1 Lineberger Comprehensive Cancer Center, The University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599-3280, USA
2 Department of Biology, The University of North Carolina at Chapel Hill, Chapel
Hill, NC 27599-3280, USA
Author for correspondence (e-mail:
mcewen{at}uthscsa.edu)
Accepted 21 June 2005
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SUMMARY |
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Key words: Apoptosis, JNK, Phosphatase, Drosophila
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Introduction |
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Three evolutionarily conserved MAPK pathways have been described. These
cascades relay information about the environment to the nucleus via the
sequential phosphorylation of a series of kinases, with terminal activation of
the MAPKs ERK, Jun N-terminal kinase (JNK) or p38 eliciting changes in gene
transcription (reviewed by Schaeffer and
Weber, 1999).
MAPKs are key modulators of cell proliferation, differentiation and
apoptosis during oncogenesis. The Ras-Raf-ERK pathway has received the most
attention for its role in promoting transformation. By contrast, the JNK
pathway has both oncogenic and tumor-suppressing roles depending on the
cellular context. For example, transformation of leukemia cell lines by
Bcr-Abl is dependent on JNK signaling
(Dickens et al., 1997;
Raitano et al., 1995
). By
contrast, chemotherapeutic agents promote JNK-dependent apoptosis
(Osborn and Chambers, 1996
;
Seimiya et al., 1997
;
Stadheim and Kucera, 2002
).
Thus, JNK signaling can both support and antagonize transformation; however,
the molecular mechanisms underlying this choice are not well defined.
JNK signaling is initiated by dual phosphorylation of threonine and
tyrosine residues in its activation loop, while removal of either phosphate
inactivates MAPKs (reviewed by Davis,
2000). Thus, steady-state levels of MAPK activity depend upon the
equilibrium between MEK kinases and protein phosphatases. MAPK phosphatases
(MKPs) are one class of proteins tempering MAPK activity (reviewed by
Keyse, 1998
). At least nine
vertebrate MKPs, all very similar in their phosphatase domains, have been
identified. One class, typified by MKP-1/CL100, are immediate-early genes:
their transcription is activated by JNK signaling and they limit the peak
level and duration of MAPK activity by completing a negative-feedback loop. As
differences in peak amplitude and duration of MAPK signaling can elicit
distinct cellular responses, MKP activity may play an important role in
regulating biological responses to stimuli.
Functional redundancy among the three JNK isoforms complicates analysis of
JNK signaling in mammals (reviewed by
Davis, 2000). By contrast, a
single JNK [basket (bsk)] exists in Drosophila and
plays an important role in embryonic development
(Riesgo-Escovar et al., 1996
;
Sluss et al., 1996
). Likewise,
little is known about the in vivo function of vertebrate MKPs, as functional
redundancy among MKPs (Dorfman et al.,
1996
) and overlapping substrate specificity of distinct MKPs
(Franklin and Kraft, 1997
;
Sun et al., 1993
) hinder the
assessment of phenotypes in vivo. Again, a single MKP, Puckered (Puc),
antagonizes JNK signaling in Drosophila
(Martin-Blanco et al., 1998
;
Rintalen et al., 2003). Thus, Drosophila has emerged as an excellent
model system for characterizing both the function and regulation of JNK
signaling in vivo.
Drosophila JNK signaling coordinates dorsal closure, a
morphogenetic event that unites the two lateral epidermal sheets. Mutations in
any component of the JNK pathway, like hemipterous (the JNK kinase),
bsk, kayak (kay; Drosophila fos) and Djun
(Jra FlyBase) disrupt dorsal closure (reviewed by
Noselli and Agnes, 1999;
Stronach and Perrimon, 1999
).
This may be an evolutionarily conserved mechanism, as JNK signaling regulates
vertebrate cell migration and epithelial wound healing (reviewed by
Martin and Parkhurst, 2004
).
puc zygotic mutants also exhibit defects in dorsal closure; increased
JNK signaling in puc mutants disrupts alignment of the lateral
epidermal sheets (Martin-Blanco et al.,
1998
). Thus, the tempering of JNK signaling by Puc is crucial
during dorsal closure.
In addition to regulating morphogenesis, JNK signaling is also activated by
other signaling pathways; in this context, JNK activation triggers apoptosis.
In Drosophila, these include TNF
(Igaki et al., 2002
;
Moreno et al., 2002b
),
TGFß (Adachi-Yamada et al.,
1999
; Adachi-Yamada and
O'Connor, 2002
; Adachi-Yamada
and O'Connor, 2004
) and Myc
(de la Cova et al., 2004
;
Moreno and Basler, 2004
).
However, the function of Puc during the induction of apoptosis is less
clear.
Previous analysis of Puc focused on zygotic mutants retaining maternally contributed Puc. Here, we extend this analysis by completely removing both maternal and zygotic Puc from embryos, and by eliminating Puc function in imaginal disc cells. We find that Puc is required continuously to antagonize JNK-dependent apoptosis in a cell-autonomous manner, suggesting that basal JNK signaling is poised just below a threshold required to eliminate epithelial cells. Second, we find that JNK signaling also plays a key role in promoting apoptosis in response to a diverse array of signals, including both the p53-dependent response to DNA damage and developmentally regulated apoptosis. Finally, we demonstrate that when apoptosis is blocked, Puc prevents tissue overgrowth by preventing unbridled JNK activation. Thus, JNK signaling can promote apoptosis or proliferation in different cellular contexts, and Puc plays an important role in controlling this balance.
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Materials and methods |
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Irradiation
Wandering 3rd-instar larvae were given 4000 rads of -radiation from
a 137Cs source, and allowed to recover for 4 hours at 25°C
before dissection.
Immunofluorescence
Dechorionated embryos were fixed for 20 minutes in 1:1 3.7%
formaldehyde/PBS:heptane and methanol-devitellinized. Discs were fixed with
3.7% formaldehyde/PBS+0.5% TritonX-100 (PBT5) for 20 minutes on ice. Tissues
were blocked for 3 hours at room temperature in PBS+0.1% TritonX-100 (PBT)
(embryos) or PBT5 (discs) + 10% BSA, incubated with primary antibody overnight
at 4°C, washed in PBT or PBT5, incubated for 1 hour at room temperature
with fluorescent secondary antibodies (1:1000; Molecular Probes) and
washed.
Antibodies
Rat--DE-cad2 (1:250; DSHB), mouse-
-phosphotyrosine (4G10
1:1000; Upstate),
-Engrailed (4D9 1:50; DSHB),
-ß-galactosidase (Promega; 1:1000) and
-Myc (9E10; 1:5)
were used. Alexa647- or Alexa568-phalloidin (Molecular
Probes) and Hoechst (Sigma) were used at 1:1000.
TUNEL labeling
Samples were post-fixed for 15 minutes in 3.7% formaldehyde in PBT5,
permeabilized in 100 mM sodium citrate/0.1% TritonX-100 at 65°C for 30
minutes, rinsed in PBT5, and free DNA ends fluorescein-labeled using the In
Situ Cell Death Detection Kit (Roche). Samples were mounted in Aqua Poly/mount
(Polysciences).
Image collection and processing
Images of embryos, wings and legs were captured on a Nikon OPTIPHOT-2 with
Kodak Technical Pan film and digitized with a Polaroid SprintScan35. Eyes,
thorax and genitalia were photographed with a stereomicroscope-mounted Nikon
Coolpix4500 digital camera after immersion in 70% ethanol. z-series
were collected with a Zeiss 510 confocal microscope, and brightest-point
projections generated with ZeissLSM software. Brightness, contrast and levels
were adjusted in Photoshop 7.0.
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Results |
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Maternally contributed mRNA and protein often mask requirements for genes
during early Drosophila development; thus, we used the
hs-flp/DFS technique (Chou and
Perrimon, 1992) to remove both maternal and zygotic Puc function.
Mothers with germlines homozygous for the strongest allele,
pucR10, failed to produce eggs, but females with germlines
homozygous for pucA251.1, a strong allele, lay reduced
numbers of small eggs, allowing us to assess the effect of complete loss of
Puc function.
pucA251.1 maternal/zygotic (M/Z) mutant embryos have
severe defects in embryonic development, secreting only fragmented scraps of
cuticle (compare Fig. 1B with
1A; henceforth, unless noted, puc refers to
pucA251.1). The same phenotype was noted using different
paternally contributed puc alleles, suggesting it is due to
puc and not another second-site mutation (see Table S1 in the
supplementary material). Similar `scraps'-like phenotypes are observed when
epithelial cells initiate apoptosis (e.g. crb mutants)
(Tepass et al., 1990). Thus,
we used TUNEL labeling to assess whether the pucM/Z
phenotype resulted from widespread apoptosis. Late-stage
pucM/Z mutant embryos have disorganized epithelia with
highly elevated numbers of TUNEL-positive cells
(Fig. 1G,H) relative to
similarly staged wild-type controls (Fig.
1E,F). A similar increase in apoptosis was observed in living
pucM/Z mutants stained with Acridine Orange (data not
shown). Thus, Puc normally prevents the induction of apoptosis throughout the
embryonic epidermis.
The cuticle defects and widespread apoptosis of pucM/Z mutants are suppressed if the embryo receives paternal wild-type Puc (Fig. 1C,I,J,), but this fails to rescue embryo viability. puc maternal-only mutants have patterning defects distinct from those of zygotic mutants, exhibiting segmental deletions and fusions, as evidenced by loss of Engrailed stripes (compare Fig. 1L with 1K) and the corresponding loss of denticle belts (compare Fig. 1C with 1D). Thus, maternal Puc plays a distinct role in establishing segmentation, but we have not pursued this role further.
Given the clear role for Puc in preventing apoptosis in
pucM/Z mutants, we re-examined the ventral
segment-polarity phenotype of puc zygotic mutants
(McEwen et al., 2000) to see
if this might also be, in part, the result of apoptosis. Increased numbers of
apoptotic cells were observed in a segmentally repeated pattern in both the
ventral and lateral epidermis (compare Fig.
1M with 1F); JNK signaling is upregulated in a similar segmental
pattern (Fig. 1M). Thus, the
puc zygotic mutant phenotype also reflects an increase in
JNK-dependent apoptosis.
|
|
Next, we assessed whether Puc plays a similar role in eye discs. Removal of Puc function from the entire eye imaginal disc results in nearly complete loss of eye tissue (pucA251.1, Fig. 2F; pucR10, Fig. 2G). To assess whether loss of puc mutant eye tissue resulted from inappropriate JNK activation, puc mutant eyes were generated in flies with reduced levels of JNK. Heterozygosity for bsk2 partially rescued puc mutant eye tissue (Fig. 2H).
Together, these data demonstrate a crucial role for Puc in preventing apoptosis in embryonic and larval epithelial cells. Apoptosis occurred in the absence of any outside stress (other than the loss of Puc) that might stimulate JNK activity, suggesting that Puc prevents apoptosis by buffering basal levels of JNK signaling.
JNK and MKP activities regulate radiation-induced apoptosis in a p53-dependent manner
Next, we explored whether Puc plays a broader role in regulating
JNK-induced apoptosis by examining -radiation-induced cell death. To
assess whether JNK activity is upregulated in response to
-irradiation,
ß-galactosidase (ß-gal) expression from an enhancer trap in
puc (Martin-Blanco et al.,
1998
) was monitored. puc enhancer traps have been widely
used to monitor JNK activity in vivo (e.g.
Adachi-Yamada et al., 1999
;
Igaki et al., 2002
;
Tateno et al., 2000
). For
simplicity we subsequently refer to this readout as JNK reporter
(JNKREP) activity. In the wing disc, JNKREP activity is
normally limited to the peripodial membrane overlying the notal region of the
wing disc (Fig. 3B). However,
-irradiation strongly induced both JNKREP activity
(Fig. 3C) and apoptosis
(Ollmann et al., 2000
)
throughout the wing disc within 4 hours.
To confirm that JNKREP activation reflects JNK activation in situ, we used Myc-tagged Puc (Myc-Puc) to antagonize JNK activity. Expression of Myc-Puc in the posterior compartment of the wing disc (Fig. 3A) prevented radiation-induced JNKREP activation (Fig. 3D). Interestingly, it also substantially reduced radiation-induced apoptosis (Fig. 3G,G'). By contrast, expression of a catalytically-inactive form of Puc (Myc-PucDEAD) failed to block either JNKREP activity (Fig. 3E) or apoptosis (Fig. 3H,H'). Thus, in Drosophila, radiation-induced apoptosis depends, at least in part, on JNK signaling.
Discontinuities in morphogen gradients induce JNK-dependent apoptosis,
perhaps by promoting discontinuities in a JNK activation gradient
(Adachi-Yamada et al., 1999;
Adachi-Yamada and O'Connor,
2002
). However, lowering JNK activity by expressing Myc-Puc in the
posterior compartment did not trigger apoptosis along the anteroposterior (AP)
compartment border (Fig. 3F).
Furthermore, expression of Myc-Puc in a stripe anterior to the A-P compartment
boundary using patched-Gal4 or in clonal patches throughout the wing
disc by flp-out mediated gene induction did not induce autonomous or
non-autonomous apoptosis, but it blocked radiation-induced apoptosis (data not
shown).
p53 is a key regulator of cell cycle progression and apoptosis in mammals,
and is mutated in more than half of all tumors. Like its ortholog,
Drosophila p53 is required for radiation-induced apoptosis and can
induce apoptosis when overexpressed
(Brodsky et al., 2000;
Brodsky et al., 2004
;
Ollmann et al., 2000
;
Jassim et al., 2003
;
Lee et al., 2003
;
Sogame et al., 2003
). However,
unlike mammalian p53, overexpression of fly p53 does not induce G1
arrest (Ollmann et al., 2000
)
and it is not required for G2/M arrest in response to radiation
(Brodsky et al., 2000
).
|
Although p53 is not required for cell viability, it is thought to monitor genomic integrity during normal development. We thus asked whether the p53 and JNK pathways interact in the developing eye disc in the absence of stress. Although inactivation of puc in the eye disc results in ablation of the adult eye (Fig. 2F,G), concomitant loss of p53 suppressed cell death to some extent (Fig. 2I). As loss of p53 alone has no effect on normal eye development, this may suggest that p53 plays a more general role in cell viability by regulating basal JNK activation.
Reaper functions downstream of JNK signaling
Reaper (Rpr), Head Involution Defective (Hid) and Grim (collectively
referred to as RHG proteins) are key regulators of apoptosis that promote
caspase activation via IAP degradation. Transcriptional upregulation of RHG
genes precedes developmental and stress-induced apoptosis, whereas
mis-expression of any these genes induces apoptosis. In developing embryos,
rpr-11-lacZ has been used to assess rpr induction in
response to both developmental defects as well as radiation
(Nordstrom et al., 1996).
To test whether JNK signaling can regulate rpr reporter expression
in wing discs, we examined the effects of JNK activation on
rpr-11-lacZ expression. In un-irradiated rpr-11-lacZ/+ wing
discs, basal levels of reporter are seen, with ß-gal expression highest
at the wing margin and along the anterior side of the AP compartment boundary
(Fig. 4A). -Radiation
triggers a substantial increase in ß-gal expression throughout the wing
pouch (Fig. 4B), consistent
with previous work. To determine whether this induction by radiation is JNK
dependent, we overexpressed Myc-Puc throughout the posterior compartment of
rpr-11-lacZ/+ larvae. Inactivation of JNK signaling by Myc-Puc
overexpression in the posterior compartment blocked induction of the reporter
by irradiation (Fig. 4D; it
also blocked ß-gal expression along the wing margin in un-irradiated
discs; Fig. 4C). Together,
these results suggest that JNK signaling may promote apoptosis by
transcriptionally upregulating rpr expression; this is subject to the
caveat that the rpr-11-lacZ reporter does not perfectly reflect
endogenous rpr expression (e.g.
Nordstrom et al., 1996
).
However, JNK-induced upregulation of hid was observed in eye imaginal
discs (Moreno et al.,
2002b
).
Previous work suggested that caspase activation might regulate JNK
signaling in Drosophila (Kuranaga
et al., 2002; Ryoo et al.,
2004
). To test directly whether RHG protein-mediated induction of
apoptosis triggers JNK activation, we examined JNKREP activity in
response to rpr and hid mis-expression. When we used
en-Gal4 to direct rpr expression in embryos, cell death was
observed in en stripes, without concomitant JNK activation
(Fig. 1O,P). As
en-GAL4-driven rpr is embryonic lethal, we could not use
this to examine wing discs. We thus used heat-shock to express hid in
wing discs. A one-hour heat-shock of
pucA251.1/hs-hid larvae induced apoptosis
throughout the wing disc (data not shown), but did not elevate
JNKREP activity (Fig.
4G,H; a slight increase in JNKREP was sometimes seen in
the overlying peripodial membrane, as determined by the superficial position
and size of the nuclei in H). This contrasts with the effect of
-irradiation (Fig. 3C). Heat-shock of control pucA251.1/+ wing discs did not
induce JNKREP (compare Fig.
3B to Fig. 4E,F) or
apoptosis (data not shown). Thus, RHG-induced apoptosis can occur without JNK
activation.
|
JNK signaling regulates programmed cell death during normal development
In Drosophila, apoptosis plays crucial roles in establishing
tissue architecture and removing excess cells during normal development. For
example, inactivation of hid leads to mis-orientation of the external
male genitalia (Abbott and Lengyel,
1991), and inactivation of either dark
(Drosophila Apaf) or Traf1 (Drosophila
tumor-necrosis factor receptor-associated factor1) leads to extra scutellar
bristles (Kanuka et al., 1999
;
Kuranaga et al., 2002
;
Rodriguez et al., 1999
). To
test whether JNK signaling is required for developmentally regulated
apoptosis, we blocked JNK signaling in the genital and wing discs.
Inhibition of apoptosis by expressing the baculovirus caspase inhibitor p35
resulted in genital mis-orientation (Fig.
5B; quantified in 5D), phenocopying reduction in Hid function.
Mis-expression of Myc-Puc had a similar effect
(Fig. 5C, quantified in 5D),
while control constructs (GFP or Myc-PucDEAD) failed to affect
genital orientation (Fig. 5D).
Extra scutellar bristles were produced when we prevented apoptosis in the
scutellar region of the notum by expressing p35 with ptc-Gal4
(Fig. 5F; mean=5.4
bristles/fly, range=4-10, compared with four bristles in wild-type).
ptc-Gal4-mediated expression of Myc-Puc also increased bristle number
(Fig. 5G; mean=6.71
bristles/fly; range=4-10), whereas neither GFP nor Myc-PucDEAD
altered the number of scutellar bristles
(Fig. 5H; means=4.07 and 4.15
bristles/fly, respectively). While our manuscript was in preparation, Macias
et al. (Macias et al., 2004)
also reported that Puc or p35 mis-expression both block genital rotation.
Together, these results suggest that JNK signaling is required for
developmentally regulated, caspase-dependent apoptosis.
When cell death is blocked, inappropriate activation of JNK signaling can promote overgrowth
Growth of different compartments of the wing disc is strictly coordinated
so that the adult wing has a defined shape and precisely matched surfaces. In
normal wings, proliferation and apoptosis are coordinated to correct
experimental perturbations, and presumably to correct defects arising during
normal development (de la Cova et al.,
2004; Neufeld et al.,
1998
). However, apoptosis does not appear to play an essential
role in patterning of the wild-type wing, as over-expression of the caspase
inhibitor p35 in the posterior compartment has little or no effect on wing
pattern (over 96% of the wings of en-Gal4/UAS-p35 flies appear
normal; Fig. 6B) (see also
Pérez-Garijo et al.,
2004
), although altering apoptosis may affect wing size
(de la Cova et al., 2004
).
By contrast, when apoptosis is blocked in the posterior compartment of puc heterozygotes (en-Gal4/UAS-p35; puc/+), we observed tissue overgrowths at random positions throughout the posterior compartment in >90% of the flies (Fig. 6C-G). Overgrowths arising in the interior of the wing blade resemble wing blisters; however, there appear to be excess cells in only one compartment, as outgrowths project from the dorsal or ventral surfaces, but not both (Fig. 6C,E). When overgrowths arise along the wing margin, excess tissue is readily apparent as an extension of the wing (Fig. 6D,F). Cells within overgrowths respond to developmental cues, adopting appropriate cell fates (Fig. 6E-G). Similar but less frequent outgrowths were observed in posterior regions of adult legs (Fig. 6H). Thus, when cell death is blocked and restraints on JNK activation are relaxed by puc heterozygosity, small groups of cells escape regulation by the machinery that normally synchronizes growth of different cell populations in wing discs.
|
While our manuscript was in preparation, three groups reported that when
they induced apoptosis by various stimuli and blocked caspase activity, it
also triggered overgrowth (Ryoo et al.,
2004; Pérez-Garijo et
al., 2004
; Huh et al.,
2004
). Each group found that some of the `undead' cells expressed
the morphogen and disc growth factor Wg. We examined whether this was also the
case in our situation. In wing discs from en-Gal4/UAS-p35;puc/+
larvae, a subset of cells in JNKREP-positive foci in the wing pouch
ectopically expressed Wg (Fig.
6P); however, in control discs Wg is only expressed in a stripe at
the dorsal/ventral compartment boundary
(Fig. 6O). Ectopic Wg
expression, like JNKREP activation, was confined to the posterior
compartment where p35 was expressed.
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Discussion |
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Puc restrains apoptosis in the absence of stress
Mammalian JNK signaling promotes apoptosis in response to both extrinsic
(e.g. TNF) and intrinsic (e.g. DNA damage) cues
(Davis, 2000
).
Drosophila JNK signaling plays a similar role in apoptosis triggered
by extrinsic cues (Adachi-Yamada et al.,
1999
; Adachi-Yamada and
O'Connor, 2002
; Burke and
Basler, 1996
; Igaki et al.,
2002
; Kuranaga et al.,
2002
; Martin et al.,
2004
; Moreno et al.,
2002a
; Moreno et al.,
2002b
), but its role in response to intrinsic cues remained to be
established. The role of Puc in regulating responses to either sort of signal
was also unclear.
We 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
(Brondello et al., 1999;
Lin et al., 2003
). Thus, cell
signals may influence apoptosis by modulating MKP accumulation.
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
(Martin-Blanco et al., 1998).
However, this JNK signaling does not trigger apoptosis
(Fig. 1N). 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, as Dpp
is a survival factor in wing discs (e.g.
Moreno et al., 2002a
).
Interplay between death and survival signals may also explain the strongly
synergistic stimulation of apoptosis in the embryonic epidermis we previously
observed when we simultaneously eliminated zygotic Puc (lowering the threshold
for JNK signaling) and removed the survival signal Wg
(McEwen et al., 2000
).
Fitting JNK into the circuitry regulating radiation-induced apoptosis
Studies of JNK signaling in mammalian cells suggest that it plays a role in
radiation-induced apoptosis (Davis,
2000). We tested this hypothesis. When we block JNK activation 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
(Tournier et al., 2000
).
In mammalian cells, prolonged JNK activity correlates with apoptosis
(Chen et al., 1996), while
transient activation induces cell proliferation
(Sabapathy et al., 1999
).
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.
Our data provide an instance of how this may regulate the DNA damage response.
puc is upregulated by
-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.
p53 is a key regulator of decisions between life and death in response to
DNA damage. Thus, we investigated how JNK signaling and p53 are integrated.
Expression of p53 activated JNK, as revealed by its effect on the
JNKREP, while loss of p53 prevented 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 (Adachi-Yamada et al.,
1999; McEwen et al.,
2000
).
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 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 (Bulavin et al.,
1999; Huang et al.,
1999
), and JNK can regulate p53 stability/activity via direct
phosphorylation (Buschmann et al.,
2001
). 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.
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
(reviewed by Martin, 2002).
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
(Graves et al., 1998
), while
other data suggest that Mst1 activates caspases via a JNK-dependent pathway
(Ura et al., 2001
). Likewise,
in Drosophila, initiation of an apoptotic response by inactivation of
DIAP1 may lead to caspase-independent induction of a JNKREP
(Kuranaga et al., 2002
;
Ryoo et al., 2004
).
We directly assessed regulatory relationships between JNK activation, RHG
proteins and caspase activation. Our 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, promotes hid expression and apoptosis in eye
discs (Moreno et al.,
2002b).
To assess whether JNK signaling can be triggered by caspase activation, we examined JNKREP activity in response to Rpr or Hid expression. Both induced apoptosis without concomitant JNK activation. Furthermore, our 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.
JNK-induced apoptosis during normal development
Work on JNK-induced apoptosis in Drosophila has focused on its
role in the complex processes shaping organ size. Wing discs 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 TGFß family member Dpp
(Burke and Basler, 1996;
Martin et al., 2004
;
Moreno et al., 2002a
). 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 (e.g. Adachi-Yamada
and O'Connor, 2002
).
Our data clarifies roles for JNK signaling in adult development
(Agnes et al., 1999). Our
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, as JNK inactivation in the posterior
compartment (by Myc-Puc mis-expression) did not have drastic consequences. We
did identify roles for JNK signaling in genitalia and thoracic bristles, where
it regulates developmentally programmed apoptosis; this was also reported by
Macias et al. (Macias et al.,
2004
).
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- (Behrens et al.,
2000; Smeal et al.,
1991
) or BCR-Abl-mediated
(Dickens et al., 1997
;
Raitano et al., 1995
)
transformation. As 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.
Inhibition of apoptosis is one prerequisite for tumorigenesis. We thus examined the consequences of JNK activation 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.
Ryoo et al. (Ryoo et al.,
2004), Pérez-Garijo et al.
(Pérez-Garijo et al.,
2004
), and Huh et al. (Huh et
al., 2004
) recently reported related results, inducing apoptosis
by diap inactivation, irradiation or Hid expression, while
simultaneously blocking caspase activation. This triggered 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. Ryoo et al. show that
Wg signaling is required for compensatory proliferation. Our results extend
theirs, as our experiment differed in one significant way: we 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 our experiment), but JNK-induced
apoptosis rapidly eliminates them. We also found that at least a subset of the
undead cells activated 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.
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 TGFß, suggesting how JNK signaling can be both pro- and anti-tumorigenic. Future experiments should address mechanisms by which JNK activation triggers Wnt and TGFß signaling. Our data also suggest that runaway JNK activation can promote cell-autonomous proliferation, as 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.
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. As mitotic recombination in
somatic tissue can occur (Baker et al.,
1978), Puc might act in a manner analogous to classic tumor
suppressor genes. Additional studies will be required to distinguish between
these possibilities.
In summary, our 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. Our
results position JNK signaling in the hierarchy of events regulating
radiation-induced apoptosis. Finally, our data support the possibility
previously suggested by cytogenetics (Armes
et al., 2004; Furukawa et al.,
2003
) 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.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/17/3935/DC1
* Present address: Children's Cancer Research Institute, Universiity of Texas
Health Science Center at San Antonio, San Antonio, TX 78229, USA
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