A role for caspase-8 and c-FLIPL in proliferation and cell-cycle progression of primary hepatocytes
David Gilot *,
Anne-Laure Serandour 1,
Guennady P. Ilyin 1,
Dominique Lagadic-Gossmann,
Pascal Loyer 1,
Anne Corlu 1,
Alexandre Coutant 1,
Georges Baffet 1,
Marcus E. Peter 2,
Olivier Fardel and
Christiane Guguen-Guillouzo 1
INSERM U620, Rennes, F-35043, France; Université de Rennes 1, Rennes, F-35043, France, 1 INSERM U522, Hôpital Pontchaillou, Rennes, F-35033, France and 2 The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL 60637, USA
* To whom correspondence should be addressed. Tel: +33 2 23 23 44 41; Fax: +33 2 23 23 47 94; E-mail: david.gilot{at}rennes.inserm.fr
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Abstract
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Growth factors are known to favor both proliferation and survival of hepatocytes. In the present study, we investigated if c-FLIPL (cellular FLICE-inhibitory protein, long isoform) could be involved in epidermal growth factor (EGF)-stimulated proliferation of rat hepatocytes since c-FLIPL regulates both cell proliferation and procaspase-8 maturation. Treatment with MEK inhibitors prevented induction of c-FLIPL by EGF along with total inhibition of DNA replication. However, EGF failed to inhibit processing of procaspase-8 in the presence of EGF suggesting that c-FLIPL does not play its canonical anti-apoptotic role in this model. Downregulation of c-FLIP expression using siRNA oligonucleotides strongly reduced DNA replication but did not result in enhanced apoptosis. Moreover, intermediate cleavage products of c-FLIPL and caspase-8 were found in EGF-treated hepatocytes in the absence of caspase-3 maturation and cell death. To determine whether the Fas/FADD/caspase-8/c-FLIPL complex was required for this activity, Fas, procaspase-8 and Fas-associated death domain protein (FADD) expression or function was inhibited using siRNA or constructs encoding dominant negative mutant proteins. Inhibition of any of these components of the Fas/FADD/caspase-8 pathway decreased DNA replication suggesting a function of these proteins in cell-cycle arrest. Similar results were obtained when the IETD-like caspase activity detectable in EGF-treated hepatocytes was inhibited by the pan-caspase inhibitor, z-ASP. Finally, we demonstrated co-immunoprecipitation between EGFR and Fas within 15 min following EGF stimulation. In conclusion, our results indicate that the Fas/FADD/c-FLIPL/caspase-8 pathway positively controls the G1/S transition in EGF-stimulated hepatocytes. Our data provide new insights into the mechanisms by which apoptotic proteins participate to mitogenic signals during the G1 phase.
Abbreviations: c-FLIPL, cellular FLICE-inhibitory protein, long isoform; CaMKII, calmodulin kinase II; DED, death effector domain; DEVD-AMC, Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; DISC, death-inducing signalling complex; EGF, epidermal growth factor; EGFR, EGF receptor; FADD, Fas-associated death domain protein; FCS, fetal calf serum; GFP, green fluorescent protein; IAP, inhibitor of apoptosis protein; IKK, inhibitor of NF-kB kinase complex; MKP1, MAPK phosphatase 1; NIK, NF-kB inducing kinase; RIP, receptor interacting protein; siRNA, small interference RNA; TCR, T cell receptor; TNF, tumor necrosis factor
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Introduction
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During liver regeneration, quiescent hepatocytes undergo one or two rounds of replication and then return to a non-proliferative state when liver mass is restored (1). At early stages of liver regeneration, tumor necrosis factor-
(TNF
) functions as a priming agent for hepatocyte replication and increases the sensitivity of hepatocytes to growth factors, such as epidermal growth factor (EGF), which trigger G1/S transition and completion of the cell cycle (2). The proliferative response of hepatocytes can also be induced in vitro. It has previously been established that hepatocytes undergo G0 to G1 transition during cell isolation and are able to progress in culture from early to mid-late G1 phase, regardless of mitogen stimulation, but remain blocked at this stage. Isolated rat hepatocytes stimulated with EGF or hepatocyte growth factor (HGF) undergo a single round of division via activation of the Raf-1/MEK/ERK/cyclin D1 pathway (3,4). These growth factors have pleiotropic activities. EGF is capable of regulating cell proliferation (4), apoptosis (5) and cell spreading (3,6) through different pathways, such as MEK/ERK, PI3K/AKT, JNK and p38. Although the proliferative signal induced by EGF is well understood, its anti-apoptotic activity remains poorly documented (5). It has been recently demonstrated that the HGF receptor/c-Met (c-Met) directly binds to and sequesters the death receptor Fas in hepatocytes (7) and it was proposed that c-Met and Fas preexist as a complex, and that engagement of either of these two receptors by their cognate ligand at high concentrations disrupts their association with one another. Independently, Reinehr et al. (8,9) demonstrated a direct binding between Fas and EGF-receptor (EGFR). They proposed that hydrophobic bile salts induce EGFR activation and EGFR-dependent Fas tyrosine phosphorylation, leading to hepatocyte apoptosis via the death-inducing-signaling complex (DISC) formation (8). Together, these data suggest a novel link between growth factor receptors and death receptors. Recent reports suggest that proteins of the DISC, such as Fas (10,11), FADD (Fas-associated death domain protein) (12,13), c-FLIPL (1416) and caspase-8 (1719) can couple to both cell proliferation and cell death. Among them, c-FLIPL was initially considered as an inhibitor of the extrinsic pathway notably from data on MEFs (mouse embryonic fibroblasts) isolated from mice deficient in c-FLIP (20). Indeed, these cells were shown to be more sensitive to death-receptor-induced apoptosis.
Full-length, 55-kDa, c-FLIPL shows an overall structural homology to procaspase-8. It contains two death effector domains (DED) that interact with FADD, but bears a mutation in the caspase-like domain (21). Following Fas ligation, FADD, c-FLIPL and procaspase-8 are recruited into the DISC. However, the activity of c-FLIPL depends on its expression levels. Three recent reports demonstrated that c-FLIPL is not only an inhibitor of apoptosis, but also an activator of procaspase-8 (2224). Despite some discrepancies, all three reports proposed that in the absence of c-FLIP, active caspase-8 (p18) is released from the DISC and mediates apoptosis. If c-FLIPL is expressed at a sufficient concentration, it efficiently activates procaspase-8, but does not allow full processing to the mature enzyme. First cleavage intermediates (p41/43) of procaspase-8 and (p43) of c-FLIPL may stay tethered to the DISC, allowing only DISC-proximal substrates to be cleaved by p41/43 caspase-8 (25). Yang et al. (26) have demonstrated the existence of three isoforms of c-FLIPL p55 and have suggested that only one form is phosphorylated by calmodulin kinase II (CaMKII) and recruited to the Fas-mediated DISC. The p43 phosphorylated c-FLIPL interrupts caspase-8 second cleavage step, and, thus inhibits caspase-8-initiated apoptosis. The duality of proteins initially described as being strictly associated with apoptosis has mostly been studied in the context of apoptosis of lymphoid cells induced by death receptors, such as Fas (25).
In the present study, we investigated how EGF and EGFR promote both proliferation and inhibition of apoptosis in rat hepatocytes in primary culture. Our data suggest that progression from G1 to S phase in hepatocyte following EGF stimulation requires induction of c-FLIPL expression, cleavage and activity of caspase-8 and participation of FADD and Fas.
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Materials and methods
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Reagents
Anti-caspase-3 (H277), anti-MKP1 (M18), anti-Fas (A20), anti-FADD (M19) and anti-ERK1/2, a mixture of ERK1 (K23) and ERK2 antibodies (C14), were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-EGFR was purchased from UpState Biotechnology (Euromedex, France). Anti-c-FLIPL (AAP440) was from StressGen Biotechnologies (Tebu, France) and monoclonal anti-phospho-ERK was from Cell Signaling Technology (Ozyme, France). The rabbit anti-caspase-8 polyclonal antibody (552038) was purchased from BD Biosciences (Tebu, France). Secondary antibodies conjugated to horseradish peroxidase were from DAKO (SA, France). Fluorimetric substrates of caspases (Ac-DEVD-AMC, Ac-IETD-AMC) were from BACHEM (BACHEM, France) and were prepared at 100 mM in recommended solvent. z-Asp-2,6-dichlorobenzoloxymethylketone (z-Asp, Alexis, France) was prepared at 100 mM in methanol, as recommended. 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). Methyl-[3H]thymidine (5 Ci/mmol) was obtained from Amersham (Les Ulis, France). MEK inhibitors U0126 and PD98059 and recombinant human EGF were from Promega (Charbonnieres, France).
Isolation and primary culture of hepatocytes
Hepatocytes were isolated and purified from male SpragueDawley rats (Elevage Janvier, France) by in situ perfusion with the highly active purified collagenase Liberase (Roche, France), as previously described (27). Hepatocytes were seeded at 52 x 103 cells/cm2 on plastic dishes in a mixture of 75% minimum essential medium (MEM) and 25% medium 199, supplemented with 10% fetal calf serum (FCS), 1 ml of medium containing 100 IU of penicillin, 100 µg of streptomycin, 1 mg of bovine serum albumin (BSA), 2 µmol L-glutamine and 5 µg of bovine insulin. After plating for 4 h, the medium was removed and cultures were maintained in EGF medium, corresponding to plating medium, deprived in FCS and supplemented with 1.4 x 107 M hydrocortisone hemisuccinate and EGF (25 ng/ml). Appropriate media were renewed every day.
Methyl-[3H]thymidine incorporation
The rates of DNA synthesis were measured in primary cultures by adding 2 µCi of methyl-[3H]thymidine for given periods of time before cell harvesting and precipitation with ice-cold 5% trichloroacetic acid (4).
Cellular protein preparation and immunoblotting analysis
Adherent and floating hepatocytes were scraped off dishes, washed with PBS and stored as pellets at 80°C. Cells were sonicated in a western blot lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 15 mM MgCl2, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween-20, 0.1 mM sodium orthovanadate, 1 mM NaF, 10 mM ß-glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride and 5 µg/ml aprotinin and leupeptin. The protein concentration was evaluated by the Biorad protein assay (Bio-Rad, Belgium). Aliquots of 100 µg of proteins were then resolved on 7.512.5% SDSPAGE and transferred onto polyvinylidene difluoride membranes (PVDF, Bio-Rad). Membranes were stained with Ponceau red to ensure equivalent amounts of protein loading and electrophoretic transfer among samples. Subsequently, non-specific binding sites were blocked with Tris buffer saline (TBS) containing 4% BSA, for 1 h at room temperature. Then, filters were incubated overnight at 4°C with appropriate primary antibody in TBS containing 4% BSA. Filters were thereafter washed three times with TBS and incubated with appropriate secondary antibody conjugated to horseradish peroxidase, for 1 h at room temperature. Following 45 washes with TBS, antibody-labeled proteins were visualized with SupersignalTM according to the manufacturer's instructions (Pierce Chemical, IL). All experiments described have been done at least three times.
DNA extraction and agarose gel electrophoresis
Adherent and non-adherent hepatocytes from 60 mm dish cultures were harvested and centrifuged at 2000 r.p.m. for 2 min at 4°C. DNA was then isolated from cells with the High Pure PCR Template Preparation Kit (Roche Diagnostics, France) according to the manufacturer's instructions. After purification, samples were analyzed by electrophoresis on 1% agarose gel and observed under UV light.
Caspase activity assay
Hepatocytes were lysed in the caspase activity buffer containing 20 mM piperazine-N,N'-bis-(2-ethanesulfonic acid) (PIPES) (pH 7.2), 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% 3-[3-cholamidopropyl-dimethylammonio]-2-hydroxy-1-propanesulfonic acid (CHAPS) and 10% sucrose as previously described (28). Aliquots of 100 µg of crude cell lysate were incubated with 80 µM substrate-AMC at 37°C for 1 h. Caspase-mediated cleavage of peptide-AMC was measured using a fluorescence plate reader (Gemini, Molecular Devices, France) at the excitation/emission wavelength pair (ex/em) of 380/440 nm. The caspase activity was expressed as arbitrary units of fluorescence (per 100 µg of total proteins) or in Vmax.
Transfection and RNA interference (RNAi)
Small interfering RNA (siRNA) oligonucleotides (Eurogentec, Belgium) were designed as recommended (29) with two base 2'-deoxythymidine overhands on each strand. The following sequences were used successfully: GFP siRNA (iGFP: sense 5'-GCUGACCCUGAAGUUCAUC-3'), rat c-FLIPL (icFLIP: sense 5'-AUAUGAAUGCUCUCCAGGC-3'). siRNA oligonucleotide annealing was performed according to the manufacturer's recommendations. siRNA oligonucleotides directed against Fas (iFas1 sens: 5'-GGGUUUGGAGUUGAAGAGG-3', iFas2 sens: 5'-CGUAUCGUUUUUGCAUUUC-3') and Caspase-8 (iCasp8 1 sens: 5'-GCAGAGUCCUAAAAAGCAG-3', iCasP8 2 sens: 5'-CCUUUAAGGAGCUUCAUUU-3') were purchased from Ambion (Ambion, UK). Rat c-FLIP was amplified by PCR using 5'-GAACATGGCCCTGAGCACTGTGTCTG-3' and 5'-TGCTGATATTCCACACACTGGCTCCAG-3' from mRNA extracted from LPS-stimulated rat macrophages. Amplicon was cloned into pcDNA3.1V5HisTopo (InVitrogen, France) and the construct was verified by DNA sequencing.
Briefly, transfection of siRNA or co-transfection of expression plasmid along with siRNA were performed in 6-well plates in the presence of Lipofectamine 2000 (InVitrogen). Aliquots of 3 µg of expression plasmid and/or siRNAs (30 or 300 nM) and 5 µl Lipofectamine 2000 per well were applied in a final volume of 1.2 ml Opti-MEM. After 4 h, the medium was renewed with a mixture of 75% MEM and 25% medium 199, supplemented with 5% FCS, serum albumin, insulin and hydrocortisone hemisuccinate as described above (30). After 12 h, hepatocytes were stimulated by EGF. Plasmids encoding FADD-DN, MC159 and E8 were kindly provided by Drs M.T.Dimanche-Boitrel and J.I.Cohen.
Fas immunoprecipitation
Hepatocytes were cultured on plastic culture plates (diameter, 15 cm; Falcon) at a density of 8 x 106 cells/dish. After plating for 24 h, cells were stimulated with EGF (25 ng/ml) during 0, 15 or 240 min. They were harvested in buffer containing 136 mmol/l NaCl, 20 mmol/l TrisHCl, 10% (v/v) glycerol, 0.1 mM sodium orthovanadate, 1 mM NaF, 10 mM ß-glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride and 5 µg/ml aprotinin and leupeptin, and 1% (v/v) Triton X-100. The lysates were kept for 10 min on ice and then centrifuged at 10 000 g for 20 min at 4°C, and aliquots were taken for protein determination using the Bio-Rad protein assay. The supernatants containing equal protein amounts (400 µg) were incubated for 18 h at 4°C with a polyclonal rabbit anti-rat Fas antibody (3 µg), and then 50 µl of slurry protein G sepharose (Zymed) was added and incubated at 4°C for 3 h. Immunoprecipitates were washed six times with lysis buffer [0.1% (v/v) Triton X-100] and resuspended in 30 µl of sample buffer for western blot analysis.
Statistical analysis
Results are expressed as the mean ± SD. Data were analyzed with Student's t-test. The level of significance was P < 0.05 (*) or P < 0.005 (**).
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Results
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To understand better how EGF promotes both hepatocyte proliferation and cell survival, we investigated the expression and role of c-FLIPL during hepatocyte proliferation in primary cultures. As we previously described (4), this well-established model allows a synchronous cell proliferation in response to EGF stimulation as assessed by methyl-[3H]thymidine incorporation into DNA (Figure 1A). DEVD-AMC caspase activity, reflecting global activity of executioner caspases, was only enhanced on days 56 (Figure 1B) and a DNA ladder was not observed until day 7 (Figure 1C) in primary EGF-stimulated hepatocytes. In contrast, untreated hepatocytes rapidly underwent apoptosis as demonstrated by DEVD-AMC caspase activity and DNA ladder formation (Figure 1B and C). This dual effect of EGF correlated with a strong induction of c-FLIPL (p55) protein expression in EGF-treated hepatocytes when compared with their EGF-untreated controls as assessed by western blot analysis (Figure 2A). Three isoforms of c-FLIPL could be detected after a long exposure (60 s) (Figure 2A) as it was previously described in malignant glioma cells (26). The appearance of the cleaved form of c-FLIPL (p43) was detectable as soon as 24 h after EGF addition. Based upon the fact that we have previously demonstrated that MEK/ERK activities are required for hepatocyte proliferation in vitro (4) and in vivo (2) and that other groups correlated c-FLIPL expression with ERK activation (3133), EGF-stimulated hepatocytes were treated with chemical inhibitors of MEK activity, UO126 and PD98059, in order to test the contribution of the ERK1/2 pathway to c-FLIPL induction. UO126, which abolished both hepatocyte proliferation and phosphorylation of ERK1/2 (Figure 2B and C), was found to strongly reduce c-FLIPL expression (Figure 2C); treatment by PD98059 gave similar results (data not shown).

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Fig. 1. EGF induces both proliferation and increases survival rates in normal rat hepatocytes. Kinetics of DNA replication in hepatocytes measured by a 12 h-incorporation of methyl-[3H]thymidine (A) in normal rat hepatocytes stimulated or not with EGF (25 ng/ml), 4 h after plating. Kinetics of programmed cell death (B) evaluated by caspase activity assay [hydrolysis of DEVD-AMC (100 µg) of cell lysate] in normal rat hepatocytes stimulated or not with EGF (25 ng/ml), in freshly isolated (T0) and cultured hepatocytes, 4 h after plating (4 h) and on different days of culture. Data represent the mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.005 when compared with unstimulated hepatocytes. Kinetics of DNA degradation (C) in normal liver (L), in freshly isolated (T0) and cultured hepatocytes on different days of culture. M: molecular weight markers.
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Fig. 2. c-FLIPL is induced by phosphorylated ERK (P-ERK) and is cleaved during hepatocyte proliferation. Western blot analysis of c-FLIPL on different days of culture (A) in normal rat hepatocytes stimulated or not by EGF. The films were exposed for 15 or 60 s. Evaluation of methyl-[3H]thymidine incorporation in hepatocytes between days 3 and 4 (B) and western blot analyses (C) of c-FLIPL, P-ERK and total ERK1/2 were performed using normal rat hepatocytes stimulated or not with EGF and treated or not with 20 µM of UO126, a specific inhibitor of MEK, for 3 days. Data represent the mean ± SD of three independent experiments. **P < 0.005 when compared with EGF-stimulated hepatocytes.
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Considering the major role played by c-FLIPL in regulating apoptosis and proliferation in T lymphocytes (21,34), we next investigated whether its upregulation in EGF-treated hepatocytes may link it to one of these two process in the model of proliferating hepatocytes. We, therefore, knocked down expression of c-FLIPL using the siRNA technology. First, efficiency of the c-FLIP siRNA oligonucleotide (icFLIP) was monitored in co-transfected hepatocytes with V5-tagged-c-FLIPL or empty vector and c-FLIP siRNA or GFP specific siRNA (Figure 3A). Expression of V5-c-FLIPL protein was abolished only in cells treated with the icFLIPL (Figure 3A). Second, c-FLIP siRNA treatment of primary rat hepatocytes markedly inhibited both c-FLIPL expression (Figure 3C) and methyl-[3H]thymidine incorporation in EGF-treated cells (Figure 3D) without inducing caspase-3 activity (Figure 3B) or cell death (data not shown). In addition, a slight decrease of the phosphorylated forms of ERK1/2 (Figure 3C) was observed in EGF-stimulated hepatocytes transfected with icFLIP. The siRNA treatment did not lead to a global decrease of protein expression since induction of the MAP kinase phosphatase (MKP1) was detected in the presence of icFLIP. Altogether, our results demonstrate a strong induction of c-FLIPL (p55) and its cleaved form (p43) by EGF; they further indicate that such an induction is necessary for DNA replication in hepatocytes in primary culture.

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Fig. 3. The DNA replication of EGF-stimulated hepatocytes is dependent on c-FLIPL protein. Western blot analysis of overexpressed V5-tagged-c-FLIPL (A) in EGF-stimulated hepatocytes using anti-V5 antibody. Hepatocytes were co-transfected with empty vector (pcDNA) or a vector encoding c-FLIPL (pcDNAV5FLIP) and 0 or 100 nM of siRNA (c-FLIP specific siRNA; icFLIP or GFP directed control siRNA oligonucleotides; iGFP). Rat hepatocytes were transfected with 0, 30 or 300 nM of icFLIP or iGFP for 4 h after 1 day of culture, as indicated. Quantification of cell death using caspase activity assay [hydrolysis of DEVD-AMC (100 µg) of cell lysate] (B). Western blot analyses (C) of c-FLIPL, phosphorylated-, total-forms of ERK1/2 and MKP1 were performed 36 h after transfection, as indicated. Quantification of hepatocyte proliferation (incorporation of methyl-[3H]thymidine for 24 h) (D). Data represent the mean ± SD of three independent experiments. **P < 0.005 when compared with unstimulated hepatocytes (for B) and to iGFP transfected hepatocytes (for D).
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As the cleavage of c-FLIPL was described to occur in the DISC by caspase-8 (35,36), we tested the possible involvement of FADD, Fas and procaspase-8 in DNA replication in EGF-treated hepatocytes. To interfere with the ability of c-FLIPL to bind to FADD and to be recruited into the DISC, a dominant negative mutant of FADD (FADD-DN) and two v-FLIP proteins, equine herpesvirus type 2 E8 and Molluscum contagiosum virus MC159 were transiently transfected in hepatocytes before addition of EGF (Figure 4A). MC159 and E8 block the signal from FADD to caspase-8: MC159 binds to FADD and prevents caspase-8 processing, whereas E8 binds to caspase-8 and inhibits FADD interactions (37). In each case, the methyl-[3H]thymidine incorporation in EGF-treated cells was reduced by
50%, which corresponded to transfection efficiency under our conditions (38). Subsequently, knock-down of the expression of Fas and procaspase-8 were performed and hepatocellular proliferation was evaluated using methyl-[3H]thymidine incorporation in EGF-treated cells. iFas1 and iCasp8 1 gave the best reduction of Fas and procaspase-8 expression, respectively (Figure 4C). These decreases correlated with significant inhibition of DNA synthesis (Figure 4B). We next hypothesized that the link between the Fas/FADD/c-FLIPL/caspase-8 and the EGFR pathway could be a binding of Fas to EGFR. Co-immunoprecipitation studies showed an EGF-induced association between EGFR and Fas within 15 min that persisted for up to 240 min (Figure 4D). Fas and EGFR expression levels were not affected despite a shift in migration of EGFR detected in lysates of EGF-treated hepatocytes (15 and 240 min).

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Fig. 4. Fas, FADD and procaspase-8 are required for DNA synthesis of EGF-treated hepatocytes in primary culture. Rat hepatocytes were transiently transfected with pcDNA3.1 LacZ cDNA or with FADD dominant negative mutant (FADD-DN) cDNA or MC159 cDNA or E8 cDNA for 4 h after 1 day of culture. After 18 h, transfected hepatocytes were further treated by EGF and quantification of methyl-[3H]thymidine incorporation in hepatocytes (A) was performed 36 h after transfection as indicated. Rat hepatocytes were transfected with 0 or 100 nM of Fas (iFas1 or iFas2) or procaspase-8 (iCasp8 1 or iCasp8 2) or GFP (iGFP) siRNAs oligonucleotides for 4 h after 1 day of culture, as indicated. Quantification of hepatocyte proliferation was performed as in Figure 3 (B). Data represent the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.005 when compared with lacZ (for A) or iGFP (for B) transfected hepatocytes. Western blot analyses (C) of Fas and procaspase-8 were performed 36 h after transfection, as indicated. Cells extracts and immunoprecipitated samples collected from unstimulated (0) and EGF-stimulated cells (15 or 240 min) were analyzed as described in Materials and methods (D). Briefly, the samples were immunoprecipitated with rabbit anti-Fas and analyzed by western blot analysis using sheep anti-EGFR. Heavy chains detection serves as immunoprecipitation control. Expression of Fas and EGFR was detected by western blot analysis in lysates of EGF-treated hepatocytes.
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Since the cleavage of c-FLIPL p55 into p43 was specifically attributed to active caspase-8 in the DISC, we next assessed by western blotting the maturation of procaspase-8 in EGF- treated and untreated hepatocytes. In the presence of EGF, the appearance of cleaved forms of procaspase-8 (p41/p43) coincided with the decrease of procaspase-8 (Figure 5A). By contrast, cleaved fragments of the executioner procaspase-3 (p17) were only detected on day 5 in EGF-stimulated hepatocytes, whereas they were detected from day 3 in unstimulated hepatocytes (Figure 5A), thus confirming the kinetics of the DEVDase activity (Figure 1B). Since activation of procaspase-3 was undetectable during the first 4 days of EGF-stimulation despite an early maturation of the initiator caspase-8, these data may suggest a sequestration of cleavage intermediates (p41/p43) of procaspase-8 into the DISC (23).

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Fig. 5. Caspase-8 cleavage and activity are required for cell-cycle progression of EGF-treated hepatocytes in primary culture. Western blot analyses of caspases (8 and 3) (A) in cultured hepatocytes (15 days). M: molecular weight markers. DEVD-AMC (B) and IETD-AMC (C) caspase activities measured in 4-day cultured hepatocytes, cultured with or without EGF. CasputinTM corresponds to a peptidic inhibitor of executioner caspases. Caspase activities were expressed in arbitrary units (A.U.) of fluorescence or as Vmax. Data represent the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.005 when compared with EGF-stimulated hepatocytes. Quantification of methyl-[3H]thymidine incorporation in EGF-treated hepatocytes (D) or co-treated with EGF plus a pan-caspase inhibitor (z-Asp) as indicated. Data represent the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.005 when compared with control (0 µM z-Asp). Western blot analysis of cyclin D1 (E) in lysates of cultured hepatocytes treated during 3 days with EGF alone or EGF plus z-Asp (50 µM).
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It has been demonstrated that active caspase-8 is responsible for the cleavage of c-FLIPL (20,35) and is important for proliferation of T lymphocytes (18,19). In this context, we asked whether the cleavage and protease activities of caspase-8 were required for hepatocyte proliferation. Activity of caspase-8 was, therefore, measured in cell lysates obtained from EGF-treated and untreated 4-day-old hepatocytes using the fluorogenic tetrapeptide IETD-AMC (Figure 5B and C). To avoid a misinterpretation of this activity, through a possible hydrolysis of this substrate by executioner caspases (3 and 7), analyses were performed in the presence of casputin, a recombinant peptide specifically inhibiting the activities of caspases (3 and 7) (39), thus abolishing DEVD-AMC caspase activity in hepatocyte lysates (Figure 5B). Regardless of whether casputin was added or not, IETD-AMC caspase activity was found to be lower in EGF-stimulated hepatocytes than in their unstimulated counterparts (Figure 5C), indicating that cleaved fragments of caspase-8 found in EGF-exposed cells were less active than in cells not treated with EGF. Finally, as initiator caspase activity remained detectable during hepatocyte proliferation, we decided to culture EGF-stimulated hepatocyte in the presence of a pan-caspase inhibitor (z-Asp-CH2-DCB), previously shown to efficiently inhibit hepatocyte apoptosis (4042), to test whether the z-Asp-sensitive caspase activity was involved in the proliferative response of hepatocytes. Evaluation of hepatocyte DNA replication was quantified through incorporation of methyl-[3H]thymidine into hepatocytes stimulated or not with EGF (25 ng/ml) or co-treated with EGF plus the caspase inhibitor (Figure 5D). In the presence of EGF, a dose-dependent inhibition of hepatocyte DNA replication was found with z-Asp (Figure 5D). To characterize the blockage of cell cycle in EGF-stimulated hepatocytes by z-Asp, we evaluated the protein level of a critical cell-cycle regulator, cyclin D1. We have previously shown that induction of cyclin D1 is strictly associated with the G1 to S phase progression in primary cultures of hepatocytes and during liver regeneration (4,30). The expression of cyclin D1 was significantly decreased in hepatocytes treated with both EGF and z-Asp (50 µM) in comparison with hepatocyte treated with EGF alone (Figure 5E).
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Discussion
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The present study was designed to investigate the role of c-FLIPL in normal proliferating cells, such as EGF-treated rat hepatocytes. First, we demonstrated by western blot analysis that c-FLIPL expression was markedly induced in these cells when compared with their untreated controls. This upregulation was fully prevented by MEK/ERK inhibitors, such as UO126, indicating that the ERK1/2 pathway, whose activation is also required for hepatocyte proliferation (2,4), may effectively control c-FLIPL expression levels in hepatocytes. Other factors, such as activation of PI3K/Akt and NF-
B, have also been described to be involved in c-FLIPL induction (reviewed in Ref. 43). In this context, it is noteworthy that EGF has been reported to activate both pathways (6). We found that Ly294002, an inhibitor of PI3K, also inhibits c-FLIPL expression (data not shown) in our model. Whether NF-
B-related pathway may play a role in c-FLIPL upregulation in EGF-treated rat hepatocytes deserves further investigation.
Increased expression of c-FLIPL in proliferating EGF-treated hepatocytes was associated with the development of a resistance toward apoptosis when compared with EGF-untreated hepatocytes, as assessed by DNA ladder analysis and DEVD-AMC caspase activity. Surprisingly, partial maturation of procaspase-8 occurred in both EGF-treated and unexposed cells, indicating that c-FLIPL upregulation in EGF-treated hepatocytes was not capable of preventing the first cleavage step of procaspase-8. Moreover, measurement of caspase-8 activity, using a specific substrate in the presence of recombinant inhibitors of executioner caspases, revealed that this caspase was active in EGF-treated cells. The inhibition of this weak caspase activity by cell permeable pan-caspase inhibitor or the knock-down of procaspase-8 led to a blockage of the hepatocyte G1 to S phase transition.
Altogether our results suggest that c-FLIPL does not exert its canonical anti-apoptotic function in proliferating hepatocytes, i.e., the inhibition of procaspase-8 processing. These data are in accordance with three reports: (i) Chang et al. (24) were the first to propose that the long isoform of c-FLIP can promote procaspase-8 activation through heterodimerization. c-FLIPL exerts its effect through its protease like domain which associates efficiently with the procaspase-8 protease domain and induces the enzymatic activity of the zymogen. (ii) Micheau et al. (23) have demonstrated that caspase-8, in the presence of c-FLIP long isoform, might only cleave DISC proximal substrates, such as RIP, which would explain the lack of activation of the executioner procaspase-3 and the default of apoptosis in our model. (iii) Recently, Boatright et al. (22) provided further evidence for the long splice form of c-FLIP being an activator of caspases (8 and 10), and demonstrated that the resulting heterodimer is enzymatically active.
In the present study, p43/41 cleaved fragments of caspase-8 and p43 cleaved fragment of c-FLIPL were detected soon after 20 h of EGF addition. These data suggest the possible involvement of an FADD/caspase-8/c-FLIPL complex during hepatocyte proliferation in primary culture since, to our knowledge, c-FLIPL is described to be exclusively cleaved in the DISC by caspase-8. As methyl-[3H]thymidine incorporation in EGF-stimulated hepatocytes is altered by FADD-DN, z-Asp and siRNA (against c-FLIPL, Fas and procaspase-8), components of the Fas/FADD/caspase-8/c-FLIPL complex appear necessary for the G1 to S phase progression (Figure 6). The association between EGFR and Fas (Figure 4D) in response to EGF treatment also argues for such a complex driving caspase-8/c-FLIPL cleavage. Moreover, these results are in accordance with a possible role of Fas during liver and neurite regeneration (44,45). It has been demonstrated that liver regeneration is delayed in mice with decreased Fas expression.

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Fig. 6. Schematic diagram. Hypothetical model of Fas/FADD/Caspase-8/c-FLIPL dependent EGF-induced DNA synthesis in primary hepatocytes. Epidermal growth factor (EGF) binds to its receptor, which recruits Fas to form a heterodimer. Subsequently, ERK becomes phosphorylated and it regulates c-FLIPL protein induction. Procaspase-8 and c-FLIPL are likely to be recruited with FADD which allows the cleavage and the accumulation of p43Caspase-8 and p43c-FLIPL. The inhibition of this cascade at different steps prevents DNA synthesis in primary hepatocytes.
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Although all three isoforms of c-FLIPL p55 are detected in hepatocytes, their expression differs in presence or absence of EGF. Yang et al. (26) have suggested that only one form is phosphorylated by CaMKII and recruited to the Fas DISC. They showed that immediately upon recruitment, phosphorylated c-FLIPL protein was cleaved into the p43 cleavage fragment in the DISC, which prevents further cleavage of caspase, and thus inhibits caspase-8-initiated apoptosis.
It is tempting to speculate that cleaved caspase-8, activated during cell proliferation, might regulate crucial cell cycle regulators by proteolytic cleavage. Among them, the cyclin dependent kinase inhibitor p27 represents a potential target in hepatocyte since: (i) p27 has been described to be cleaved by caspases 8 and 3 (46); (ii) we have recently shown that EGF-stimulated hepatocytes contain the p23 truncated form of p27 (30); (iii) we detected an IETD-like caspase activity in proliferating hepatocytes without appreciable caspase-3 activity; and (iv) other groups (47,48) have demonstrated that mice lacking p27 gene display liver hyperplasia, a consequence of an elevation of DNA replication and Cdk2-associated activity.
Our data based on results obtained with cell permeable caspase inhibitors demonstrate that a z-Asp-sensitive caspase activity is required for hepatocyte proliferation. However, it is important to stress that these peptide caspase inhibitors are not absolutely specific as pointed out by Thornberry et al. (49,50). We cannot exclude that these inhibitors abolish another protease activity involved in the regulation of cell cycle. Recently, a novel protease named separase, with a high level of similarity to caspases, has been identified to tightly control mitosis (51,52).
Cellular and molecular mechanisms involved in c-FLIPL control of hepatocyte proliferation remain to be precisely determined. The phosphorylated forms of ERK1/2, responsible for c-FLIPL induction in our model (Figure 2) and known to be required for hepatocyte growth (2), were slightly decreased in cells treated with the c-FLIPL specific siRNA. The induction of MKP1, an MAP kinase phosphatase, might explain this result. The upstream activator of MEK/ERK, the Raf-1 kinase, could play a crucial role in this proliferative process through c-FLIPL binding, subsequently leading to cell growth (32,33) assigning a central role for Raf-1 in cell fate determination (53,54). Other identified binding partners of c-FLIPL, such as FADD (55), TRAF-1 and -2 (56), RIP (57), p38 MAPK (58), NIK and IKK-2 (59), could also be part of the link between cell proliferation and c-FLIPL.
In summary, we have demonstrated that the Fas/FADD/c-FLIPL/caspase-8 complex controls the G1 to S phase progression of EGF-stimulated hepatocytes in primary culture. This role for c-FLIPL in modulating cell growth may also be important for tumor growth. c-FLIP is frequently upregulated in human tumors (43,60), especially in hepatocellular carcinomas (34). Our data along with others suggest that c-FLIPL protein represents a therapeutic target in oncology since its inhibition might lead to a cell-cycle arrest and might render tumor cells sensitive to death receptor-induced clearance.
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Acknowledgments
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We thank M.T.Dimanche-Boitrel for the stimulating discussions. A.L.S. and A.C. are recipients of doctoral fellowships from the French Research Ministry.
Conflict of Interest Statement: None declared.
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Received March 3, 2005;
revised June 23, 2005;
accepted July 15, 2005.