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INTRODUCTION |
Control of apoptosis is essential to development and homeostasis
in mammals. Progression of the apoptotic signal can be inhibited at
different levels depending on death inducers and cell types. Thus,
endogenous caspase inhibitors such as cellular FLICE-like inhibitory
protein (c-FLIP) (1) have been shown to prevent apoptotic transduction
through inhibition of the initiator caspase-8 maturation. Bcl-2 and
Bcl-Xl (2, 3) are also capable of blocking apoptosis, but this time at
mitochondrion level by preventing cytochrome c release (4)
and hence the downstream maturation of caspase-9. In addition,
inactivation of caspases might even occur after their cleavage through
AKT-dependent phosphorylation of caspase-9 (5) or by
inhibitors of apoptosis proteins
(IAPs)1 that bind to cleaved
caspase-3, -7, and -9 (6). Moreover, nitric oxide (NO), generated by NO
donors or NO synthases, has been reported to block activity of several
members of the caspase family via S-nitrosylation of their
catalytic site (7, 8). All these mechanisms are aimed at preventing
inappropriate procaspase maturation or inhibiting activity of matured caspases.
In TNF
-induced apoptosis, upon TNF binding,
oligotrimerization of TNF receptors occurs and results in
aggregation of death domain-containing proteins, allowing recruitment
of TRADD (TNF receptor I-associated death domain protein). TRADD binds
FADD (Fas-associated death domain-containing protein) and TRAF-2 (TNF receptor I-associated protein 2) proteins, which in turn lead to
activation of procaspase-8 and ASK1 (apoptosis signal-regulating kinase
1), respectively (9). ASK1 is a MAP kinase kinase kinase (MAPKKK),
ubiquitously expressed, that activates the MKK4/MKK7-JNK (c-Jun
N-terminal kinase) and MKK3/MKK6-p38 signaling cascades (10). JNK and
p38 MAP kinase are preferentially activated by stress agents such as UV
radiation, osmotic shock, and proinflammatory cytokines, including
TNF. The disruption of the ASK1 gene in mouse (11) strongly reduces
cell death induced by these activators indicating that ASK1 is a key
element in cytokine- and stress-induced apoptosis (9). In addition,
overexpression of wild-type or constitutively active form of ASK1
induces apoptosis in various cell types (10, 12, 13), whereas the
kinase-inactive mutant of ASK1 inhibits it (10, 12, 14). ASK1 activity
is negatively regulated through phosphorylation by AKT (15) or
dephosphorylation by CDC25 (16) or protein phosphatase 5 (17), or
through sequestration by proteins such as thioredoxin (12, 18), 14-3-3 (19), or Raf-1 (20). Intriguingly, a recent report shows that mouse
glutathione S-transferase mu1 (GST M1) (21) can also bind
ASK1.
GST M1 is a member of the GST protein family that catalyzes the
conjugation of reduced glutathione (GSH) to a variety of electrophiles (22). In addition to this role, these proteins have also been shown to
serve as nonenzymatic binding proteins interacting with various
lipophilic compounds that include steroid and thyroid hormones (23).
GSTs have been grouped into eight classes, with the most abundant ones
being the alpha, mu, and pi classes (22). In rat hepatocytes, GSTP1
have been associated to cell proliferation and decreased level of
differentiation in contrast to GST A1/2, which is related to high level
of differentiation. GST M1/2 seemed to be expressed under both
situations (24, 25). In addition, GST M1 and P1 appear to act as
direct inhibitors of ASK1 and JNK, respectively, independently of their
catalytic detoxication activity and of cellular GSH level (21, 26).
This strongly suggests that GSTs might be involved in cell protection
against apoptotic signals, such as osmotic stress, TNF, or UV
radiation, through both detoxication and inhibition of the
stress-signaling cascade ASK1-JNK. However, the contribution of GSTs,
proteins highly expressed in liver parenchymal cells, to apoptosis of
these cells has never been investigated.
Liver injuries can be induced by a variety of apoptotic factors such as
TNF or Fas ligand (27, 28), as well as hepatotoxins such as
chloroform (29), acetaminophen (30), and thioacetamide (THA) (31, 32).
However the mechanisms by which they induce cell damage are poorly
documented. In addition, numerous physiological factors such as IL-6
(33) or soluble TNF receptors (34) and free radical scavengers such
as Me2SO and dimethylthiourea (31), have been reported to
be able to protect the liver against injuries induced by apoptotic signals.
Me2SO protects, in vivo, hepatocytes from cell
death in acute hepatitis induced by THA (31) or acetaminophen (30).
In vitro, Me2SO is used as a powerful inducer
for long term survival and differentiation in primary hepatocyte
cultures (35). Moreover, TNF is known to induce apoptosis in
Me2SO-treated hepatocyte cultures, but only after
Me2SO removal (36). It has been then postulated that
Me2SO might protect liver from hepatotoxin by scavenging
hydroxyl radicals (31) but the mechanism by which Me2SO
would inhibit apoptosis pathway in hepatocytes has not yet been elucidated.
In this study, we have investigated the mechanism by which liver
protection is controlled using Me2SO as hepatoprotector by focusing our attention on the apoptotic transduction inhibition. In vivo, THA-induced liver failure known to induce
hepatocyte apoptosis was chosen, and cultures of hepatocytes undergoing
apoptosis were designed as an in vitro model. We provide
evidence that hepatoprotection may result from both inhibition of
caspase cascade, through an efficient inactivation of cleaved initiator
caspases and prevention of ASK1-JNK activities via GST regulation.
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EXPERIMENTAL PROCEDURES |
Animals and Treatments--
Male Lewis rats and BALB/c mice
(Elevage Janvier) were injected intraperitoneally with a single dose of
thioacetamide (THA, Sigma), 100 mg/kg. In some experiments,
Me2SO (2 ml/kg, Sigma) was also injected 1 h prior to
THA, then at 12 and 24 h after the THA injection according to the
protocol of Bruck et al. (31). Animals were sacrificed at
different times after THA injection (for animals receiving only THA)
and 30 h after THA administration when considering animals treated
with both THA and Me2SO; this latter time corresponded to
maximal DEVD-AMC caspase activity in THA-treated animals.
Reagents--
Rabbit anti-GST mu1/2, alpha1/2 and pi1 antibodies
were from Biotrin International (Ireland). Anti-ASK1 (DAV) was
previously described (12). Anti-caspase-3, -MKP1 and -JNK were from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-caspase-8 (APP-108) and
anti-caspase-9 were from StressGen Biotechnologies Corp. (Tebu, France), and anti-P-JNK, -P-p38, and -p38MAPK from Cell Signaling Technology (Tebu, France). Secondary antibodies conjugated to horseradish peroxidase were from DAKO (DAKO SA, France). Fluorigenic substrates were from BACHEM (BACHEM) and prepared at 100 mM
in the recommended solvent. The CasputinTM reagent, a
specific inhibitor of caspases-3 and -7, corresponding to an
Escherichia coli-expressed recombinant fusion protein, which comprises BIRs (baculovirus inhibitor of apoptosis protein Repeats) 2 and 3 from human XIAP (X-linked inhibitor of apoptosis protein), was
purchased from Biomol (Tebu, France). Transforming growth factor 
1
(TGF 1, R&D Systems) and TNF (PromoCell, Heidelberg, Germany)
were prepared according to the manufacturer's instructions.
Isolation and Primary Culture of Hepatocytes--
Hepatocytes
were isolated and purified from male Sprague-Dawley rats (Elevage
Janvier, France) as described previously (37). Hepatocytes were seeded
at 7 × 104 cells/cm2 on plastic dishes in
a mixture of 75% minimum essential medium and 25% medium 199, supplemented with 10% fetal calf serum, and per ml: 100 IU of
penicillin, 100 µg of streptomycin, 1 mg of bovine serum albumin, 2 µmol of L-glutamine, and 5 µg of bovine insulin.
Four hours after plating, the medium was removed, and cultures were
maintained in different media: 1) basal medium, corresponding to
plating medium, deprived in fetal calf serum and supplemented with
1.4 × 10
7 M hydrocortisone
hemisuccinate (Roussel-UCLAF), 2) Me2SO medium, corresponding to basal medium supplemented with 2% Me2SO;
3) TGF1 or TNF medium, TGF1 (1 or 2.5 ng/ml) or TNF (20 ng/ml) was added to basal medium at 24 h after cell plating.
Treatment was then performed for 2 days. Appropriate media were renewed
every day.
Immunoblotting Analysis--
Freshly isolated hepatocytes and
cultured cells were harvested, washed with phosphate-buffered saline,
and stored as pellets at 
80 °C. Hepatocytes were lysed in a
Western blot lysis buffer, and protein content was measured as
previously described (38). 100 µg of proteins were resolved on
7.5-12.5% SDS-PAGE and transferred onto polyvinylidene difluoride
membranes (PVDF, Bio-Rad). Subsequently, nonspecific binding sites were
blocked with Tris-buffered saline (TBS) containing 4% bovine serum
albumin, for 1 h at room temperature. Then, filters were incubated
overnight at 4 °C with primary antibody in TBS containing 4% bovine
serum albumin. Filters were washed three times with TBS and incubated
with appropriate secondary antibody conjugated to horseradish
peroxidase, for 1 h at room temperature. Following 4-5 washes
with TBS, the proteins of interest were visualized with
SupersignalTM (Pierce Chemical Co.).
DNA Extraction and Agarose Gel Electrophoresis--
Adherent and
non-adherent hepatocytes from 60-mm dishes were harvested and
centrifuged at 2000 rpm for 2 min at 4 °C. DNA was isolated from
cultured cells or fresh biopsies with High Pure PCR template
preparation kit (Roche Diagnostics). After purification, samples were
analyzed by electrophoresis on a 1% agarose gel and observed under UV light.
Caspase Activity Assay--
Hepatocytes and liver biopsies were
lysed in the caspase activity buffer (39). 100 µg of crude cell
lysate were incubated with 80 µM substrate-AMC for 1 h at 37 °C. Caspase-mediated cleavage of peptide-AMC was measured by
spectrofluorimetry (Molecular Devices) at the excitation/emission
wavelength pair (ex/em) of 380/440 nm. The caspase activity was given
in arbitrary units of fluorescence (per 100 µg of total protein).
Detection of S-Nitrosylation--
Caspase activity was measured
after preincubation of cytosol with or without 10 mM DTT
for 10 min at room temperature. In these experiments, caspase activity
buffer did not contain DTT.
Transfection--
24 h after Me2SO addition,
hepatocytes were transfected using GB12 as previously described (40).
Per dish, a total of 4 µg of DNA plasmid was mixed with 10 µg of
GB12. Human GST M1, A1, and P1 cDNAs were obtained by PCR from
liver tissue RNA and subcloned in pClneo (Promega). pcDNA3.1
HA-ASK1 and HA-ASK1 (Km, kinase mutant) were previously described
(21).
ASK1 Activity--
Analysis was performed as previously
described (21) with minor modifications. The substrate of ASK1, 0.5 µg of GST-MKK4 per reaction was purchased from Upstate Biotechnology
(Tebu, France).
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RESULTS |
Massive Apoptosis Involving Caspase-9, -8, and -3 Occurs in Basal
Primary Culture of Hepatocytes--
To investigate the mechanism by
which free radical scavengers like Me2SO modulate the
apoptotic process in hepatocytes, a study of such a process in basal
primary culture of adult hepatocytes was first performed. A minimal
medium composed of a mixture of MEM/M199 medium supplemented with
insulin and a low concentration of glucocorticoids (107
M) was chosen, limiting hepatocyte life-span to a few days.
Apoptotic process undergone by hepatocytes under this condition was
characterized (Fig. 1) and compared with
that induced in the presence of TGF1 used as a potent apoptotic
inducer in hepatocytes (41).
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Fig. 1.
Kinetics of caspase expression and maturation
during apoptosis in rat hepatocytes. Kinetics of cell death
evaluated by protein content per dish (A). Cells were
maintained under basal medium (basal) or treated with
TGF1 (2.5 ng/ml). Kinetics of DNA degradation (B) and
Western blot analysis of caspase-9, -8, -3, and PARP (C) in
normal liver (NL), freshly isolated (T0), and
cultured hepatocytes, 4 h after plating (4h), and at
different days of culture. M, molecular weight markers.
DEVD- and IETD-AMC (D) caspase activities in freshly
isolated hepatocytes (T0), and cultured hepatocytes at
4 h (4h) and during 6 days of culture in basal medium.
DEVD-AMC caspase activity (inset) measured in cells
maintained in basal medium (basal) or treated with TGF1
(2.5 ng/ml). DEVD- and IETD-AMC (E) caspase activities in
cell extracts in 3-day-old cultured hepatocytes, in the presence or
absence of CasputinTM, a peptidic inhibitor of executioner
caspases. Caspase activities were expressed in arbitrary units (A.U.)
of fluorescence.
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Cell viability loss, measured by the decrease in protein content per
dish was approximately 50% as early as 3-4 days of culture (Fig.
1A) paralleled by a clear degradation of DNA into
oligonucleosomes (Fig. 1B). TGF1 treatment induced more
rapid and extensive cell damages.
Western blot analysis was next performed to look for
expression of pro- and cleaved forms of the caspase-9, -8, and -3 (Fig. 1C). The proforms and cleaved forms of both caspase-9 and -3 were observed in freshly isolated hepatocytes. Then, from days 2 to 3, cleaved forms of caspase-9, -8, and -3 were detected and accumulated with time of culture. In addition, appearance of cleaved forms of
caspases was concomitant with the proteolytic cleavage of the endogenous substrate poly-ADP-ribose polymerase (PARP). As in untreated
cells, cleaved caspase-3 and -9 and PARP were found in TGF1-treated
cells but no cleaved caspase-8 was detected (Fig. 1C).
To confirm that cleaved caspases were active, assays using fluorogenic
substrates were carried out. They were based on the differential
efficiency of each caspase at processing three fluorogenic tetrapeptide
substrates: 1) DEVD-AMC known to be essentially cleaved by caspase-3
and to a lesser extent by caspase-7, 2) LEHD- and IETD-AMC, two main
substrates of the initiator caspases, mainly caspase-9 and -8, respectively.
DEVD-AMC caspase activity was very low in freshly isolated cells and
increased thereafter, displaying a biphasic induction, moderate at 1, and sharp at 3 days after plating (Fig. 1D). IETD- (Fig.
1D) and LEHD-AMC (data not shown) caspase activity values displayed a similar biphasic profile but always remained lower than
DEVD-AMC caspase activities. Addition of TGF1, led to a dramatic
induction of activity with both substrates, 24 and 48 h after
treatment, reaching in the DEVD-AMC assay a 4-5-fold higher level than
that measured in cells maintained in basal medium (Fig. 1D,
inset).
Moreover, we have verified that the DEVD-AMC activity, in 3-day-old
cultured cells, was strongly inhibited in the presence of the
CasputinTM reagent, a recombinant protein of the endogenous
caspase inhibitor XIAP, which specifically binds to and inhibits
cleaved caspase-3 and -7 with negligible effects on other caspases (6).
This indicated that the cleavage of DEVD-AMC was essentially due to caspase-3 and/or -7 (Fig. 1E) in contrast to IETD-AMC (Fig.
1E).
These results demonstrated that the apoptotic process of hepatocytes
cultured under basal conditions involved maturation and activities of
caspase-9, -8, and -3.
Addition of Me2SO to Basal Hepatocyte Culture
Constantly Prevents Maturation of Executioner Caspase-3 and Blocks Late
Stage of Apoptosis--
To determine the mechanism involved in the
inhibition of apoptosis by Me2SO, 2% Me2SO was
added to the basal medium 24 h after plating according to Isom
et al. (35); then, cell morphology, DNA degradation, and
expression and activation of the three caspases were studied (Fig.
2).
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Fig. 2.
Me2SO invariably prevents
maturation of executioner caspase and apoptosis. Morphology in
phase contrast of hepatocytes maintained 6 days under basal
(A) or Me2SO (B) conditions
(Magnification, ×40). Absence of DNA degradation into oligonucleosomes
under Me2SO-differentiated hepatocytes (C) and
in freshly isolated cells (To), in contrast with hepatocytes
cultured under basal conditions. DEVD-AMC caspase activities
(D) in cell extracts of hepatocytes cultured in basal medium
with or without Me2SO, 2%. Western blot analysis of
caspases 3 (E) in hepatocytes cultured in the presence of
Me2SO for 6 days. Controls, cells maintained 2 days in the presence of TGF1 (2.5 ng/ml). Western blot analysis of
caspase-8 and -9 (F) at days 1-6 in hepatocytes cultured in
the presence of Me2SO (DMSO). Caspase activities
were expressed in arbitrary units (A.U.) of fluorescence. Caspase
activities are the results of three independent experiments.
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Typical differentiated hepatocyte morphology was maintained in
Me2SO-treated hepatocytes (Fig. 2B), and no DNA
ladder was observed under this condition (Fig. 2D) compared
with hepatocytes exhibiting characteristics of apoptosis under basal
conditions (Fig. 2, A and C).
DEVD-AMC caspase activity strongly decreased at day 2 and then remained
very low for at least 6 days (Fig. 2C) while no cleaved fragment of caspase-3 was detected by Western blotting (Fig.
2E). Surprisingly, the expression of the cleaved caspase-8
became detectable from 72 h and gradually accumulated from days
4-6 of culture (Fig. 2F). Similarly, the cleaved form of
caspase-9 was detected as early as 24 h and its level increased
with time.
These results indicated that Me2SO prevented hepatocyte
apoptosis occurrence by inhibiting the caspase-3 maturation,
indispensable to cell death although it failed to inhibit
maturation of caspase-8 and -9.
Inhibition of Cleaved Initiator Caspases Involves Mechanism(s)
Distinct from Nitrosylation, AKT-dependent Phosphorylation,
or XIAP Binding--
As observed above, maturation of procaspase-3 was
constantly inhibited despite the presence of cleaved caspase-8 and -9. This prompted us to evaluate whether these cleaved caspase-8 and -9 were active. Caspase-8 and -9 activities (Fig.
3A) were thus measured from
cell lysates using fluorogenic tetrapeptides IETD- and LEHD-AMC, respectively, in Me2SO-treated hepatocytes or not, 4 days
after plating. For each activity, Me2SO-treated hepatocytes
exhibited a weak activity in contrast to control cells, indicating
that, in the presence of Me2SO, the cleaved caspase-8 and
-9 were poorly active.
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Fig. 3.
Me2SO inhibits cleaved caspase-8
and -9 and induces reversible anti-apoptotic signal distinct from
caspase nitrosylation, AKT-dependent phosphorylation, or
XIAP caspase inhibition. IETD- and LEHD-AMC caspase activities
(A) in cell extracts of hepatocytes cultured under basal
condition or in the presence of 2% Me2SO during 4 days.
Reversibility of Me2SO inhibition; hepatocytes were
cultured in basal medium supplemented with 2% Me2SO
between days 1 and 3 after plating, then Me2SO was removed
and IETD-AMC (B) caspase activity was measured at days 3 and
4. LEHD-AMC caspase activities (C) in cell extracts of
4-day-old cultured hepatocytes containing various concentrations of
Me2SO (from 0 to 4%) added prior to caspase assay. DEVD-
(D) and IETD-AMC (E) caspase activities were
measured in cell lysates of 4-day-old hepatocytes cultured under basal
conditions or 2% Me2SO-supplemented medium. Lysates were
incubated with or without DTT 10 mM at room temperature for
10 min prior to caspase assays. For E, IETD-AMC caspase
assays were performed in the presence of CasputinTM. Assays
are expressed in percent of the highest activity (cell extract of basal
condition treated with DTT). LEHD- and DEVD-AMC (F) caspase
assays in cell extracts of 7-day-old Me2SO-cultured
hepatocytes in the presence or absence of inhibitor of PI 3-kinase,
Ly294002 (12 µM), or vehicle, added at day 1 of primary
culture. Western blot analysis of XIAP (G) in hepatocytes
cultured in the presence of 2% Me2SO for 6 days.
Controls, cells maintained 1 and 2 days in the presence of
TGF (2.5 ng/ml). Caspase activities were expressed in arbitrary
units (A.U.) of fluorescence or in percentage of basal level.
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To address the question as to whether the inhibition by
Me2SO of cleaved initiator caspase activities and
maturation of executioner caspases was a reversible phenomenon,
hepatocytes were first cultured in the presence of 2%
Me2SO between days 1 and 3 after plating and then without
Me2SO for the 2 following days. IETD-AMC caspase activity
was measured at days 3 and 4 (Fig. 3B). This activity was
very low in hepatocytes constantly stimulated by Me2SO but strongly increased at day 4 after Me2SO removal.
We also tested whether or not Me2SO, per se, was
capable of inhibiting active mature caspases in a cell extract. To this
aim, both IETD- (data not shown) and LEHD-AMC (Fig. 3C)
caspase activities were estimated in hepatocyte extracts of 4-day-old
cultures maintained under basal conditions, i.e. exhibiting
high caspase activities, to which increasing concentrations (0.5-4%)
of Me2SO were added. Caspase activities were not affected
by the addition of Me2SO to the caspase activity assays,
thus demonstrating that this molecule was not directly responsible for
the inactivation of the enzymatic activity of active initiator caspases
in cell extracts.
To identify the mechanism underlying inhibition by Me2SO of
initiator caspase activity, we determined whether initiator caspases were S-nitrosylated (42), phosphorylated by AKT (5), and/or associated with active fragment(s) of IAP (6), which corresponded to
the three previously identified mechanisms known to inhibit cleaved caspases.
To estimate the level of caspase nitrosylation, DEVD- (Fig.
3D) and IETD-AMC (Fig. 3E) caspase activities
were measured in the presence or absence of DTT, a reducing agent used
to evidence S-nitrosylation of proteins (8, 42).
Preincubation of 100 µg of cell lysates of 4-day-old hepatocytes
cultured under basal condition with DTT led to an increase of DEVD- and
IETD-AMC caspase activities (Fig. 3, D and E)
thus indicating that hepatocytes undergoing apoptosis in culture
contained caspases in both S-nitrosylated and
non-nitrosylated forms. In Me2SO-treated cells, the
DEVD-AMC activity was slightly increased in the presence of DTT but
remained much lower than under basal conditions. Moreover, the IETD-AMC caspase activity was not affected by the denitrosylation agent DTT
(Fig. 3E), leading us to conclude that the mature initiator caspases were not nitrosylated in the presence of Me2SO and
that nitrosylation was not responsible for their low activities.
Possible inhibition of cleaved caspase-9 through its phosphorylation by
AKT (5) was also investigated. The specific inhibitor, Ly294002, of the
upstream AKT activator PI3K (43) was added to culture medium to test
whether inhibition of this survival pathway could restore DEVD- and
LEHD-AMC caspase activities in the presence of Me2SO (Fig.
3F). Both activities were similar in hepatocyte extracts
from cultures maintained in the presence or absence of Ly294002
indicating that PI3K/AKT survival pathway was not essential for
apoptosis inhibition by Me2SO.
Then, we examined if caspase inhibition was based on a direct binding
of IAP fragment to the caspase-9 catalytic site (6). By Western
blotting, we investigated if XIAP was processed and able to counteract
cleaved caspase-9 in Me2SO-treated hepatocytes or
maintained under the TGF1 condition (Fig. 3G). Cleaved
fragments of XIAP were easily detectable under TGF1-treated cells in
contrast to hepatocytes cultured in the presence of Me2SO,
suggesting that mature initiator caspase-9 was not inhibited by binding
to XIAP fragments.
Altogether, these results indicated that inhibition by
Me2SO of cleaved initiator caspase activities and of
maturation of executioner caspase-3 is mediated by one or several
anti-apoptotic mechanism(s) distinct from caspase nitrosylation, XIAP
binding, or AKT-dependent phosphorylation.
Me2SO Prevents Apoptosis Through Inhibition of ASK1
Transduction Pathway--
Meanwhile, evidence was provided that
inhibition of caspase activities would not be sufficient to allow cell
survival (9), suggesting that other apoptotic transduction pathway(s)
might play important roles in the cell decision to undergo apoptosis or
survival. In parallel, recent data have shown that the ASK1/JNK-p38 protein kinase pathway is a transduction signal also activated during
apoptosis. We therefore postulated that the ASK1/JNK-p38 pathway might
contribute to the Me2SO-mediated apoptosis regulation (Fig. 4). To address this issue, we used
TNF, a cytokine requiring both caspase and apoptotic kinase
activation such as ASK1/JNK-p38, to elicit cell death (9, 44), and we
investigated the ability of Me2SO to counteract
TNF-induced apoptosis in cultured hepatocytes (Fig. 4A).
DEVD-AMC caspase activities were measured in cell extracts of
hepatocytes cultured in the presence or absence of Me2SO
and treated with TNF or TGF1 (Fig. 4A). DEVD-AMC
caspase activity was strongly increased in cultures treated with either
cytokine alone but not in cells cultured in the presence of both one
cytokine and Me2SO, demonstrating that Me2SO
could prevent apoptosis induced by TNF. Then, we analyzed whether
Me2SO regulated the activity of the ASK1/MKK4-7/JNK/c-Jun
pathway by measuring ASK1 activity under these conditions. ASK1 was
immunoprecipitated with a specific antibody (DAV), and its activity was
estimated through its ability to phosphorylate recombinant MKK4
in vitro (Fig. 4B). As expected, ASK1 was active
in TNF-treated cells as demonstrated by the phosphorylation of MKK4
substrate. In contrast, in cells treated with Me2SO alone or Me2SO and TNF, the activity of ASK1 was strongly
inhibited (Fig. 4B), demonstrating that Me2SO
inhibited this kinase apoptotic pathway, which in turn resulted in
hepatocyte survival promotion.
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Fig. 4.
Inhibition of ASK1 apoptotic signal in
Me2SO-treated hepatocytes. DEVD-AMC caspase activity
(A) in hepatocytes cultured under basal conditions treated
or not with TNF (20 ng/ml) or TGF1 (2.5 ng/ml) in the absence or
presence of 2% Me2SO. Me2SO was added at
24 h of culture, and cytokine treatment was started at 48 h;
all the cultures were then harvested at 80 h and the DEVD-AMC
caspase assay (A) and ASK1 activity assay (B)
were performed. ASK1 activity was analyzed by immune complex-coupled
kinase assay (21) using anti-ASK1 (DAV) antibody and recombinant
GST-MKK4 as substrate. Reversibility of Me2SO inhibition:
Western blot analysis (C) of phosphorylated p38 and p38 MAPK
forms were performed in lysates from hepatocytes cultured in basal
medium supplemented with 2% Me2SO between days 1 and 3 after plating, then Me2SO was removed. Western blot
analysis of GST M1/2, A1/2, and P1, (D) in normal liver
(NL), and cultured hepatocytes, in the presence or absence
of 2% Me2SO, at different days of culture. Transfection of
differentiated hepatocytes with plasmids containing -galactosidase
(LacZ), wild-type ASK1, kinase mutant ASK1(KM) forms, and different GST
isoforms alone or in combination (1:1, w/w of DNA). 24-h later,
apoptosis was evaluated using DEVD-AMC caspase activity (E)
and Hoechst staining (inset).
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To emphasize the role of ASK1 inactivation, observed in
Me2SO-treated hepatocytes, we checked if other targets to
the downstream ASK1 pathway (10) were inactivated. We thus studied the
expression of phospho-p38 MAPK and its inhibition reversibility.
Hepatocytes were cultured in the presence of 2% Me2SO
between days 1 and 3 after plating, then without Me2SO for
two more days (Fig. 4C). As previously observed with caspase
activities (see Fig. 3B), p38 phosphorylation was inhibited
in the presence of Me2SO. Moreover, Me2SO
removal was associated with a strong induction of phosphorylation of
p38 MAPK.
To further assess the role of ASK1/JNK pathway in the apoptotic
inhibition mediated by Me2SO, hepatocytes cultured in the presence of Me2SO were transfected with wild type ASK1 (WT)
or an inactive kinase mutant ASK1(KM) form. Twenty-four hours after transfection, apoptosis was evaluated by DEVD-AMC caspase activity measurement (Fig. 4E) and Hoechst staining. ASK1 (WT)
strongly induced DEVD-AMC caspase activity (Fig. 4E) and DNA
fragmentation (Fig. 4E, insets), while ASK1 (KM)
and the control plasmid LacZ did not. Together, these results pointed
to the ability of ASK1 to overcome inhibition of apoptosis and
executioner caspase maturation mediated by Me2SO, thus
strongly suggesting that ASK1 might play a key role in apoptotic
transduction in hepatocytes. This led us to analyze regulation of its activity.
Because the activities of ASK1/JNK protein kinases are inhibited by GST
M1 and P1 isoforms, we investigated if Me2SO-mediated inhibition of ASK1 might involve GST. First, GST M1/2, P1, and A1/2
expression was analyzed in the presence or absence of Me2SO (Fig. 4D). GST M1/2 and A1/2 level rapidly decreased under
basal conditions as compared with normal liver and
Me2SO-treated cells. GST P1 was detected only in
hepatocytes cultured under basal conditions (Fig. 4D).
Second, we tested whether GSTs were capable of inhibiting
ASK1-dependent apoptotic pathway. Me2SO-treated
hepatocytes were transiently transfected (Fig. 4E) with ASK1
in combination with GSTM1, A1, or P1. Surprisingly, GSTs M1, P1, as
well as GST A1, inhibited ASK1-induced DEVD-AMC caspase activity. This
demonstrated that the different GSTs expressed in hepatocytes could
contribute to the ASK1/MKK4-7/JNK pathway regulation. These results
indicate that Me2SO might efficiently prevent ASK1 activity
by maintaining a high level of GST proteins in hepatocytes.
Me2SO Prevents THA-mediated Liver Damage through
Inhibition of Caspases and ASK1-JNK Activities--
To confirm that
Me2SO was also able to prevent apoptosis and liver failure
in vivo through inhibition of both caspase activities and
ASK1-JNK pathway, intraperitoneal injection of THA in mice and rats,
known to induce a severe hepatocyte apoptosis and fulminant hepatitis
(30, 31), was used as a model of liver injury.
DNA fragmentation and expression of caspase-3, -8, and -9 were analyzed
in liver of rats treated with THA injected or not with
Me2SO prior to THA (Fig. 5).
Degradation of DNA in oligonucleosomes was evidenced as early as
12 h after THA injection and strongly increased at 24 h
before disappearance at 36 h (Fig. 5A). Histological examination evidenced a massive centrilobular cell death in livers of
THA-treated animals (data not shown) while co-treatment with Me2SO prevented DNA degradation (Fig. 5A) as
well as histological lesions in accordance with previous reports
(31).
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Fig. 5.
Inhibition of caspase-9 activity by
Me2SO in THA injected animals. Kinetics of DNA
fragmentation (A) in normal liver (NL) and liver
extracts of rats obtained at different times after THA injection and
30 h after co-treatment with THA and Me2SO
(THA+DMSO). Western blotting of caspase-3, -9, and -8 (B) in normal liver (NL) and livers of rats
30 h after treatments with THA or THA and Me2SO
(THA+DMSO). T+, hepatocytes cultured in basal medium over 4 days. Kinetics of DEVD-AMC (C) in liver extracts of rats at
different times after THA injection and level after 30 h of
co-treatment with THA and Me2SO (THA+ DMSO).
LEHD-AMC caspase activity (D) in normal liver
(NL) and in liver of rats 30 h after treatments with
THA or THA plus Me2SO. Caspase activities were expressed in
arbitrary units (A.U.) of fluorescence. Caspase activities are the
results of three independent experiments.
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In liver extracts obtained 30 h postinjection, expression of pro-
and mature caspase-3, -8, and -9 was examined (Fig. 5B). As
expected, the cleaved form of caspase-3 was found in liver of rats
injected with THA but not in normal and THA plus
Me2SO-treated animals. The level of pro-caspase-8 was
affected neither by THA nor by THA plus Me2SO treatments
compared with normal liver, and the cleaved caspase-8 was undetectable.
In contrast, the pro-caspase-9 was strongly decreased under THA and THA
plus Me2SO conditions, compared with untreated animals, and
cleaved forms of caspase-9 were clearly evidenced in both conditions
(Fig. 5B).
To demonstrate that different cleaved caspases detected by Western
blotting were active in THA-injured livers, caspase activities were
monitored using different fluorogenic tetrapeptide substrates (Fig. 5,
C and D). DEVD-AMC caspase activities were
measured in liver extracts at different times after THA administration
to the animals (Fig. 5C). A slight increase was first
detected within 16 h postinjection, followed by a strong induction
between 20 and 36 h peaking at 30 h concomitantly with DNA
degradation (Fig. 5A). Co-treatment with Me2SO
totally prevented induction of DEVD-AMC caspase activity measured at
30 h (Fig. 5C). In addition, we showed that THA
strongly induced LEHD-AMC caspase activity (Fig. 5D) while
under the THA plus Me2SO condition, it remained at a
similar level as in normal liver, indicating the absence of caspase-9 activity in the presence of Me2SO.
Interestingly, as previously observed in vitro under basal
conditions, we showed that the expression of GST M1 and A1 (Fig. 6A) was strongly decreased in
THA-treated animals, while Me2SO prevented this
down-regulation. This decrease in GST M1/2 and A1/2 expression was
correlated with a sharp increase of ASK1 activity (Fig. 6B)
and phosphorylated form of JNK (Fig. 6A), which demonstrated an activation of the ASK1/JNK pathway during THA-induced apoptosis in
liver. Importantly, in animals co-injected with THA and
Me2SO, the level of phospho-JNK was significantly reduced
and, as expected, the activity of ASK1 was barely detectable (Fig.
6B). Moreover, the increase of ASK1 and JNK activities in
THA-treated animals was not due to variations of the JNK phosphatase
(MKP1) or ASK1 protein levels (Fig. 6A).
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Fig. 6.
Inhibition of ASK1 activity by
Me2SO in THA-injected animals. Western-blot analysis
(A) of phosphorylated-JNK, total-JNK forms, MKP1, GSTs M1/2,
A1/2, P1, and ASK1 in liver extracts of mice 24 h after indicated
treatments. ASK1 activity (B) analyzed by immune
complex-coupled kinase assay (21) using anti-ASK1 (DAV) antibody or
non-immune serum (Ig) and recombinant GST-MKK4 as substrate.
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Altogether, these results demonstrate that THA-induced apoptosis is
dependent upon activation of caspase-9 and -3 but not of caspase-8, and
is accompanied by the induction of the ASK1/JNK pathway and a strong
decrease in GST A1/2 and M1/2 expression. In addition,
Me2SO inhibits THA-mediated apoptosis by both
preventing the down-regulation of GSTs and decreasing activities of
cleaved caspase-9 and ASK1.
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DISCUSSION |
Hepatoprotection remains one of the major challenges of
clinical therapy to limit liver injuries such as chronic hepatitis and
cholestasis. Many molecules have been described as liver protectors including the modified bile acid, ursodeoxycholic acid (UDCA) (45),
currently used in primary treatment of cholestasis, free radical
scavengers such as Me2SO and dimethylthiourea (31), and
cytokines such as IL-6 (33). However, the precise mechanisms controlling liver protection remain poorly documented. The previous data demonstrating the inhibition by Me2SO of cell death
occurring in liver during acute hepatitis induced by THA (31),
supported the view that the protective effect of this agent was related to its free radical scavenging property. Nevertheless, the molecular mechanisms responsible for this scavenging protection remain to be
investigated. In addition, whether this Me2SO-protective
effect requires one or several complementary mechanisms has to be elucidated.
In this report, we have attempted to highlight the key mechanisms
involved in inhibition of apoptosis by Me2SO using both hepatocytes undergoing apoptosis in primary culture and THA-induced liver failure as an in vivo model. We provide evidence that
Me2SO prevented hepatocyte apoptosis occurrence, in
vitro, by inhibiting the caspase-3 maturation, but failed to
inhibit maturation of caspase-8 and -9. In addition, these
Me2SO-treated hepatocytes exhibited weak initiator caspase
activities in contrast to apoptotic cells, indicating that in the
presence of Me2SO the cleaved caspase-8 and -9 were poorly
active. In vivo, Me2SO totally protected against liver failure induced by THA injection as demonstrated by: 1) absence
of histological lesions, 2) inhibition of pro-caspase-3 maturation and
activity, and 3) absence of DNA fragmentation. In addition, cleaved
caspase-9 was also detected in livers of both THA and THA plus
Me2SO-treated animals but its activity in the presence
of Me2SO was very low.
It is noteworthy that Me2SO is able to inhibit spontaneous
as well as TGF1- or TNF-induced apoptosis in primary cultures of
hepatocytes, suggesting that Me2SO may protect from cell
death regardless of the nature of apoptotic stimulus. This emphasizes the potent protection ability of Me2SO against caspase
pathway. However, the signal involved in this inhibition remains to be specified.
Among possible mechanisms of caspase inhibition, induction or
activation of endogenous proteins such as heat shock proteins (HSP)
(46), NO synthases (NOS) (7, 47), AKT (5), or IAPs (6), have been
described. The involvement of HSP27, 70, and/or 90, known to prevent
maturation of caspase-9 or -3 (46) was excluded since caspase-9 was
cleaved in our in vivo and in vitro models.
Moreover, Western blot analysis confirmed the absence of HSP27
induction in Me2SO-treated hepatocytes (data not shown). Considering the three other inhibitory mechanisms, they were unlikely to be responsible for inhibition of apical caspase activities under our
conditions (see Fig. 3). Since treatments with actinomycin D or
cycloheximide overrode this apoptosis blockage (data not shown),
the induction of one or several gene products exhibiting anti-apoptotic
effects is strongly suggested.
K. Tobiume et al. (11). recently reported that efficient
TNF-induced apoptosis blockage required a dual inhibition of caspase and kinase activities (9). In this context, a
Me2SO-mediated inhibition of the ASK1/JNK pathway was
therefore postulated. The observations that Me2SO inhibits
ASK1 activity and that overexpression of ASK1 restores apoptosis in
Me2SO-treated hepatocytes, provide strong evidence that
ASK1 plays a key role in apoptotic transduction in hepatocytes.
An important issue to be elucidated is then the relationship between
the ASK1/JNK cascade and the caspase pathway. Indeed, it is currently
unclear whether the ASK1/JNK cascade is required for caspase activation
and/or activity, or vice versa. K. Tobiume et al.
(11) previously reported that caspase-8-like activity was present in
TNF-treated MEFs ASK1+/+ mice in a similar manner as
ASK1/, whereas MEFs ASK1/ mice did not
undergo cell death. In addition, H. Nishitoh et al. (48)
hypothesized that ASK1 may lie upstream of caspase-12 in ER
stress-induced apoptosis and neurotoxicity by amyloid- proteins
(49). These data together with our present results led us to
hypothesize that ASK1-JNK might be indispensable for maturation of
executioner caspases in our model of protection by Me2SO.
They also suggest that a direct regulation of caspases by the ASK1/JNK
pathway might occur. However, complexity is increased by the fact that
MEFs ASK1/ mice died in response to Fas ligand (11),
indicating that two cascades might act independently and in parallel
(11).
The most intriguing results concern the potent role of GSTs on
apoptosis. Interesting reports show that the activities of ASK1 and JNK
protein kinases are inhibited by GST M1 and P1 isoforms, respectively
(21, 26). Since Me2SO is known to maintain the expression
of liver-specific genes including GSTs, we investigated if
Me2SO was protecting hepatocytes through the modulation of the expression of GSTs M1/2, A1/2, and P1. We demonstrated that endogenous inhibitor of ASK1, namely GST M1, was constantly maintained in Me2SO-treated hepatocytes despite apoptotic signals such
as THA, TNF, and TGF1, and correlated with hepatocyte survival both in vitro and in vivo. Furthermore,
transfection of GSTs M1, P1 as well as GST A1, counteracted
ASK1-induced DEVD-AMC caspase activity, confirming the ability of these
GSTs to regulate the activity of the ASK1/MKK4-7/JNK pathway and
demonstrating the key role of GSTs in controlling hepatocyte apoptosis.
Our results are in agreement with those by S. G. Cho et al.
(21), who recently described that ASK1 was associated with GST M1 in
normal liver, preventing its oligomerization and autophosphorylation
(18, 50).
S. Dorion et al. (51) recently suggested that both TRX and
GST M1, two endogenous inhibitors of ASK1, may compete for the same
N-terminal region. They demonstrated the release of GST M1 from ASK1,
which correlates with its subsequent activation. They hypothesized (51)
that during heat shock, small lipophilic molecules might be released or
produced thus favoring the release of GST M1 from ASK1. In our hepatic
cell models, we demonstrated that ASK1 activation was strictly
correlated with a decrease of GSTs M1/2 and A1/2. In this context, it
is tempting to postulate that Me2SO may ensure
hepatoprotection by preventing the decrease of ASK1 inhibitors such as
GST M1.
In normal tissue, GST would represent two percent of cytosolic proteins
(22). The liver constantly exposed to endogenous toxins and
xenobiotics, expresses the largest panel of GSTs, a major class of
detoxifying enzymes. The GST P expression differs in liver tissues
between rodent species. In normal quiescent hepatocytes, two isoforms
of the GST P class are found in mouse while they are absent in rat. In
contrast, mouse and rat hepatocytes constantly expressed GST Ms in this
status (22, 52). Since ASK1 and JNK are inhibited by GST M1 and GST P1,
respectively, it is tempting to postulate that the inhibition of
ASK/JNK pathway might occur at distinct levels in function of the
isoforms expressed in rodent species. However, we cannot exclude an
isoform compensation in rat.
Moreover, it has been shown that mutation and/or overexpression of GSTs
(53), frequently occur upon transformation to malignancy and,
consequently, cells acquire resistance to electrophilic anticancer drugs (54). V. Adler (26) also suggested that tumor cells overexpressing GST may escape from apoptosis through high intrinsic JNK
inhibitory activity.
In conclusion, besides its free radicals scavenging properties known to
afford hepatoprotection, Me2SO is mediating apoptosis inhibition through a dual blockage of cleaved caspase and ASK1/JNK activities. Evidence was provided that GSTs should play a key role in
ASK1 regulation and thus on apoptosis. These data raise the question of
the controlling mechanisms that determine the reciprocal GST pools
involved in detoxication or apoptosis transduction processes for
reaching efficient hepatoprotection.
,
, and
TOP
ABSTRACT
INTRODUCTION
Recipient of a doctoral fellowship from La Ligue Nationale Contre
le Cancer. To whom correspondence should be addressed. Tel.: 33-2-99-54-37-37; Fax: 33-2-99-54-01-37; E-mail:
david.gilot@rennes.inserm.fr.
