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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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
-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-
) 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 DH5
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
-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.
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RESULTS |
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).
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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.
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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.
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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.
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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 -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.
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DISCUSSION |
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-
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.