures containing c-Fos-deficient fibroblasts, it is unlikely that the regulatory function of c-Jun is mediated through heterodimerization with c-Fos (Szabowski, 2000 and references therein).
In fibroblasts lacking JunB, basal level expression of both KGF and GM-CSF is enhanced and cocultivation with keratinocytes or treatment with IL-1 superinduces expression of both genes. JunB obviously acts as a negative regulator of KGF and GM-CSF. The existence of AP-1 target genes that are antagonistically regulated by c-Jun and JunB has been postulated. Using genetically defined loss-of-function mutant cells, KGF and GM-CSF have been identified as endogenous target genes, whose expression depends on the antagonistic regulatory activity of c-Jun and JunB. The mechanism of repression of KGF and GM-CSF by JunB is not clear, but might be explained by the formation of transcriptionally inactive c-Jun/JunB heterodimers. Alternatively, JunB may exhibit its repressor function through binding to a c-Jun-independent cis-acting element, or indirectly through protein/protein interaction with other transcription factors acting on the KGF and GM-CSF promoters (Szabowski, 2000 and references therein).
The data suggest a critical role of the ratio of cytokines with mitogenic potential (KGF), and those exhibiting both proliferation and differentiation inducing (GM-CSF) activity to allow normal epidermal tissue reconstitution. In skin, homeostasis of the epidermis relies on a tightly regulated balance between proliferation and differentiation, where a continuous proliferation of keratinocytes in the stratum basale is a prerequisite to replace the shedded material of the stratum corneum. During wound healing, this balance has to be temporally altered to stimulate keratinocyte proliferation to achieve regeneration of the epidermis. Inappropriate adjustment will result in the development of a hyperplastic skin phenotype, as observed in KGF and GM-CSF transgenic mice. An aberrant adjustment of the equilibrium between cytokines may also be involved in pathological diseases in human, such as lichen planus, psoriasis, or dermatofibrosis. Histologically cutaneous lichen planus is characterized by epidermal hypergranulosis (a pronounced stratum granulosum with high amounts of keratohyalin granules), dermal fibrosis, and a dense infiltrate. However, psoriatic epidermis exhibits hyperproliferation and absence of granular cells. There is evidence that the epidermal alterations in psoriasis and dermatofibroma are caused by changes in the underlying dermal fibroblasts. In vitro, normal keratinocytes develop psoriatic-skin-like epidermal phenotypes when growing on fibroblasts from psoriatic lesions. In dermatofibromas, a significant hyperplasia of the epidermis can be observed overlying the benign fibroblastic tumor nodule, indicating interplay between fibroblasts and keratinocytes. This is explained by fibrotic cells stimulating the epidermis in a fashion similar to that of embryonic mesenchyme (Szabowski, 2000 and references therein).
The in vitro skin model described herein, using genetically defined mutant fibroblasts, will be a powerful system to study functional complementation of deficiencies in order to identify critical cytokines, such as KGF and GM-CSF, and to define their specific activity to promote keratinocyte proliferation, differentiation, or both. The data on the role of AP-1 in skin biology and the identification of AP-1 regulated cytokines may serve as the basis for the dissection of disease mechanisms and will shed new light on the molecular mechanism of dermal-epidermal interactions in skin physiology and pathology (Szabowski, 2000).
Mice lacking the AP-1 transcription factor c-jun die at mid-gestation showing heart defects and impaired hepatogenesis. To inactivate c-jun in hepatocytes, mice carrying a floxed c-jun allele were generated. Perinatal liver-specific c-jun deletion causes reduced hepatocyte proliferation and decreased body size. After partial hepatectomy, half of the mutants died and liver regeneration was impaired. This function of c-jun is independent of Jun N-terminal phosphorylation (thought to increase transcription by stimulating the recruitment of the coactivator proteins CBP and p300 to target gene promoters), since liver regeneration was normal in mice harboring a mutant c-jun allele lacking the JNK phosphoacceptor serines 63 and 73. The failure of jun mutants to regenerate is accompanied by increased cell death and lipid accumulation in hepatocytes. Moreover, cyclin-dependent kinases and several cell cycle regulators are affected, resulting in inefficient G1- S phase progression.
These studies identify c-Jun as a critical regulator of hepatocyte proliferation and survival during liver development and regeneration (Behrens, 2002).
Jun is a major component of the heterodimeric transcription factor AP-1 and is essential for embryonic development, since fetuses that lack Jun die at mid-gestation. Ubiquitous mosaic inactivation of a conditional Jun allele by cre/LoxP-mediated recombination was used to screen for novel functions of Jun and revealed that its absence results in severe malformations of the axial skeleton. More specific Jun deletion by collagen2a1-cre demonstrated the essential function of Jun in the notochord and sclerotome. Mutant notochordal cells show increased apoptosis, resulting in hypocellularity of the intervertebral discs. Subsequently, fusion of vertebral bodies causes a scoliosis of the axial skeleton. Thus, Jun is required for axial skeletogenesis by regulating notochord survival and intervertebral disc formation (Behrens, 2003).
To investigate the function of c-Jun during skin development and skin tumor formation, c-jun was conditionally inactivated in the epidermis. Mice lacking c-jun in keratinocytes develop normal skin but express reduced levels of EGFR in the eyelids, leading to open eyes at birth, as observed in EGFR null mice. Primary keratinocytes from c-jun deficient mice proliferate poorly, show increased differentiation, and form prominent cortical actin bundles, most likely because of decreased expression of EGFR and its ligand HB-EGF. In the absence of c-Jun, tumor-prone K5-SOS-F transgenic mice develop smaller papillomas, with reduced expression of EGFR in basal keratinocytes. Thus, using three experimental systems, it has been shown that EGFR and HB-EGF are regulated by c-Jun, which controls eyelid development, keratinocyte proliferation, and skin tumor formation (Zenz, 2003).
Hindbrain development is a well-characterized segmentation process in vertebrates. The bZip transcription factor MafB/kreisler is specifically expressed in rhombomeres (r) 5 and 6 of the developing vertebrate hindbrain and is required for proper caudal hindbrain segmentation. Evidence is provided that the mouse protooncogene c-jun, which encodes a member of the bZip family, is coexpressed with MafB in prospective r5 and r6. Analysis of mouse mutants suggests that c-jun expression in these territories is dependent on MafB but independent of the zinc-finger transcription factor Krox20, another essential determinant of r5 development. Loss- and gain-of-function studies, performed in mouse and chick embryos, respectively, demonstrate that c-Jun participates, together with MafB and Krox20, in the transcriptional activation of the Hoxb3 gene in r5. The action of c-Jun is likely to be direct, since c-Jun homodimers and c-Jun/MafB heterodimers can bind to essential regulatory elements within the transcriptional enhancer responsible for Hoxb3 expression in r5. These data indicate that c-Jun acts both as a downstream effector and a cofactor of MafB and belongs to the complex network of factors governing hindbrain patterning (Mechta-Grigoriou, 2003).
Thus, c-Jun is involved in the early phase of Hoxb3 expression in r5 and it acts through the previously identified Hoxb3 r5 enhancer. This enhancer has been shown to carry both MafB and Krox20 binding sites that are essential for its activity. Furthermore, MafB and Krox20 have been shown to synergistically cooperate in regulating enhancer activity and Hoxb3 expression. The domain of Hoxb3 expression in the hindbrain reflects these requirements, since it corresponds precisely to the intersection of the territories where MafB (r5 and r6) and Krox20 (r3 and r5) are expressed. These data add further complexity to the regulation of Hoxb3, since it is shown that the expression of Hoxb3 in r5 can be divided in two phases -- an early phase (around 5 s stage) that is dependent on c-Jun, and a late phase (around 10-12 s stage) that is c-Jun-independent. Other AP-1 members, such as JunB and JunD, are not expressed in the hindbrain during these stages, suggesting that they are not involved in the late upregulation of Hoxb3 in c-jun mutant embryos. Moreover, it has been shown that these AP-1 members are not upregulated in the c-jun knock-out. Electroporation studies in chick embryos confirm and extend the mouse data. They demonstrate that c-Jun can cooperate with Krox20 in the activation of the Hoxb3 r5-enhancer (Mechta-Grigoriou, 2003).
It is unclear why c-Jun is required for the early phase of Hoxb3 expression, while c-jun is itself under the control of MafB. However, the following points are noteworthy: (1) although one of the two MafB binding sites (the Kr2 site) in the Hoxb3 r5 enhancer is absolutely required for activity, it interacts relatively poorly with MafB homodimers; (2) during the early phase of Hoxb3 expression in r5, the level of MafB in this rhombomere is likely to be much lower than at later stages (judging from the levels of mRNA). (3) MafB can form heterodimers with c-Jun, as shown by coimmunoprecipitations and bandshift experiments. On the basis of these observations, the following model is proposed for Hoxb3 regulation in r5: at the onset of Hoxb3 expression, the level of MafB in r5 is sufficient to allow transcriptional activation of c-jun, but not to sustain Hoxb3 activation. In contrast, c-Jun itself has sufficient affinity to bind efficiently to the Kr2 site of the Hoxb3 r5-enhancer, either as a homodimer or heterodimer with MafB, and to activate Hoxb3 expression in r5 synergistically with Krox20. At later stages, the level of MafB in r5 increases dramatically and can replace c-Jun, either as a homodimer or a heterodimer with other bZip proteins. At this stage, c-Jun is no longer required for expression of Hoxb3 in r5 (Mechta-Grigoriou, 2003).
In conclusion, c-Jun appears to accelerate the activation of Hoxb3 by MafB, allowing transcriptional activation when MafB alone is insufficient. In addition, the identification of c-Jun as a novel upstream regulator of Hoxb3 supports an important role for MafB partners in hindbrain patterning. The complexity of interactions involved in this latter process may offer additional levels of regulation for fine tuning of gene expression (Mechta-Grigoriou, 2003).
The delay in Hoxb3 activation in r5 is the only phenotype so far identified associated with c-jun inactivation at early developmental stages. The normal expression of r5 markers, such as Hoxa3, MafB, Krox20, and EphA4, indicates that the patterning of r5 and r6 is not dramatically affected and that the restored expression of Hoxb3 in r5 at later stages does not result from a delay in maturation of this rhombomere. Furthermore, the lack of modification in the Phox2B and neurofilament expression patterns suggests that specification of regional identity and neurogenesis in r5 and r6 are not dramatically perturbed. Hoxb3-/- mice survive until adulthood, but show minor defects in the cervical vertebrae. The early lethality of the c-jun mutants at 13.5 dpc prevents determination of whether the delayed expression of Hoxb3 leads to similar defects (Mechta-Grigoriou, 2003).
Outside of the CNS, several of the early sites of c-jun expression are correlated with phenotypic consequences. The c-jun gene is expressed in the foregut and the septum transversum. This latter structure is critical for liver formation since it affects the outgrowth of hepatic ducts and also influences the vascular organization of the foregut/midgut junction. The impaired hepatogenesis observed in c-jun-/- mutants might reflect a premature deficiency in the foregut and/or result from an early defect in the septum transversum. c-jun expression is also observed in the future sclerotomal compartment of the recently formed somites. Consistently, conditional inactivation of c-jun in the sclerotome and notochord has been shown to perturb the development of intervertebral bodies. The demonstration of a causal link between these different sites of early c-jun expression and the observed phenotypes deserves further investigation (Mechta-Grigoriou, 2003).
The AP-1 transcription factor JunB is a transcriptional regulator of myelopoiesis. Inactivation of JunB in postnatal mice results in a myeloproliferative disorder (MPD) resembling early human chronic myelogenous leukemia (CML). JunB regulates the numbers of hematopoietic stem cells (HSC). JunB overexpression decreases the frequency of long-term HSC (LT-HSC), while JunB inactivation specifically expands the numbers of LT-HSC and granulocyte/macrophage progenitors (GMP) resulting in chronic MPD. Further, junB inactivation must take place in LT-HSC, and not at later stages of myelopoiesis, to induce MPD and that only junB-deficient LT-HSC are capable of transplanting the MPD to recipient mice. These results demonstrate a stem cell-specific role for JunB in normal and leukemic hematopoiesis and provide experimental evidence that leukemic stem cells (LSC) can reside at the LT-HSC stage of development in a mouse model of MPD (Passegue, 2004).
The iterative formation of nephrons during embryonic development relies on continual replenishment of progenitor cells throughout nephrogenesis. Defining molecular mechanisms that maintain and regulate this progenitor pool is essential to understanding nephrogenesis in developmental and regenerative contexts. Maintenance of nephron progenitors is absolutely dependent on BMP7 signaling, and Bmp7-null mice exhibit rapid loss of progenitors. However, the signal transduction machinery operating downstream of BMP7 as well as the precise target cell remain undefined. Using a novel primary progenitor isolation system, signal transduction and biological outcomes elicited by BMP7 were investigated. It was found that BMP7 directly and rapidly activates JNK signaling in nephron progenitors resulting in phosphorylation of Jun and ATF2 transcription factors. This signaling results in the accumulation of cyclin D3 and subsequent proliferation of PAX2(+) progenitors, inversely correlating with the loss of nephron progenitors seen in the Bmp7-null kidney. Activation of Jun and ATF2 is severely diminished in Bmp7-null kidneys, providing an important in vivo correlate. BMP7 thus promotes proliferation directly in nephron progenitors by activating the JNK signaling circuitry (Blank, 2009).
Using a BMP-reporter mouse, it has been shown that the nephrogenic zone (NZ) is essentially unresponsive to SMAD-mediated transcription in vivo. Owing to the strong effect of Bmp7 inactivation on nephron progenitor maintenance, this surprising finding prompted an investigation of whether BMP7 acts through a SMAD-independent signaling mechanism in the NZ. The most extensively described alternative pathway downstream of TGFβ and BMP is activation of MAPK signaling through TGFβ-activated kinase 1 (TAK1). Activation of p38 and JNK pathways have both been reported downstream of TAK1, and immortalized Tak1 mutant embryonic fibroblasts exhibited impaired
phosphorylation of JNK. Mice deficient in Tak1 die at mid-gestation, precluding analysis of TAK1-mediated signaling in metanephric kidney development. However, TAK1 is expressed in the NZ of the developing kidney, suggesting that activation of MAPK signaling downstream of BMPs might occur in this region of the kidney. Previous work has indicate that BMP7 can activate p38 signaling in collecting duct cells in vitro. Similarly, conditional deletion of Bmp7 using a podocyte-specific Cre, resulted in defective p38 signaling, but no effect on phosphorylation of SMAD1/SMAD5/SMAD8 was observed. Using primary cell purification system, this study found no evidence that BMP7 activates p38 in the NZ. Instead, several JNK isoforms were rapidly phosphorylated in response to BMP7, resulting in downstream phosphorylation of both Jun and ATF2. Importantly, it was shown that TAK1 is required for efficient activation of JNK in response to BMP7. SIX2 immunostaining shows that JNK activation takes place directly in the nephron progenitor compartment, indicating that these cells indeed do respond directly to BMP7. Interestingly, phosphorylated forms of the JNK-activated transcription factors Jun and ATF2 are both localized to cap mesenchyme in the developing E17.5 kidney. Furthermore, E12.5 Bmp7-null kidneys displayed sharply reduced JNK-Jun-ATF2 signaling in metanephric mesenchyme compared with the wild type, demonstrating that Bmp7 is required for activation of this signaling axis in nephron progenitors in vivo. Interestingly, phosphorylation of Jun or ATF2 does not appear to be significantly reduced in collecting duct cells of Bmp7-deficient embryos, suggesting that other growth factors function to activate the pathway in this cellular compartment (Blank, 2009).
The migration of epithelial layers requires specific and coordinated organization of the cells at the leading edge of the sheet. Mice that are conditionally deleted for the c-jun protooncogene in epidermis are born at expected frequencies, but with open eyes and with defects in epidermal wound healing. Keratinocytes lacking c-Jun are unable to migrate or elongate properly in culture at the border of scratch assays. Histological analyses in vitro and in vivo demonstrate an inability to activate EGF receptor at the leading edge of wounds; it is possible to rescue this phenotype by supplementation with conditioned medium or the EGF receptor ligand HB-EGF. Lack of c-Jun prevents EGF-induced expression of HB-EGF, indicating that c-jun controls formation of the epidermal leading edge through its control of an EGF receptor autocrine loop (Li, 2003).
In mice, eyelid fusion begins at E13.5 and requires migration of periderm-derived epithelium to cover the surface of cornea. As development progresses, the leading edge epithelial cells of the eyelid start changing morphology and form protruding tips. Fusion of the elongated epithelium is then followed by dermis extension. Mutation of a number of genes have been shown to cause the open-eye phenotype in mice, including gene disruption of EGFr, fibroblast growth factor receptor 2 (Fgfr2), and MEK kinase 1 (MEKK1). Upregulation of TGFalpha transcription is observed in the protruding eyelid tip and has been suggested as a controlling element in the migration of elongated epithelium. MEKK1 expression is also localized to the protruding eyelid tip and MEKK1 can regulate JNK signaling in response to EGFr stimulation. In combination with observations that eyelid protruding epithelium have increased c-Jun expression and EGFr activation, these results strongly suggest that an EGFr-MEKK1-c-Jun cascade is critical for eyelid development (Li, 2003).
Skin development is normal in both strains of mutant mice described in this study, which suggests functional redundancy of other members of the Jun family in skin formation. However, the process of epidermal differentiation, which initiates with cell proliferation at the basal layer and a gradual migration upward toward differentiation into suprabasal layers, is fairly slow. Hence, it may be that c-Jun is more important in conditions where the proliferation and motility of epithelium are rapidly altered, such as eyelid fusion and wound healing. Consistent with this notion, replacing c-Jun with JunB can rescue the lethality of c-Jun null fetuses, but still gives the open eye phenotype described in this study; this may be evidence of the requirement specifically for c-Jun in this process (Li, 2003).
Reepithelialization is an important stage of wound healing, in which epidermal integrity is restored following cutaneous injury. During this process, keratinocytes at the wound margin undergo morphology changes prior to detachment from the basement membrane and formation of a migratory leading front. Guided by chemotactic factors produced in the provisional extracellular matrix of the wound, migrating keratinocytes invade into fibrin clot by producing matrix metalloproteases and plasminogen activator. All of these events are regulated by altered effector gene expression, which is mediated in turn by transcription factors (Li, 2003).
There is growing support for the role of AP-1 as a wound-induced transcription factor. AP-1 activation can be induced by growth factors and cytokines produced by damaged cells. Elevation of c-Fos, another AP-1 factor, is observed in both fetal and adult rat skin during wounding. In corneal epithelium, the rapid induction of c-Fos, fosB, c-Jun, and JunB was detected shortly post-wounding, with their peaks between 30 min to 1 hr (Li, 2003).
Reconstitution of the epidermis during wound healing is associated with reprogramming of keratin expression patterns, so as to change cell destiny from differentiation to migration. AP-1 binding sites have been identified in the promoter region of several of the integrin and keratin genes. The finding that the keratinocyte activation marker K6 is reduced in c-Jun null mutant mice provides in vivo evidence that AP-1 is functionally involved in keratinocyte activation. EGF is one of the major components in regulated epithelial cell proliferation and migration, and activation of EGF receptor is observed at the neoepidermal leading edge; defects in forming a proper keratinocyte migration leading edge and suppression of EGFr activation in conditional knockout K14cre:c-jun+f/+f keratinocytes strongly suggest that c-Jun is involved in EGFr activation in the leading edge cells. Consistent with this notion, a study of wound healing in Drosophila reveals that JNK signaling is required to initiate cell shape change at the leading edge of the wound (Li, 2003).
This study shows that epidermal deletion of c-Jun can affect the activation of EGF receptor signaling, which in turn is essential for epidermal cell migration in eyelid development and wound healing. Based on studies in vitro, a model of c-Jun function in mammalian LE formation is proposed that parallels c-Jun function seen in Drosophila dorsal closure. It should be noted that in this model, the JNK signaling cascade is not directly involved in cytoskeleton rearrangement and cell motility in leading edge keratinocytes. Instead, the production of HB-EGF through c-Jun activation provides a positive feedback loop for maintaining activation of EGFr signaling at the leading edge, which in turn controls cell shape changes. Several lines of evidence support this model: (1) in Drosophila, defective AP-1 signaling does not affect initial LE cell stretching, but terminates initial cell elongation prematurely; (2) constitutively activated forms of RhoGTPases can induce actin remodeling independently of the JNK cascade; (3) actin patterning and FA formation are different in c-Jun null and c-Jun/AA keratinocytes, suggesting that defective JNK signaling does not totally abolish actin remodeling and FA turnover. This model thus suggests that EGF receptor signaling is a critical regulatory pathway in eyelid development and wound healing (Li, 2003and references therein).
Reactive oxygen species (ROS) are implicated in the pathophysiology of various diseases, including cancer. In this study, JunD, a member of the AP-1 family of transcription factors, is shown to reduce tumor angiogenesis by limiting Ras-mediated production of ROS. Using junD-deficient cells, JunD is demonstrated to regulate genes involved in antioxidant defense, H2O2 production, and angiogenesis. The accumulation of H2O2 in junD-/- cells decreases the availability of FeII and reduces the activity of HIF prolyl hydroxylases (PHDs) that target hypoxia-inducible factors-alpha (HIFalpha) for degradation. Subsequently, HIF-alpha proteins accumulate and enhance the transcription of VEGF-A, a potent proangiogenic factor. This study uncovers the mechanism by which JunD protects cells from oxidative stress and exerts an antiangiogenic effect. Furthermore, new insights are provided into the regulation of PHD activity, allowing immediate reactive adaptation to changes in O2 or iron levels in the cell (Gerald, 2004).
Production of ROS and hypoxic response are key players in the occurrence and progression of cancers. The junD-/- adult mice do not develop tumors spontaneously, suggesting that the protective effect of JunD may only be uncovered under stress conditions. A protective effect of JunD has been demonstrated in cells transformed by the Ras oncogene, one of the most frequently mutated oncogenes in human cancers. Ras-mediated transformation enhances ROS production, and treatment with antioxidant molecules decreases the proliferation rate of Ras-transformed cell lines. JunD has been shown to antagonize Ras-mediated transformation by modulating cell proliferation. The present study shows that overexpression of JunD decreases ROS production in Ras-transformed cells. Thus, it is proposed that the inhibitory effect of JunD on the proliferation of Ras-transformed cell lines is mediated in part through the decreased level of ROS. Moreover, Ras oncogene contributes to the growth of solid tumors by a direct effect on cell proliferation and by facilitating tumor angiogenesis. Indeed, transformation by Ras stabilizes HIF-1α and upregulates VEGF-A expression as well as other HIF target genes. Furthermore, Ras-induced stabilization of HIF-1α is mediated through inhibition of HIF hydroxylation. The data argue that ROS accumulation in Ras-transformed cells triggers PHD inhibition. JunD has a major effect on this process. Indeed, JunD decreases ROS production, restores PHD activity, and subsequently reduces significantly Ras-dependent tumor angiogenesis in vivo. Thus, JunD displays a protective role against Ras-mediated transformation by buffering cells to maintain the redox balance (Gerald, 2004).
DJun:
Biological Overview
| Regulation
| Developmental Biology
| Effects of Mutation
| References
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