The hindsight gene regulates cell morphology, cell fate specification, planar cell polarity and epithelial integrity during Drosophila retinal development. In the third instar larval eye imaginal disc, Hnt protein expression begins in the morphogenetic furrow and is refined to cells in the developing photoreceptor cell clusters just before their determination as neurons. In hnt mutant larval eye tissue, furrow markers persist abnormally, posterior to the furrow; there is a delay in specification of preclusters as cells exit the furrow; there are morphological defects in the preclusters, and recruitment of cells into specific R cell fates often does not occur. Additionally, genetically mosaic ommatidia with one or more hnt mutant outer photoreceptor cell, have planar polarity defects that include achirality, reversed chirality and misrotation. Mutants in the JNK pathway act as dominant suppressors of the hnt planar polarity phenotype, suggesting that Hnt functions to downregulate JUN kinase (JNK) signaling during the establishment of ommatidial planar polarity. Hnt expression continues in the photoreceptor cells of the pupal retina. When an ommatidium contains four or more hnt mutant photoreceptor cells, both genetically mutant and genetically wild-type photoreceptor cells fall out of the retinal epithelium, indicating a role for Hnt in maintenance of epithelial integrity. In the late pupal stages, Hnt regulates the morphogenesis of rhabdomeres within individual photoreceptor cells and the separation of the rhabdomeres of adjacent photoreceptor cells. Apical F-actin is depleted in hnt mutant photoreceptor cells before the observed defects in cellular morphogenesis and epithelial integrity. The analyses presented here, together with previous studies in the embryonic amnioserosa and tracheal system, show that during development Hnt has a general role in regulation of the F-actin-based cytoskeleton, JNK signaling, cell morphology and epithelial integrity (Pickup, 2002).
The fact that all of the symmetrical ommatidia along the borders of hnt clones are of the R3/R3 conformation suggests that Hnt function is necessary for correct R4 fate and orientation. It has been suggested that, owing to its closer proximity to the polarizing signal from the equator, a stronger activation of the JNK pathway occurs in the R3 precursor cell. Activated JUN would then be responsible for the upregulation of the target gene, Delta, in the R3 precursor cell relative to the R4 precursor cell. Since results in the eye and results in the embryo imply that Hnt is necessary for downregulating JNK function, it is proposed that the wild-type function of Hnt is to downregulate JNK activity in the R4 precursor cell. Such downregulation would enhance JNK signaling differences between the R3 and R4 cells. In the absence of the Hnt gene product, JNK signaling would be inappropriately elevated in the R4 precursor cell, thereby upregulating the transcription of JNK targets such as Delta, leading that cell to behave more like an R3 precursor cell. Consistent with this model, it has been found that Delta hypomorphs act as enhancers of the hntpeb rough eye phenotype. R3/R3 symmetric clusters are observed both when the R4 cell is mutant for hnt and the R3 precursor is hnt+, and when the R3 cell is mutant for hnt and the R4 precursor is Hnt+. In the latter case, the above model would lead one to expect normal R3/R4 clusters. Since only R3/R3 clusters are observed, it is speculated that Hnt can affect the R4 precursor cell when expressed only in the neighboring R3 precursor cell (i.e. that there may be some communication feedback between these cells leading to local non-autonomy of the hnt phenotype) (Pickup, 2002).
These results clearly implicate Hnt in regulation of several types of cellular events that are common to the different contexts in which Hnt functions. These include establishment or maintenance of the morphology of individual cells within an epithelium, as well as maintenance of the integrity of the epithelium per se. There are also shared molecular correlates of these Hnt functions. In particular, Hnt is required for establishment of localized F-actin- and phosphotyrosine-rich complexes in the leading edge epidermal cells, as well as in the photoreceptor cells. Hnt functions to regulate two JNK signaling dependent processes (planar polarity in the eye and dorsal closure of the embryo), possibly by downregulating JNK signaling in time and space. A fuller understanding of whether the functions of Hnt in different tissues and at different times during development derive from control of the same molecular pathway will await genetic and molecular analyses of the genes regulated by Hnt (Pickup, 2002).
Much of what is known about apoptosis in human cells stems from pioneering genetic studies in the nematode C. elegans. However, one important way in which the regulation of mammalian cell death appears to differ from that of its nematode counterpart is in the employment of TNF and TNF receptor superfamilies. No members of these families are present in C. elegans, yet TNF factors play prominent roles in mammalian development and disease. The cloning and characterization of Eiger, a unique TNF homolog in Drosophila, is described. Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution (Moreno, 2002).
Another pathway activated by mammalian TNF death factors is the JNK pathway, although its role in inducing apoptosis upon TNF signaling is less well defined. JNK signaling in Drosophila is reflected by the expression levels of puckered (puc), a gene encoding a dual-specificity phosphatase that forms a negative feedback loop by downregulating the activity of JNK. High levels of puc expression are induced by Eiger. Due to its function as a negative regulator of JNK, Puc can also be used as a powerful means to repress JNK activity if overexpressed by a constitutive promoter that is no longer dependent on this activity. Coexpression of Eiger and Puc completely blocks Eiger activity, strikingly reverting the eye and wing phenotypes to wild-type and blocking Eiger-induced elimination of cell clones. Forced expression of Puc does not prevent all forms of cell death. For example, when tested in the situation of polyglutamine repeat-induced neurodegeneration, which is also caused by apoptosis, coexpression of puc has no discernible protective effect. Taken together, these results are interpreted as firm evidence that Eiger induces the JNK signaling pathway and that Eiger-induced apoptosis is critically dependent on JNK activity (Moreno, 2002).
Consistent with this interpretation, several components of the Drosophila JNK pathway are rate limiting in mediating or preventing Eiger-induced apoptosis. The removal of one wild-type copy of either DTRAF1 (encoding the homolog of human TRAF2), misshapen (encoding a Ste20 kinase that binds to DTRAF1), or basket (encodes Drosophila JNK) suppresses Eiger-induced apoptosis. Conversely, animals heterozygous for a mutation in puc display an enhanced phenotype (Moreno, 2002).
The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).
Although Jun amino-terminal kinase (JNK) is known to mediate a physiological stress signal that leads to cell death, the exact role of the JNK pathway in the mechanisms underlying intrinsic cell death is largely unknown. Through a genetic screen, it has been shown that a mutant of Drosophila tumor-necrosis factor receptor-associated factor 1 (DTRAF1) is a dominant suppressor of Reaper-induced cell death. Reaper modulates the JNK pathway through Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), which negatively regulates DTRAF1 by proteasome-mediated degradation. Reduction of JNK signals rescues the Reaper-induced small eye phenotype, and overexpression of DTRAF1 activates the Drosophila ASK1 (apoptosis signal-regulating kinase 1; a mitogen-activated protein kinase kinase kinase) and JNK pathway, thereby inducing cell death. Overexpresson of DIAP1 facilitates degradation of DTRAF1 in a ubiquitin-dependent manner and simultaneously inhibits activation of JNK. Expression of Reaper leads to a loss of DIAP1 inhibition of DTRAF1-mediated JNK activation in Drosophila cells. Taken together, these results indicate that DIAP1 may modulate cell death by regulating JNK activation through a ubiquitin-proteasome pathway (Kuranaga, 2002).
The molecular circuitry underlying innate immunity is constructed of multiple, evolutionarily conserved signaling modules with distinct regulatory targets. The MAP kinases and the IKK-NF-kappaB molecules play important roles in the initiation of immune effector responses. The Drosophila NF-kappaB protein Relish plays a crucial role in limiting the duration of JNK activation and output in response to Gram-negative infections. Relish activation is linked to proteasomal degradation of TAK1, the upstream MAP kinase kinase kinase required for JNK activation. Degradation of TAK1 leads to a rapid termination of JNK signaling, resulting in a transient JNK-dependent response that precedes the sustained induction of Relish-dependent innate immune loci. Because the IKK-NF-kappaB module also negatively regulates JNK activation in mammals, thereby controlling inflammation-induced apoptosis, the regulatory cross-talk between the JNK and NF-kappaB pathways appears to be broadly conserved (Park, 2004).
The JNK and IKK/Relish branches of the Imd pathway mediate distinct gene induction responses in Drosophila innate immunity. After diverging downstream from TAK1, these two signaling cascades regulate two separate groups of target genes that are distinct in their induction kinetics and function. The IKK/Relish targets have been extensively characterized and most encode products whose role in innate immunity is relatively well established. In contrast, the JNK-regulated, LPS-responsive genes represent a largely uncharacterized set of loci whose function in innate immunity is not clear. These genes exhibit transient induction kinetics, reaching a maximum ~1 h after induction. It was found that the transient kinetics of the JNK target genes is controlled by the transient kinetics of the JNK module of the Imd pathway and that the IKK/Relish branch plays an active role in turning off JNK activity. Hence, the two seemingly independent branches of the Imd pathway are wired in such a way as to coordinate the temporal order of individual responses (Park, 2004).
The evidence indicates that Relish-mediated JNK inhibition involves proteasomal degradation of TAK1, the MAPKKK responsible for JNK activation in response to LPS. Treatment with proteasomal inhibitors or RNAi against a component of the proteasome complex results in sustained JNK activation during the LPS response. Furthermore, in cells expressing constitutively active Relish, the stability of TAK1 is greatly decreased. Based on these findings, it is suggested that certain targets of Relish that are induced during immune responses facilitate destruction of TAK1 and switch off the JNK cascade. The fact that cycloheximide and actinomycin D also block the down-regulation of JNK activity indicates the involvement of targets of Relish rather than Relish itself. The Relish target involved in this cross-talk likely increases the susceptibility of TAK1 to proteasomal degradation by direct targeting of TAK1 or by antagonizing factors responsible for TAK1 stabilization (Park, 2004).
In considering this model, it should be noted that TAK1 is critically required for activating both IKK and JNK. Elimination of TAK1 during the LPS response thus turns off both of the downstream signaling cascades. Yet IKK/Relish target genes do not show a transient expression pattern, whereas JNK targets do. One possible explanation for this discrepancy lies in the nature of JNK and Relish activation. JNK is activated through its phosphorylation, a modification that is highly reversible. Thus, termination of the input that contributes to JNK phosphorylation is sufficient to result in its rapid inactivation, especially when one of the JNK targets encode a JNK phosphatase, Puckered. Relish, in contrast, is activated through proteolysis, an irreversible modification. Once activated Relish enters the nucleus, it may remain bound to its target genes for some time even after termination of the upstream signal (Park, 2004).
Interestingly, the antagonism between NF-kappaB and JNK signaling is evolutionarily conserved. In mice, inactivation of either IKKß or NF-kappaB RelA (p65) in cells leads to a sustained JNK activation in response to TNFalpha. The sustained JNK activation by TNFalpha has been associated with TNFalpha-induced apoptosis. Several independent studies have proposed different molecules as mediators for the NF-kappaB inhibition of JNK signaling in the TNFalpha pathway including XIAP, GADD45ß, and reactive oxygen species. Nevertheless, the underlying mechanism remains largely unknown. As the Drosophila Imd pathway and the signaling pathway downstream of mammalian TNF receptor share many conserved features, a similar mechanism may govern the antagonistic relationship between IKK/NF-kappaB and JNK in both flies and mammals. Thus, the mechanistic insights gained from studies in Drosophila should be relevant when elucidating the mechanism that connects NF-kappaB to JNK signaling in mammals (Park, 2004).
MAPK phosphatases (MKPs) are important negative regulators of MAPKs in vivo, but ascertaining the role of specific MKPs is hindered by functional redundancy in vertebrates. MKP function was characterized by examining the function of Puckered (Puc), the sole Drosophila Jun N-terminal kinase (JNK)-specific MKP, during embryonic and imaginal disc development. Puc is a key anti-apoptotic factor that prevents apoptosis in epithelial cells by restraining basal JNK signaling. Furthermore, JNK signaling plays an important role in gamma-irradiation-induced apoptosis, and this study examined how JNK signaling fits into the circuitry regulating this process. Radiation upregulates both JNK activity and puc expression in a p53-dependent manner; apoptosis induced by loss of Puc can be suppressed by p53 inactivation. JNK signaling acts upstream of both Reaper and effector caspases. JNK signaling directs normal developmentally regulated apoptotic events. However, if cell death is prevented, JNK activation can trigger tissue overgrowth. Thus, MKPs are key regulators of the delicate balance between proliferation, differentiation and apoptosis during development (McEwen, 2005).
Mammalian JNK signaling promotes apoptosis in response to both extrinsic (e.g. TNF) and intrinsic (e.g. DNA damage) cues. Drosophila JNK signaling plays a similar role in apoptosis triggered by extrinsic cues, but its role in response to intrinsic cues remains to be established. The role of Puc in regulating responses to either sort of signal has also been unclear (McEwen, 2005).
This study found that removal of Puc triggers apoptosis in epithelia, even in the absence of stress. Thus, basal JNK signaling exists in the absence of stress in embryonic and larval epithelia, and if Puc is not present to restrain this intrinsic JNK activity, it exceeds a threshold and triggers apoptosis. This suggests cells may regulate apoptosis without exogenous JNK stimulation, by regulating MKP levels. Biochemical studies suggest a possible mechanism. Several MKPs are labile proteins, whose half-lives can be shortened or lengthened by post-translational modification. Thus, cell signals may influence apoptosis by modulating MKP accumulation (McEwen, 2005).
Although Puc is crucial to restrain JNK activity and prevent apoptosis in most epithelia, high-level JNK signaling does not always induce apoptosis. JNK signaling is required for embryonic dorsal closure. High JNK activity is normally restricted to the dorsal-most epithelial cells, though in puc mutants it extends into adjacent cells. However, this JNK signaling does not trigger apoptosis. By contrast, ectopic JNK signaling in ventral and lateral epithelial cells in puc mutants does trigger apoptosis. Thus, cells where JNK activity is normally high are somehow refractory to JNK-induced apoptosis. JNK signaling normally activates Dpp in dorsal epithelial cells, with ectopic Dpp activation in more lateral cells in puc mutants -- perhaps this is anti-apoptotic, since Dpp is a survival factor in wing discs. Interplay between death and survival signals may also explain the strongly synergistic stimulation of apoptosis in the embryonic epidermis observed when zygotic Puc (lowering the threshold for JNK signaling) and the survival signal Wg are simultaneously eliminated (McEwen, 2005).
Studies of JNK signaling in mammalian cells suggest that it plays a role in radiation-induced apoptosis. This hypothesis was tested in Drosophila. When JNK activation was blocked by expressing Myc-Puc using a JNK-independent promoter, radiation-induced apoptosis is attenuated. Thus, JNK signaling plays a crucial role in promoting apoptosis in response to radiation in Drosophila, paralleling its essential role in mammalian UV-induced apoptosis (McEwen, 2005).
In mammalian cells, prolonged JNK activity correlates with apoptosis, while transient activation induces cell proliferation. Thus, differences in both signal amplitude and duration are key determinants of the biological outcome. One mechanism modulating signal amplitude is a negative feedback loop; JNK signaling induces expression of MKPs such as Puc. These data provide an instance of how this may regulate the DNA damage response. puc is upregulated by gamma-irradiation in a JNK-dependent manner, and artificially prolonged Myc-Puc expression prevents radiation-induced apoptosis. Thus, the initial increase in Puc expression following irradiation may create a 'grace period' during which Puc elevates the threshold of JNK activation required to induce apoptosis. During this time, cells could attempt to repair radiation-induced damage. However, if damage persists and JNK stimulation continues, the Puc-defined threshold may be exceeded. Indeed, artificially prolonged p53 overexpression overcomes the anti-apoptotic effects of Puc overexpression, perhaps overwhelming the ability of Puc to restrain JNK (McEwen, 2005).
p53 is a key regulator of decisions between life and death in response to DNA damage. Thus, how JNK signaling and p53 are integrated was investigated. Expression of p53 activates JNK, as revealed by its effect on a JNK enhancer trap reporter JNKREP, while loss of p53 prevents radiation-induced JNK activation. These results suggest that p53 acts upstream of JNK signaling in response to cellular stress. Several mechanisms are possible; e.g., p53 might upregulate transcription of JNK pathway components, whose overexpression can induce apoptosis (McEwen, 2005).
p53 normally monitors genome integrity. Loss of p53 significantly, although not completely, suppresses apoptosis induced by Puc inactivation. One model to explain this suggests that basal levels of DNA damage or replication errors act through p53 to regulate basal JNK activity during normal development. This basal activity is kept below the apoptotic threshold by Puc. In p53 mutants, basal JNK activity would be lowered enough so that it could not exceed the apoptotic threshold, even in the absence of Puc. Alternatively, p53 may also function downstream of JNK. Consistent with this, p38 can activate p53 in response to UV, and JNK can regulate p53 stability/activity via direct phosphorylation. This would set up a positive-feedback loop: DNA damage-triggered activation of p53 would induce JNK activation, which would further elevate p53 activity. Additional experiments are required to test these alternate hypotheses (McEwen, 2005).
Signal transduction pathways regulate apoptosis, at least in part, by regulating transcription of rpr, hid and grim (the RHG proteins), the key developmental effectors of apoptosis in Drosophila. The relationship between JNK signaling, RHG proteins and caspase activation are not well understood. Caspase 3 cleavage of Mst1, an upstream regulator of JNK and p38, has been suggested to amplify apoptotic responses, while other data suggest that Mst1 activates caspases via a JNK-dependent pathway. Likewise, in Drosophila, initiation of an apoptotic response by inactivation of DIAP1 may lead to caspase-independent induction of a JNKREP (McEwen, 2005).
Regulatory relationships between JNK activation, RHG proteins and caspase activation were directly assessed. The data suggest that JNK signaling acts through RHG proteins and caspases to induce apoptosis. JNK signaling is required for rpr-reporter induction in response to radiation. Thus, RHG proteins may be JNK-responsive target genes upregulated to elicit apoptosis. Consistent with this hypothesis, mis-expression of eiger, a known JNK activator, has been shown to promote hid expression and apoptosis in eye discs (McEwen, 2005).
To assess whether JNK signaling can be triggered by caspase activation, JNKREP activity was examined in response to Rpr or Hid expression. Both induce apoptosis without concomitant JNK activation. Furthermore, these data suggest that, in at least some contexts, caspases act downstream of JNK: p35-mediated caspase blockade allows cells with elevated JNK signaling to survive in imaginal discs, but does not prevent JNK activation in response to irradiation. Thus, RHG proteins elicit caspase-mediated apoptosis downstream of JNK activation (McEwen, 2005).
Work on JNK-induced apoptosis in Drosophila has focused on its role in the complex processes shaping organ size. Wing disc cells make decisions about whether to die or proliferate by integrating levels of different developmental signals they receive, and comparing their status with that of their neighbors. This occurs in part by competition for survival signals like the TGFbeta family member Dpp. Complex crosstalk among this and other signaling pathways precisely regulates the size and pattern of the wing, in a process that is very resistant to tissue damage or developmental errors. Discontinuities in smooth morphogen gradients, which may arise from errors in patterning or tissue injury, are corrected in part by JNK-dependent apoptosis (McEwen, 2005).
These data clarify roles for JNK signaling in adult development. Failure to recover puc clones anywhere in the wing disc suggests that basal JNK activity is sufficient to promote cell-autonomous apoptosis independent of other signals activating JNK. However, JNK signaling does not play a crucial role in wing patterning; JNK inactivation in the posterior compartment (by Myc-Puc mis-expression) does not have drastic consequences. Roles for JNK signaling can be identified in genitalia and thoracic bristles, where it regulates developmentally programmed apoptosis (McEwen, 2005).
Can MKPs act as tumor suppressors? JNK signaling plays complex roles during oncogenesis. It can prevent tumorigenesis by promoting apoptosis, and it can promote tumorigenesis by supporting Ras-mediated or BCR-Abl-mediated transformation. Since each tumor type has a unique set of mutations in oncogenes and/or tumor suppressors, the phenotypic effects of JNK activation probably differ depending on the activity of other pathways (McEwen, 2005).
Inhibition of apoptosis is one prerequisite for tumorigenesis. Thus the consequences of JNK activation were examined when apoptosis was blocked. When cell death was blocked in the posterior compartment of the wing and the restraints on JNK activation were relaxed by puc heterozygosity, tissue overgrowth occurred. Groups of posterior cells, presumably of clonal origin, exhibited elevated levels of JNK activation, and formed small overgrowths both in developing imaginal discs and in the resulting adult wings and legs. Thus, when apoptosis is suppressed, JNK activation can lead to tissue over-growth (McEwen, 2005).
Related results have been reported with respect to inducing apoptosis by diap inactivation, irradiation or Hid expression, while simultaneously blocking caspase activation. This triggers non-autonomous proliferation of neighboring cells, presumably to compensate for cell lost by apoptosis. Furthermore, some of the 'undead' cells produced by these treatments show JNK-dependent upregulation of Wg or Dpp. Wg signaling has been shown to be required for compensatory proliferation. The current results extend previous studies, since the current experiment did not actively induce apoptosis, but simply blocked caspase activation in puc heterozygotes. Thus, when apoptosis is inhibited and Puc repression is reduced, cells become susceptible to runaway JNK activation. It is likely that analogous focal JNK activation occurs in cells in which apoptosis is not blocked (e.g., anterior compartment cells in these experiments), but JNK-induced apoptosis rapidly eliminates them. At least a subset of the undead cells activate expression of Wg, which may promote excess growth. Interestingly, however, this ectopic Wg expression does not alter cell fates, at least in those situations where overgrowths survive to be observed in the adult wing (McEwen, 2005).
Together, these data have interesting implications. In tumor cells in which apoptosis is prevented, JNK signaling might switch from promoting apoptosis to promoting proliferation by inducing Wnt or TGFbeta, suggesting how JNK signaling can be both pro- and anti-tumorigenic. Future experiments should address mechanisms by which JNK activation triggers Wnt and TGFβ signaling. These data also suggest that runaway JNK activation can promote cell-autonomous proliferation, since groups of cells with elevated JNK activity are associated with overgrowths. Thus, JNK activation or loss of MKP-mediated JNK repression may play multiple roles in tumors whose cells have lost the ability to die (McEwen, 2005).
Ectopic JNK activation occurred in small groups of cells in random positions throughout the posterior compartment. The event(s) that initiate unrestrained JNK activation in these cells remain to be defined. Perhaps there are stochastic variations in JNK signaling that are normally below the threshold triggering apoptosis. However, when Puc activity is reduced, some cells may exceed this threshold, triggering runaway JNK activation in the initial cell and its descendents. Variations in JNK activity may also be induced by spontaneous DNA damage. Normally, such damage would trigger JNK-dependent apoptosis; however, if apoptosis is blocked, the cells proliferate. Finally, loss-of-heterozygosity at the puc locus could create cells lacking restraints on JNK signaling. Since mitotic recombination in somatic tissue can occur, Puc might act in a manner analogous to classic tumor suppressor genes. Additional studies will be required to distinguish between these possibilities (McEwen, 2005).
In summary, this work establishes that Puc is a key negative regulator of
apoptosis throughout Drosophila development. In its absence, basal JNK activity
is poised to eliminate cells from the developing epithelium. The results
position JNK signaling in the hierarchy of events regulating radiation-induced
apoptosis. Finally, the data support the possibility previously suggested by
cytogenetics that MKPs may act as
tumor suppressor genes. These data prompt many new mechanistic questions
regarding the role of JNK signaling in apoptosis and oncogenesis (McEwen, 2005).
Imd-mediated innate immunity is activated in response to infection by
Gram-negative bacteria and leads to the activation of Jun amino-terminal kinase
(JNK) and Relish, a NF-kappaB transcription factor responsible for the
expression of antimicrobial peptides. Plenty of SH3s (POSH) has been shown to
function as a scaffold protein for JNK activation, leading to apoptosis in
mammals. This study reports that POSH controls Imd-mediated immunity signalling in
Drosophila. In POSH-deficient flies, JNK activation and Relish induction were
delayed and sustained, which indicated that POSH is required for properly timed
activation and termination of the cascade. The RING finger of POSH, possessing
ubiquitin-ligase activity, is essential for termination of JNK activation.
POSH binds to and degrades TAK1, a crucial activator of both the JNK
and the Relish signalling pathways. These results establish a novel role for
POSH in the Drosophila immune system (Tsuda, 2005).
Among the components of the Imd pathway, TAK1 plays a crucial role as an
activator of both the JNK and the Relish pathways (Vidal 2001; Silverman, 2003; Park, 2004). Flies that are deficient in TAK1 do
not produce antibacterial peptides and are therefore highly susceptible to
Gram-negative bacterial infection (Vidal, 2001).
In Drosophila S2 cells, TAK1 has been shown to be required for the
peptidoglycan-induced activation of IkappaB
kinase (IKK) and JNK (Leulier, 2003; Silverman, 2003; Kaneko,
2004). TAK1 is also important for limiting the duration of JNK activation,
thereby resulting in a transient JNK-dependent response that precedes the
sustained induction of the Relish-dependent genes (Tsuda, 2005).
Although it has been shown that proteasomal degradation of
TAK1 leads to the rapid termination of JNK signalling (Park, 2004),
the degradation mechanism has yet to be entirely clarified,
and a ubiquitin ligase that promotes TAK1 degradation has not been reported.
This study identifies Plenty of SH3s (POSH) as a crucial component that controls the termination of immunity signalling in Drosophila. POSH, which was
initially isolated as a Rac1-interacting protein, consists of a RING-finger
domain and four SH3 domains, and has been shown to function as a scaffold
protein for JNK signalling components leading to apoptosis in mammals (Tapon, 1998; Xu, 2003). In Drosophila, overexpression of POSH in imaginal discs
activates JNK, which results in various defects in adult morphology (Seong, 2001). Based on flies that are deficient in
POSH, evidence was provided that POSH is required for the properly timed
activation and termination of Imd-mediated immune responses, and that the RING
finger of POSH, which shows ubiquitin-ligase activity, is essential for this
regulation (Tsuda, 2005).
To assess the role of POSH in vivo, a null allele of POSH
was created by the excision of a flanking P-element in EP(2)1026. A mutant was obtained
bearing a deletion that included the start codon and extended 1,282
nucleotide bases into the coding region of POSH. Northern blot analysis
indicated that POSH transcripts were not detectable in
POSH74 homozygous animals. Most
POSH74 homozygous individuals survived embryogenesis,
and the JNK cascade operating during dorsal closure remained intact. Furthermore,
POSH74 showed no genetic interaction with the JNK
pathway components in embryos. Taken together, these results
indicate that not every instance of JNK signalling requires POSH as a scaffold.
Although mutants showed no obvious developmental defects, adult
flies did not live as long as control flies, and older cultures often
contained dead larvae and pupae that were heavily melanized.
In addition, the viability of POSH-deficient flies
after injection with Escherichia coli was significantly lower than that
of wild type. A possible cause for such phenotypes is defective
immune function, and therefore a role for POSH in the immune
response was sought (Tsuda, 2005).
Whether the TAK1-mediated immune response was compromised by the
POSH74 mutation was tested. Infection with E. coli
activates TAK1, which consequently phosphorylates JNK and induces the expression
of Relish/NF-kappaB, resulting in the subsequent induction of a
battery of genes encoding antimicrobial peptides, such as Diptericin.
Northern blot analysis indicated that
the induction of relish and diptericin (dpt) messenger RNAs
after E. coli infection was significantly delayed in
POSH74 flies compared with that in controls.
Interestingly, it was also observed that expression of
relish was sustained for a longer period after infection in
POSH74 flies (Tsuda, 2005).
To investigate the effects of the POSH mutation more quantitatively,
real-time PCR analysis of immune response genes was performed.
Consistent with Northern blot analysis, the
induction of dpt was delayed, and relish induction was sustained,
with a maximum expression at 2 h after infection. The
expression pattern of attacin A (attA), which is dependent on
Relish, was also analyzed. Induction of attA was almost normal, but unlike
in the control flies, its expression level continued to increase up to 4 h after
infection. The maximum level of expression was twice that of control flies,
consistent with Relish activity being elevated in POSH mutant flies. The
expression profile of puckered (puc), a target
gene of the JNK pathway, was also analyzed. Induction of puc was slightly delayed, with the
highest level of expression at 1 h after infection, and expression was sustained
for a longer period in POSH74 flies compared with that
in controls. These results indicate that POSH is required for properly timed
activation and rapid termination of both JNK- and Relish-mediated immune
response pathways (Tsuda, 2005).
TAK1 has been shown to be required for the maintenance
of relish mRNA expression, and its
proteasomal degradation is responsible for the rapid termination of Relish and
JNK signalling (Park, 2004). POSH contains a
CH4C4-type RING-finger domain near its N terminus. It has been shown that the
RING-finger domain of mammalian POSH possesses E3 ubiquitin-ligase activity and
regulates the level of POSH protein through the proteasomal pathway (Xu, 2003).
Drosophila POSH also had E3 ubiquitin-ligase activity;
its auto-ubiquitination has been demonstrated. To assess the functional importance of the RING domain, POSHmR was constructed, bearing point mutations in the RING domain, and POSHDeltaR
lacking the RING-finger domain altogether. Neither of these
proteins was able to auto-ubiquitinate after expression in cultured
Drosophila cells (S2). It is conceivable, therefore, that POSH is
responsible for the feedback regulation of the immune response through TAK1
ubiquitination and its subsequent degradation. Observations indicate that
the RING-finger function of POSH is essential for the degradation of TAK1 (Tsuda, 2005).
To characterize further the role of
POSH in the regulation of JNK signalling, immunocompetent S2 cells were used in an
in vitro model system used to study the immune response triggered by
peptidoglycan in a commercial lipopolysaccharide preparation. Consistent with
puc expression results in POSH mutant flies, the reduction of POSH
by RNA interference (RNAi) significantly decreased the level of JNK activation,
but it was sustained for a longer period than in control cells.
In contrast, overexpression of POSH induced JNK
activation at a high level, but then inactivated it rapidly. When
POSHmR was transfected into S2 cells, phosphorylated JNK persisted
for a longer period than in control cells. The effects of POSH
overexpression and the role of the RING-finger function were examined
in vivo using
armadillo-GAL4 (arm-GAL4) as a ubiquitous expression driver.
Following bacterial infection, the expression level of
puc, a target of JNK signalling, rapidly declined in flies overexpressing
POSH (arm>POSH). In contrast, puc expression was sustained in
flies overexpressing POSHmR (arm>POSHmR),
which suggests that POSHmR has a dominant-negative effect on JNK
signalling. Together, these results indicate that POSH affects both activation
and inactivation of JNK signalling induced by microbial infection, and that the
RING-finger domain of POSH is essential for the negative feedback regulation of
JNK signalling. The profile of puc expression in
POSH74 flies is remarkably similar to that seen in
relish flies, which supports the idea that POSH mediates Relish-dependent inactivation of JNK in the immune system (Tsuda, 2005).
These results show that
POSH modulates the TAK1-mediated innate immune response. Following microbial
infection, POSH facilitates rapid JNK activation and the induction of
relish/NF-kappaB expression, both of which are mediated by TAK1
(Silverman, 2003). Conversely, POSH is also
required for the rapid termination of both JNK activation and relish
transcription through the E3 ubiquitin-ligase activity of the RING-finger
domain. The results indicate that POSH is involved in a mechanism of negative
feedback regulation of JNK and NF-kappaB
signalling. The Drosophila TAK1-mediated innate immune response is
similar to the tumour necrosis factor (TNF) signalling cascade in mammals.
Imd protein contains a death domain
with homology to that of mammalian TNF-receptor-interacting protein (RIP).
Most of the pathway
components (such as FADD, TAK1) and Drosophila homologues of
IKKgamma and IKKbeta are conserved in mammalian TNF signalling. The
activation of Relish involves its phosphorylation and cleavage by DREDD, the
Drosophila homologue of caspase 8. Eiger, a
Drosophila homologue of the TNF-alpha
superfamily ligand, has recently been identified. Overexpression of Eiger induces
cell death by activating the JNK signalling pathway, as in mammals. Although the
Eiger signalling pathway has not been fully elucidated in Drosophila, it
has been clearly shown that TAK1 is essential to transduce the cell-death
signal. It is possible that POSH is involved in Eiger signalling and modulates
TAK-mediated JNK signalling as in the immune system. It will be of interest to
determine whether POSH functions in mammalian TNF signalling (Tsuda, 2005).
Plenty of SH3s (POSH) functions as a scaffold protein for the Jun N-terminal kinase (JNK) signal transduction pathway, which leads to cell death in mammalian cultured cells and Drosophila. This study shows that POSH forms a complex with Apoptosis-linked gene-2 (ALG-2) and ALG-2-interacting protein (ALIX/AIP1) in a calcium-dependent manner. Overexpression of ALG-2 or ALIX in developing imaginal eye discs results in roughened or melanized eyes, respectively. These phenotypes are enhanced by co-overexpression of POSH. It was found that overexpression of either gene could induce ectopic JNK activation, suggesting that POSH/ALG-2/ALIX may function together in the regulation of the JNK pathway (Tsuda, 2006).
The present study demonstrates that POSH can form a complex with ALG-2 and ALIX both in vitro and in Drosophila cultured cells. Transgenic studies revealed that overexpression of ALG-2 in imaginal eye discs using GMR-GAL4 resulted in a roughened eye, similar to that induced by POSH. However, when both ALG-2 and POSH are overexpressed, the eye size is dramatically reduced, suggesting that ALG-2 in combination with POSH can kill the cells during development. Overexpression of ALIX produced eyes with melanotic pigments, which is indicative of neuronal degeneration. Flies overexpressing both ALIX and POSH show increased amounts of melanotic pigments, with no effect on the eye size. The phenotypic differences between ALG-2 and ALIX suggest that the cell death occurs more rapidly in eye discs overexpressing ALG-2/POSH than in those overexpressing ALIX/POSH (Tsuda, 2006).
It was further demonstrated that ALG-2 and ALIX can activate the JNK signaling pathway, similar to the case for POSH. Since neither ALIX nor ALG-2 has a putative kinase domain, overexpression of either protein together with POSH may facilitate ectopic JNK activation by affecting the subcellular localizations of JNK components. In fact, ALG-2 has been shown to interact with ASK1, thereby regulating its subcellular localization and JNK activation in mammalian cells (Tsuda, 2006).
ALG-2 and ALIX are both thought to be involved in membrane trafficking. ALG-2 interacts with Annexin XI and VII, both of which play roles in vesicular trafficking and exocytosis. In contrast, ALIX binds to CHMP4b, a human homolog of yeast Snf7, which is involved in multivesicular body (MVB) sorting. Furthermore, ALG-2 and ALIX both bind to Tsg101, a component of ESCRT-I. ESCRT-I cooperates with two other complexes, ESCRT-II and ESCRT-III, to drive MVB formation. MVB sorting is thought to be topologically identical to the budding of HIV and other retroviruses from the plasma membrane. Indeed, ALIX has been shown to associate with HIV-1 Gag protein, which is required for promoting membrane fission events, and recruit the ESCRT machinery to permit budding. Recently, POSH was also shown to be required for sorting of HIV-1 Gag protein to the plasma membrane. Furthermore, POSH interacts with hepatocyte growth factor-regulated tyrosine kinase substrate, which is known to play a central role in MVB formation, and modulates its stability. These findings suggest that the POSH/ALIX/ALG-2 complex may have a role in membrane trafficking and virus budding (Tsuda, 2006).
Although recent progress has unveiled the diverse in vivo functions of LKB1, detailed molecular mechanisms governing these processes still remain enigmatic. This study shows that Drosophila LKB1 negatively regulates organ growth by caspase-dependent apoptosis, without affecting cell size and cell cycle progression. Through genetic screening for LKB1 modifiers, the JNK pathway was discovered as a novel component of LKB1 signaling. The JNK pathway is activated by LKB1 and mediates the LKB1-dependent apoptosis. Consistently, the LKB1-null mutant is defective in embryonic apoptosis and displayed a drastic hyperplasia in the central nervous system: these phenotypes are fully rescued by ectopic JNK activation as well as wild-type LKB1 expression. Furthermore, inhibition of LKB1 results in epithelial morphogenesis failure, which is associated with a decrease in JNK activity. Collectively, these studies unprecedentedly elucidate JNK as the downstream mediator of the LKB1-dependent apoptosis, and provide a new paradigm for understanding the diverse LKB1 functions in vivo (Lee, 2005).
Tumor suppressor LKB1 is a serine/threonine kinase that is heterozygotically mutated in the germ line of Peutz-Jegher syndrome (PJS) patients. PJS patients have a highly increased risk of malignant tumors, especially in the gastrointestinal tract, breast, uterine cervix, and ovary. Many of these tumors acquire somatic mutations in the remaining wild-type allele of LKB1. Moreover, various sporadic cancers are also associated with the loss of LKB1, implicating the general role of LKB1 in tumor suppression. Since point mutations of LKB1 found in PJS mostly reside in the highly conserved kinase domain, it is very likely that the kinase activity of LKB1 is essential for its tumor suppressive function (Lee, 2005 and references therein).
Since the discovery of LKB1 in 1998, various functions of LKB1 molecule have been suggested to be responsible for its tumor-suppressing activity, such as inhibition of cell cycle progression, cell growth retardation, apoptotic cell death, and cell polarity control. The LKB1 protein was originally characterized as a cell cycle inhibitor, and its putative downstream targets such as Brg1 and p21 were suggested to mediate LKB1-dependent cell cycle arrest. It has been recently proposed that LKB1 also regulates cellular growth by controlling another tumor suppressor, tuberous sclerosis complex (TSC), via the AMP kinase (AMPK)-dependent pathway. In addition, studies showing the absence of apoptosis in PJS polyps reveal that LKB1 is an inducer of apoptosis in vivo. Another tumor suppressor, p53, the first identified in vitro substrate of LKB1, was proposed as a mediator of this apoptosis. Finally, LKB1 was shown to be necessary for the polarization of intestinal epithelial cells and Drosophila ovary cells, demonstrating another important function of LKB1 in regulating cell structure (Lee, 2005).
Although considerable progress has been made to characterize the in vivo function of LKB1, only a limited amount of information concerning its molecular mechanisms has been found in detail; the major biological pathway responsible for the tumor suppressive function of LKB1 remains to be clarified (Lee, 2005).
The c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) family that mediates various cellular responses including programmed cell death, epithelial sheet movement, and planar polarity. The activity of JNK is tightly regulated by reversible phosphorylation, which is stimulated by a sequential cascade of protein kinases and inhibited by JNK-specific phosphatases. Activation of the JNK pathway is required for the release of cytochrome c from the mitochondria and the subsequent activation of the caspase cascade. Therefore, abrogation of the JNK signaling pathway causes various defects in developmental or stress-induced apoptosis. In addition to controlling apoptosis, regulation of epithelial morphogenesis is another well-known function of the JNK signaling pathway. Depletion of JNK activity results in various epithelial defects, including those of dorsal closure and planar polarity in Drosophila, and of the optic and neuronal system in mice (Lee, 2005 and references therein).
Since JNK is required for apoptosis and epithelial organization, disruption of the JNK signaling pathway is thought to be a prerequisite of tumorigenesis by enabling tumor cells to evade programmed cell death and to acquire a metastatic potential. Indeed, JNK1 and JNK2 are both required for the suppression of oncogenic transformation and tumorigenesis. Loss of JNK3, which is selectively expressed in neuronal cells, is also closely associated with human brain tumors. Furthermore, the Mkk4 gene, which encodes an upstream kinase of JNK, has been identified as a tumor suppressor gene and a metastasis suppressor gene (Lee, 2005 and references therein).
This paper shows a genetic connection between tumor suppressor LKB1 and the JNK signaling pathway. The results indicate that LKB1 activates the JNK pathway in vivo and that the JNK pathway mediates the LKB1-dependent apoptotic cell death (Lee, 2005 )
Using various Gal4 drivers, a high-level expression was obtained of both wild-type and kinase-dead LKB1 in the specific region of various tissues: the posterior region of the eye imaginal disc by the glass multiple reporter-Gal4 driver (gmr-Gal4), the dorsal region of the wing disc by the apterous-Gal4 driver (ap-Gal4), and the central pouch region of the wing disc by the ms1096-Gal4 driver). The expression levels of the LKB1 protein were determined by immunostaining using Drosophila LKB1-specific antisera. Eye-specific expression of wild-type LKB1 by the gmr-Gal4 driver induced a slight reduction in overall eye size, and this reduction became severer when the LKB1 expression level was elevated. However, the kinase-dead LKB1 overexpression did not induce this specific phenotype, showing that the kinase activity of LKB1 is important in inducing tissue-size reduction. The dorsal region-specific expression of wild-type and kinase-dead LKB1 by the ap-Gal4 driver displayed more dramatic phenotypes. Since Drosophila wing discs are composed of two layers of tissues, dorsal and ventral, alterations in size of only the dorsal layer give rise to bent-up or bent-down wing phenotypes. Although the dorsal tissue-specific expression of kinase-dead LKB1 did not induce any alterations, wild-type LKB1 induced a dramatic bent-up wing phenotype in a dose-dependent manner. Consistently, the ms1096-Gal4-driven expression of wild-type LKB1 dose-dependently reduced the overall wing size, although kinase-dead LKB1 did not. Therefore, it is concluded that Drosophila LKB1 negatively regulates the tissue and organ size in a kinase activity-dependent manner (Lee, 2005).
To examine the effect of LKB1 in individual cells, clonal analyses was performed by inducing LKB1 overexpression in a specific subset of cells within wing imaginal discs. Compared to the control clones expressing only GFP, the LKB1-expressing clones expanded very poorly. Similarly, overexpression of the TSC complex, another well-known tumor suppressor, also decreased the clone size, although with a milder severity than the overexpression of LKB1 (Lee, 2005).
Since the LKB1-dependent reduction of tissue size appropriately reflected the
tumor-suppressing activity of LKB1, whether this tissue-size reduction is caused by cell cycle arrest or apoptotic cell death was assessed. Both these phenomena have been proposed as major cellular processes underlying the tumor-suppressing function of LKB1. To check the G1-S cell cycle progression of LKB1-expressing tissues, the bromodeoxyuridine (BrdU) incorporation assay, which specifically labels S-phase cells, was performed. Although LKB1 is highly expressed in these tissues, no significant alterations in the S-phase cell number were detected with respect to the control tissues. Similarly, there was no change in the antiphosphospecific histone 3 (PH3) antibody staining pattern between the wild-type and the LKB1-expressing tissues, demonstrating that G2-M cell cycle transition was also unaffected. However, when the equivalent tissues were subjected to acridine orange (AO) staining, which specifically labels the dying cells with disrupted membrane integrity, they displayed prominent death signals, specifically in the region of LKB1 overexpression. These results strongly implied that the LKB1-induced organ size reduction is caused by apoptotic cell death, not by cell cycle arrest. Furthermore, TUNEL staining of the eye and wing discs also confirmed the existence of extensive apoptosis in the LKB1-expressing cells. Therefore, it was concluded that LKB1 is a potent apoptosis instigator in Drosophila (Lee, 2005).
Tumor suppressor LKB1 is a unique serine/threonine kinase with no other close relatives in the mammalian genome. Likewise, only a single LKB1 orthologue exists in the Drosophila genome, implying well-conserved functional characteristics between the mammalian and Drosophila LKB1. This study has discovered a novel functional connection between LKB1 and the JNK pathway: the JNK pathway is present downstream of the LKB1-dependent signaling pathway and controls apoptosis and organ size in response to LKB1 activities (Lee, 2005).
Previous studies on classical tumor suppressors in the Drosophila model system showed that hyperactivation of each tumor suppressor negatively regulates organ size by cell size reduction, cell cycle inhibition, or cell death promotion. LKB1 negatively regulates organ size in Drosophila in a kinase activity-dependent manner. Recent reports on the TSC tumor suppressor suggested that LKB1 suppresses TOR activity via the TSC complex, ultimately resulting in inhibition of cell growth and proliferation. Likewise, it was observed that TSC overexpression dramatically reduces cell size in Drosophila clonal overexpression analyses. However, unexpectedly, LKB1 overexpression did not induce any cell size reduction, in stark contrast to the case of TSC. Moreover, LKB1 overexpression in the clones of endoreplicating tissues altered neither cell size nor DNA content, confirming that the tumor-suppressing activity of LKB1 is not related to the negative regulation of cell growth. Therefore, it is concluded there is no physiological connection between LKB1 and TSC in the context of cell growth control in Drosophila (Lee, 2005).
However, histological analyses clearly demonstrate that the LKB1-overexpressing tissues suffer caspase-dependent programmed cell death, without apparent alteration in G1/S and G2/M cell cycle transition. The cell death induced by LKB1 overexpression displays the most typical phenotypes of apoptosis, such as membrane integrity disruption, DNA fragmentation, and caspase activation. Furthermore, endogenous LKB1 was physiologically required for normal embryonic apoptosis, and also essential for the size control of the central nervous system. Consistent with the results, LKB1 is reported to be highly expressed in apoptotic cells of the small intestine in humans, and PJS polyps that lack the functional LKB1 protein possessed fewer apoptotic cells than in adjacent normal tissues. Collectively, these results strongly suggested that instigation of apoptosis is a critical function of tumor suppressor LKB1 (Lee, 2005).
Surprisingly, in contradiction to previous research done with the mammalian system, the LKB1-induced apoptosis in Drosophila is a p53-independent event; it is not blocked by the expression of a dominant-negative version of p53. Moreover, apoptosis induced by DNA damage or p53 overexpression is also unaffected by downregulation of LKB1 activity. Furthermore, diagnostic symptoms and major tumor types of PJS patients are highly distinct from those of Li-Fraumeni syndrome patients Therefore, it is very likely that LKB1 and p53 are indirectly related since they both have caspase activation as a common downstream signaling event (Lee, 2005).
Genetic screening using the Drosophila model system enabled discovery or the JNK pathway as a downstream effector of the LKB1-dependent signaling pathway. Through simple genetic screening, more than 500 genes for interaction with LKB were found; this study covered various signal transduction pathways including the Ras/ERK, JNK, PI3K, TOR, Wnt, TGF-beta, and NF-kappaB pathways. Among them, some components of the JNK signaling pathway showed outstanding genetic interactions with LKB1, strongly enhancing the tissue-size reduction phenotype of LKB1. This result implied the specific involvement of the JNK pathway in LKB1-dependent signal transduction, prompting additional studies carried out concerning the relationship between LKB1 and the JNK signaling pathway (Lee, 2005).
These biochemical and histological analyses clearly demonstrated that JNK can be activated by LKB1 overexpression. Since the kinase-dead mutant of LKB1 did not induce this JNK activation, this phenomenon seems to be a direct consequence of LKB1 phosphotransferase activity. Moreover, blockage of the JNK signaling pathway strongly suppresses the LKB1-induced apoptosis, as well as the tissue-size reduction. Furthermore, the activation of JNK signaling is shown to be involved in the physiological apoptosis induced by endogenous LKB1. Therefore, it is concluded that the JNK pathway acts as a downstream effector of LKB1 by mediating the LKB1-induced apoptosis. Accordingly, any signals upregulating the kinase activity of LKB1 would also activate the JNK pathway, ultimately leading to apoptosis in the cell. In fact, some microtubule-disrupting agents that induce the LKB1-dependent cell death also activate the JNK pathway to promote apoptosis (Lee, 2005).
Another interesting point of this research is that inhibition of LKB1 results in defective epithelial morphogenesis, which is associated with low JNK activity. Several previous studies already demonstrated the requirement of LKB1 and JNK for epithelial integrity and polarity, both in the mammalian and Drosophila systems. Consistently, it was observed that inhibition of LKB1 activity during development causes various morphogenesis defects including malformation of adult thoraxes and failure in embryonic dorsal closure. Furthermore, LKB1 is shown to be required for maintaining adequate JNK activity during embryonic and larval development. Since loss of tissue integrity is a common feature of PJS polyps and cancer, the role of LKB1 in epithelial organization seems to be also important for mediating its tumor-suppressing activity (Lee, 2005).
This study has demonstrated that the JNK pathway is involved in the LKB1-dependent apoptosis and epithelial morphogenesis in Drosophila. Since deregulation of both LKB1 and the JNK pathways is highly implicated in cancer development, the novel connection between LKB1 and the JNK signaling pathway provides a new direction for future studies in better understanding the complex functions of tumor suppressor LKB1 (Lee, 2005).
Jun N-terminal kinase (JNK) signaling is a highly conserved pathway that controls both cytoskeletal remodeling and transcriptional regulation in response to a wide variety of signals. Despite the importance of JNK in the mammalian immune response, and various suggestions of its importance in Drosophila immunity, the actual contribution of JNK signaling in the Drosophila immune response has been unclear. Drosophila TAK1 has been implicated in the NF-kappaB/Relish-mediated activation of antimicrobial peptide genes. However, this study demonstrates that Relish activation is intact in dTAK1 mutant animals, and that the immune response in these mutant animals is rescued by overexpression of a downstream JNKK. The expression of a JNK inhibitor and induction of JNK loss-of-function clones in immune responsive tissue revealed a general requirement for JNK signaling in the expression of antimicrobial peptides. The data indicate that dTAK1 is not required for Relish activation, but instead is required in JNK signaling for antimicrobial peptide gene expression (Delaney, 2006).
Innate immune responses are critical for a rapid host defense against pathogens. The signaling pathways that control these responses are present in all multicellular organisms, ranging from humans to flies, and are remarkably well conserved. Although the innate response lacks the antigen recognition capacity of vertebrate adaptive immunity, it is nevertheless complex and crucial for host survival. Drosophila is a proven genetic model organism for the study of innate immunity and has provided invaluable insights into the control of responses to infection (Delaney, 2006).
Toll and Imd are the founding members of two principal innate immune response signaling pathways in Drosophila. Toll signals through two NF-kappaB/Rel family transcription factors, Dif and Dorsal, and is required for responses to fungal and Gram+ bacterial infections. Imd signaling controls primarily Gram- bacteria-specific responses through the cleavage and activation of a third Rel family transcription factor, Relish, by the Drosophila caspase Dredd. Relish activation also requires an IkappaB kinase (IKK) complex that is itself activated by Imd signaling. The transcriptional targets of Dif and Relish are not entirely distinct. For example, cecropinA expression requires either Relish or Dif, or both, depending on the type and strain of infecting microorganism. More than 20 Drosophila genes have been implicated in these signaling pathways and nearly all of them have mammalian homologues with conserved immune functions (Delaney, 2006 and references therein).
Jun N-terminal kinase (JNK) signaling has been linked to stress responses, cell migration, apoptosis, and immune responses in both insects and mammals. JNK activity can be induced by infection, lipopolysaccharide, and inflammatory cytokines such as tumor necrosis factor (TNF) in flies and mammals. Null mutations in JNK signaling components are typically embryonic lethal in flies and thus unlikely to appear as targets of mutagenesis screens designed to detect immune response genes in living animals. An exception to this rule is dTAK1. Overexpression and dominant-negative studies indicated that dTAK1 can act as a JNK kinase kinase (Delaney, 2006 and references therein).
Previously characterized dTAK1 mutations, however, showed no apparent JNK-like phenotype, but failed to express Relish-dependent antimicrobial peptides, suggesting a role in the Imd pathway (Vidal, 2001). Previous epistasis analysis using the UAS/GAL4 overexpression system to ectopically express dTAK1 placed dTAK1 downstream of imd and upstream of the IKK complex in the Relish signaling pathway (Vidal, 2001). In vitro experiments implicated dTAK1 in the IKK-dependent phosphorylation of Relish in S2 cells (Delaney, 2006).
Evidence has been uncovered for a Relish-independent function of dTAK1 in the control of antimicrobial peptide gene expression. Several aspects of Relish activation appeared normal in infected dTAK1 mutant animals, including cleavage, nuclear localization, and promoter binding. Therefore whether JNK pathway components mediate dTAK1 function in the immune response was examined. Several lines of evidence are reported for dTAK1 acting through the JNK cascade in the innate immune response. First, overexpression of Hemipterous, a JNKK, rescued attacin and diptericin expression in dTAK1 mutant animals, whereas overexpression of the downstream Imd component Dredd did not. Second, it was found that expression of the Puckered (Puc) phosphatase, an inhibitor of JNK activity, suppressed the expression of antimicrobial peptide genes. To directly test for a JNK requirement in immune signaling, JNK mutant clones were induced in the fat body of larvae. Strikingly, diptericin, attacin, Metchnikowin, and Drosomycin expression was lost in the mutant tissue (Delaney, 2006).
It is concluded that the JNK pathway is required to mediate dTAK1 signaling during the Drosophila immune response. Furthermore, a model is proposed where the JNK and NF-kappaB signaling are both required to activate antimicrobial peptide gene expression during the immune response in the Drosophila fat body (Delaney, 2006).
The function of TAK1 in vertebrates has remained enigmatic. It was originally identified as a TGFβ-activated kinase, hence the name, in mammalian cell culture assays. However, follow-up work in multicellular contexts and in vivo analyses in vertebrates, C. elegans, and Drosophila have shown no clear link to TGFβ signaling, but rather suggest a role for TAK family kinases in JNK activation or as upstream activators of Nemo-like kinases. In mammalian systems, TAK1 is one of a number of kinases that can activate IKK complexes and, consequently, NF-kappaB signaling in vitro. In vitro studies of human cells have shown that targeting of TAK1 by RNAi reduces NF-kappaB activation by TNFalpha and IL-1 stimulation. Recent studies using fibroblasts derived from TAK1 mutant mouse embryos and mice with a B-cell-specific deletion of TAK1 showed that JNK activation was impaired in response to all stimuli tested in TAK1 mutant cells. Although NF-kappaB activation was impaired in response to stimulation by IL-1β, TNF, and TLR3 and TLR4 ligands, NF-kappaB activation by B-cell receptor or LT-β stimulation remained intact, suggesting a specific role for TAK1 upstream of IKKβ and JNK, but not IKKalpha. Interestingly, IKKalpha activation leads to the phosphorylation and processing of NF-kappaB2 from the p100 to the active p52 form, reminiscent of Relish activation in Drosophila (Delaney, 2006 and references therein).
Biochemical analyses in mammalian systems have demonstrated that TAK1 functions in multimeric protein complexes that can include TAB1, TAB2, and different TRAF proteins. The exact composition of these complexes seems to determine TAK1 responsiveness and downstream effects. In the fly, genetic studies found an interaction between dTRAF1 and dTAK1 in the activation of JNK signaling and apoptosis. Gain- and loss-of-function analyses indicate that dTRAF2, but not dTRAF1, is necessary for the activation of Relish-dependent gene expression; however, no interaction between dTRAF2 and dTAK1 in the activation of antimicrobial peptides has been reported (Delaney, 2006).
Genome-wide analyses that examined in vivo responses in Drosophila identified dJun and puc as genes potentially regulated by Toll and Imd signaling, suggesting a cross-regulation between these pathways and the JNK signaling pathway. A study recently reported that RNAi knockdown of kayak, msn, hep, or aop blocked E. coli-induced attacin and drosomycin expression in S2 cells. Furthermore, in related studies, it was also observed that, although dTAK1 RNAi-treated S2 cells failed to express an attacin reporter gene, Relish cleavage and nuclear localization remain intact in these cells. Other RNAi analyses in S2 cells have concluded that JNK signaling does not have a significant role in antimicrobial peptide gene expression. However, RNAi against hep or bsk seemed to partially block antimicrobial peptide induction, especially of attacin and cecropinA and, accordingly, attacinD levels were lower in microarrays when the JNK pathway was blocked. The current results confirm a positive role for JNK signaling in the antimicrobial peptide response in vivo (Delaney, 2006).
The placement of dTAK1 function upstream of JNK, rather than IKK, requires a remodeling of the signaling pathways that activate the antimicrobial peptide genes. Earlier models were based on studies that showed that dTAK1 mutations blocked the constitutive activation of diptericin by Imd overexpression (Vidal, 2001). In turn, IKK mutations blocked dTAK1-induced diptericin expression. One interpretation of these data places IKK directly downstream of dTAK1. However, if the activation of both JNK and IKK signaling pathways is required, then a disruption in either branch would be sufficient to suppress any upstream activation (Delaney, 2006).
Overexpression of dTAK1 is sufficient to induce antimicrobial peptide expression (Vidal, 2001). However, dTAK1 is an extremely potent activator of JNK signaling and apoptosis, and overexpression of dTAK1 could activate proteins that are not normal phosphorylation targets. Based on RNAi studies in S2 cells, dTAK1 is required for dIKK complex-dependent phosphorylation of Relish in vitro. This could reflect a stringent requirement for dTAK1 in blood cell-derived S2 cells that is different in fat body tissue (Delaney, 2006).
The new model would predict that overexpression of the Dredd caspase would be insufficient to activate fully the antimicrobial peptides in dTAK1 mutant animals and this is indeed the case. Overexpression of Dredd may be sufficient to induce antimicrobial peptide gene expression in a wild-type background because of inadvertent JNK pathway activation by ectopic caspase activity or by the heat-shock protocol itself. Alternatively, an additional role for Dredd has been proposed in the ubiquitin-mediated activation of dTAK1 and the dIKK complex (Zhou, 2005). The suppression by dTAK1 mutants of ectopic Dredd expression is consistent with this model as well, and does not distinguish between the two potential functions of Dredd. The current data are consistent with a model that places dTAK1 activity in a pathway parallel to the functions of IKK and Relish and in which both these pathways are required for the activation of antibacterial peptide genes such as diptericin and attacin (Delaney, 2006).
Promoter analyses of most antimicrobial peptide genes have not revealed any obvious binding sites for activator protein-1 (AP-1) complexes, the Jun/Fos heterodimer, and transcriptional mediator of JNK signaling. However, AP-1 binding sites can be quite diverse and are not always predictable directly from DNA sequence. Nevertheless, a recent study identified a functional AP-1 binding site in the attacinA promoter. These data suggest that AP-1 binding represses attacinA transcription by recruiting histone deacetylase 1 (dHDAC1) to the promoter. In contrast, in mammalian studies, c-Jun function is itself repressed by association with HDAC3. This repression is relieved upon JNK signaling. A similar mechanism may be employed in the Drosophila fat body. Accordingly, the sustained expression of attacin and other antimicrobial peptide genes in vivo would require an activation (or de-repression) of AP-1 function at the onset of the immune response. Such positive cooperation between AP-1 and NF-kappaB transcription factors was also seen in molecular studies of the human β-defensin-2 promoter (Delaney, 2006).
AP-1-dependent gene expression is normally rapid. Thus, if AP-1 activity is not directly required for diptericin expression, it could act indirectly through the activation of other genes. Alternatively, JNK could phosphorylate some targets other than the AP-1 complex proteins Jun and Fos. In mammalian studies, it has been shown that JNK can phosphorylate, and thereby inhibit, Insulin Receptor Substrate-1. However, the recent finding that RNAi against kayak/dFos can block antimicrobial peptide expression and the current dJun loss-of-function studies in vivo suggest that JNK does indeed signal through AP-1 to control expression of these genes (Delaney, 2006).
It is intriguing that overexpressed Puc not only blocks Relish-dependent antimicrobial peptide gene expression, but it also strongly blocks drosomycin expression, which is not true in dTAK1 mutants. This suggests that JNK or JNK-related proteins, for example, p38a, p38b, and MPK2, may also be important for other aspects of the immune response, for example, the Toll/Dif-dependent antimicrobial genes. The clonal analysis of JNK mutant tissue confirms that JNK is required not only for the expression of Gram--specific peptides diptericin and attacin, but also for Metchnikowin (Gram+/fungal specific) and drosomycin (fungal specific). Mutations in dTAK1 had less of an impact on Metchnikowin or drosomycin expression than on attacin, for example. Furthermore, reduced dJun activation occurred in dTAK1 mutant animals, indicating that other upstream kinases may be involved in the control of these genes. JNK is a member of a large family of mitogen-activated protein kinases (MAPKs). In the fly, there are at least five MAPKKKs, four MAPKKs, and five MAPKs, and so the potential redundancies are many. If these other proteins contribute to the immune response, how they do so has yet to be tested in genetic loss-of-function in vivo studies in the fat body (Delaney, 2006).
How JNK and NF-kappaB signals integrate to positively control gene expression is a critical question. This study has demonstrated that both are required for the expression of a particular set of immune responsive genes in vivo. Through the use of Drosophila genetics, it should be possible to identify novel immune response genes that are controlled cooperatively by JNK and NF-kappaB signaling. From promoter analysis of these genes, it may be possible to predict additional genes that are important for other biological processes. Both the JNK and NF-kappaB signaling pathways have been implicated many times in many different contexts. Continued analysis in Drosophila may lead to a general understanding of their roles in normal biological processes and developmental malignancies (Delaney, 2006).
Apparent defects in cell polarity are often seen in human cancer. However, the underlying mechanisms of how cell polarity disruption contributes to tumor progression are unknown. Using a Drosophila genetic model for Ras-induced tumor progression, a molecular link has been shown between loss of cell polarity and tumor malignancy. Mutation of different apicobasal polarity genes activates c-Jun N-terminal kinase (JNK) signaling and downregulates the E-cadherin/β-catenin adhesion complex, both of which are necessary and sufficient to cause oncogenic RasV12-induced benign tumors in the developing eye to exhibit metastatic behavior. Furthermore, activated JNK and Ras signaling cooperate in promoting tumor growth cell autonomously, since JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras. The finding that such context-dependent alterations promote both tumor growth and metastatic behavior suggests that metastasis-promoting mutations may be selected for based primarily on their growth-promoting capabilities. Similar oncogenic cooperation mediated through these evolutionarily conserved signaling pathways could contribute to human cancer progression (Igaki, 2006).
Most human cancers originate from epithelial tissues. These epithelial tumors, except for those derived from squamous epithelial cells, normally exhibit pronounced apicobasal polarity. However, these tumors commonly show defects in cell polarity as they progress toward malignancy. Although the integrity of cell polarity is essential for normal development, how cell polarity disruption contributes to the signaling mechanisms essential for tumor progression and metastasis is unknown. To address this, a recently established Drosophila model of Ras-induced tumor progression triggered by loss of cell polarity has been used. This fly tumor model exhibits many aspects of metastatic behaviors observed in human malignant cancers, such as basement membrane degradation, loss of E-cadherin expression, migration, invasion, and metastatic spread to other organ sites (Pagliarini, 2003). In the developing eye tissues of these animals, loss of apicobasal polarity is induced by disruption of evolutionarily conserved cell polarity genes such as scribble (scrib), lethal giant larvae (lgl), or discs large (dlg), three polarity genes that function together in a common genetic pathway, as well as other cell polarity genes such as bazooka, stardust, or cdc42. Oncogenic Ras (RasV12), a common alteration in human cancers, causes noninvasive benign overgrowths in these eye tissues (Pagliarini, 2003). Loss of any one of the cell polarity genes somehow strongly cooperates with the effect of RasV12 to promote excess tumor growth and metastatic behavior. However, on their own, clones of scrib mutant cells are eliminated during development in a JNK-dependent manner; expression of RasV12 in these mutant cells prevents this cell death (Igaki, 2006).
To better quantify the metastatic behavior of tumors in different mutant animals, the analysis focused on invasion of the ventral nerve cord (VNC), a process in which tumor cells leave the eye-antennal discs and optic lobes (the areas where they were born) and migrate to and invade a different organ, the VNC. It was further confirmed that the genotypes associated with the invasion of the VNC in this study also resulted in the presence of secondary tumor foci at distant locations, although the number and size of these foci were highly variable (Pagliarini, 2003; Igaki, 2006).
In analyzing the global expression profiles of noninvasive and invasive tumors induced in Drosophila developing eye discs, it was observed that expression of the JNK phosphatase puckered (puc) was strongly upregulated in the invasive tumors. Upregulation of puc represents activation of the JNK pathway in Drosophila. Therefore an enhancer-trap allele, puc-LacZ, was used to monitor the activation of JNK signaling in invasive tumor cells. Strong ectopic JNK activation was present in invasive tumors, while only a slight expression of puc was seen in restricted regions of RasV12-induced noninvasive overgrowth. Intriguingly, more intense JNK activation was seen in tumor cells located in the marginal region of the eye-antennal disc and tumor cells invading the VNC. Analysis of clones of cells with a cell polarity mutation alone revealed that JNK signaling was activated by mutation of cell polarity genes. Notably, JNK signaling was not activated in a strictly cell-autonomous fashion. JNK activation in these cells was further confirmed by anti-phospho-JNK antibody staining that detects activated JNK (Igaki, 2006).
To examine the contribution of JNK activation to metastatic behavior, the JNK pathway was blocked by overexpressing a dominant-negative form of Drosophila JNK (BskDN). As previously reported (Pagliarini, 2003), clones of cells mutant for scrib, lgl, or dlg do not proliferate as well as wild-type clones, while combination of these mutations with RasV12 expression resulted in massive and metastatic tumors. Strikingly, inhibition of JNK activation by BskDN completely blocked the invasion of the VNC, as well as secondary tumor foci formation. Drosophila has two homologs of TRAF proteins (DTRAF1 and DTRAF2), which mediate signals from cell surface receptors to the JNK kinase cascade in mammalian systems. It was found that RNAi-mediated inactivation of DTRAF2, but not DTRAF1, in the tumors strongly suppressed their metastatic behavior. Inactivation of dTAK1, a Drosophila JNK kinase kinase (JNKKK), or Hep, a JNKK, also suppressed metastatic behavior. Drosophila has two known cell surface receptors that act as triggers for the JNK pathway, Wengen (TNF receptor) and PVR (PDGF/VEGF receptor). Intriguingly, it was found that RNAi-mediated inactivation of Wengen partially suppressed tumor invasion. Inactivation of PVR, in contrast, did not show any suppressive effect on metastatic behavior. It was also found that the metastatic behavior of RasV12-expressing tumors that were also mutated for one of three other cell polarity genes, bazooka, stardust, or cdc42, was also blocked by BskDN. These data indicate that loss of cell polarity contributes to metastatic behavior by activating the evolutionarily conserved JNK pathway (Igaki, 2006).
Next, whether JNK activation is sufficient to trigger metastatic behavior in RasV12-induced benign tumors was examined. Two genetic alterations can be used to activate JNK in Drosophila. First, JNK signaling can be activated by overexpression of Eiger, a Drosophila TNF ligand. While mammalian TNF superfamily proteins activate both the JNK and NFκB pathways, Eiger has been shown to specifically activate the JNK pathway through dTAK1 and Hep. Indeed, the eye phenotype caused by Eiger overexpression could be reversed by blocking JNK through Bsk-IR (Bsk-RNAi). Second, overexpression of a constitutively activated form of Hep (HepCA) can also activate JNK signaling. However, the eye phenotype caused by HepCA overexpression was only slightly suppressed by Bsk-IR, suggesting that HepCA overexpression may have additional effects other than JNK activation. Therefore Eiger overexpression was used to activate JNK in RasV12-induced benign tumors, and it was found that the RasV12+Eiger-expressing tumor cells did not result in the invasion of the VNC. This indicates that loss of cell polarity must induce an additional downstream effect(s) essential for metastatic behavior. A strong candidate for the missing event is downregulation of the E-cadherin/catenin adhesion complex, since this complex is frequently downregulated in malignant human cancer cells and is also downregulated by loss of cell polarity genes in Drosophila invasive tumors (Pagliarini, 2003). In addition, it has been recently reported that higher motility of mammalian scrib knockdown cells can be partially rescued by overexpression of E-cadherin-catenin fusion protein, suggesting a role of E-cadherin in preventing polarity-dependent invasion. Furthermore, overexpression of E-cadherin blocks metastatic behavior of RasV12/scrib−/− tumors (Pagliarini, 2003), indicating that loss of E-cadherin is essential for inducing tumor invasion in this model. It was found that loss of the Drosophila E-cadherin homolog shotgun (shg), combined with the expression of both RasV12 and Eiger, induced the invasion of the VNC. Intriguingly, loss of shg in RasV12-expressing clones also showed a weak invasive phenotype at lower penetrance. In agreement with the essential role of JNK in tumor invasion, clones of shg−/− cells weakly upregulated puc expression. It was further found that JNK activation in dlg−/− clones is not blocked by overexpression of E-cadherin, suggesting that mechanism(s) other than loss of E-cadherin also exist for inducing JNK activation downstream of cell polarity disruption. The metastatic behavior of RasV12+Eiger/shg−/− tumors was completely blocked by coexpression of BskDN, indicating a cell-autonomous requirement of JNK activation for this process. Furthermore, it was found that loss of the β-catenin homolog armadillo also induced metastatic behavior in RasV12-induced benign tumors. In contrast, overexpression of HepCA in RasV12/shg−/− cells resulted in neither enhanced tumor growth nor metastatic behavior. Together, these results suggest that, although the RasV12+Eiger/shg−/− does not completely phenocopy the effect of RasV12/scrib−/−, activation of JNK signaling and inactivation of the E-cadherin/catenin complex are the downstream components of cell polarity disruption that trigger metastatic behavior in RasV12-induced benign tumors (Igaki, 2006).
Aside from its evolutionarily conserved role in cell migration and invasion, JNK signaling is also a potent activator of cell death in Drosophila and mammals. Although RasV12-expressing tissues showed a weak and restricted activation of JNK at later stages of development, mutation of cell polarity genes in combination with RasV12 expression constitutively activated JNK signaling. Striking acceleration of tumor growth occurred during days 5 and 6, and these tumors outcompeted surrounding wild-type tissues, resulting in a loss of the unlabeled wild-type cells and a dramatic increase in the GFP-expressing mutant tissue. The activated JNK was correlated with this accelerated tumor growth, suggesting that JNK signaling may play a role in tumor growth. Indeed, in addition to blocking metastatic behavior, inactivation of JNK pathway components strongly suppressed the accelerated tumor growth caused by cell polarity disruption. These results reveal that JNK signaling activated by loss of cell polarity also stimulates tumor growth (Igaki, 2006).
Since JNK signaling is required for both tumor growth and invasion, it was next asked whether these two phenotypes are separable processes. To address this, different types of tumors caused by alterations in genes involved in cell proliferation, growth, and cell polarity were analyzed. Day 6 RasV12/scrib−/− tumors showed moderate tumor growth and VNC invasion phenotypes. Loss of the Akt gene, a component of insulin growth signaling, considerably reduced the tumor load of RasV12/scrib−/− animals but did not impair metastatic behavior. In contrast, overexpression of Akt, combined with mutations in both the scrib gene and the lats gene, a potent tumor suppressor, did not cause metastatic behavior despite accelerated tumor growth comparable to RasV12/scrib−/−. In addition, although RasV12/Tsc1−/− mutant cells resulted in extremely large tumors, these tumor cells never exhibited metastatic behavior. These data indicate that tumor growth and invasion are separable processes in this model system (Igaki, 2006).
It was found that JNK signaling is indeed activated in polarity-deficient cells, and acridine orange staining revealed that most of these cells die. Interestingly, ectopic cell death was mostly blocked within clones of polarity-deficient cells also expressing RasV12, despite strong JNK activation. In addition, coexpression of RasV12 and Eiger, a potent inducer of cell death, resulted in accelerated tumor growth, although neither RasV12 alone nor Eiger alone caused dramatic overgrowth. This massive overgrowth was completely blocked by coexpression of BskDN. Moreover, stimulation of JNK signaling by expressing Eiger dramatically enhanced tumor growth of RasV12/shg−/− tissues, although Eiger/shg−/− clones were very small, probably because of cell death of these mutant clones. The accelerated growth of the RasV12+Eiger/shg−/− tumors was again completely blocked by BskDN. Together, these data indicate that, in the context of oncogenic Ras, JNK activation is the primary mediator of tumor growth downstream of cell polarity disruption. These observations suggest that JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras, and that the dramatic tumor growth is caused by cooperation between oncogenic Ras and JNK signaling (Igaki, 2006).
This study provides a molecular link between loss of cell polarity and tumor malignancy, namely activation of JNK signaling and inactivation of the E-cadherin/catenin complex in the context of oncogenic Ras activation. Although RasV12 alone only induces noninvasive overgrowth, and loss of cell polarity alone results in JNK-mediated cell death, the combination of these two alterations promotes both tumor growth and invasion through oncogenic cooperation. Thus, the tumor-promoting alterations caused by loss of cell polarity do not function alone and rather act as oncogenic Ras modifiers or “oncomodifiers” (Igaki, 2006).
The JNK signaling is essential for a variety of biological processes such as morphogenesis, cell proliferation, migration, invasion, and cell death. Genetic studies in Drosophila have demonstrated that JNK signaling is essential for epithelial cell movements and invasive behavior during normal development. A genetic study in mice revealed that TNF-triggered JNK signaling stimulates epidermal proliferation. These studies suggest that JNK may play an important role in tumorigenesis, tumor growth, and metastasis. Indeed, a substantial body of evidence indicates that JNK activation and c-Jun phosphorylation play important roles in cancer development. In mammalian cell culture systems, Ras acts cooperatively with JNK or c-Jun to enhance cellular transformation. Furthermore, knockin mice expressing a mutant form of c-Jun (JunS63A,S73A) suppress development of skin tumors in response to Ras activation and also block development of intestinal epithelial cancers caused by APC mutation. Moreover, liver-specific inactivation of c-Jun impairs development of chemically induced hepatocellular carcinomas. Furthermore, JNK signaling is activated in many tumor types. On the contrary, however, it has been also shown that JNK functions as a negative regulator for tumor development in Ras/p53-transformed fibroblasts. Thus, the role of JNK signaling seems to be highly dependent on cellular context, and, this study provides the first evidence for a cell-autonomous oncogenic cooperation between JNK and Ras signaling that promotes tumor growth and malignancy (Igaki, 2006).
How is JNK signaling activated? Loss of cell polarity may directly influence activity of a JNK pathway component. Alternatively, cell polarity defects may activate a cell surface receptor that triggers JNK signaling. The genetic analysis of multiple JNK pathway components suggests that the pathway is activated through a cell surface receptor, Wengen. It would be interesting to further investigate whether mislocalization or disregulation of Wengen, which should be normally tightly regulated in polarized epithelial cells, results in stimulation of JNK pathway signaling (Igaki, 2006).
The discovery that metastasis-promoting alterations (i.e., JNK activation) also increase tumor growth may explain why tumor cells acquire such mutations; that is, they primarily provide a selective advantage in tumor growth. Given that cell polarity defects are frequently associated with human tumor malignancy, and that the pathways identified in Drosophila are evolutionarily conserved, similar molecular mechanisms could be involved in human tumor progression. It would be particularly interesting to study these processes in human tumors with high frequencies of Ras mutations. If such processes prove conserved, components of these pathways, especially JNK signaling, could serve as potential therapeutic targets against such cancers (Igaki, 2006).
Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, null and hypomorph alleles of the Drosophila Ral gene have been generated. Ral null animals are not viable. Reduced Ral expression in cells of the sensory organ lineage has no effect on cell division but leads to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggest that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst, a protein complex involved in vesicles trafficking, specifically the tethering and spatial targeting of post-Golgi vesicles to the plasma membrane prior to vesicle fusion, is a Ral effector. Epistasis between mutants of Ral and of misshapen (msn), the fly ortholog of HGK, suggests the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. It is conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. It is proposed that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK (Balakireva, 2006).
The Ral pathway is an essential component of physiological Ras signaling as well as Ras-driven oncogenesis. It can be instrumental in oncogenic transformation, and an activated form of a Ral exchange factor, Rlf, recapitulates the capacity of Ras to transform immortalized human cell cultures, either alone or together with other Ras effectors. Reciprocally, the lack of RalGDS, another Ral exchange factor, reduces tumorigenesis in a multistage skin carcinogenesis model and transformation by Ras in tissue culture. The molecular basis of the Ral contribution to oncogenesis remains to be elucidated (Balakireva, 2006).
None of the Ral effectors and their attributed cellular functions are obvious actors in oncogenesis. One of the two well-documented Ral effectors, RLIP76/RalBP1, is involved in endocytosis. The other, the exocyst complex, is involved in secretion, polarized exocytosis, and migration and can be found at the tip of filopods and at tight junctions. The exocyst complex is composed of eight proteins, which have been initially identified via mutants of secretion in the budding yeast. Exocyst complexes are bound to vesicles and are supposed to participate in vesicle trafficking and tethering to the plasma membrane. Globally, Ral appears to be a regulator of vesicle trafficking with consequences on cell proliferation, cell fate, and cell signaling (Balakireva, 2006).
In order to gain insight into Ral function, a genetic and cell biology approach was undertaken using Drosophila, which has a single Ral gene. Null and hypomorph alleles of Ral were generated, and Ral was shown to be an essential gene. Ral loss-of-function has dramatic effects on the differentiation of sensory organ precursor cells and leads to caspase-8-independent cell death by releasing ectopic tumor necrosis factor (TNF) receptor-associated factor 1-c-Jun N-terminal kinase (TRAF1-JNK) signaling. Sensory organ cell survival in Ral mutants is rescued by an activation of p38 mitogen-activated protein (MAP) kinase, revealing an antiapoptotic function of this latter. The influence of Ral on sensory organ cell fate is directly mediated by the exocyst complex together with a novel interaction partner, the MAP4K4 (also known as hepatocyte progenitor kinase-like/germinal center kinase-like kinase [HGK] in mammals and Misshapen [MSN] in flies). This suggests that a Ral/exocyst/JNK regulatory axis may represent a key component of developmental regulatory programs (Balakireva, 2006).
Hypomorph mutations of Ral displayed a loss-of-bristle phenotype with sockets without shafts, as do flies expressing dominant negative alleles of Ral). Whereas Ral is expressed in many if not all tissues, the only situation where a decreased level of Ral appears compatible with adult viability leads to a developmental phenotype in the bristle sensory organs. In Ral mutants, the pI precursor cells undergo the right number of divisions with a correct timing, but afterward shaft cells die by apoptosis, showing that death hits after cell division and determination has taken place, during the subsequent differentiation stage (Balakireva, 2006).
The various pathways that lead to apoptosis for their interactions with Ral have been explored. The caspase-8-mediated pathway did not contribute to the Ral phenotype, as opposed to a caspase-9-mediated pathway. The JNK pathway, a cascade of four kinases starting with MSN (MAP4K4 or HGK in human), which requires formation of a complex with TRAF1 for its full activity, and ending at the Jun N-terminal kinase, was tested. Puckered is a phosphatase that dephosphorylates and deactivates JNK (Balakireva, 2006).
Loss-of bristle and apoptosis phenotypes due to decrease of Ral signaling were suppressed by down-regulation of the JNK pathway and enhanced by its up-regulation. Symmetrically, a phenotype due to a hyperactivation of the Ral pathway by the overexpression of RalG20V was suppressed and enhanced by enhancing or decreasing JNK signaling, respectively (Balakireva, 2006).
The fact that the enhancement and suppression can be induced by genetic alterations of TRAF and MSN as well as of JNK proteins suggests that Ral is a general negative regulator of this cascade. Dominant negative alleles of transcriptional effectors of the JNK, Jun itself but also Fos, suppress the Ral phenotype, suggesting that Ral regulates transcriptional events involved positively or negatively in apoptosis (Balakireva, 2006).
Down-regulating the JNK pathway is not only suppresses apoptosis in Ral-defective cells but also rescues normal bristle development. Together with data in S2 cells, where Ral behaves also as a negative regulator of JNK in the absence of any cell death (Sawamoto, 1999), the results suggest a functional relationship between Ral and the JNK pathway wherein Ral activation keeps JNK down. Data using activated and dominant negative alleles of Ral in mammalian cell culture support a positive effect of Ral on JNK activation. The source of this discrepancy, which might be due to cell- and/or context-specific interactions of Ral with the JNK pathway, is not understood. However, the current data obtained by RNA interference in HeLa cells are consistent with the fly model (Balakireva, 2006).
Epistatic relationships between Ral and p38 MAP kinase mutants revealed another actor in Ral-dependent apoptosis: the p38 MAP kinase behaves as an antiapoptotic kinase, which could be positively regulated by Ral (Balakireva, 2006).
A control of the basic JNK activity might serve two purposes: (1) it minimizes JNK activity and avoids undesirable cell death in normal conditions; (2) a low level of basal JNK activity allows better differential in activation of JNK when this activation happens in response to stresses that lead eventually to apoptosis (Balakireva, 2006).
The molecular basis of Ral action on the JNK pathway was addressed genetically and biochemically. The model that emerges is that the exocyst complex is the matchmaker between Ral and the JNK pathway, and the simplest interpretation of genetic data is that the exocyst works like a negative regulator of HGK activity. Finally, the exocyst complex was found to bind in vivo to HGK, providing a biochemical basis for the functional effect of Ral on JNK (Balakireva, 2006).
Decreasing the JNK pathway seems to favor the oncogenic capacity of Ras in mouse primary fibroblasts. The current results can explain one of the contributions of the Ral pathway to oncogenesi: cancer cells have to sustain proliferative signals and relieve proapoptotic signals, and Ral via the exocyst complex might be in charge, at least, of this latter task in oncogenesis. Finally, it has been recently shown that the exocyst complex carries enzymatic activities working in the NF-kappaB pathway. These data together with the present report widen the role of the exocyst to functions other than directing vesicle traffic and contributing to exocytosis (Balakireva, 2006).
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