Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, No. 1 section 1 Jen-Ai Road, Taipei 100, Taiwan, Republic of China
* Author for correspondence (e-mail: zfchang{at}ha.mc.ntu.edu.tw)
Accepted 7 May 2003
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Summary |
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Key words: Rho kinase, Myosin light chain, Phosphorylation, Apoptosis, Caspase
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Introduction |
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Similar to other members of the Rho family of small GTPases, RhoA cycles
between a GDP-bound inactive form and a GTP-bound active form that is
necessary for interaction with and activation of their effectors. When cells
are stimulated with LPA or serum, RhoA is converted to the GTP-bound form
(Goetzl and An, 1998), which
binds to specific effectors and exerts its biological functions, including
cell adhesion, motility, enhancement of the contractile response, cytokinesis
and transcriptional regulation (Bishop and
Hall, 2000
). The best-characterized function of RhoA is the
regulation of the architecture of the actin cytoskeleton. Activated RhoA
mediates the formation of stress fibers, which are elongated actin bundles
that traverse the cells and promote cell attachment to the extracellular
matrix through focal adhesions. Unlike that in fibroblasts, activated RhoA in
hematopoietic cells prevents cell adhesion induced by PMA
(Aepfelbacher, 1995
;
Kaibuchi et al., 1999
;
Lai et al., 2001
). For TF-1
cells, we have established that the PMA treatment induces apoptosis in cells
that do not display adhesion, which involves upregulation of RhoA activity
(Lai et al., 2001
). Two forms
of serine/threonine kinase (Rho kinases, ROCKI and ROCKII) have been
identified as effectors of RhoA (Amano et
al., 2000
; Matsui et al.,
1996
). RhoA in GTP-bound form interacts with ROCK proteins and
activates their kinase activity by disrupting their autoinhibition. Activation
of ROCK regulates the phosphorylation of myosin light chain (MLC) by directly
phosphorylating MLC at Thr18 and Ser19, and by the inactivation of myosin
phosphatase (Amano et al.,
1996
; Kimura et al.,
1996
). Phosphorylation of MLC activates myosin ATPase activity,
which couples with actin-myosin filaments to the plasma membrane, thus
increasing the actin-myosin force generation and cell contractility. In
addition, recent studies have linked this contraction event to membrane
blebbing and apoptotic body formation observed in apoptotic cells. ROCKI, a
substrate of caspase-3, is activated by cleavage in its C-terminal region,
which is involved in its negative regulation
(Coleman et al., 2001
;
Sebbagh et al., 2001
). The
cleaved form of ROCKI activates MLC phosphorylation, thus resulting in
formation of membrane blebbing in apoptotic cells. These studies suggest that
caspase-3 activation mediates Rho-independent ROCKI activation, which
generates contraction force as the characteristic of apoptotic phenotype.
Distinctly, in this study, we demonstrated that ROCK-mediated MLC
phosphorylation acts as an upstream event required for membrane contraction
and the subsequent caspase-3 activation during PMA stimulation.
Caspases comprise a family of different cysteine proteases that are
synthesized as inactive zymogens and are activated by proteolysis. It is well
established that different initiator pathways can activate caspase-3 in
response to various apoptotic stimuli
(Thornberry and Lazebnik,
1998). In general, a pro-apoptotic signal generated from a death
receptor or mitochondria can activate an initiator or upstream caspase, which
usually possesses a long NH2-terminal prodomain such as found in
caspases-8, -9 and -10 (Muzio et al.,
1996
; Strasser et al.,
2000
; Yang et al.,
1998
). In turn, these initiator caspases can activate the effector
caspases, such as caspases-3 and -6, which result in apoptotic execution. One
of the best-defined apoptotic pathways is mediated by death receptors such as
CD95 or tumor necrosis factor receptors (TNFRs). Upon ligand binding, the
intracellular death domain of the death receptor recruits Fas-associated death
domain (FADD) through protein-protein interaction. FADD links the receptor to
the apoptotic caspase, procaspase-8 or -10, through homotypic interactions of
death effector domains (DED), to form a death-inducing signaling complex
(DISC); this, in turn, leads to oligomerization and activation of these two
zymogens by self cleavage and the subsequent apoptotic cascade
(Ashkenazi and Dixit, 1998
;
Kischkel et al., 2001
;
Vincenz and Dixit, 1997
;
Wang et al., 2001
).
In this study, we also provided the first evidence that PMA-induced activation of caspase-8 and -10 in TF-1 cells is controlled by ROCK-mediated activation of myosin motor activities, by which effector caspase-3 is activated to trigger this apoptotic pathway. In particular, we showed that DISC formation is enhanced in PMA-induced apoptotic cells. On the basis of these data, we propose that activation of the RhoA/ROCK/MLC phosphorylation pathway in cooperation with PMA signaling in cells provides a cellular context that generates an initial membrane contraction, which in turn leads to activation of caspase-8 and -10 through a mechanism involving membrane receptor-mediated signaling.
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Materials and Methods |
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Cell culture
TF-1 cells were maintained in RPMI-1640 supplemented with 10%
heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 u/ml
penicillin G, 100 u/ml streptomycin and 1 ng/ml of granulocyte-macrophage
colony stimulation factor (GM-CSF). Human GM-CSF was purchased from R & D
Systems (Minneapolis, MN).
Constructs
RhoAV14 in the pcDNA3 vector was constructed as described previously
(Lai et al., 2001). The cDNA
of ROCK(CAT) in pEF-BOS-myc vector was from K. Kaibuchi (Nara Institute of
Science and Technology, Ikoma, Japan) and was further subcloned to pcDNA3
vector. The pEGFP expression plasmid was purchased from Clontech Laboratories
(Palo Alto, CA). The protease-inactive mutant of procaspase-10L
C401S was a gift from M. J. Lenardo (National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, USA). The fusion
construct of procaspase-10 C/S-flag was generated by inserting
procaspase-10L C401S into the pRK5 expression vector to have
flag-tag in-frame fused to the C-terminus of procaspase-10L
C401S.
Caspases-3 assay
Caspases-3 activity was assayed in 100 µl of reaction mixtures with
Acetyl-DEVD-pNA (CalbioChem-Novabiochem). Briefly, cells were lysed
in the buffer [20 mM Hepes, pH 7.8, 100 mM NaCl, 1 mM EDTA, 10 mM EGTA, 1 mM
DTT, 0.1% Chaps, 10% sucrose, 1 mM phenyl methylsulfonic fluoride, 1 µg/ml
each of leupeptin and aprotinin], after which equal amounts of lysates
containing 100 µg protein were incubated with the substrate peptide (0.4
mM) at 37°C for 2 hours, followed by reading the absorbance at 405 nm with
a spectrophotometer.
SDS-PAGE and immunoblotting
Samples containing equal amounts of proteins (50 µg) were separated by
10% SDS-PAGE and electrophoretically transferred to a PVDF membrane
(Millipore). The antibodies used and their dilutions were as follows: rabbit
polyclonal anti-ROCKI (Santa Cruz) at 1:1000, anti-MLC (Sigma) at 1:5000,
anti-phosphoMLC at 1:250, anti-caspases-3, -8 and -10 at 1:1000 dilution,
anti-FADD (CalbioChem-Novabiochem) at 1:1000. Horse radish peroxidase
(HRP)-conjugated goat anti-rabbit IgG or anti-mouse IgG antibody (Santa Cruz)
was used for the detection of the primary antibodies. The ECL detection for
HRP reaction was performed according to the vender's instruction.
Detection of apoptosis by DNA fragmentation assay
2x106 cells were collected following treatment and the
cell pellets were then lysed in 100 µl of lysis buffer [50 mM Tris-HCl, pH
7.4, 10 mM EDTA, 1% SDS, 0.5 mg/ml proteinase K] and incubated at 50°C for
3 hours. DNase-free RNase A (final 0.5 mg/ml) was added, and incubated for
another 3 hours. After phenol/chloroform extraction, samples were mixed with
loading buffer [50% glycerol, 0.25% bromophenol blue] and were resolved on a
1.8% agarose gel.
Transient transfection
5x106 cells were washed twice with STBS buffer [25 mM
Tris-HCl, pH 7.4, 5 mM KCl, 0.7 mM CaCl2, 137 mM NaCl, 0.6 mM
Na2HPO4, 0.5 mM MgCl2]. The cell pellet was
resuspended in 250 µl of STBS containing 600 µg DEAE-dextran and 6 µg
of plasmid DNA consisting of 4.5 µg of expression plasmid plus 1.5 µg of
pEGFP. After incubation for 20 minutes at 37°C, 5 ml of STBS buffer was
added to the transfection mixture. Cells were then centrifuged and resuspended
in 10 ml of RPMI-1640 containing 10% heat-inactivated FBS and GM-CSF, and
incubated for 48 hours at 37°C prior to the treatment.
Fluorescence microscopy
After transfection for 48 hours, cells were pelleted and resuspended in
fresh RPMI-1640 medium. Following treatment without or with PMA for 8 hours,
cells that remained in suspension were collected by centrifugation at 800
g for 5 minutes. Cells expressing GFP were counted using an
Olympus AX-70 fluorescence microscope.
DISC analysis by immunoprecipitation
D2 or TF-1 cells were transfected with the expression plasmid procaspase-10
C/S-flag as described above. After transfection for 48 hours,
13x107 cells with different treatment were washed with
ice-cold PBS, and collected for the subsequent lysis with buffer [30 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM
PMSF and protease inhibitor cocktail (Sigma)] for 30 minutes at 4°C. The
post-nuclear supernatants were obtained by centrifugation at 15,000
g for 20 minutes at 4°C, and then were rotated at 4°C
overnight in the presence of 25 µl of anti-flag M2 beads (Sigma) to
immunoprecipitate procaspase-10 C/S-flag-containing complex. After six washes
with lysis buffer, the immunocomplexes were analyzed by SDS-PAGE followed by
immunoblotting analysis using antibody against FADD
(CalbioChem-Novabiochem).
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Results |
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Membrane contraction was observed for TF-1 cells as early as 1 hour after
PMA treatment (Fig. 1C). It has
been extensively studied that MLC phosphorylation plays a crucial role in
actomyosin formation and contributes to membrane contractility. Therefore, we
used antibody specific for phophorylated MLC
(Ratcliffe et al., 1999) to
examine the phosphorylation status of MLC after PMA treatment. The level of
MLC phosphorylation became detectable after 3 hours of PMA treatment and was
further increased, accompanied with the subsequent cleavage of procaspase-3 at
6 hours (Fig. 1D). It has been
shown that, by removing its C-terminal inhibitory domain, caspase-3-mediated
cleavage of ROCKI activates its intrinsic kinase activity, resulting in
myosin-driven membrane blebbing (Coleman et
al., 2001
; Sebbagh et al.,
2001
). However, in our case, a short form of ROCKI, corresponding
to a cleaved active form, was not detected until 9 hours in PMA-induced
apoptotic cells, where procaspase-3 was already extensively cleaved.
Consistently, MLC became further heavily phosphorylated concomitantly with
ROCKI cleavage. Probably, the PMA treatment induces initial MLC
phosphorylation and membrane blebbing prior to the onset of caspase-3
activation.
Correlation between effects of serum or LPA on ROCK-mediated MLC
phosphorylation and cell death during PMA treatment
When TF-1 cells were incubated in serum-free medium, cells were attached to
the plate and remained viable following PMA treatment. By contrast, the
presence of serum or LPA promotes PMA-induced apoptosis, which can be
prevented with pretreatment of cells with a specific inhibitor of ROCK, Y27632
(Narumiya et al., 2000)
(Fig. 2A). We next tested
whether the ROCK/MLC phosphorylation pathway is absent in TF-1 cells incubated
in serum-free medium. As shown in Fig.
2B, MLC phosphorylation was indeed undetectable in these survived
and adherent cells, and could be seen in the cells incubated in the medium
containing either LPA or serum, which underwent subsequent cell death.
Treatment of cells with Y27632 abrogated PMA-induced apoptosis and MLC
phosphorylation for those cells incubated in LPA or serum-containing medium
(Fig. 2B). Whereas the extent
of PMA-induced phosphorylation of ERK was similar in the cells incubated in
the medium regardless of the presence or absence of serum or LPA
(Fig. 2B), it is likely that
MLC phosphorylation is specifically associated with upregulation of the
LPA/RhoA pathway in a ROCK-dependent manner and correlates well with
PMA-induced apoptosis.
|
MLC phosphorylation and membrane contraction occur upstream of
caspase activation during PMA induction
Since PMA-induced apoptosis is associated with early contraction, we then
addressed the questions of whether membrane contraction is a direct result of
caspase activation. We found that cells pretreated with a general caspase
inhibitor (Boc-D-FMK) still displayed the contraction phenotype following PMA
treatment, whereas cell death was significantly inhibited. This result implied
not only that caspase activity is responsible for the major execution of
PMA-induced cell death, but also that membrane contraction is not a downstream
event of caspase activation (Fig.
3A). The involvement of ROCK in cell contraction and cell death
was further assessed by pretreatment of cells with Y27632. Following PMA
treatment, these Y27632-pretreated cells remained rounded or spread without
the appearance of membrane contraction and were viable. Pretreatment of cells
with latrunculin B, an actin polymerization inhibitor
(Spector et al., 1989), also
abolished contraction and resulted in cell survival during PMA stimulation,
confirming the necessary role of actin in generating contraction. Clearly, a
ROCK-dependent contraction force during the early stage of PMA treatment is
necessary for activation of the apoptotic signal.
|
In addition to ROCK, MLC kinase (MLCK) is the major kinase that
phosphorylates MLC (Gallagher et al.,
1997; Kohama et al.,
1996
). To ascertain whether ROCK plays a necessary role for MLC
phosphorylation during PMA-induced apoptosis, we then tested the effects of
ML-7, an inhibitor of MLCK (Itoh et al.,
1992
), and Y27632 on MLC phosphorylation in TF-1 cells during PMA
treatment. As expected, PMA-induced MLC phosphorylation was diminished by
Y27632 pre-treatment, but not by the MLCK inhibitor ML-7, ranging from 1 to 20
µM (Fig. 3B). In addition,
DNA fragmentation analysis also showed that PMA-induced apoptosis is inhibited
by Y27632 but not ML-7 (Fig.
3C). Similar results could be observed for the cells treated with
another MLCK inhibitor, ML-9 (Ishikawa et
al., 1988
) (data not shown). Thus, these experimental results
exclude the possibility of MLCK in generating a contraction force for
PMA-induced apoptosis.
PMA-induced activation of caspase-3 is controlled by myosin motor
activity
To prove the necessary role of myosin motor activity in caspase-3
activation, we further examined the effect of Y27632, latrunculin B and
2,3-butanedione monoxime (BDM), a myosin ATPase inhibitor
(Soeno et al., 1999), on
PMA-induced activation of caspase-3 activity in TF-1 cells. Similar to the
results obtained from cells treated with Y27632, latrunculin B treatment also
abolished the increase in caspase-3 activity after PMA induction, indicating
that the involvement of the actin filament in the myosin-actin interaction is
required for this apoptotic process (Fig.
4). To examine the dependency on myosin function, cells were
treated with PMA in the presence of BDM. The results demonstrated that
treatment of cells with BDM at 5 µM decreased caspase-3 activity induced by
PMA, suggesting that the motor activity of myosin-ATPase is involved in
caspase-3 activation during PMA induction. Together, these results strengthen
the idea that activation of actin-myosin motor activity is required for
caspase-3 activation and that ROCK-mediated MLC phosphorylation contributes to
myosin motor activation during PMA treatment.
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Inhibition of ROCK abrogates the requirement of adhesion for
survival
It should be mentioned that TF-1 cells in serum-free medium always survive
with the adhesion property during PMA exposure. We have previously proven that
adhesion can act to prevent the occurrence of the apoptotic signal triggered
by PMA treatment, and RhoA signaling from serum or LPA may interfere with the
adhesion process to allow PMA-induced apoptosis to be triggered
(Lai et al., 2001). Therefore,
it is possible that inhibition of ROCK may switch cells to become adherent,
thereby preventing apoptosis. We then plated cells onto a hydrogel-coated dish
to prevent cell adhesion during PMA treatment in serum-free medium, as
compared with those plated onto the regular culture dish to allow adhesion. It
appeared that pretreatment of cells with Y27632 rescued cells from PMA-induced
apoptosis even when cell adhesion was blocked
(Fig. 5), indicating that the
inhibition of ROCK that abolishes the apoptotic process is not through the
adhesion event. Most importantly, this result also implied that ROCK-mediated
contraction occurs downstream of loss of adhesion to exert its effect in
PMA-induced apoptosis.
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Effects of expression of dominant-active forms of RhoA and ROCK on
PMA-induced apoptosis in serum-free conditions
We have previously shown that over-expression of RhoAV14, a dominant-active
form of RhoA, causes cells to become apoptotic in response to PMA in
serum-free medium. As the effector pathways of the RhoA signal are diverse, we
then assessed whether activation of the ROCK-mediated signal is a solely
effector pathway of RhoA involved in PMA-induced apoptosis. For this purpose,
the expression vector of ROCK(CAT), which contains only an
NH2-terminal kinase domain (catalytic domain, amino acids 6 to 553)
(Amano et al., 1997), or
RhoAV14 was co-transfected with the EGFP expression vector into TF-1 cells.
Cells were incubated in serum-free medium during PMA treatment. The
successfully transfected cells were revealed by fluorescent microscopic
observation. The suspension cells before and after PMA treatment were
collected, and GFP-positive cells were counted. All PMA-treated cells in
suspension were apoptotic, as judged by Trypan Blue staining after 15 hours of
PMA treatment. Consistent with our previous observation, expression of RhoAV14
significantly increased PMA-induced apoptosis under serum-free conditions.
However, expression of the dominant-active form of ROCK(CAT) did not increase
PMA-induced apoptosis in serum-free medium to an extent similar to that by
RhoAV14 transfection (Fig. 6).
As the effect of RhoAV14 on PMA-induced apoptosis in serum-free medium can be
consistently abolished by Y27632 treatment (data not shown), it is clear that
the RhoA/ROCK pathway is necessary for this apoptotic induction. Given the
fact that activation of RhoA interferes with phorbol ester-induced adhesion,
it is conceivable that another downstream effector pathway of RhoA is involved
in prevention of adhesion, and that a ROCK-mediated signal by itself is not
sufficient to result in membrane contraction and to turn cells apoptotic.
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Activation of initiator caspases requires ROCK-dependent contraction
in PMA-induced apoptotic cells
We next determined which caspase initiator is responsible for caspase-3
activation in this apoptotic process. By western blot analysis, we could
clearly see the processed form of caspase-8 and -10 at 6 hours after PMA
treatment (Fig. 7A). To know
whether caspase-8 and -10 are the apical caspases responsible for the
PMA-induced activation of caspase-3, we further pretreated cells with
different caspase inhibitors. We found that caspase-3 activity in PMA-treated
cells was completely blocked by pretreatment of cells with caspase-3 inhibitor
(z-DEVD-fmk). Activation of caspase-3 in PMA-treated cells was clearly
decreased by caspase-8 inhibitor (z-IETD-fmk) or caspase-10 inhibitor
(z-AEVD-fmk), as compared with a slight decrease by caspase-9 inhibitor
(z-LEHD-fmk) (Fig. 7B).
Consistent results could be seen in D2 cells, a cytokine-independent
derivative from TF-1, which also displayed apoptosis and differentiation in
response to PMA treatment (Lai et al.,
2002).
|
We further incubated cells with caspase-3 inhibitor (z-DEVD-fmk) during PMA treatment to examine whether activation of caspase-8 and -10 is a secondary event of caspase-3 activation. As shown in Fig. 7C, neither capase-8 nor -10 activation was affected by inhibition of caspase-3, whereas ROCKI cleavage was prevented. This result excludes the possibility that the activation of these two caspases is a result of caspase-3 activation and provides further evidence that ROCKI does serve as a substrate of caspase-3 in the latter stage of PMA-induced apoptosis. Taken together, caspase-8 and -10 are likely the initiator caspases in PMA-induced apoptosis.
We further tested whether the cleavage of caspase-8 and -10 in PMA-treated TF-1 cells depends on the ROCK-mediated activation of actin-myosin motor activity. As expected, pretreatment of cells with either Y27632 or latrunculin B prevented PMA-induced cleavage of caspases-10, -8 and -3. As a comparison, pretreatment of cells with MLCK inhibitor, ML-9, did not affect this activation process (Fig. 8). Clearly, ROCK-mediated MLC phosphorylation and the subsequent myosin-actin interaction is a requisite process for activation of caspase-8 and -10 that act as the initiator caspases in this apoptotic process.
|
Involvement of FADD in PMA-induced apoptosis
Given the fact that caspase-8 and -10 are always recruited to the
cytoplasmic death domain of the death receptor via FADD, by which a DISC at
the cell membrane is formed to facilitate their activation, we then assessed
the involvement of DISC formation in PMA-induced apoptosis. Since FADD often
plays an adaptor role in death receptor-mediated activation of caspase-8 and
-10, we expressed procaspase-10 C/S-flag, a protease-dead mutant, in D2 and
TF-1 cells to perform DISC co-immunoprecipitation to examine whether there is
an increased association between FADD and procaspase-10 in response to PMA
treatment. It appeared that a significant increase of FADD is associated with
the immunocomplex of procaspase-10 C/S-flag expressed in D2 and TF-1 cells in
PMA-induced pro-apoptotic cells that remained in suspension, but not in the
attached and survived population (Fig.
9A). To substantiate the role of ROCK in PMA-induced DISC
formation, D2 cells with procasapse-10 C/S-flag transfection were treated with
PMA for 30 minutes. Cells remained in suspension, representing the early
pro-apoptotic stage, were collected and re-plated into a new culture dish in
the presence or absence of Y27632 and incubated for another 2.5 hours in the
PMA-containing medium. Data shown in Fig.
9B revealed that Y27632 treatment diminished complex formation
between pro-caspase-10 C/S-flag and FADD, indicating that inhibition of ROCK
reduced the recruitment of FADD to form the DISC complex in PMA-treated cells.
Taken together, there is a good possibility that ROCK-mediated membrane
contraction can provide a mechanism to stimulate a death receptor-mediated
pathway during PMA induction.
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Discussion |
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Membrane blebbing has been considered as a morphological change in the
execution phase of apoptosis. In particular, it has been demonstrated that
ROCK activation by caspase-3-mediated cleavage at its C-terminus inhibitory
domain results in MLC phosphorylation
(Coleman et al., 2001;
Sebbagh et al., 2001
), which
contributes to myosin motor activity and membrane blebbing in apoptosis
(Coleman and Olson, 2002
).
Here, we found that this process does occur at the late stage in PMA-induced
apoptotic cells; however, the initial membrane contraction in PMA-induced
apoptotic cells is via the RhoA/ROCK pathway rather than through
caspase-3-dependent cleavage of ROCKI. Relevant to our results, it has been
shown that membrane blebbing occurs in Rat-1 and PC-12 cells after serum
withdrawal (McCarthy et al.,
1997
; Mills et al.,
1998
), and that the extent of MLC phosphorylation by MLCK or the
Rho-dependent pathway is increased in the population of blebbing cells when
caspase is inhibited. However, in our system, serum or LPA provides signal via
the ROCK pathway to stimulate MLC phosphorylation independent of MLCK. In
addition, it should be noted that MLC phosphorylation is a PMA-induced event.
It has been shown that activity of myosin phosphatase can be inhibited by CPI
(PKC-potentiated inhibitory phosphoprotein of myosin phosphatase), whose
inhibitory activity is potentiated by PKC-mediated phosphorylation
(Watanabe et al., 2001
). From
this view, it is possible that MLC phosphorylation in PMA-induced
pro-apoptotic cells is a result of ROCK and PKC activation. Still, it remains
to be determined whether CPI is involved in MLC phosphorylation in PMA-treated
TF-1 cells or whether activation of ROCK requires PKC activation.
It has been reported that MLCK-mediated MLC phosphorylation increases
translocation of tumor necrosis factor receptor (TNFR) to the plasma membrane
independently of a TNF signal, and this in turn activates caspase-8 to
initiate the apoptotic pathway (Jin et
al., 2001). Another study has also shown that treatment of cells
with the cytoskeleton-disturbing reagent cytochalasin B increases clustering
of the CD95 receptor to activate caspase-8 and enhances UV-induced apoptosis
(Kulms et al., 2002
). Here,
our results indicate that both caspase-8 and -10 are the apical caspases in
PMA-induced apoptosis and their activation is a result of membrane contraction
dependent on ROCK. The obligatory role of caspase-8 and -10 in apoptosis
initiation by a death receptor-mediated pathway has prompted us to examine
whether the PMA-induced death signal involves the enhancement of the
receptor-mediator adaptor recruitment. Indeed, a complex containing endogenous
FADD with procaspase-10 is preferentially formed in PMA-induced pro-apoptotic
cells, but not the survived cells. Distinct from our result, one study
(Meng et al., 2002
) has
demonstrated that PMA inhibits Fas-mediated apoptosis by disturbing the
receptor-mediated adaptor molecule recruitment, suggesting a mechanism
conferred by PMA-induced signal for survival. Again, together with our
results, it is conceivable that the different concurrent pathways can
cooperate with the diverse signals from PMA stimulation to have different
cellular fates. Previously, another study has shown that phorbol ester induces
apoptosis in U937 cells, in part through a pathway that requires endogenous
production of TNF-
depending on activation of MEK/ERK during
stimulation (Takada et al.,
1999
). Since PMA-induced apoptosis in TF-1 cells did not require
newly synthesized protein, it is unlikely that induction of a particular gene
expression is required for the apoptotic signal in this case. To test the
possibility that release of the death receptor ligand TNF-
is involved,
we have pre-incubated cells with recombinant TNF receptor R1 protein prior to
PMA induction. However, this pre-treatment experiment did not affect
PMA-induced apoptosis, excluding the possibility that TNF ligand binding is
involved in this apoptotic process (data not shown). In this study, we also
found that PMA-induced apoptosis was decreased, but was not completely
abolished by expression of a dominant-negative form of FADD (data not shown),
implying that the death receptor-mediated pathway probably only plays a
partial role in PMA-induced apoptosis. It has been demonstrated that unligated
integrins recruit caspase-8 to the membrane and form the DISC without FADD,
suggesting the presence of a death receptor-independent caspase-8 activation
mechanism (Stupack et al.,
2001
). Therefore, it is possible that another pathway independent
of the death receptor is also involved in this apoptotic process. In summary,
we propose that the first phase of membrane contraction leads to the
subsequent random clustering of cell-surface death receptors or other membrane
receptosr, such as unligated integrins, which in turn activate the apoptotic
signaling via caspase-8 and -10.
Our previous study has demonstrated that expression of a dominant-active
form of RhoAV14 increases PMA-induced apoptosis significantly in TF-1 cells
under serum-free conditions (Lai et al.,
2001). We proposed that, in serum-free medium, PMA induces only
adhesion, which provides the survival signal, whereas activation of RhoA by
LPA or serum may promote PMA-induced apoptosis by interfering with the
adhesion process. In this study, we further found that inhibition of ROCK
abrogates the requirement of adhesion for cell survival following PMA
treatment by preventing myosin-mediated contraction, so that the death signal
is abolished; under this circumstance, the survival signal from adhesion is no
longer required. However, the effect of expressing a dominant-active form of
ROCK(CAT) on PMA-induced apoptosis in serum-free medium was not as dramatic as
that of expressing RhoAV14 (Fig.
5). Probably, membrane contraction induced by PMA in TF-1 cells
indeed requires cells remaining in the suspension status, and adhesion can
provide a signal to prevent the occurrence of ROCK-dependent contraction. This
finding led us to propose that another downstream pathway of RhoA is involved
in preventing TF-1 cells from PMA-induced adhesion, by which the concurrent
activation of ROCK can confer contraction force. In other words, under
serum-free conditions, cell adhesion may still provide an inhibitory mechanism
to prevent membrane contraction despite the presence of a dominant-active form
of ROCK. According to these results, we propose a model for PMA-induced
apoptosis in TF-1 cells as depicted in
Fig. 10, in which there is an
interplayed relationship between cell adhesion, PKC activation and the
serum/RhoA/ROCK pathway in MLC phosphorylation that results in myosin-mediated
contractility, thus switching-on the death receptor-mediated or death
receptor-independent activation of the caspase cascade.
|
A growing number of examples have shown that the coordinated activation and
functional cooperation between members of the Ras and Rho GTPase families
regulate cellular proliferation and actin-based cell motility (reviewed by
Bar-Sagi and Hall, 2000).
Specifically, it has been shown that Rho suppresses the induction of the
cell-cycle inhibitor p21Cip1/Waf1, thus enabling Ras to stimulate
cell-cycle progression in 3T3 fibroblasts
(Olson et al., 1998
), and this
mechanism is important for the role of Rho in uncontrolled proliferation
during Ras-induced transformation (Qiu et
al., 1995
; Sahai et al.,
2001
). Interestingly, we have recently shown that a ROCK-mediated
signal may cause cytosolic retention of activated ERK in PMA-induced apoptotic
cells, thus impairing ERK-mediated gene expression such as
p21Cip1/Waf1 (Lai et al.,
2002
). The enhancement of PMA-induced apoptosis by the
LPA/RhoA/ROCK pathway in TF-1 cells intriguingly exemplifies a situation in
which upregulated ROCK from RhoA signaling in some cells not only impairs gene
expression required for growth arrest or differentiation, but also has a
potential in changing the extent of myosin-mediated contraction to trigger
apoptosis. Thus, the RhoA/ROCK pathway can act as a molecular switcher by
cooperating with other pathways depending on the cellular context to determine
cell fate during hematopoiesis.
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References |
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