* Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Québec, G1R 2J6, Canada; and Laboratoire de recherche sur le cancer de la peau CHUL, RC-9700, Sainte-Foy, Québec, Canada
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Abstract |
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In endothelial cells, H2O2 induces the rapid formation of focal adhesion complexes at the ventral face of the cells and a major reorganization of the actin cytoskeleton into dense transcytoplasmic stress fibers. This change in actin dynamics results from the activation of the mitogen-activated protein (MAP) kinase stress-activated protein kinase-2/p38 (SAPK2/p38), which, via MAP kinase-activated protein (MAPKAP) kinase-2/3, leads to the phosphorylation of the actin polymerization modulator heat shock protein of 27 kD (HSP27). Here we show that the concomitant activation of the extracellular signal-regulated kinase (ERK) MAP kinase pathway by H2O2 accomplishes an essential survival function during this process. When the activation of ERK was blocked with PD098059, the focal adhesion complexes formed under the plasma membrane, and the actin polymerization activity led to a rapid and intense membrane blebbing. The blebs were delimited by a thin F-actin ring and contained enhanced levels of HSP27. Later, the cells displayed hallmarks of apoptosis, such as DEVD protease activities and internucleosomal DNA fragmentation. Bleb formation but not apoptosis was blocked by extremely low concentrations of the actin polymerization inhibitor cytochalasin D or by the SAPK2 inhibitor SB203580, indicating that the two processes are not in the same linear cascade. The role of HSP27 in mediating membrane blebbing was assessed in fibroblastic cells. In control fibroblasts expressing a low level of endogenous HSP27 or in fibroblasts expressing a high level of a nonphosphorylatable HSP27, H2O2 did not induce F-actin accumulation, nor did it generate membrane blebbing activity in the presence or absence of PD098059. In contrast, in fibroblasts that expressed wild-type HSP27 to a level similar to that found in endothelial cells, H2O2 induced accumulation of F-actin and caused bleb formation when the ERK pathway was inhibited. Cis-platinum, which activated SAPK2 but induced little ERK activity, also induced membrane blebbing that was dependent on the expression of HSP27. In these cells, membrane blebbing was not followed by caspase activation or DNA fragmentation. We conclude that the HSP27-dependent actin polymerization-generating activity of SAPK2 associated with a misassembly of the focal adhesions is responsible for induction of membrane blebbing by stressing agents.
Key words: SAPK2/p38; HSP27; F-actin; blebbing; apoptosis ![]() |
Introduction |
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MEMBRANE blebbing is an early manifestation of
toxicity. In vivo, it has been observed after cell
injury in liver, brain, heart, and kidney. In vitro,
it is frequently found after exposure of cells to stressful
stimuli (for review see Gores et al., 1990). The physiopathological consequence of bleb formation in vivo may be dramatic. Surface blebbing of endothelial cells results
in a narrowing of the vascular lumen, which would increase the vascular resistance, leading ultimately to cardiac failure (Becker and Ambrosio, 1987
). Bleb shedding
from ischemic renal tubule cells may lead to tubular obstruction (Phelps et al., 1989
). In liver, bleb shedding from
hepatocytes seems to be responsible for the release in the
blood stream of viral antigens, such as hepatitis B, as well
of the hepatic enzymes that are routinely used clinically as
an indication of hepatic injury (Gores et al., 1990
). The
mechanisms of blebbing are not well understood. Membrane blebbing has been associated with various biochemical alterations (including a decrease in ATP levels, increase in cellular calcium, activation of nonlysosomal proteolytic system, and activation of phospholipase A2) as
well as with cytoskeletal alterations affecting the dynamics
of actin and myosin and the formation of focal adhesion
complexes (Fishkind et al., 1991
; Cunningham, 1995
; Martin et al., 1995
; Miyoshi et al., 1996
; Mills et al., 1998
).
Apoptosis defines a type of regulated cell death associated with various morphological features that include cell
shrinkage, chromatin condensation, nuclear/cytoplasmic
fragmentation, and formation of dense bodies that are
quickly removed via phagocytosis by neighboring cells
(Martin et al., 1994). Apoptosis can result from a genetic
program aimed at regulating the cell number in different
physiological and pathological conditions. Apoptosis is
also induced in response to environmental changes, namely
growth factor withdrawal (Xia et al., 1995
), loss of normal
adhesion to extracellular matrix during anoikis (Frisch and
Francis, 1994
; Re et al., 1994
), or exposures to a variety of
stresses and cytotoxic drugs (Verheij et al., 1996
; Zanke et
al., 1996
; Lee et al., 1997
). Once initiated, the apoptotic
program involves activation of a series of biochemical
events that underlie the morphological hallmarks of apoptosis. This includes increased intracellular Ca2+ concentrations, uncoupling of mitochondrial electrical potential, internucleosomal DNA fragmentation, and proteolytic
degradation of specific substrates, such as poly-(ADP-ribose)-polymerase (PARP)1 and lamin. Proteolysis is
achieved through activation of cysteine proteases called
caspases, a family of enzymes related to the interleukin
1
-converting enzyme (ICE) or ICE/CED3 proteases
(Juo et al., 1997
). In vitro and in vivo studies showed that
caspases act in a cascade to convey the apoptotic signal
(Darmon et al., 1995
; Na et al., 1996
).
In most cases, the signaling pathways that lead to membrane blebbing as well as those that link the apoptotic signal to activation of the death execution programs remains
to be clarified. The mitogen-activated protein (MAP) kinase ERK1/2 (extracellular signal-regulated kinase) and
the stress-activated protein kinases-1 (SAPK1/JNK) and
-2 (SAPK2/p38) are central elements of homologous pathways used by mammalian cells to orchestrate the signals
generated by environmental stimuli (Waskiewicz and
Cooper, 1995; Cohen, 1997
). These MAP kinases are activated by many agents that induce apoptosis, such as oxidative stress, inflammatory cytokines, UV light, and anticancer drugs. SAPK1 and SAPK2 appear as important
modulators of the apoptotic program. For example, SAPK1
modulates apoptosis initiated by Fas/APO, tumor necrosis
factor
(TNF
), oxidative stress, heat shock, and the anticancer drug cis-platinum (Wei et al., 1995
; Cahill et al.,
1996
; Verheij et al., 1996
; Zanke et al., 1996
). In some cell
lines and in certain contexts, activation of SAPK1 lies upstream of caspase-3-mediated apoptosis (Seimiya et al.,
1997
), while in others, activation of SAPK1/JNK is downstream of ICE-like protease (Mosser et al., 1997
). In
anoikis, for example, caspases activate MEKK-1, an activator of SAPK1, suggesting that SAPK1 lies downstream of caspase (Cardone et al., 1997
). SAPK2 modulates
apoptosis induced by sodium salicylate in Rat-1 cells
(Schwenger et al., 1997
), glutamate in rat cerebellar granule cells (Kawasaki et al., 1997
), withdrawal of trophic factors in Rat-1 fibroblasts and differentiated PC12 cells
(Kummer et al., 1997
), and Fas activation in Jurkat clone
A3 (Brenner et al., 1997
; Juo et al., 1997
). Fas induction of
SAPK2 activity requires the action of ICE/CED3 in Jurkat
clone A3, whereas etoposide activation of SAPK2-mediated apoptosis relies on protease-independent mechanisms or on proteases unrelated to caspases-3 (Juo et al.,
1997
). Recently, apoptosis signal-regulating kinase (ASK)
1 has been identified as a redox-sensitive MAP kinase kinase kinase that activates SAPK1 and SAPK2 during apoptosis induced by TNF
(Saitoh et al., 1998
). Whereas
SAPK1 and SAPK2 appear to be involved in modulating
apoptosis, ERK is generally considered as a survival factor
(Xia et al., 1995
). In fact, growth factor withdrawal downregulates ERK and induces apoptosis in several cell systems, whereas activation of ERK during stress can be protective (Xia et al., 1995
; Guyton et al., 1996
). Apoptosis
has been reported to be regulated by a balance between
activation of SAPKs and ERK (Xia et al., 1995
).
Vascular endothelial cells are in heavy contact with
circulating oxyradicals produced or released from normal
oxygen metabolism, oxidizable xenobiotics, or the respiratory burst of activated phagocytic cells, monocytes,
macrophages, and neutrophils (Hardy et al., 1994). Endothelial cells themselves produced oxyradicals after abnormal oxygenation, such as intermittent hypoxia or hyperoxia alternating with hypoxia (Zulueta et al., 1997
). Under
these circumstances, they produce large amounts of superoxide anions, at least partially because of the conversion of
xanthine dehydrogenase to xanthine oxidase during hypoxia (Michiels et al., 1992
). Nitric oxide, another type of
oxyradical, is released from endothelial cells in response to
various agonists, including histamine and VEGF (Van de
Voorde et al., 1987
; van der Zee et al., 1997
). We have previously shown that exposure to H2O2 induced in endothelial cells a SAPK2-dependent physiological-like reorganization of the F-actin cytoskeleton that rearranges from
cortical ruffles into trans-cytoplasmic stress fibers (Huot
et al., 1997
). This process was shown to be dependent on
the SAPK2/MAPKAPK-2/3-induced phosphorylation of
the actin polymerization modulator HSP27. Exposures to
similar oxidative stress resulted in deleterious fragmentation of F-actin in fibroblasts and to intense surface
blebbing in hepatocytes (Huot et al., 1996
; Mirabelli et al.,
1988
). This illustrates the high level of intrinsic resistance
of endothelial cells to oxidative stress.
In the present study, we show that membrane blebbing is induced when the ERK pathway is not appropriately coactivated with SAPK2 in endothelial cells exposed to oxidative stress. We provide several lines of evidence suggesting that membrane blebbing results from the strong F-actin polymerization activity generated by activation of SAPK2 and from a defect in the assembly of focal adhesions. A similar SAPK2 and HSP27 phosphorylation-dependent cell blebbing was observed in fibroblastic cells treated with the antineoplastic drug cis-platinum, suggesting that activation of the SAPK2/MAPKAPK-2/HSP27 pathway represents, at least in some cellular context, a mechanism for the early formation of blebs. We also show that membrane blebbing can be dissociated from caspase activation and internucleosomal DNA fragmentation.
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Materials and Methods |
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Materials
[-32P]ATP (3,000 Ci/mmol) was purchased from NENTM Life Science
Products, Boston, MA. H2O2, VEGF165 and endothelial cell growth supplement (ECGS) were from Sigma Chemical Co. (St. Louis, MO). Recombinant HSP27 was purified from Escherichia coli transformed with a
plasmid containing the coding sequence for Chinese hamster HSP27.
SB203580 and PD098059 were purchased from Calbiochem (La Jolla,
CA) and Research Biochemical International (Natick, MA). These inhibitors were diluted in DMSO to make stock solutions of 40 and 20 mM, respectively. Cytochalasin D was purchased from Sigma Chemical Co.,
zVAD-fmk was purchased from Enzyme System Products (Livermore,
CA), and DEVD-AMC was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Chemicals for electrophoresis were obtained from Bio-Rad Labs (Hercules, CA) and Fisher Scientific (Pittsburgh, PA).
Antibodies
Hu27ab is a rabbit antiserum that specifically recognizes human HSP27
(Landry et al., 1989). Anti-GST-MAPKAP kinase-2/3 is a rabbit polyclonal antibody raised in rabbit after injecting a glutathione S-transferase
(GST) fusion protein containing the 223 COOH-terminal amino acids of
Chinese hamster MAPKAP kinase-2 (Huot et al., 1995
). Anti-ERK2 is a
rabbit polyclonal antibody raised against a synthetic peptide that corresponds to the 14 COOH-terminal amino acids of rat ERK2 (Huot et al.,
1995
). The anti-caspase-3 R#MF393 is a rabbit polyclonal antibody that
recognizes both the entire form and the p17 active subunit of caspase-3. This antibody was obtained from Dr. D. Nicholson (Merck Centre for
Therapeutics Research, Pointe Claire, Québec, Canada). Anti-PARP antibody C-2-10 was a kind gift from Dr. G. Poirier (Université Laval,
Québec). hVin-1 is an anti-human vinculin monoclonal antibody purchased from Sigma Chemical Co.
Cells
Human umbilical vein endothelial cells (HUVECs) were isolated by collagenase digestion of umbilical veins from undamaged sections of fresh
cords (Huot et al., 1997). In brief, the umbilical vein was cannulated,
washed with Earle's balanced salt solution (EBSS), and perfused for 10 min with collagenase (1 mg/ml) in EBSS at 37°C. After perfusion, the detached cells were collected, the vein was washed with 199 medium, and the
wash-off was pooled with the perfusate. The cells were washed by centrifugation and plated on gelatin-coated 75-cm2 culture dishes in 199 medium
containing 20% heat-inactivated FBS, ECGS (60 µg/ml), glutamine, heparin, and antibiotics. Replicated cultures were obtained by trypsinization
and were used at passages <5. The identity of HUVECs as endothelial
cells was confirmed by their polygonal morphology and by detecting their
immunoreactivity for factor VIII-related antigens. HU27 cell line B12 is a
CCL39 transformant that constitutively expresses 4.8 ng/µg protein of human HSP27 (Huot et al., 1996
), while the CCL39 transformant cell line
HU27 V expresses 3.3 ng/µg protein of a nonphosphorylatable form of
human HSP27 (Huot et al., 1996
). Clone 3 is a CCL39 transfectant that expresses the selection gene, neo, and endogenous hamster HSP27 (~1.5 ng/
µg protein). The transfectants were maintained in DME containing
NaHCO3 (2.2 g/liter) and glucose (4.5 g/liter) and were supplemented with
5% FBS (Lavoie et al., 1995
; Huot et al., 1996
). Cultures were incubated
at 37°C in a humidified atmosphere containing 5% CO2.
Immunoprecipitation
After treatments, cells were scraped and extracted in lysis buffer containing 20 mM MOPS, pH 7.0, 10% glycerol, 80 mM -glycerophosphate, 5 mM
EGTA, 0.5 mM EDTA, 1 mM Na3VO4, 5 mM Na4P2O7, 50 mM NaF, 1%
Triton X-100, 1 mM benzamidine, 1 mM DTT, and 1 mM PMSF. The extracts were vortexed and centrifuged at 17,000 g for 12 min at 4°C. The
clarified supernatants were either immediately used for immunoprecipitation or stored at
80°C. The further steps were carried out at 4°C. The
clarified supernatant was diluted four times in buffer I (20 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 1 mM EGTA, 1 mM MgCl2, 1 mM
Na3VO4, 1% Triton, 1 mM PMSF). Undiluted antibodies were added in
limiting concentrations, and the mixtures were incubated for 1 h. 10-15 µl
of protein A-Sepharose (Pharmacia Biotech, Piscataway, NJ) 50% vol/vol
in buffer I was added, and the mixtures were incubated for 30 min. Samples were centrifuged for 15 s, and washed three times with 300 µl of
buffer I. Immunoprecipitates were directly used for the kinase assays.
Immunocomplex Kinase Assays
Kinase activities were assayed in immune complexes. SAPK2 activation
was measured by assessing the activity of its substrate MAPKAP kinase-2.
The activity of immunoprecipitated MAPKAP kinase-2 was measured using recombinant HSP27 (Huot et al., 1995). The assays were carried out in
25 µl of kinase buffer K (100 µM ATP, 3 µCi of [
-32P]ATP [3,000 Ci/
mmol], 40 mM p-nitrophenyl phosphate, 20 mM MOPS pH 7, 10% glycerol, 15 mM MgCl2, 0.05% Triton X-100, 1 mM dithiothreitol, 1 mM leupeptin, 0.1 mM PMSF, and 0.3 µg of protein kinase A inhibitor. The kinase activity was assayed for 30 min at 30°C and was stopped by the
addition of 10 µl SDS-PAGE loading buffer. Immunoprecipitated ERK2
was assayed analogously using myelin basic protein as substrate (Huot et al.,
1997
) in buffer K containing 10 mM MgCl2. Kinase activities were evaluated by measuring incorporation of the radioactivity into the specific substrates after resolution by SDS-PAGE and quantification using PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
PARP Cleavage Assay
After treatments, floating cells (F1) were collected in PBS and centrifuged, and the pellet was frozen on dry ice. Adherent cells were incubated
for an additional 6 h. The floating cells (F2) were collected and pelleted.
The remaining adherent cells were scraped in PARP extraction buffer
(62.5 mM Tris, pH 6.8, 2% SDS, 6 M Urea, 10% glycerol, 0.00125% bromophenol blue, 720 mM -mercaptoethanol) and pooled with F1 and F2.
Proteins were separated on 8.5% SDS-PAGE and transferred onto nitrocellulose membrane. PARP was immunodectected by standard Western
blotting procedure with the C2-10 antibody (Shah et al., 1995
, 1996
).
DEVDase Cleavage Activity
DEVD cleavage activity was determined according to Enari et al. (1996)
with some modifications. In brief, after treatments or incubation periods,
floating and adherent cells were pooled, and cytosolic extracts were prepared by eight repeated cycles of freezing and thawing in 100 µl of DEVDase extraction buffer (50 mM MOPS, pH 7.0, 50 mM KCl, 5 mM EGTA,
2 mM MgCl2, 1 mM DTT, 20 µM cytochalasin D, 1 mM PMSF, 1 µg/ml
leupeptin, 1 µg/ml pepstatin A, 50 µg/ml antipain). Extracts were clarified
by centrifugation for 12 min in a microfuge at 4°C. Protein extracts (or
buffer only for blank) were mixed with 500 µl of ICE standard buffer (100 mM Hepes-KOH, pH 7.5, 10% sucrose, 0.1% CHAPS, 10 mM DTT, 0.1 mg/ml ovalbumin) containing 1 µM DEVD-AMC and incubated at 30°C for 30 min. The fluorogenic product substrate AMC was detected by
excitation at 380 nm and emission at 460 nm with a luminescence spectrometer (model LS50B; Perkin-Elmer Corp., Norwalk, CT). Caspase activities were normalized for protein concentrations of individual extracts
and then were calculated relatively to the activity of the control samples.
Caspase-3 activity was evaluated by Western blotting on cell extracts prepared as for PARP cleavage. After extraction, samples containing 35 µg of proteins were separated by SDS-PAGE and transferred on nitrocellulose membrane. Rabbit polyclonal anti-caspase-3 R#MF393 antibody was used to detect the p17 subunit of caspase-3. Anti-rabbit IgG-HRP was used to reveal the antigen-antibody complex.
Internucleosomal DNA Fragmentation
Cells prepared as for PARP cleavage assay were lysed in TENS-proteinase K buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% SDS, 0.2 mg/ml proteinase K) at 37°C for 16 h. After phenol-chloroform extraction, DNA was precipitated with ethanol and resuspended in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 0.5 mg/ml RNAse). The DNA was separated into an 1.0% agarose gel.
Confocal Fluorescence Microscopy
Confocal microscopy was used for immunofluorescent visualization of
F-actin, vinculin, and HSP27 (Huot et al., 1995). The cells were plated on
gelatin-coated LabTek dishes (Naperville, IL). After treatment, they were
fixed with 3.7% formaldehyde and permeabilized with 0.1% saponin in
PBS, pH 7.5. F-actin was detected using FITC-conjugated phalloidin (33.3 µg/ml) diluted 1:50 in phosphate buffer. hVIN-1 monoclonal antibody and
HU27 antibody were used to detect vinculin and human HSP27, respectively. Vinculin and HSP27 antigen antibody complexes were detected
with biotin-labeled anti-mouse IgG (vinculin) or anti-rabbit IgG (HSP27)
and were revealed with Texas red-conjugated streptavidin. The cells were examined as previously reported by confocal microscopy with a Bio-Rad
MRC-1024 imaging system mounted on a Nikon Diaphot-TDM (Melville,
NY) equipped with a 60× objective lens with a 1.4 numerical aperture
(Huot et al., 1997
).
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Results |
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Activation of ERK and SAPK2 by H2O2
In primary cultures of exponentially growing HUVECs,
oxidants induced a 10-12- and 4-fold activation of SAPK2
and ERK2, respectively (Fig. 1 and Huot et al., 1997).
Maximal activation was observed after 5 min in both cases.
In contrast, SAPK1/JNK was not activated during the first
hour of exposure to H2O2. Some activity was induced after
6 h, suggesting an indirect mechanism of activation (data not shown). Exposure of cells to 10 µM SB203580 inhibited H2O2-induced SAPK2 activity as reflected by the inhibition of MAPKAP kinase-2/3 activation, an immediate
downstream physiological substrate of SAPK2 (Fig. 1 a),
but had no effect on ERK activation (Fig. 1 b). Similarly,
exposure of cells to PD098059 (25-50 µM for 1 h) inhibited
ERK activation (Fig. 1 b) but had no effect on SAPK2 activity (Fig. 1 a). Moreover, inhibition of SAPK2 by SB203580 was not modified by PD098059 (Fig. 1 a), nor was the inhibition of ERK by PD098059 modified by SB203580 (Fig. 1
b). These findings confirm the high specificity of SB203580
and PD098059 as inhibitors of SAPK2 activity and of ERK
activation, respectively (Cuenda et al., 1995
; Dudley et al.,
1995
; Kumar et al., 1996
; Rousseau et al., 1997
).
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ERK Activation Prevents H2O2-induced Apoptosis
HUVECs in primary cultures are extremely resistant to
cytotoxicity and apoptosis induced by oxidative stress. Exposures to 0.5 mM H2O2 for 1 h induce little cell blebbing,
no DEVD cleavage activities, and no DNA fragmentation
(Fig. 2). In several cell systems, activation of ERK during
stress confers survival advantages. Treatment of cells with
activators of the ERK pathways induces protection against
stress, whereas inhibiting activation of ERK enhances toxicity (Xia et al., 1995; Guyton et al., 1996
; Katoh et al.,
1995
). Consistent with the notion that ERK activation has protective functions and antiapoptotic properties, we
found that blocking the H2O2-induced ERK activation
with PD098059 resulted in an intense blebbing activity in
up to 60% of the cells immediately after treatment (Figs. 2
a and 3 c), a progressive increase in zVAD-fmk-sensitive
caspase activity (Fig. 2 b), PARP cleavage that started
around 3 h (Fig. 2 e), and after 6 h, a major zVAD-fmk- inhibitable internucleosomal DNA fragmentation (Fig. 2
d). PD098059 by itself did not induce any of these manifestations of cytotoxicity. Because activation of SAPK2 has
often been associated with activation of apoptosis, we analyzed the contribution of H2O2-induced SAPK2 activity on
apoptotic events. In the presence of SB203580, membrane blebbing was inhibited and found in less than 10% of the
cells (Fig. 2 a). The inhibition occurred without any significant modification in the number of adherent cells (data
not shown). SB203580 had no effect on DEVDase and
caspase-3 activities (Fig. 2, b and c) or on DNA fragmentation (Fig. 2 d), but it did inhibit the cleavage of PARP
(Fig. 2 e). Overall, these results indicate that activation of
ERK contributed to prevent induction of several manifestations of H2O2-induced cytotoxicity and apoptosis, among
which, some were dependent on SAPK2 activity, like cell
blebbing. Subsequent investigations reported in this manuscript focused on SAPK2-dependent mechanisms of cell
blebbing.
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Membrane Blebbing Is Modulated by the SAPK2/HSP27-dependent Regulation of F-Actin Dynamics and Involves Misassembly of Focal Adhesions
One major consequence of SAPK2 activation in HUVECs
is a reorganization of the actin filaments. In control
HUVECs, actin microfilaments are found mostly in cortical structures forming characteristic ruffles (Fig. 3 a). Exposures to 0.5 mM H2O2 for 1 h induced a dramatic
SAPK2-dependent reorganization of F-actin, which rearranged from cortical microfilaments into long stress fibers
that traversed the cells (Fig. 3 b). This reorganization was
totally inhibited by SB203580 (data not shown and Huot et
al., 1997). Intriguingly, inhibiting ERK activation was associated with dramatic changes in the H2O2-induced microfilament organization. Instead of forming transcytoplasmic stress fibers, F-actin accumulated at the periphery of the cell, where actin filaments formed a dense spherical
network separating the blebs from the cytoplasm. F-actin
also accumulated at the perimeter of the blebs (Figs. 3 c
and 6 c). In presence of SB203580, the inhibition of membrane blebbing was associated with an almost total reversion of the F-actin reorganization toward control cells
(Fig. 3 d). To determine whether membrane blebbing was
a direct consequence of the SAPK2-mediated F-actin polymerization activities, we looked at the effect of the actin
polymerization inhibitor cytochalasin D using a very low
concentration (10 nM), just sufficient to inhibit microfilament reorganization without significantly affecting the
filament organization of uninduced cells. At this concentration, cytochalasin D blocked the formation of blebs induced by the combination of PD098059 and H2O2 and partially restored the normal pattern of F-actin as seen in
untreated cells (Fig. 3 e). Conversely, membrane blebbing
was not blocked by treating the cells with zVAD-fmk,
which totally inhibited caspase activities and DNA fragmentation (Figs. 3 f and 2, b and d). Furthermore, cytochalasin D, like SB203580, had no influence on the induction of DEVDase activities and internucleosomal DNA fragmentation (data not shown and Fig. 2, b and d). We next
verified whether inhibition of ERK was a sufficient condition to induce membrane blebbing. HUVECs pretreated
(Fig. 4, b, d, and f) or not (Fig. 4, a, c, and e) with
PD098059 were treated with FGF (Fig. 4, c and d) to activate ERK but not SAPK2 or VEGF (Fig. 4, e and f) to activate both ERK and SAPK2 (Huot et al., 1997
; Rousseau
et al., 1997
). Consistent with a role of SAPK2 in actin reorganization, we found that VEGF but not FGF induced
stress fiber formation in HUVECs (Fig. 4, c and e). However, neither FGF (Fig. 4, c and d) nor VEGF (Fig. 4, e and
f) induced membrane blebbing in presence of PD098059.
Moreover, stress fiber formation after VEGF was maintained even in presence of PD098059.
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The observation that the SAPK2-mediated actin reorganization in presence of PD098059 plus H2O2 results in membrane blebbing, whereas it still drives stress fiber formation in presence of PD098059/VEGF, suggests that oxidative stress induced some specific alterations that caused a misassembly of actin into transcytoplasmic stress fibers. Accordingly, we investigated the possibility that inhibiting ERK activation in the presence of oxidative stress but not VEGF was associated with an early defect in the assembly of the focal adhesion plaques to which stress fibers anchor. We found, within the first 5 min of treatments, a marked alteration in the distribution of focal adhesion plaques in cells treated with PD098059 and H2O2. In these cells, vinculin accumulated at the periphery of the cells instead of forming the typical elongated pattern found at the ventral face in cells treated with H2O2 only (Fig. 5, a and b). In contrast, pretreating the cells with PD098059 did not affect the VEGF-induced ventral distribution of vinculin into elongated focal adhesions (Fig. 5, c and d). The rapidity at which the redistribution of focal adhesion occurs after treatment with PD098059/H2O2 suggests that it may underlie the accumulation of F-actin required at the periphery of the cells to trigger bleb formation.
|
Regulation of actin dynamics by SAPK2 activation depends on the phosphorylation of HSP27, an F-actin polymerization factor that has a general cytoplasmic localization but is found at particularly high level at the leading
edge of lamellipodia and membrane ruffles where actin
polymerization is very intense (Lavoie et al., 1993; Pietrowicz and Levin, 1997
; Fig. 6, a and b). Intriguingly, we found that membrane blebs, which were delimited by a
thin ring of F-actin, contained an enhanced concentration
of HSP27 (Fig. 6, c and d). Since inhibition of bleb formation by SB203580 was accompanied by an inhibition of
HSP27 phosphorylation, this suggested that HSP27 may
be involved in modulating the local polymerization of
F-actin that was required for bleb formation. Endothelial
cells constitutively express high levels of HSP27 (6 ng/µg
total protein). To verify the role of HSP27 in bleb formation, we used three distinct clonal cell lines derived from
the CCL39 Chinese hamster fibroblasts. They express, in
addition to a low level of endogenous Chinese hamster
HSP27 (1.5 ng HSP27/µg total proteins), either a wild-type human HSP27 (clone B12, 4.8 ng HSP27/µg total proteins), a nonphosphorylatable HSP27 (clone V, 3.3 ng
HSP27/µg total proteins), or the neo gene only (clone 3)
(Fig. 7, a-i). Clone 3 and clone V respond similarly to
H2O2. In both cases, the oxidant induced a severe fragmentation of F-actin, but no bleb was formed whether
PD098059 was present or not (Fig. 7, a, d, and g, and Fig.
7, c, f, and i). In contrast, the clone B12, similarly to endothelial cells, formed stress fibers when exposed to H2O2
(Fig. 7, b and e) and membrane blebbing when H2O2 was
added together with PD098059 (Fig. 7, b and h). In addition, as in HUVECs, the blebbing activity was inhibited by
treating B12 cells with SB203580 (data not shown). ERK
inhibition in the presence of H2O2 was not associated
(even after 10 h) with DEVDase activation or, after 24 h,
with internucleosomal DNA fragmentation (Fig. 8, a and
b) in any of these three cell lines. These results identify
HSP27 phosphorylation as being involved in coupling
SAPK2 activity to bleb formation, and they clearly show
that surface blebbing can be dissociated from apoptosis.
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Cis-platinum-induced SAPK2-dependent Membrane Blebbing
The ERK pathway is mostly activated by physiological agonists that induce growth or differentiation responses, and in contrast to H2O2, most toxic agents induce little ERK activity. This raises the possibility that SAPK2-dependent membrane blebbing may be a general phenomenon that could be associated with the stress response generated by anticancer drug treatment. To test this possibility, the CCL39 clones were treated with 100 µM cis-platinum, a concentration that weakly activates ERK but increases SAPK2 activity up to five times (data not shown). Cis-platinum treatments of clone B12, which overexpresses the wild-type form of HSP27, resulted in the relocalization of F-actin at the cell cortex and a marked cell blebbing activity (Fig. 9, c and d). Again, this blebbing activity was inhibited by inhibiting SAPK2 with SB203580 (Fig. 9, e and f). In comparison, very little cell blebbing was observed in the control clone 3 (Fig. 9, a and b) and in the clone V that expresses the nonphosphorylatable form of HSP27 (data not shown).
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Discussion |
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Both SAPK2/p38 and ERK, two members of the MAP kinase family, are activated by oxidative stress in vascular
endothelial cells. Activation of SAPK2 leads to phosphorylation of the actin polymerization factor HSP27, which in
turn modulates stress fiber formation and recruitment of
vinculin to focal adhesions (Guay et al., 1997; Huot et al.,
1997
). This cytoskeleton reorganization is involved in
changing the shape of the endothelial cell and in modulating properties of the endothelium, such as permeability to
fluids, macromolecules, and inflammatory cells. In the
present study, we found that activation of the ERK pathway also had a major effect on the cell response to H2O2.
In the presence of PD098059, actin filaments, instead of
bundling into stress fibers, rather accumulated under the
plasma membrane, generating within 1 h intense membrane blebbing activities. Several hallmarks of apoptotic
cell death also became apparent at later times, namely
caspase activation, cleavage of PARP, and internucleosomal DNA fragmentation.
The rapidity and synchrony at which membrane blebbing occurred suggested a direct mechanism of induction.
Our results indicated that membrane blebbing lies in a
pathway distinct from apoptosis and can even be dissociated from it. Membrane blebbing was not downstream of
any of the apoptotic events measured in HUVECs. It occurred very early, well before DEVDase activation, PARP
cleavage, and internucleosomal DNA fragmentation, and
it occurred independently of zVAD-fmk-sensitive caspase
activation. Membrane blebbing was not upstream of these
events since cytochalasin D and SB203580 inhibited bleb
formation but did not affect DEVDase activities or internucleosomal DNA fragmentation. Moreover, in CCL39
cells expressing high levels of HSP27, membrane blebbing
was induced without detectable caspase activation or
DNA fragmentation. In addition to membrane blebbing,
PARP cleavage was also inhibited by SB203580. However,
PARP cleavage did not result from membrane blebbing
since it was not inhibited by cytochalasin D. This suggests that PARP cleavage and membrane blebbing are not required in the process of apoptosis. The existence of distinct independent pathways mediating the execution of
apoptosis has been suggested in several recent studies. In
KB cells, it was shown that Fas-mediated chromatin condensation and DNA fragmentation occur independently
of SAPK1- and SAPK2-mediated membrane blebbing,
cell shrinkage, and induction of Apo2.7 staining in mitochondria (Toyoshima et al., 1997). Moreover, expression
of a constitutively active Rac mutant induces membrane blebbing but not apoptosis in NIH-3T3 cells (Alexander et
al., 1998
). In other studies, it was demonstrated that cells
in which apoptosis is induced by serum deprivation, c-myc,
etoposide, or Bak accumulate in a doomed blebbing state
when caspases are inhibited with zVAD-fmk (McCarthy
et al., 1997
; Mills et al., 1997). It was proposed that membrane blebbing is a discrete subprogram operating during
mammalian apoptosis that can lead to cell death via
caspase-independent mechanism (McCarthy et al., 1997
).
Our data further suggest the corollary that surface
blebbing is not essential for apoptosis completion and can
even be dissociated from it. Membrane blebbing is thus a
primary manifestation of cell toxicity that is not necessarily linked to apoptosis.
In various cell types, caspase-3 is the major caspase involved in PARP cleavage. Intriguingly, we found that inhibiting SAPK2 activity with SB203580 inhibited PARP
cleavage but not caspase-3 or other DEVDase activities.
One possibility is that in endothelial cells, cleavage of
PARP involves a SAPK2-dependent protease unrelated to
DEVDases. Indeed, it is known that PARP can be cleaved
by proteases different from caspases (de Murcia and Ménissier-de Murcia, 1994). Another possibility is that SAPK2
collaborates with caspase-3 to the cleavage of PARP. For
example, PARP and/or caspase-3 might require phosphorylation downstream of SAPK2 to interact to each other.
Whereas there is no evidence that caspases are phosphorylated, phosphorylation of PARP has been observed both
in vitro and in vivo (Bauer et al., 1992
; Ruscetti et al.,
1998
). The protective mechanism of SB203580 on PARP
cleavage is under investigation.
The intense membrane blebbing activity that resulted
from inhibition of ERK in cells exposed to H2O2 was associated with an accumulation of F-actin at the periphery of
the cells and at the perimeter of the blebs. In fact, membrane blebbing was strictly dependent on actin polymerization activity, being blocked by cytochalasin D at a concentration that has no effect of the organization of the
actin filaments in control cells. The concentration of cytochalasin D (10 nM) was 100 times lower than that generally used to disrupt the actin filaments and is thought to
only compensate for new actin polymerization activity.
This finding suggests that the inhibition of membrane
blebbing by blocking SAPK2 activity with SB203580 is
also likely due to the inhibition of the actin polymerization activity generated by SAPK2 activation. Modulation of
actin dynamics by SAPK2 requires phosphorylation of
HSP27 by MAPKAP kinase-2/3, a physiological substrate
of SAPK2 (Rouse et al., 1994; McLaughlin et al., 1996
;
Guay et al., 1997
; Huot et al., 1997
). Activation of SAPK2
and phosphorylation of HSP27 are involved in an important pathway regulating F-actin dynamics, membrane ruffling, and pinocytosis in both endothelial cells and other
cells stimulated by stress and growth factors (Lavoie et al.,
1993
; Huot et al., 1996
, 1997
; Pietrowicz and Levin, 1998).
HSP27 behaves in vitro as an F-actin cap binding protein
and has a phosphorylation-modulated inhibitory function
on F-actin polymerization (Benndorf et al., 1994
). A pool
of HSP27 has been found associated with the basolateral
membrane of endothelial cells (Pietrowicz and Levin,
1998). Its rapid phosphorylation upon activation of
SAPK2 would release its binding to actin, allowing polymerization.
We found that blebs contained enhanced amounts of
HSP27, suggesting that this local concentration of HSP27
may be involved in modulating the SAPK2-actin-dependent formation of membrane blebbing. The role of HSP27
phosphorylation-mediated changes of F-actin dynamics in
membrane blebbing formation was more firmly demonstrated by showing that fibroblasts, which expressed
HSP27 in amounts comparable to HUVECs, responded
similarly to endothelial cells when they were treated with
H2O2. As in HUVECs, F-actin reorganized in these cells
into transcytoplasmic cables after exposures to H2O2. Furthermore, they formed blebs when they were exposed to
H2O2 and PD098059. This contrasts with the effect observed in control fibroblasts, where H2O2 treatment resulted in the fragmentation of preexisting stress fibers but
not in bleb induction. These data strongly suggest that the
SAPK2 activation by H2O2 modulates membrane blebbing
formation through HSP27 phosphorylation. In corollary,
these results indicate that the strong actin polymerization- generating activity of SAPK2/HSP27, which in normal
conditions may be useful for mediating endothelial remodeling, can be toxic during H2O2 treatment unless appropriate ERK activity is concomitantly induced. Interestingly,
in most cases, the ERK pathway is not activated strongly
by stressing agents, and most apoptotic stimuli induce
preferentially the stress-activated kinase pathways involving SAPK1 and SAPK2 (Verheij et al., 1996; Juo et al.,
1997
; Sanchez-Perez et al., 1998
). In fact, cis-platinum also
induced a SB203580-sensitive blebbing activity at a concentration that did not affect or weakly increased ERK activity (data not shown and Sanchez-Perez et al., 1998
) expanding the concept that activation of the SAPK2/HSP27 pathway can, depending on the cell context, be responsible
for induction of membrane blebbing activities in response
to stress. Inhibition of the ERK pathway was not, however, sufficient by itself to induce membrane blebbing in
HUVECs. FGF, which strongly activated ERK without affecting SAPK2 (Huot et al., 1997
), did not induce bleb formation when ERK was blocked with PD098059. Furthermore, inhibiting ERK activation during cell stimulation with VEGF, which like H2O2 activates both ERK and
SAPK2 and produced a SAPK2-mediated actin reorganization (Rousseau et al., 1997
), did not produce cell blebbing. Thus, a strong imbalance between activation of
SAPK2 and ERK signaling pathways was also not sufficient to induce membrane blebbing. Membrane blebbing
would therefore require, in addition to high SAPK2 activity and low ERK activity, some stress-induced alterations
that prevent proper organization of F-actin into transcytoplasmic stress fibers.
Although several reports have illustrated the role of
F-actin in the membrane blebbing process, very little is
known on the exact mechanisms involved. Membrane
blebbing induction may share mechanistic features with
the formation of membrane protrusions, like lamellipodia
and filopodia involved in cell motility (Mitchison and
Cramer, 1996). The formation of motile protrusions involves actin polymerization, which coupled with the action of unconventional myosin pushes the membrane forward. It also involves conventional myosin that drives the
retraction of the cytoplasm (Mitchison and Cramer, 1996
).
Recently, strong evidence has been presented implying a
role of conventional and unconventional myosins in the
formation of blebs in serum-deprived cells (Mills et al., 1998
). It has been proposed that the centripetal force generated by myosin II would cause extrusion of the cytoplasm in regions where there exist weak anchor points between the membrane and actin. Similarly, unconventional
myosin, which in normal conditions pushes F-actin outwards in the protrusions, would be responsible for bleb retraction in regions where contact of F-actin with the substratum are not appropriate. Bundling of actin into
transcytoplasmic stress fiber formation as well as formation of motile cell protrusion are tightly associated with
formation of focal adhesions to which actin filaments are
anchored (Nobes and Hall, 1995
). Focal adhesions are
sites of tight adhesion between the membrane and the extracellular matrix on one hand, and the membrane and the
cytoskeleton on the other. They are assembled after the
recruitment of signaling molecules such as FAK and paxillin, and of structural and membrane actin-anchoring proteins such as talin, vinculin, tensin, and
-actinin, which link the microfilament network to the adhesive molecule
integrins at their sites of clustering (Burridge et al., 1997
).
These proteins provide a structural link allowing the anchorage of stress fibers to the membrane and to integrins.
We found here that H2O2 and VEGF, which both activate
ERK and SAPK2, induce a SAPK2-dependent formation
of transcytoplasmic stress fibers associated with recruitment of vinculin to ventral adhesion plaques. In the presence of PD098059, H2O2 but not VEGF, induces a major
defect in the assembly of focal adhesions, which was characterized by the accumulation of vinculin at the periphery
of the cells instead of forming the typical elongated pattern at the ventral face. This quick redistribution of focal adhesions induced by H2O2 suggests that it may be causal
to membrane blebbing. Interestingly, oxidants have been
shown to impair formation of focal adhesion by stimulating the activity of calpain leading to the degradation of
talin and
actinin. In hepatocytes, this effect has been
proposed to underlie membrane blebbing (Miyoshi et al.,
1995). Moreover, H2O2 treatments induce a rapid translocation from the membrane to the cytoplasm of filamin
a
protein involved in anchoring actin to membrane proteins
(Hastie et al., 1997
)
a redistribution of integrins from focal adhesion plaques to the apical and lateral surface of
the cells (Bradley et al., 1995
), and activation of MLCK
and phosphorylation of MLC (Zhao and Davis, 1998
). We
thus propose that a weakening of the membrane-actin and
actin-substratum linkage at the focal adhesions combined
with a stronger myosin contractile force and with increased actin polymerization activity generated by SAPK2
may all contribute to formation of blebs during exposure
to H2O2. Finding how inhibition or low activity of the
ERK pathway contributes to the misassembly of focal adhesions and to the increase in bleb formation by H2O2 in
endothelial cells may provide a clue for the general protective function described for the ERK pathways during exposure of cells to H2O2 and other apoptotic stimuli.
In several cell systems, activation of SAPK2 has been
correlated with cellular toxicity and with the onset of apoptosis (Kummer et al., 1997; Schwenger et al., 1997
). The
major contribution of our study is to describe a mechanism
by which SAPK2 activation can lead to membrane blebbing. The blebbing activity depends first on the induction
of a stress-sensitive misassembly of focal adhesions, which
is amplified in the absence of ERK activation, and second on a strong F-actin reorganization activity generated by
phosphorylation of the F-actin cap binding protein HSP27.
Endothelial cells, smooth muscle cells, and many tumor
cells express high levels of HSP27, suggesting that the described mechanisms of SAPK2-induced cell blebbing may
be of wide occurrence. In other cellular contexts, activation by stress of other actin signaling pathways may similarly activate blebbing activity. For example, RhoA also
leads to membrane blebbing through a stimulation of actin
polymerization activity and through an increase in myosin-dependent contractile forces (Watanabe et al., 1997
; Mills
et al., 1998
). In summary, our results suggest a key role
played by actin organization and dynamics in modulating
membrane blebbing generated by stresses, and they illustrate how the intersignaling balance between biological
transduction pathways can modulate the fine tuning regulation of survival and toxic responses.
![]() |
Footnotes |
---|
Address correspondence to Dr. Jacques Huot, Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, 11 côte du Palais, Québec, G1R 2J6, Canada. Tel.: (418) 691-5553. Fax: (418) 691-5439. E-mail: Jacques.Huot{at}phc.ulaval.ca
Received for publication 15 June 1998 and in revised form 8 October 1998.
We thank Dr John Grose for providing the anti-ERK2. We also thank Dr. François Marceau and the staff of the Pathology Laboratory of the Hôpital St-François d'Assise, Québec, for providing the umbilical cords and Éric Pellerin for his help in confocal microscopy. We also thank Dr. Joseé Lavoie for helpful discussion.
This study was supported by the Medical Research Council of Canada (grant MT-13177) and the Cancer Research Society, Inc. Simon Rousseau owns a studentship from the CRS.
![]() |
Abbreviations used in this paper |
---|
EBSS, Earle's balanced salt solution;
ECGS, endothelial cell growth supplement;
FGF, fibroblast growth factor;
ERK, extracellular signal-regulated kinase;
GST, glutathione S-transferase;
HSP27, heat shock protein of 27 kD;
HUVEC, human umbilical
vein endothelial cells;
ICE, interleukin 1-converting enzyme;
IL, interleukin;
JNK/SAPK1, jun NH2-terminal kinase/stress-activated protein kinase-1;
MAP kinase, mitogen-activated protein kinase;
MAPKAP K2, MAP kinase-activated protein kinase-2;
PARP, poly-(ADP-ribose)-polymerase;
SAPK2, stress-activated protein kinase-2/p38;
TNF
, tumor necrosis factor
;
VEGF, vascular endothelial growth factor.
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References |
---|
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---|
1. | Alexander, M.A., J.E. Meredith, and W.K. Kiosses. 1998. An activated Rac mutant functions as a dominant negative for membrane ruffling. Oncogene. 17: 625-629 |
2. | Bauer, P.I., G. Farkas, L. Buday, G. Mikala, G. Meszaros, E. Kun, and A. Farago. 1992. Inhibition of DNA binding by the phosphorylation of poly ADP-ribose polymerase protein catalyzed by protein kinase C. Biochem. Biophys. Res. Commun. 187: 730-736 |
3. | Becker, L.C., and G. Ambrosio. 1987. Myocardial consequences of reperfusion. Progr. Cardiovasc. Dis. 30: 23-44 |
4. | Benndorf, R., K. Haye, S. Ryazantsev, M. Wieske, J. Behlke, and G. Lutsch. 1994. Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerizion-inhibiting activity. J. Biol. Chem. 269: 255-261 . |
5. | Bradley, J.R., S. Thiru, and J.S. Pober. 1995. Hydrogen peroxide-induced endothelial retraction is accompanied by a loss of the normal spatial organization of endothelial cell adhesion molecules. Am. J. Pathol. 147: 627-641 [Abstract]. |
6. |
Brenner, B.,
U. Koppenhoefer,
C. Weinstock,
O. Linderkamp,
F. Lang, and
E. Gulbins.
1997.
Fas- or ceramide-induced apoptosis is mediated by a Rac1-regulated activation of Jun N-terminal kinase/p38 kinases and GADD153.
J.
Biol. Chem.
272:
22173-22181
|
7. | Burridge, K., M. Chrzanowska-Wodnicka, and C. Zhong. 1997. Focal adhesion assembly. Trends Cell Biol. 7: 342-347 . |
8. | Cahill, M.A., M.E. Peter, F.C. Kischkel, A.R. Chinnaiyan, V.D. Dixit, P.H. Krammer, and A. Nordheim. 1996. CD95 (APO-1/Fas) induces activation of SAP kinases downstream of ICE-like proteases. Oncogene. 13: 2087-2096 |
9. | Cardone, M.H., G.S. Salvesen, C. Widmann, G. Johnson, and S.M. Frish. 1997. The regulation of anoikis: MEKK-1 activation requires cleavage by caspases. Cell. 90: 315-323 |
10. | Cohen, P.. 1997. The search for physiological substrates of MAP and SAP kinases in mammalian cells. Trends Cell Biol. 7: 353-361 . |
11. | Cuenda, A., J. Rouse, Y.N. Doza, R. Meier, P. Cohen, T.F. Gallagher, P.R. Young, and J.C. Lee. 1995. SB203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 364: 229-233 |
12. | Cunningham, C.C.. 1995. Actin polymerization and intracellular solvent flow in cell surface blebbing. J. Cell Biol. 129: 1589-1599 [Abstract]. |
13. | Darmon, A.J, D.W. Nicholson, and R.C. Beackley. 1995. Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. Nature. 377: 446-448 |
14. | de Murcia, G., and J. Ménissier-de Murcia. 1994. Poly (ADP-ribose) polymerase: a molecular nick sensor. Trends Biochem. Sci. 19: 172-176 |
15. | Dudley, D.T., L. Pang, S.J. Decker, A.J. Bridges, and A.L Saltiel. 1995. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA. 92: 7686-7689 [Abstract]. |
16. | Enari, M., R.V. Talanian, W.W. Wong, and S. Nagata. 1996. Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature. 380: 723-726 |
17. | Fishkind, D.J., L. Cao, and Y. Wang. 1991. Microinjection of the catalytic fragment of myosin light chain kinase into dividing cells: effects on mitosis and cytokinesis. J. Cell Biol. 114: 967-975 [Abstract]. |
18. | Frisch, S.M., and H. Francis. 1994. Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol. 124: 619-626 [Abstract]. |
19. | Gores, G.J., B. Herman, and J.J. Lemasters. 1990. Plasma membrane bleb formation and rupture: a common feature of hepatocellular injury. Hepatology. 4: 609-698 . |
20. |
Guay, J.,
H. Lambert,
G. Gingras-Breton,
J.N. Lavoie,
J. Huot, and
J. Landry.
1997.
Regulation of actin filament dynamics by p38 map kinase mediated
phosphorylation of heat shock protein.
J. Cell Sci.
110:
357-368
|
21. |
Guyton, K.Z.,
Y. Liu,
M. Gorospe,
Q. Xu, and
N.K. Holbrook.
1996.
Activation
of mitogen-activated protein kinase by H2O2.
J. Biol. Chem.
271:
4138-4142
|
22. |
Hardy, M.M.,
A.G. Flickinger,
D.P. Riley,
R.H. Weiss, and
U.S. Ryan.
1994.
Superoxide dismutase mimetics inhibit neutrophil-mediated human aortic
endothelial cell injury in vitro.
J. Biol. Chem.
269:
18535-18540
|
23. | Hastie, L.E., W.F. Patton, H.B. Hechtman, and D. Shepro. 1997. Filamin redistribution in an endothelial cell reoxygenation injury model. Free Radic. Biol. Med. 22: 955-966 |
24. | Huot, J., H. Lambert, J.N. Lavoie, A. Guimond, F. Houle, and J. Landry. 1995. Characterization of 45-kDa/54-kDa HSP27 kinase, a stress-sensitive kinase which may activate the phosphorylation-dependent protective function of mammalian 27-kDa heat shock protein HSP27. Eur. J. Biochem. 227: 416-427 [Abstract]. |
25. | Huot, J., F. Houle, D.R. Spitz, and J. Landry. 1996. HSP27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Res. 56: 273-279 [Abstract]. |
26. |
Huot, J.,
F. Houle,
F. Marceau, and
J. Landry.
1997.
Oxidative stress-induced
actin reorganization mediated by the p38 mitogen-activated protein kinase/
heat shock protein 27 pathway in vascular endothelial cells.
Circ. Res.
80:
383-392
|
27. | Juo, P., C.J. Kuo, S.E. Reynolds, R.F. Konz, J. Raingeaud, R.J. Davis, H.P. Biemann, and J. Blenis. 1997. Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-family proteases. Mol. Cell. Biol. 17: 24-35 [Abstract]. |
28. | Katoh, O., H. Tauchi, K. Kawaishi, A. Kimura, and Y. Satow. 1995. Expression of the vascular endothelial growth factor (VEGF) receptor gene, KDR, in hematopoietic cells and inhibitory effect of VEGF on apoptotic cell death caused by ionizing radiation. Cancer Res. 55: 5687-5692 [Abstract]. |
29. |
Kawasaki, H.,
T. Morooka,
S. Shimohama,
J. Kimura,
T. Hirano,
Y. Gotoh, and
E. Nishida.
1997.
Activation and involvement of p38 mitogen-activated protein kinase in glutamate-induced apoptosis in rat cerebellar granule cells.
J.
Biol. Chem.
272:
18518-18521
|
30. |
Kumar, S.,
M.J. Orsini,
J.C. Lee,
P.C. McDonnell,
C. Debouck, and
P.R. Young.
1996.
Activation of the HIV-1 long terminal repeat by cytokines and
environmental stress requires an active CSBP/p38 MAP kinase.
J. Biol.
Chem.
271:
30864-30869
|
31. |
Kummer, J.L.,
P.R. Rao, and
K.R. Heidenreich.
1997.
Apoptosis induced by
withdrawal of trophic factors is mediated by p38 mitogen-activated protein
kinase.
J. Biol. Chem.
272:
20490-20494
|
32. | Landry, J., P. Chrétien, H. Lambert, E. Hickey, and L.A. Weber. 1989. Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells. J. Cell Biol. 109: 93-101 [Abstract]. |
33. |
Lavoie, J.N.,
E. Hickey,
L.A. Weber, and
J. Landry.
1993.
Modulation of actin
microfilament dynamics and fluid phase pinocytosis by phosphorylation of
heat shock protein 27.
J. Biol. Chem.
268:
24210-24214
|
34. | Lavoie, J.N., H. Lambert, E. Hickey, L.A. Weber, and J. Landry. 1995. Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27. Mol. Cell. Biol. 15: 505-516 [Abstract]. |
35. | Lee, J.U., R. Hosotani, M. Wada, R. Doi, T. Kosiba, K. Fujimoto, C. Mori, N. Nakamura, K. Shiota, and M. Imamura. 1997. Mechanism of apoptosis induced by cisplatin and VP16 in PANC-1 cells. Anticancer Res. 17: 3445-3450 |
36. |
Martin, S.J.,
G.A. O'Brien,
W.K. Nishioka,
A.J. McGahon,
A. Mahboubi,
T.C. Saido, and
D.R. Green.
1995.
Proteolysis of fodrin (non erythroid spectrin)
during apoptosis.
J. Biol. Chem.
270:
6425-6428
|
37. | Martin, S.M., D.R. Green, and T.G. Cotter. 1994. Dicing with death: dissecting the components of the apoptosis machinery. Trends Biol. Sci. 19: 26-30 . |
38. |
McCarthy, N.J.,
M.K.B. Whyte,
C.S. Gilbert, and
G.I. Evan.
1997.
Inhibition of
Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak.
J. Cell Biol.
136:
215-227
|
39. |
McLaughlin, M.M.,
S. Kumar,
P.C. McDonnell,
S. Van Horn,
J.C. Lee,
G.P. Livi, and
P.R. Young.
1996.
Identification of mitogen-activated protein
(MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38
MAP kinase.
J. Biol. Chem.
271:
8488-8492
|
40. | Michiels, C., T. Arnoud, A. Houbion, and J. Remacle. 1992. Human umbilical vein endothelial cells submitted to hypoxia-reoxygenation in vitro: implication of free radicals, xanthine oxidase, energy deficiency. J. Cell. Physiol. 153: 53-61 |
41. |
Mills, J.C.,
N.L. Stone,
J. Erhardt, and
R.N. Pittman.
1998.
Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation.
J. Cell
Biol.
140:
627-636
|
42. | Mirabelli, F., A. Salis, V. Marinoni, G. Finardi, G. Bellomo, H. Thor, and S. Orrenius. 1988. Menadione-induced bleb formation in hepatocytes is associated with the oxidation of thiol groups in actin. Arch. Biochem. Biophys. 264: 261-269 |
43. | Mitchison, T.J., and L.P. Cramer. 1996. Actin-based cell motility and cell locomotion. Cell. 84: 371-379 |
44. | Miyoshi, H., K. Umeshita, M. Sakon, S. Imajoh-Ohmi, K. Fujitani, M. Gotoh, E. Oiki, J. Kambayashi, and M. Monden. 1996. Calpain activation in plasma membrane bleb formation during tert-butyl hydroperoxide-induced rat hepatocyte injury. Gastroenterology. 110: 1897-1904 |
45. | Mosser, D.D., A.W. Caron, L. Bourget, C. Denis-Larose, and B. Massie. 1997. Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol. Cell. Biol. 17: 5317-5327 [Abstract]. |
46. |
Na, S.,
T.H. Chuang,
A. Cunningham,
T.G. Turi,
J.H. Hanke,
G.M. Bokoch, and
D.E. Danley.
1996.
D4-GDI, a substrate of CPP32, is proteolyzed during
Fas-induced apoptosis.
J. Biol. Chem.
271:
11209-11213
|
47. | Nobes, C.D., and A. Hall. 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81: 53-62 |
48. | Phelps, P.C., M.W. Smith, and B.F. Trump. 1989. Cytosolic ionized calcium and bleb formation after acute cell injury of cultured rabbit renal tubule cells. Lab. Invest. 60: 630-642 |
49. |
Pietrowicz, R.S., and
E.G. Levin.
1997.
Basolateral membrane-associated 27-kDa heat shock protein and microfilament polymerization.
J. Biol. Chem.
272:
25920-25927
|
50. | Re, F., A. Zanetti, M. Sironi, N. Polentarutti, L. Lanfrancone, E. Dejana, and F. Colotta. 1994. Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J. Cell Biol. 127: 537-546 [Abstract]. |
51. | Rouse, J., P. Cohen, S. Trigon, M. Morange, A. Alonso-Llamazares, D. Zamanillo, T. Hunt, and A.R. Nebrada. 1994. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell. 78: 1027-1037 |
52. | Rousseau, S., F. Houle, J. Landry, and J. Huot. 1997. p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene. 15: 2169-2177 |
53. |
Ruscetti, T.,
B.E. Lehnert,
J. Halbrook,
H. Le Trong,
M.F. Hoekstra,
D.J. Chen, and
S.R. Peterson.
1998.
Stimulation of the DNA-dependent protein
kinase by poly (ADP-ribose) polymerase.
J. Biol. Chem.
273:
14461-14467
|
54. |
Saitoh, M.,
H. Nishitoh,
M. Fujii,
K. Takeda,
K. Tobiume,
Y. Sawada,
M. Kawabata,
K. Miyazono, and
H. Ichijo.
1998.
Mammalian thioredoxin is a direct
inhibitor of apoptosis signal-regulating kinase (ASK) 1.
EMBO (Eur. Mol.
Bio. Organ.) J.
17:
2596-2606
|
55. | Sanchez-Perez, I., J.R. Murguia, and R. Perona. 1998. Cisplatin induces a persistent activation of JNK that is related to cell death. Oncogene. 16: 533-540 |
56. |
Schwenger, P.,
P. Bellosta,
I. Vietor,
C. Basilico,
E.Y. Skolnik, and
J. Vilcek.
1997.
Sodium salicylate induces apoptosis via p38 mitogen-activated protein
kinase but inhibits tumor necrosis factor-induced c-Jun N-terminal stress-activated protein kinase activation.
Proc. Natl. Acad. Sci. USA.
94:
2869-2873
|
57. |
Seimiya, H.,
T. Mashima,
M. Toho, and
T. Tsuruo.
1997.
c-Jun NH2-terminal kinase-mediated activation of interleukin-1![]() |
58. | Shah, G.M., S.H. Kaufman, and G.G. Poirier. 1995. Detection of poly-(ADP-ribose) polymerase and its apoptosis-specific fragment by a nonisotopic activity-Western blot technique. Anal. Biochem. 232: 251-254 |
59. | Shah, G.M., R.G. Shah, and G.G. Poirier. 1996. Different cleavage pattern for poly(ADP-ribose) polymerase during apoptosis and necrosis in HL-60 cells. Biochem. Biophys. Res. Commun. 229: 838-844 |
60. |
Toyoshima, F.,
T. Moriguchi, and
E. Nishida.
1997.
Fas induces cytoplasmic responses and activation of the MKK7-JNK/SAPK and MKK6-p38 pathways
independent of CPP32-like proteases.
J. Cell Biol.
139:
1005-1015
|
61. | Van de Voorde, J., H. Vanderstichele, and I. Levsen. 1987. Release of endothelium-derived relaxing factor from human umbilical vessels. Circ. Res. 60: 517-522 [Abstract]. |
62. | van der Zee, R., T. Murohara, Z. Luo, F. Zollmann, J. Passeri, C. Lekutat, and J.M. Isner. 1997. Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium. Circulation. 18: 1030-1037 . |
63. | Verheij, M., R. Bose, X.H. Lin, B. Yao, W.D. Jarvis, S. Grant, M.J. Birrer, E. Szabo, L.J. Zon, J.M. Kyriakis, et al . 1996. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature. 380: 75-79 |
64. | Waskiewicz, A.J., and J.A. Cooper. 1995. Mitogen and stress response pathways: Map kinase cascades and phosphatase regulation in mammals and yeast. Curr. Opin. Cell Biol. 7: 798-805 |
65. |
Watanabe, N.,
P. Madaule,
T. Reid,
T. Ishizaki,
G. Watanabe,
A. Kakizuka,
Y. Saito,
K. Nakao,
B.M. Jockusch, and
S. Narumiya.
1997.
p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small
GTPase and is a ligand for profilin.
EMBO (Eur. Mol. Biol. Organ.) J.
16:
3044-3056
|
66. | Wei, Y.Q., X. Zhao, Y. Karya, K. Teshigawara, and A. Uchida. 1995. Inhibition of proliferation and induction of apoptosis by abrogation of heat shock protein (HSP)70 expression in tumor cells. Cancer Immunol. Immunother. 40: 73-78 |
67. | Xia, Z., M. Dickens, J. Raingeaud, R.J. Davis, and M.E. Greenberg. 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 270: 1326-1331 [Abstract]. |
68. | Zanke, B.W., K. Boudreau, E. Rubie, E. Winnett, L.A. Tibbles, L. Zon, J. Kyriakis, F.F. Liu, and J.R. Woodgett. 1996. The stress activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation or heat. Curr. Biol. 6: 606-613 |
69. | Zhao, Y., and H.W. Davis. 1998. Hydrogen peroxide-induced cytoskeletal rearrangement in cultured pulmonary endothelial cells. J. Cell. Physiol. 174: 370-379 |
70. |
Zulueta, J.J.,
R. Sawhney,
F.S. Yu,
C.C. Cote, and
P.M. Hassoum.
1997.
Intracellular generation of reactive oxygen species in endothelial cells exposed to
anoxia-reoxygenation.
Am. J. Physiol.
272:
L897-L902
|