Bile Acids Stimulate cFLIP Phosphorylation Enhancing TRAIL-mediated Apoptosis*

Hajime Higuchi, Jung-Hwan Yoon, Annette Grambihler, Nathan Werneburg, Steven F. Bronk, and Gregory J. GoresDagger

From the Division of Gastroenterology and Hepatology, Mayo Medical School, Clinic, and Foundation, Rochester, Minnesota 55905

Received for publication, September 12, 2002, and in revised form, October 28, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bile acids induce hepatocyte injury by enhancing death receptor-mediated apoptosis. In this study, bile acid effects on TRAIL-mediated apoptosis were examined to gain insight into bile acid potentiation of death receptor signaling. TRAIL-induced apoptosis of HuH-7 cells, stably transfected with a bile acid transporter, was enhanced by bile acids. Caspase 8 and 10 activation, bid cleavage, cytosolic cytochrome c, and caspase 3 activation by TRAIL were all increased by the bile acid glycochenodeoxycholate (GCDCA). GCDCA (100 µM) did not alter expression of TRAIL-R1/DR4, TRAIL-R2/DR5, procaspase 8, cFLIP-L, cFLIP-s, Bax, Bcl-xL, or Bax. However, both caspase 8 and caspase 10 recruitment and processing within the TRAIL death-inducing signaling complex (DISC) were greater in GCDCA-treated cells whereas recruitment of cFLIP long and short was reduced. GCDCA stimulated phosphorylation of both cFLIP isoforms, which was associated with decreased binding to GST-FADD. The protein kinase C antagonist chelerythrine prevented bile acid-stimulated cFLIP-L and -s phosphorylation, restored cFLIP binding to GST-FADD, and attenuated bile acid potentiation of TRAIL-induced apoptosis. These results provide new insights into the mechanisms of bile acid cytotoxicity and the proapoptotic effects of cFLIP phosphorylation in TRAIL signaling.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bile acids, amphipathic molecules synthesized from cholesterol within hepatocytes, are secreted across the hepatocyte canalicular membrane into bile. Bile acids accumulate within the liver in cholestasis, a pathologic condition characterized by impaired bile acid canalicular secretion. Elevated bile acid concentrations within the liver promote liver injury and the development of liver cirrhosis and liver failure (1-3). For example, children with the progressive familial intrahepatic cholestasis (PFIC) subtype 2 syndrome lack the canalicular transport protein for bile acid secretion, and develop a progressive liver disease caused by the inability to excrete bile acids from hepatocytes (4, 5). Numerous studies have shown that bile acids mediate their cytotoxicity by inducing cellular apoptosis. Bile acid apoptosis appears to be death receptor-dependent. For example, toxic bile acids trigger oligomerization of Fas in a ligand-independent manner by promoting trafficking of intracellular Fas to the plasma membrane (6). In the absence of Fas, toxic bile acids enhance TRAIL-R21/DR5 expression and aggregation also promoting a death receptor-dependent apoptosis (7). The in vivo relevance of these in vitro observations has been amply demonstrated. Liver injury and fibrosis are significantly diminished in Fas-deficient lpr mice during cholestasis following bile duct ligation (8, 9). Likewise, hepatic DR5/TRAIL-R2 expression is increased in the bile duct-ligated mouse sensitizing the liver to TRAIL-mediated hepatotoxicity (10). Thus, bile acid modulation of death receptor signaling plays a critical role in cholestatic liver injury. Further insights into how toxic bile acids promote death receptor cytotoxic signaling would, therefore, be of scientific and potentially therapeutic relevance.

Once aggregated, both death receptors Fas and DR5/TRAIL-R2 signal cell death by inducing a death-inducing signaling complex (DISC) composed of the cytoplasmic adapter protein FADD (Fas-associated death domain) and initiator caspases, procaspase 8 and 10 (11-13). Procaspases are recruited to the DISC via homotypic interactions between death effector domains (DED) of both FADD and the initiator caspases. Once recruited to the DISC, these procaspases undergo autocatalytic processing within the DISC (14, 15). Procaspase 8, which exists as p55 and p53 isoforms, is first cleaved to p43/41 forms releasing a C-terminal p10 subunit and then subsequently processed to produce p23, p18, and p10 subunits. The p18 and p10 subunits combine to form a heterotetramer, the active form of this protease (14, 15). Likewise, procaspase 10, which exists as a p54 and two p59 isoforms with active protease domains, is initially cleaved to p47/43 forms releasing a C-terminal p12 subunit, and then processed to produce the active p25 and p22/17, and p12 subunits (13). In hepatocytes, which undergo death receptor-mediated apoptosis by the so-called type-II signaling pathway (16), activated caspase 8 cleaves Bid, a proapoptotic BH3 domain-only protein (17, 18). The cleaved or truncated form of Bid (tBid) translocates to mitochondria and induces cytochrome c release into the cytosol, which then binds to apoptosis-activating factor-1 (Apaf-1), resulting in activation of caspase 9, followed by activation of effector caspases, caspase 3, 6, and 7 (14, 15, 17). The effector caspases cleave a variety of cellular targets inducing the stereotypic morphologic features of apoptosis. Although both caspase 8 and 10 activation occurs in death receptor signaling, caspase 8 is essential for cell death in mouse embryonic fibroblasts (19). The ability of caspase 10 to substitute for caspase 8 in mammalian cells remains controversial (20). Thus, recruitment and processing of procaspase 8 and perhaps caspase 10 within the DISC is essential for death receptor-mediated apoptosis.

The cellular FLICE inhibitory protein (cFLIP, also called Flame-1/I-FLICE/caspase/CASH/MRIT/CLARP/usurpin) (21-28) regulates both recruitment and processing of procaspases within the DISC (29-31). On the mRNA level, several cFLIP splice variants exist; however, only two protein forms, cFLIP-L (55 kDa) and cFLIP-s (26 kDa), have been identified (21, 24, 28, 32). 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. The short form of cFLIP, cFLIP-s, is also composed of two death effector domains, a structure resembling the N-terminal half of procaspase 8, but lacks the entire caspase domain (29). Both cFLIP-L and cFLIP-s bind to FADD within the DISC via DED-DED homotypic interactions. cFLIP-s directly inhibits caspase 8 activation within the DISC. Interestingly, cFLIP-L is first cleaved within the DISC in a caspase dependent manner to a p43 polypeptide (33); this cleaved form of cFLIP-L inhibits complete processing of caspase 8 to its active subunits (29, 32, 33). cFLIP isoforms, which are expressed by hepatocytes (34, 35), are therefore, potent negative regulators of death receptor cytotoxic signaling.

A potential mechanism by which toxic bile acids may promote death receptor cytotoxic signaling is by modulating the composition of the DISC. Thus, the overall objective of this study was to examine the effects of bile acids on the TRAIL DISC. We employed HuH-7 cells stably transfected with the sodium-dependent transporting polypeptide to ensure bile acid transport and GCDCA as the cytotoxic bile acid as its concentrations are increased in cholestasis. TRAIL-mediated death receptor signaling was examined as a relevant model of death receptor signaling modulated by bile acids (7). The results demonstrate that bile acids sensitize cells to TRAIL-induced apoptosis by stimulating cFLIP phosphorylation, which reduces its translocation to the DISC thereby facilitating activation of caspases 8 and 10.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Materials-- Reagents were purchased from the following suppliers: DCA, GDCA, TDCA, CDCA, GCDCA, TCDCA, ursodeoxycholic acid (UDCA);, chelerythrine, anti-FLAG M2 mouse monoclonal antibody (IgG1), and Sepharose-CL 4B were obtained from Sigma Chemicals Co.; DAPI was from Molecular Probes Inc. (Eugene, OR); rabbit polyclonal anti-caspase 8, mouse monoclonal (IgG2b) anti-cytochrome c, rabbit polyclonal anti-caspase 3, HRP-conjugated anti-mouse IgG1 and HRP-conjugated anti-mouse Ig kappa -chain were from PharMingen (San Diego, CA); rabbit polyclonal anti-DR4, goat polyclonal anti-DR5, mouse monoclonal (IgG1) anti-DR5, and rat monoclonal (IgG2a) anti-cFLIP were from Alexis (San Diego, CA); mouse monoclonal (IgG1-kappa ) anti-caspase 10 was from MBL (Nagoya, Japan); mouse monoclonal (IgG1) anti-FADD was from Transduction Laboratories (San Diego, CA); goat polyclonal anti-Bid was from R&D systems (Minneapolis, MN); mouse monoclonal (IgG2a) anti-Bcl-xL was purchased from Exalpha Biologicals (Boston, MA); mouse monoclonal (IgG1) anti-Bcl-2, rabbit polyclonal anti-Bax, and goat polyclonal anti-actin were from Santa Cruz Biotechnology (Santa Cruz, CA); HRP-conjugated anti-goat Ig, rabbit Ig, and mouse Ig were from Biosource (Camarillo, CA); protein G-Sepharose was from Zymed Laboratories, Inc. (San Francisco, CA); Sepharose-coupled glutathione was obtained from Amersham Biosciences; Alexa Fluor 633-conjugated donkey anti-goat IgG was obtained from Molecular Probes Inc.

Cell Culture-- HuH-7 cells, a human hepatocellular carcinoma cell line stably transfected with the sodium-dependent taurocholate co-transporting polypeptide (Ntcp), were employed for this study (7). Established clones (HuH-BAT for HuH-Bile Acid Transporting) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100,000 units/liter), streptomycin (100 mg/liter), gentamycin (100 mg/liter), and G418 (1,200 mg/liter).

Preparation of FLAG-TRAIL-- The extracellular portion of human TRAIL (amino acids 95-281) was subcloned into the pFLAG expression plasmid (Sigma Chemicals Co.) between XhoI and XbaI restriction sites. The N-terminal FLAG epitope-tagged TRAIL was expressed in Escherichia coli and purified over an anti-FLAG monoclonal antibody (M2)-agarose column (Sigma Chemicals Co.) (36). Purity of the recombinant protein was determined by SDS-PAGE and silver nitrate staining.

Quantitation of Apoptosis-- Apoptosis was quantitated by assessing the characteristic nuclear changes of apoptosis (i.e. chromatin condensation and nuclear fragmentation) using the nuclear binding dye 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and fluorescence microscopy (37).

Immunoblot Analysis-- Cells 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 Protease Inhibitor Mixture; Roche Molecular Biochemicals). After insoluble debris was pelleted by centrifugation at 14,000 × g for 15 min at 4 °C, the supernatants were collected. Samples were resolved by 12.5% SDS-PAGE, transferred to nitrocellulose membrane, and blotted with appropriate primary antibodies at a dilution of 1:1,000. Peroxidase-conjugated secondary antibodies were incubated at a dilution of 1:2,000 to 1:10,000. Bound antibody was visualized using the chemiluminescent substrate (ECL; Amersham Biosciences) and exposed to Kodak X-OMAT film.

Subcellular Fractionation-- Cytosolic extracts for cytochrome c immunoblot assay were obtained as described by Leist et al. (38). Briefly, at the desired time points, the culture medium was exchanged with permeabilization buffer (210 mM D-mannitol, 70 mM sucrose, 10 mM HEPES, 5 mM succinate, 0.2 mM EGTA, 0.15% bovine serum albumin, 80 µg/ml digitonin, pH 7.2). The cells were incubated in this buffer for 5 min on ice. The permeabilization buffer was then removed and centrifuged for 10 min at 13,000 × g. Supernatants representing the cytosolic extract were employed for the immunoblot analysis.

Death-inducing Signaling Complex (DISC) Analysis-- HuH-BAT cells were treated with FLAG-TRAIL (200 ng/ml) plus anti-FLAG M2 antibody (2 µg/ml) in the presence or absence of GCDCA (50 µM). At desired time points, cells were washed with cold PBS and lysed by incubation on ice for 30 min in the same lysis buffer described in immunoblot analysis. Insoluble debris was removed by centrifugation at 14,000 × g for 15 min at 4 °C. For time 0, 1 ml of cell lysate from untreated cells was supplemented with 0.5 µg of FLAG-TRAIL and 1 µg of anti-FLAG M2 antibody. After the protein concentration in the extracts was determined by Bradford assay, cell lysates containing 3 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, and then aliquots of protein G-Sepharose (30 µl) were added for an additional 2 h at 4 °C. Immune complexes were pelleted by centrifugation for 5 min at 14,000 × g, washed five times with lysis buffer, and released from the beads by boiling for 5 min in SDS sample buffer. Samples were subjected to SDS-PAGE, transferred to nitrocellulose membrane, and sequentially probed with antibodies for DR4, DR5, caspase 8, caspase 10, FADD, and cFLIP.

GST-FADD Pull-down Assay-- The pGex4T-2 construct containing GST fused to full-length FADD was kindly provided by Dr. M. Peter (University of Chicago). E. coli strain DH5alpha cells transformed with this construct were grown overnight in the presence of 1 mM isopropyl-D-thiogalactoside. GST-FADD was bound to glutathione-Sepharose beads according to the manufacturer's protocol and eluted by incubation with 10 mM glutathione in 50 mM Tris-HCl (pH 9.0). The eluted GST-FADD was dialyzed against PBS overnight. After HuH-BAT cells were treated with GCDCA (100 µM) for 3 h, cell lysates were prepared by freezing and thawing cells in PBS containing the same protease and phosphatase inhibitors described above for the lysis buffer. Aliquots containing 3 mg of protein were incubated for 3 h at 4 °C with purified GST-FADD. Glutathione-Sepharose was added for an additional 2 h at 4 °C. FADD-bound proteins were then pelleted by centrifugation for 5 min at 600 × g, washed five times with PBS buffer containing the protease and phosphatase inhibitors described above for the lysis buffer, and released from the beads by boiling for 5 min in SDS sample buffer. Recovery of procaspase 8, procaspase 10, and cFLIP was assessed by immunoblot analysis.

cFLIP-Green Fluorescent Protein (GFP)-- A human cDNA for cFLIP was a gift from Dr. P. Dent (Virginia Commonwealth University, Richmond, VA). cDNA encoding amino acids 1-202 was subcloned into green fluorescent protein (GFP)-expression plasmid pEGFP-N1 (Clontech, Palo Alto, CA) at the Kpn-I site. The plasmid pEGFP-cFLIP was transfected into the HuH-BAT cells using LipofectAMINE Plus (Invitrogen, Carlsbad, CA). Forty-eight hours later, the cells were treated with FLAG-TRAIL plus M2 antibodies in the presence or absence of GCDCA (100 µM). The cFLIP-GFP fluorescence was continuously observed by employing fluorescence microscopy (TE200 Nikon Inverted Fluorescent Microscope, Nikon, Tokyo, Japan), and the cellular distribution of green fluorescence was visualized at 30-min intervals.

To visualize co-localization of the cFLIP-GFP and DR5, immunofluorescence for DR5 immunoreactivity was performed as described previously (7). In brief, cells were fixed with 3% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, and incubated with goat anti-DR5 primary antisera (1:300 dilution) for 2 h at 37 °C. After washing with PBS, the cells were incubated with Alexa Fluor 633-conjugated anti-goat IgG (Molecular Probes, 10 mg/ml) for 1 h at 37 °C. Fluorescence was visualized by laser scanning confocal microscopy (Axiovert 100 M-LSM 510, Carl Zeiss Inc., Thornwood, NY). Excitation and emission wavelengths for GFP were 488 and 505 nm, respectively, and for Alexa Fluor 633 the excitation and emission wavelengths were 633 and 650 nm, respectively.

cFLIP Phosphorylation-- HuH-BAT cells in 10-cm plates were incubated with gamma -32P (200 µCi/ml) in the presence or absence of DC or GDCA (100 µM) for 3 h. Cells were lysed with the lysis buffer described under "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 protein was immunoprecipitated by incubation with rat monoclonal (IgG2a) anti-cFLIP (Alexis) antibody (5 µg) and protein G-Sepharose (30 µl) for an additional 2 h at 4 °C. Immune complexes were pelleted by centrifugation for 5 min at 14,000 × 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 transferred to nitrocellulose membranes. Radioactivity was determined by autoradiography at -70 °C overnight.

Statistical Analysis-- All 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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bile Acids Sensitize HuH-BAT Cells to TRAIL-induced Apoptosis-- Initially, the effects of various bile acids on TRAIL-induced apoptosis was carefully characterized using the bile acid transporting HuH-BAT cells. Cellular apoptosis was minimal (<15%) following treatment of the cells with the unconjugated and conjugated forms of DCA and CDCA (50 µM) for 4 h (Fig. 1, A and B). TRAIL itself (100 ng/ml) induced only slight apoptosis over the same time period, 27 ± 3% apoptosis. In contrast, all of the bile acids (50 µM) studied markedly sensitized cells to TRAIL-mediated apoptosis. Of the bile acids examined, unconjugated and glycine conjugates of DCA and CDCA were the most potent in enhancing TRAIL-induced apoptosis, and increased TRAIL-mediated apoptosis ~4-fold (Fig. 1, A and B). The ability of bile acids to enhance TRAIL-mediated apoptosis was concentration-dependent (Fig. 2). Both GCDCA and GDCA at a concentration of 50 µM enhanced TRAIL-mediated apoptosis as the concentration of TRAIL was increased (0-1,600 ng/ml), with maximum cell killing at TRAIL concentrations of >= 400 ng/ml (Fig. 2, A and C). Likewise, cells were increasingly sensitized to TRAIL (50 ng/ml) cytotoxicity as the concentration of bile acid was increased (0-200 µM); maximum cell killing was observed with a bile acid concentration of 100 µM for GCDCA and a concentration 50 µM for GDCA (Fig. 2, B and D). At all the conditions tested, cellular apoptosis was synergistically increased by the combination of the bile acid and TRAIL. These data demonstrate that bile acids sensitize cells to TRAIL-mediated apoptosis. Because GCDCA is one of the major bile acids in human bile (39), we choose GCDCA for subsequent experiments.


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Fig. 1.   Bile acids sensitize HuH-BAT cells to TRAIL-mediated apoptosis. HuH-BAT cells were incubated with FLAG-TRAIL (100 ng/ml) plus anti-FLAG M2 antibody (2 µg/ml) or M2 antibody alone in the presence or absence of indicated bile acid (50 µM) for 12 h. Apoptosis was evaluated by DAPI staining and fluorescence microscopy. All data were expressed as mean ± S.D. from three individual experiments. All the bile acids significantly enhance TRAIL (100 ng/ml)-induced apoptosis as compared with TRAIL treatment alone (p < 0.01).


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Fig. 2.   Glycine-conjugated bile acids potently sensitize cells to TRAIL-mediated apoptosis. HuH-BAT cells were incubated with indicated concentrations of FLAG-TRAIL plus anti-FLAG M2 antibody (2 µg/ml) or M2 antibody alone in the presence or absence of either GCDCA (panels A and B) or GDCA (panels C and D) for 12 h. Apoptosis was evaluated by DAPI staining and fluorescence microscopy. All data were expressed as mean ± S.D. from three individual experiments. p < 0.01 for no bile acid group versus either GCDCA (panel A) or GDCA-treated group at all the TRAIL concentrations tested. p < 0.01 for TRAIL-treated group versus no TRAIL group at all the bile acid concentrations tested (GCDCA in panel B, GDCA in panel D).

GCDCA Enhances TRAIL-mediated Cell Death Signaling Upstream of Mitochondria-- TRAIL-mediated apoptosis in HuH-BAT cells, a hepatocyte-derived cell line, occurs via the Type II pathway (Bid cleavage, cytochrome c release, and subsequent activation of caspase 3). Bile acids, therefore, could sensitize cells to TRAIL cytotoxicity by either enhancing initiator caspase activation and Bid cleavage and/or promoting mitochondrial dysfunction with cytochrome c release. To distinguish between these two possibilities, immunoblot analysis for apoptotic effector proteins upstream (caspase 8/10, tBid) and downstream of mitochondria (cytosolic cytochrome c and caspase 3) was examined. GCDCA (50 µM) alone did not activate this signaling cascade. After treatment of HuH-BAT cells with TRAIL (100 ng/ml) for 0-8 h, immunoblot analysis of cell lysates demonstrated processed polypeptides for both initiator caspases, caspase 8 and 10, appearance of cytochrome c in the cytosol, and processed forms of caspase 3, an effector caspase (Fig. 3). TRAIL plus GCDCA, however, further enhanced activation of the initiator procaspases 8 and 10, and the associated cleavage of Bid. Because these finding could be explained by increased expression of TRAIL death receptors, the effect of bile acids on TRAIL-R2/DR4 and TRAIL-R2/DR5 was examined. At the concentrations (GCDCA 0-100 µM) and time periods of this study (6-8 h), GCDCA did not alter TRAIL-R1/DR4 nor TRAIL-R2/DR5 protein expression (Fig. 4A). As Bcl-2 family proteins such as Bcl-2, Bcl-xL, or Bax have also been implicated in the regulation of TRAIL-induced apoptosis (30, 40-43), expression of these proteins was next assessed in the presence of GCDCA (50 µM). Neither Bcl-2, Bcl-xL, nor Bax cellular protein levels were altered by GCDCA treatment of HuH-BAT cells (Fig. 4B). Collectively, the above results demonstrating enhanced caspase 8 and 10 processing with increased cleavage of the endogenous caspase 8 substrate, Bid, support a role for GCDCA in enhancing TRAIL-mediated cell death signaling upstream of mitochondria.


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Fig. 3.   GCDCA enhances TRAIL-mediated apoptosis upstream of mitochondria. HuH-BAT cells were incubated with either GCDCA (50 µM), FLAG-TRAIL (100 ng/ml) plus anti-FLAG M2 antibody or GCDCA plus FLAG-TRAIL plus M2 antibody. At indicated time points, cells were lysed and the lysates containing 60 µg of protein were subjected to SDS-PAGE and immunoblot analysis using the indicated antibodies. For the analysis of cytochrome c release, a cytosolic fraction without mitochondria was procured as described under "Experimental Procedures."


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Fig. 4.   GCDCA does not alter protein expression of TRAIL-R1/DR4, TRAIL-R2/DR5, Bcl-2, Bcl-xL, or Bax. HuH-BAT cells were incubated with 50 µM of glycochenodeoxycholic acid (GCDCA) for 8 h. A, cells were lysed at indicated time points, and the lysates containing 60 µg of protein were subjected to SDS-PAGE and immunoblot analysis using the anti-TRAIL-R1/DR4 or anti-TRAIL-R2/DR5 antisera. B, cell lysates containing 60 µg of protein were subjected to SDS-PAGE and immunoblot analysis using the anti-Bcl-2, Bcl-xL, or Bax antisera.

GCDCA Inhibits cFLIP Recruitment to the DISC Resulting in Increased Activation of Caspase 8 and Caspase 10-- The enhanced initiator caspase activation described above suggested that GCDCA modifies TRAIL signaling at the level of the DISC. To assess the potential effect of GCDCA on TRAIL DISC composition, the TRAIL DISC was immunoprecipitated and the associated polypeptides examined by immunoblot analysis (Fig. 5). Both TRAIL-R1/DR4 and TRAIL-R2/DR5 receptors coprecipitated with FLAG-TRAIL. For each receptor, the amount of immunoprecipitated protein was equivalent in TRAIL versus TRAIL plus GCDCA-treated cells. This result was consistent with the prior observation that GCDCA did not alter expression of these proteins. The recruitment of caspase 10, caspase 8, FADD, cFLIP-L, and cFLIP-s to the DISC was assessed over time (0-60 min). Only partially cleaved forms of caspase 10 (47 and 44 kDa) and cFLIP-L (43 kDa) were observed in the TRAIL DISC. Although full-length procaspase 8 (55 and 53 kDa) was identified, partially cleaved forms of caspase 8 (43 and 41 kDa) were also present. These results suggest rapid processing of initiator caspases within the TRAIL DISC. Both caspase 10 and caspase 8 recruitment were significantly enhanced in the presence of GCDCA whereas recruitment of the adapter protein FADD was similar (Fig. 5). Thus, GCDCA enhances procaspase 8/10 recruitment to and/or processing within the TRAIL DISC. In contrast to the procaspases, recruitment of the antiapoptotic proteins, cFLIP-L and cFLIP-s, to the DISC was significantly reduced in GCDCA-treated cells. Because of problems associated with extracting proteins from cells (loss of protein binding interactions due to cellular disruption), the effects of bile acid on TRAIL-induced cFLIP-DISC association was further examined using a cFLIP-GFP fusion protein. cFLIP-GFP distribution was diffuse in untreated HuH-BAT cells (Fig. 6A). However, cFLIP-GFP fluorescence became punctate after TRAIL treatment (Fig. 6A), and co-localized with TRAIL-R2/DR5 (Fig. 6C). The cellular distribution of cFLIP, however, remained diffuse when the cells were incubated with TRAIL in the presence of GCDCA (Fig. 6, A and B). Thus, treatment of HuH-BAT cells with GCDCA appears to reduce cFLIP association with the TRAIL DISC.


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Fig. 5.   GCDCA treatment of HuH-BAT cells decreases recruitment of cFLIP isoforms to the TRAIL DISC. HuH-BAT cells were incubated with FLAG-TRAIL (400 ng/ml) plus anti-FLAG M2 antibody in the presence or absence of GCDCA (100 µM). Cells were lysed after 0, 15, 30, and 60 min incubation, and the lysates containing 3 mg of protein were subjected to immunoprecipitation. For time 0, 0.5 µg/ml of FLAG-TRAIL and 1 µg/ml of anti-FLAG M2 antibody were added to the untreated samples after lysis. Immunoprecipitates were subjected to SDS-PAGE and immunoblot analysis using the indicated primary antibodies.


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Fig. 6.   GCDCA abrogates TRAIL-induced cFLIP translocation to the TRAIL DISC. HuH-BAT cells were transfected with the cFLIP-GFP expression plasmid. A, cells were cultured on commercially available 3.5-cm plastic dishes with thin glass bottoms and mounted on a heated stage of an inverted fluorescent microscope. cFLIP-GFP fluorescence was monitored using excitation and emission wavelengths of 488 and 505 nm, respectively. After adding TRAIL (500 ng/ml) or TRAIL plus GCDCA (50 µM), fluorescence images were taken at 30-min intervals. Representative fluorographs are displayed. B, inset shows expression of the cFLIP-GFP fusion protein within HuH-BAT cells. Cell lysates after transient transfection with either GFP (left lane) or cFLIP-GFP (right lane) vector were subjected to immunoblot analysis using anti-GFP primary antisera. The number of cells displaying punctate of aggregates of cFLIP-GFP fluorescence was quantitated in random microscopic fields (×400 magnification). Approximately, 100 cells from 20 individual culture dishes were counted for each experimental group. C, cells were fixed after the treatments described in A and subjected to immunocytochemistry using anti-TRAIL-R2/DR5 antisera. The green (cFLIP-GFP) and the red (TRAIL-R2/DR5) fluorescence (excitation and emission wavelengths of 555 and 580 nm, respectively) was visualized using confocal microscopy.

GCDCA Reduces FADD-cFLIP Binding Affinity-- Decreased recruitment of cFLIP isoforms to the TRAIL DISC could potentially be explained by a reduction in cFLIP-FADD binding. Therefore, the ability of GCDCA to alter binding of these molecules to FADD was next ascertained by a GST-FADD pull-down assay. Protein samples were extracted from GCDCA-treated or untreated HuH-BAT cells and incubated in the presence of the GST-FADD fusion protein. After the precipitation of GST-FADD using glutathione-coupled Sepharose beads, cFLIP-L, cFLIP-s, procaspase 10, and procaspase 8 associated with GST-FADD was examined by immunoblot analysis (Fig. 7). GCDCA treatment decreased the amount of cFLIP-L and cFLIP-s bound to GST-FADD while cellular expression level of these proteins as evaluated by immunoblot in total cell lysates was unchanged. In contrast, GCDCA altered neither procaspase 10 nor procaspase 8 binding to FADD. These results were only observed using protein extracts from GCDCA-treated cells as adding GCDCA in vitro to the binding assay did not affect the amount of c-FLIP-L and -s bound to GST-FADD (data not shown). These results suggest that GCDCA decreases cFLIP-FADD interactions by altering cFLIP in a cellular context without changing caspase 10/8-FADD binding.


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Fig. 7.   GCDCA treatment decreases cFLIP binding to FADD. Cell lysates were prepared from HuH-BAT cells incubated for 2 h in the presence or absence of GCDCA (100 µM). Samples were sequentially incubated with 5 µg of purified GST-FADD and glutathione-Sepharose beads. After washing the beads five times with ice-cold PBS, bound proteins were eluted, subjected to SDS-PAGE, and immunoblot analysis performed using the indicated primary antisera.

GCDCA Induces cFLIP Phosphorylation and Enhances Apoptosis Via a Chelerythrine-sensitive Process-- A potential mechanism by which bile acids may alter cFLIP binding to FADD in a cellular context is by inducing cFLIP phosphorylation. The protein phosphorylation may cause structural and functional alterations of cFLIP, and explain, in part, how bile acids reduce cFLIP-FADD binding. Therefore, to determine if bile acid treatment of cells results in cFLIP phosphorylation, HuH-BAT cells were incubated in the presence and absence of GCDCA and 32P incorporation by cFLIP proteins assessed by autoradiography following cFLIP immunoprecipitation (Fig. 8A). Indeed, significant 32P incorporation by both cFLIP long and short was observed following GCDCA-treated cells as compared with controls. Because bile acids are known to activate protein kinase C (PKC) cascades (44, 45), the effects of the PKC antagonist, chelerythrine on bile acid-associated cFLIP phosphorylation was examined. Indeed, chelerythrine was effective in decreasing both cFLIP long and short phosphorylation (Fig. 8A). The PKC-dependent phosphorylation status of both cFLIP isoforms was next correlated with FADD binding and bile acid potentiation of TRAIL cytotoxicity. Both cFLIP-L and cFLIP-s binding to GST-FADD was restored to basal levels when cells were incubated with GCDCA plus chelerythrine (Fig. 8B). Chelerythrine treatment also attenuated GCDCA-mediated enhancement of TRAIL-mediated apoptosis (Fig. 8C). Thus, GCDCA appears to enhance TRAIL-induced apoptosis by stimulating cFLIP phosphorylation thereby reducing the antiapoptotic functions of this protein.


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Fig. 8.   Bile acid treatment of HUH-BAT cells is associated with phosphorylation of both cFLIP isoforms in a protein kinase C-dependent manner. HuH-BAT cells were incubated with either GCDCA (100 µM) or GCDCA plus chelerythrine (1 mM). A, cells were incubated in the presence of gamma -32P (200 µCi/ml) for 3 h. After the immunoprecipitation using anti-cFLIP antibody, the immunocomplexes were resolved by SDS-PAGE, and radioactivity on the membranes detected by autoradiography. The precipitated cFLIP proteins were confirmed by immunoblot (IB) analysis using the same membrane. The density of each band was measured by densitometric analysis, and the ratio of phosphorylated/total cFLIP was calculated (the relative ratio is shown in the graph). B, cell lysates were incubated with 5 µg of purified GST-FADD and glutathione-Sepharose beads. After beads were washed five times with PBS, bound proteins were eluted, resolved by SDS-PAGE, and subjected to immunoblot analysis using the indicated primary antisera. C, HuH-BAT cells were treated with either TRAIL (50 ng/ml) or TRAIL plus GCDCA (100 µM) in the presence or absence of chelerythrine (1 µM). Apoptosis was evaluated by DAPI staining and fluorescent microscopy.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The principal findings of this study relate to the cellular mechanisms by which toxic bile acids promote death receptor signaling. The results demonstrate that: (i) bile acids, at nontoxic concentrations, enhance TRAIL-mediated apoptosis upstream of mitochondria, (ii) the bile acid GCDCA alters the TRAIL DISC composition by simultaneously decreasing cFLIP and increasing caspase 8/10 recruitment to this protein complex, (iii) the altered DISC composition leads to enhanced caspase 8/10 activation followed by Bid cleavage, mitochondrial cytochrome c release, caspase 3 activation and apoptosis, (iv) cFLIP, but not procaspase 8 or 10, obtained from GCDCA-treated cells exhibits reduced binding to GST-FADD, and (v) GCDCA treatment of cells induces cFLIP phosphorylation by a chelerythrine-sensitive process. These results provide a mechanism for bile acid modulation of death receptor signaling, namely altering the composition of the DISC. Furthermore, the data suggest cFLIP is a phosphoprotein and that its phosphorylation status may alter its engagement with the TRAIL DISC.

Previous studies regarding bile acid cytotoxicity have identified alterations in death receptor cellular trafficking, expression, and oligomerization (6, 7, 46), all in the absence of ligand. The current studies extend these observations by examining the bile acid effects on death receptor signaling in the presence of ligand, a more appropriate pathophysiologic approach. The TRAIL system was chosen as it has been previously implicated in bile acid cytotoxicity (7). The results demonstrate that sublethal concentrations of bile acids potentiate TRAIL-mediated apoptosis. Of the bile acids examined, unconjugated and glycine-conjugated forms were more potent in sensitizing cells to TRAIL-mediated apoptosis than taurine conjugates. The findings are consistent with previous reports that taurine-conjugated bile acids activate intrinsic survival pathways mediated by phosphatidylinositol 3-kinase and NF-kappa B (45, 47). The current data demonstrating that bile acids sensitize cells to TRAIL-induced apoptosis are consistent with recent in vivo studies demonstrating TRAIL is hepatotoxic during cholestasis (10) and implicate a potential role for TRAIL signaling in cholestatic liver injury. Perhaps, more importantly, these observations suggest bile acids or like compounds could be used to sensitize cancer cells to TRAIL cytotoxicity. As bile acid derivatives promote apoptosis in breast cancer cell lines (48), the concept of bile acid derivatives used to sensitize cancer cells to TRAIL deserves further attention.

Current information suggests TRAIL-induced apoptosis is regulated at the cell surface by the level of TRAIL-R1/DR4 or TRAIL-R2/DR5 expression (7, 49-51), at the DISC by expression of procaspase 8 (52, 53), FADD (52), or cFLIP (29, 30); and by the expression of propapoptotic or antiapoptotic Bcl-2 family proteins such as Bcl-2, Bcl-xL, Bid, Bax (30, 40-43), which regulate mitochondrial cytochrome c release. Recently, Burns and El-Deiry (30) have demonstrated that both cFLIP-s and Bcl-xL are sufficient to protect cells from TRAIL induced apoptosis. In addition, several studies have demonstrated that Bax expression is also essential for TRAIL-mediated apoptosis signaling (41-43). However, at the concentrations employed in the current study, bile acids did not alter the expression of TRAIL-R1/DR4, TRAIL-R2/DR5, procaspase 8, cFLIP-L, cFLIP-s, Bax, Bcl-xL, or Bax. These findings suggest bile acid-induced post-translational modifications in TRAIL signaling rather then altered expression of signaling components sensitize cells to TRAIL cytotoxicity. GCDCA potentiated initiator caspase activation and Bid cleavage, suggesting bile acids potentiate TRAIL cytotoxicity upstream of mitochondria. Based on this information, the TRAIL DISC was next examined where a decrease in both cFLIP-L and cFLIP-s recruitment was identified. In agreement with current concepts, the reduction in cFLIP recruitment to the DISC is associated with a corresponding increase in caspase 8 and 10 activation. Enhanced initiator caspase activation resulted in the predictable increase in Bid cleavage, cytochrome c release, caspase 3 activation and apoptosis. Taken together, these data suggest that selected bile acids enhance the proapoptotic composition of the TRAIL DISC, thereby, enhancing the cytotoxicity of this death ligand.

The exact mechanism by which cFLIP prevents procaspase 8 or 10 activation within the DISC still remains unclear. While cFLIP-s appears to directly inhibit initiator caspase activation (33), cFLIP-L is converted within the DISC by caspases to a 43-kD subunit that remains at the DISC and 12-kDa subunit, which is released (32). In the presence of cFLIP-L, procaspase 8 within the DISC complex is partially cleaved, leading to the generation of 43- and 41-kDa subunits and concomitant release of a C-terminal 10-kDa subunit (33). Further conversion of the initiator caspases to their active subunits is blocked by the 43-kDa form of cFLIP-L within the DISC preventing apoptosis. The current data cannot exclude the possibility that the full-length phosphorylated form of cFLIP-L is still recruited to the TRAIL DISC, but that the potential phosphorylated p43 subunit is incapable of remaining in the DISC. Thus, our overall interpretation that GCDCA effects preferential recruitment of the initiator procaspases 8/10 at the expense of the cFLIP isoforms needs to be interpreted with caution.

GCDCA increased the phosphorylation status of both cFLIP isoforms, the first time this protein has been shown to be a phosphoprotein. Phosphorylation appeared to be PKC-dependent as it was decreased by chelerythrine. Increases in cFLIP phosphorylation were associated with its decreased recruitment to the TRAIL DISC and enhanced apoptosis. Presumably, cFLIP phosphorylation decreases its binding to FADD as suggested by the observation that cFLIP in cell lysates from bile acid-treated cells bound GST-FADD less well than cFLIP from control cell lysates. Because bile acids have been shown to activate several PKC isoforms in hepatocytes (44), these data suggest that bile acids promote PKC-dependent phosphorylation of cFLIP isoforms; a signaling event enhancing apoptosis. Several potential PKC phosphorylation sites are present within the DED common to both cFLIP isoforms. Because the DED domains are critical for cFLIP binding to FADD, it is rational that phosphorylation of this domain may effect cFLIP-FADD DED-mediated homotypic interactions. Further studies identifying the phosphorylation sites of the cFLIP isoforms coupled with site-directed mutagenesis will be necessary to further examine these concepts.

Collectively, the current studies further expand the role of cFLIP in TRAIL cytotoxicity, and have implications for cholestatic liver injury. The data demonstrate that cFLIP is a phosphoprotein whose binding to FADD and, therefore, recruitment to the DISC appears to be modulated by its phosphorylation status. Because phosphorylated cFLIP has reduced antiapoptotic function, TRAIL cytotoxicity is enhanced. If bile acid-associated PKC-dependent phosphorylation of cFLIP can be confirmed, perhaps PKC inhibitors may help attenuate death receptor-induced human cholestatic liver injury. The implications of these studies for potential therapy of human cholestatic liver disease merits further investigation.

    ACKNOWLEDGEMENTS

The secretarial assistance of Sara Erickson is gratefully thanked. We thank Dr. M. Peter (University of Chicago) for the pGex4T-GST-FADD construct. We also thank Dr. P. Dent (Virginia Commonwealth University) for the cFLIP cDNA.

    FOOTNOTES

* This work was supported by Grant DK41876 from the National Institutes of Health and the Mayo Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger 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@mayo.edu.

Published, JBC Papers in Press, October 28, 2002, DOI 10.1074/jbc.M209387200

    ABBREVIATIONS

The abbreviations used are: TRAIL-R, tumor necrosis factor-related apoptosis-inducing ligand receptor; CDCA, chenodeoxycholic acid; cFLIP, cellular FLICE inhibitory protein; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; DCA, deoxycholic acid; DISC, death-inducing signaling complex; DR, death receptor; FADD, Fas-associated death domain protein; GCDCA, glycochenodeoxycholic acid; GDCA, glycodeoxycholic acid; HRP, horseradish peroxidase; Ntcp, sodium-dependent taurocholate co-transporting polypeptide; PBS, phosphate-buffered saline; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid; GST, glutathione S-transferase; PKC, protein kinase C; GFP, green fluorescent protein.

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DISCUSSION
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