From the Division of Gastroenterology and Hepatology, Mayo Medical School, Clinic, and Foundation, Rochester, Minnesota 55905
Received for publication, March 28, 2003 , and in revised form, May 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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In addition to activating death receptors, bile acids also activate mitogen-activated protein kinases (MAPKs)1 (1219). At present three major groups of MAPKs are known: the classic p42/44 MAPK or extracellular receptor kinase 1 and 2, c-Jun-N-terminal kinase (JNK 1/2), and p38 MAPK. The p42/44 MAPKs are mainly involved in cell survival and proliferation pathways, and their inhibition has been shown to potentiate bile acid-induced apoptosis in hepatocytes (4, 20). In contrast, prolonged stimulation of p38 MAPK or JNK 1/2 promotes apoptosis in a variety of other models (21, 22). Several upstream kinases have been implicated in the activation of the p38 MAPK and JNK 1/2 including apoptosis signaling kinase-1 which appears to activate both p38 MAPK and JNK 1/2, MKK 3/6 which activates p38 MAPK, and MKK 4/7 which activates JNK 1/2 (23). Although both bile acids and death receptors, such as Fas and tumor necrosis factor receptor-1, have been shown to activate MAPK pathways, it is still unknown whether bile acid MAPK activation occurs downstream of death receptor-mediated caspase activation or by a separate, death receptor-independent pathway. Moreover, the mechanistic contribution of MAPK activation in bile acid-mediated apoptosis remains unclear.
The role of death receptor caspase activation in bile acid-induced MAPK activation can be potentially delineated by inhibiting death receptor cytotoxic signaling. Death receptor-dependent caspase activation can be disrupted by interrupting formation and function of the death inducing signaling complex (DISC). Once aggregated, death receptors, such as Fas and DR5/TRAIL-R2, recruit the cytoplasmic adapter protein FADD (Fas-associated death domain) and initiator caspases, procaspase 8 and 10 to the receptor complex (2426). Once recruited to the DISC, these procaspases undergo autocatalytic processing within the DISC (27, 28). This DISC formation and function can be disrupted by inhibiting FADD expression or employing a dominant negative FADD, by blocking the activation of initiator caspases with pharmacologic caspase inhibitors, or by augmenting expression of FLICE inhibitory protein (cFLIP, also called Flame-1/I-FLICE/Casper/CASH/MRIT/CLARP/Usurpin) (2936). cFLIP inhibits death receptor cytotoxic signaling by regulating both recruitment and processing of procaspases within the DISC (3739). Interrupting DISC function should help clarify if bile acid-mediated activation of MAPK is death receptor/FADD/caspase-dependent or occurs via a parallel process.
The overall objective of this study was to test the hypothesis that MAPK activation occurs and contributes to bile acid-mediated apoptosis. To address this hypothesis, the following two questions were formulated: (i) do inhibitors of p38 MAPK, p42/44 MAPK, and/or JNK 1/2 attenuate bile acid-induced apoptosis and (ii) is MAPK activation by bile acids because of death receptor-mediated caspase activation? We employed deoxycholate (DC) for these studies as it is a potent bile acid apoptogen and strongly activates MAPK pathways (4, 40). The results indicate that p38 MAPK contributes to bile acid cytotoxicity, likely by a death receptor-independent pathway. Unexpectedly, cFLIP was observed to co-immunoprecipitate with the unphosphorylated form of p38 MAPK, suggesting that this protein prevents p38 MAPK activation, thereby protecting cells from bile acid-induced apoptosis. These studies suggest a new anti-apoptotic mechanism for cFLIP, namely inhibition of p38 MAPK activation.
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EXPERIMENTAL PROCEDURES |
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Generation of HuH-cFLIP Cell Lines and Cell CultureHuH7 cells, a human hepatocellular carcinoma cell line, were stably transfected with cFLIP-L. An expression vector for human cFLIP-L, cDNA cloned into the multiple cloning site of a pcDNA3 plasmid, was a gift from Dr. E. Alnemri (Thomas Jefferson University, Philadelphia, PA). HuH7 cells were cultured until 30% subconfluent in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Transfection with the cFLIP pcDNA3 plasmid was performed using LipofectAMINE (Invitrogen) according to the manufacturer's instructions. Stably transfected clones were selected in medium containing 1200 mg/ml G418. Individual clones were subcloned and screened for cFLIP overexpression by immunoblot analysis. Established clones were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100,000 units/liter penicillin, 100 mg/liter streptomycin, 100 mg/liter gentamycin, and 1200 mg/liter G418. Before treatment with deoxycholate or other compounds, cells were cultured in the same medium without serum for 1648 h, reagents were then added to the media according to the experimental protocol.
Plasmids and TransfectionThe plasmid for mutated MKK3
(MKK3(ala) in pRC/RSV) was a gift from Dr. R. Davis (University of
Massachusetts, Worcester, MA) and has been previously described
(41). The expression vector
MKK3(ala) in pRC/RSV encodes a mutant MAPK kinase 3 which acts as a dominant
negative and prevents p38 MAPK activation. The GFP expression vector pEGFP-N1
was purchased from Clontech. HuH7 cells were transiently transfected using
Lipofect-AMINE as previously described
(19). In brief, cells grown in
12-well dishes were transfected by adding 0.5 ml of Opti-MEM I containing 3
µl of LipofectAMINE (Invitrogen), 4 µl of PLUSReagent (Invitrogen), and
0.8 µg of DNA each, MKK3(ala) (0.6 µg) and pEGFP-N1 (0.2 µg). Control
cells were transfected with 0.8 µg of pEGFP-N1. The cells were incubated in
the above mixture for 5 h at 37 °C in a 5% CO2, 95% air
incubator. After this incubation, the media was changed to Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum. Twenty-four hours later, the
cells were incubated with deoxycholate in serum-free media for 12 h.
Transfected cells were identified by their green fluorescence, and the
transfection efficiency was 3040% for all plasmids as estimated by
the percentage of cells expressing GFP. The rate of apoptosis in transfected
cells was evaluated by 4',6-diamidino-2phenylindolehydrochloride (DAPI)
staining and counting of apoptotic nuclei under fluorescent microscopy as
described below.
Short Interfering RNA and TransfectionShort interfering RNA (siRNA), a specific double-stranded 21-nucleotide RNA sequence homologous to the target gene, was used to silence FADD expression. siRNA for FADD was designed and synthesized using the software2 and SilencerTM siRNA construction kit from Ambion (Austin, TX) according to the manufacturer's instructions. The sequence of the double stranded RNA employed to block FADD expression in the current experiments is AATGCGTTCTCCTTCTCTGTGCCTGTC. Inhibition of FADD protein expression was assessed by immunoblot analysis following transfection of HuH7 cells with FADD-siRNA. Briefly, HuH7 cells were grown in 10-cm dishes and transiently transfected with 20 nM siRNA using 8 µl of siPORT Amine (Ambion Inc.) in a total transfection volume of 0.5 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. After incubation at 37 °C, 5% CO2 for 5 h, 1.5 ml of normal growth media was added. Before incubation with deoxycholate cells were serum starved for 24 h. Samples were then prepared and analyzed for apoptosis or by immunoblot as described below.
Quantitation of ApoptosisApoptosis was quantified by assessing the characteristic nuclear changes of apoptosis (i.e. chromatin condensation and nuclear fragmentation) using the nuclear binding dye DAPI and fluorescence microscopy.
Measurement of Intracellular Caspase ActivityHuH7 and HuH-cFLIP cells were grown on 35-mm glass-bottom microwell dishes (Mattek Corp., Ashland, MA) at a density of 1000 cells/dish. Intracellular total caspase activity was measured in single cells using the fluorophore-tagged pancaspase inhibitor carboxyfluorescein-VAD-fmk (FAM-VAD-fmk, CaspaTag; Intergen Co., Purchase, NY) according to the manufacturer's instructions, and fluorescence was evaluated with an inverted fluorescence microscope (Axiovert 35M; Carl Zeiss Inc., Thornwood, NY) using excitation and emission wavelengths of 490 and 520 nm, respectively. Digital images were captured and then analyzed and quantitated using fluorescent imaging software (Metafluor Imaging System; Universal Imaging Corp., West Chester, PA). Fluorescence intensity for individual cells was quantitated and the average fluorescent intensity determined for 100 cells per group.
Immunoblot AnalysisCells were lysed by incubation on ice for 30 min in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM Na3VO4, 50 mM NaF, 100 mM phenylmethylsulfonyl fluoride, and a commercial protease inhibitor mixture (Complete-Mini Protease Inhibitor Mixture; Roche Diagnostics). After insoluble debris was pelleted by centrifugation at 14,000 x g for 15 min at 4 °C, the supernatants were collected. Samples were resolved by 12% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and blotted with appropriate antibodies at a dilution of 1:1000. Peroxidase-conjugated secondary antibodies (BIOSOURCE International, Camarillo, CA.) were used at a dilution of 1:2000 to 1:10000. Bound antibody was visualized using chemiluminescent substrate (ECL; Amersham Biosciences) and exposed to Kodak X-Omat film. Primary antibodies used included anti-p38 MAPK, anti-active p38 MAPK, anti-active JNK 1/2, anti-active p42/44 MAPK, anti-cFLIP, and anti-FADD.
Immunoprecipitation AssayHuH7 cells in 10-cm dishes were incubated with deoxycholate (100 µM) for 4 h. Cells were lysed with the lysis buffer as described above (immunoblot analysis). After the protein concentration in the extracts was determined, cell lysates containing 4 mg of protein in 0.5 ml of lysis buffer were precleared by incubation with 30 µl of Sepharose-CL 4B for 3 h at 4 °C. cFLIP and p38 protein were immunoprecipitated by incubation with rat monoclonal (IgG2a) anti-cFLIP (Alexis) antibody (5 µg) or rabbit anti-total p38 MAPK antibody (Santa Cruz) and protein G-Sepharose (30 µl) for an additional 2hat4 °C. Immune complexes were pelleted by centrifugation for 5 min at 14,000 x g, washed five times with lysis buffer, and released from the beads by boiling for 5 min in SDS sample buffer. Samples were resolved by SDS-PAGE, and immunoblot analysis was performed as described above.
Statistical AnalysisAll data represent at least three independent experiments and are expressed as the mean ± S.D. unless otherwise indicated. Differences between groups were compared using analysis of variance for repeated measures and a post-hoc Bonferroni test to correct for multiple comparisons.
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RESULTS |
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Enhanced cFLIP Expression Prevents p38 MAPK Phosphorylation and
Apoptosis following DC Treatment of HuH7 CellsHaving demonstrated
that p38 MAPK contributes to bile acid-induced apoptosis, the relationship
between p38 MAPK and bile acid-associated cytotoxic death receptor signaling
was examined. The HuH7 cells employed for these studies express TRAIL
receptors-1 and -2, tumor necrosis factor receptor-1, but not Fas
(8,
43,
44). TRAIL does not activate
p38 MAPK in these cells (data not shown). Next, we employed a blocking
antibody for TNF-R1 to abrogate signaling via this death receptor. Whereas the
antibody did strongly inhibit TNF--induced p38 phosphorylation, p38
activation after DC treatment was not reduced
(Fig. 2), indicating that
activation of TNF-R1 by bile acid was not responsible for DC-mediated
activation of this MAPK. Death receptor signaling was further perturbed using
the pharmacologic pancaspase inhibitor zVAD-fmk, FADD-specific siRNA to knock
down FADD expression, and enhanced cFLIP-L expression in a stably transfected
cell line. Neither zVAD-fmk nor siRNA-mediated reduction in FADD expression
reduced DC-associated p38 MAPK phosphorylation
(Fig. 3, A and
B). The decrease in FADD expression after transfection
with FADD-siRNA was sufficient to inhibit TRAIL-mediated apoptosis in HuH7
cells (Fig. 3C). In
contrast, in a cFLIP-L stably transfected HuH7 cell line, termed HuH-cFLIP
(Fig. 4A), DC-induced
p38 MAPK phosphorylation was attenuated
(Fig. 4B). The effect
was specific for p38 MAPK, as JNK 1/2 and p42/44 MAPK activation was not
reduced. cFLIP expressing clones were also resistant to DC-mediated apoptosis,
as assessed by morphological criteria and total caspase activity
(Fig. 5, A and
B). The discordance between the effects of cFLIP-L on
blocking p38 MAPK phosphorylation and the failure of the caspase inhibitor and
attenuated FADD expression to prevent this signaling event suggests that cFLIP
may prevent p38 MAPK activation by a FADD/caspase-independent process.
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cFLIP Interacts with p38 MAPKBased on the above data we postulated a more direct effect of cFLIP on p38 MAPK phosphorylation/activation, and therefore, determined if these two proteins interact by performing co-immunoprecipitation experiments. To investigate a potential binding interaction between p38 MAPK and cFLIP, HuH-7 cells were serum-starved for 24 h and treated with 100 µM DC for 4 h. Samples were then obtained and immunoprecipitation was performed as described under "Experimental Procedures." Indeed, in nontransfected HuH-7 cells, nonphosphorylated p38 MAPK co-immunoprecipitated with cFLIP-L (Fig. 6A), and cFLIP-L was co-immunoprecipitated with the nonphosphorylated form p38 MAPK (Fig. 6B). Although in untreated HuH7 cells and in cells treated with DC there was no difference in the amount of co-immunoprecipitated proteins, the phosphorylated/active form of p38 MAPK was never identified in the immune complexes suggesting that the cFLIP may participate in a protein-protein interaction only with the inactive form of this kinase (Fig. 6A). Although the antibody recognizes both cFLIP-L and cFLIP-S, a splicing variant of cFLIP-L, cFLIP-S was not identified in the immunocomplexes. Taken together, these observations suggest that cFLIP-L, but not cFLIP-S binds the nonphosphorylated form of p38 MAPK. This binding interaction appears to prevent bile acid-associated p38 MAPK phosphorylation/activation.
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DISCUSSION |
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Consistent with the observations of others, we observed activation of JNK
1/2, p42/44 MAPK, and p38 MAPK during exposure of HuH7 cells to deoxycholate.
Although JNK 1/2 activation has been implicated in bile acid apoptosis by
promoting trafficking of Fas to the plasma membrane
(14), a pharmacologic JNK 1/2
inhibitor did not prevent deoxycholate-induced apoptosis in our studies. As
HuH7 cells do not express Fas
(8), this may be an explanation
for the discrepancy between these prior studies and our current results.
Moreover, others have suggested a role for bile acid-induced JNK 1/2
activation in regulating bile acid synthesis in the absence of cytotoxicity
(45). These latter published
results coupled with our current observations suggest that bile acid-mediated
apoptosis is unlikely to be JNK 1/2-dependent. p42/44 MAPK inhibition also did
not attenuate apoptosis consistent with the observations of others that
activation of this MAPK leads to cell survival rather than cell death. Indeed,
inhibition of p42/44 MAPK has been shown by some but not all to potentiate
bile acid-mediated apoptosis suggesting that it may have a cytoprotective
effect (4,
20,
46,
47). The contribution of p38
MAPK to bile acid-induced apoptosis has not been extensively studied. Our
current study demonstrates that the activation of p38 MAPK is proapoptotic and
contributes to apoptosis during treatment of cells with a cytotoxic bile acid,
consistent with its proapoptotic functions in other models
(21,
22,
48,
49). Of the four isoforms of
p38 MAPK that have been cloned only p38- and p38-
are
ubiquitously expressed, whereas p38-
is predominantly found in skeletal
muscle and p38-
is enriched in lung, kidney, testis, pancreas, and
small intestine
(5053);
moreover SB203580, the inhibitor used in these studies, only inhibits
p38-
and -
, but not
and
(54). Therefore we suggest
that p38-
or -
contribute to bile acid-mediated apoptosis.
p38 MAPK activation has previously been identified in death receptor
signaling by Fas and TNF-R1. In Fas-directed activation of p38 MAPK, caspase 8
activation is required (55).
However, caspase-8 activation is not necessary for p38 MAPK activation by
TNF-R1 (56). In the current
studies, we employed HuH7 cells that lack Fas but do express TRAIL-R1 and -R2,
and TNF-R1. However, TRAIL treatment of the cells failed to elicit p38 MAPK
phosphorylation. Although TNF- treatment did result in p38 MAPK
phosphorylation, TNF-R1 blocking antisera failed to prevent bile acid-induced
p38 MAPK phosphorylation. A pancaspase inhibitor and siRNA directed knock down
in FADD expression also did not alter bile acid activation of p38 MAPK.
Collectively, these data make it unlikely that bile acid-associated p38 MAPK
activation is death receptor-mediated, at least by FADD/caspase-dependent
signaling processes, and suggest that combinatorial signaling events
contribute to bile acid cytotoxicity.
Enhanced expression of cFLIP protected against bile acid-mediated apoptosis as reported previously by Qiao et al. (4). Cytoprotection by cFLIP has been attributed to abrogation of death receptor cytotoxic signaling (29, 38, 39). cFLIP-L is structurally similar to procaspase 8 in that it contains two death effector domains and a caspase-like domain. However, the caspase domain lacks the critical active site cysteine residue essential for catalytic activity. cFLIP-S, a splicing variant of cFLIP-L, lacks a portion of the caspase domain but contains the two DED domains (37). Both cFLIP isoforms bind to FADD within the DISC via DED-DED homotypic interactions and interfere with caspase 8 processing and activation (29, 57, 58). However, in our current studies we observed that cFLIP-L overexpression also inhibits p38 MAPK activation, which appears to occur by a FADD/caspase-independent mechanism. These intriguing data suggest that cFLIP-L also has cytoprotective effects independent of its interactions with death receptor membrane complexes. Indeed, in co-immunoprecipitation experiments in nontransfected cells, we were able to show a potential binding interaction between cFLIP-L and unphosphorylated p38 MAPK. Based on these observations, we postulate that in unstimulated cells cFLIP-L constitutively participates in a protein complex consisting of inactive p38 MAPK and perhaps additional proteins, rendering it inaccessible to phosphorylation by upstream kinases. Because cFLIP-S was not identified in the immunoprecipitation experiments, this effect of cFLIP appears to be specific to the long form, and suggests that the caspase domain of this protein is important for binding in this protein complex. Whether cFLIP-L directly or indirectly modulates p38 MAPK activation within protein complexes, its inhibition of the activation of this proapoptotic kinase appears to be an additional cytoprotective mechanism for this protein.
Toxic bile acids clearly promote hepatocyte apoptosis and injury in vivo. For example, children lacking the canalicular transport protein for bile salt secretion into the bile develop a progressive liver disease because of the inability to excrete bile salts from the hepatocyte (59). Mice with gene-targeted impairments in biliary secretion also develop hepatocyte apoptosis and liver injury when fed human bile acids (60, 61). The mechanisms of bile acid-mediated apoptosis are strikingly similar in vivo when compared with in vitro studies. Indeed, mechanisms of hepatocyte apoptosis occurring in bile duct-ligated animals parallel those observed in isolated cell systems (9). More pertinent to the current study, we have also observed MAPK p38 activation in the liver of 3-day bile duct-ligated mice. Thus, the results of this in vitro study appear to be quite germaine to the mechanisms of hepatocyte apoptosis in vivo during cholestatic liver syndromes.
Collectively, the current studies define a mechanistic proapoptotic function for p38 MAPK and expand the role of cFLIP cytoprotection in bile acid cytotoxicity, a cellular model for cholestatic liver injury. The data demonstrate that attenuation of p38 MAPK activation reduces apoptosis, whereas enhanced cFLIP-L expression is cytoprotective, in part, by reducing p38 MAPK phosphorylation/activation. If bile acid-associated p38 MAPK activation can be implicated in bile acid-mediated liver injury in vivo, perhaps p38 MAPK inhibitors may help alleviate human cholestatic liver injury. Likewise, mechanistic approaches that enhance cFLIP-L expression may also be therapeutically useful (62, 63). The implications of these studies for potential therapy of human cholestatic liver disease merits further investigation.
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FOOTNOTES |
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To whom correspondence should be addressed: Mayo Medical School, Clinic, and
Foundation, 200 First St. SW, Rochester, MN 55905. Tel.: 507-284-0686; Fax:
507-284-0762; E-mail:
gores.gregory{at}mayo.edu.
1 The abbreviations used are: MAPK, mitogen-activated protein kinase; cFLIP,
cellular FLICE inhibitory protein; DAPI, 4',6-diamidino-2-phenylindole
dihydrochloride; DC, deoxycholate; DED, death effector domain; DISC, death
inducing signaling complex; DR, death receptor; FADD, Fas-associated death
domain protein; FAM-VAD-fmk, carboxyfluorescein-VAD-fmk; GFP, green
fluorescent protein; JNK, c-Jun NH2-terminal kinase; MKK,
mitogen-activated protein kinase kinase; siRNA, short interfering RNA;
TNF-, tumor necrosis factor-
; TNF-R1, tumor necrosis factor
receptor 1; TRAIL, tumor necrosis factor-related apoptosis inducing ligand;
TRAIL-R, tumor necrosis factor-related apoptosis inducing ligand-receptor.
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ACKNOWLEDGMENTS |
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REFERENCES |
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