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J. Biol. Chem., Vol. 276, Issue 42, 38610-38618, October 19, 2001
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From the
Received for publication, June 8, 2001, and in revised form, August 2, 2001
Toxic bile salts induce hepatocyte apoptosis by
both Fas-dependent and -independent mechanisms. In this
study, we examined the cellular mechanisms responsible for
Fas-independent, bile acid-mediated apoptosis. HuH-7 cells, which are
known to be Fas deficient, were stably transfected with the
sodium-dependent bile acid transporting polypeptide. The
toxic bile acid glycochenodeoxycholate (GCDC)-induced apoptosis in
these cells in a time- and concentration-dependent manner.
Apoptosis and mitochondrial cytochrome c release were inhibited by transfection with dominant negative FADD, CrmA
transfection, or treatment with the selective caspase 8 inhibitor
IETD-CHO. These observations suggested the Fas-independent apoptosis
was also death receptor mediated. Reverse
transcriptase-polymerase chain reaction demonstrated tumor
necrosis factor-R1, tumor necrosis factor-related apoptosis inducing
ligand (TRAIL)-R1/DR4, -R2/DR5, and TRAIL, but not tumor necrosis
fator- In cholestasis (a pathophysiologic condition of the liver defined
as an impairment in bile formation), the enterohepatic circulation is
interrupted and bile acids accumulate within the liver because they
cannot be secreted into bile (1). Elevated bile acid concentrations within the liver promote liver injury and the development of liver cirrhosis and liver failure. For example, children lacking the canalicular transport protein for bile acid secretion develop a
progressive liver disease due to the inability to excrete bile acids
from the hepatocyte (2). Numerous studies indicate that toxic bile
acids induce hepatocyte injury in vitro by inducing apoptosis (3-5). Moreover, hepaptocyte apoptosis has also been shown
in animal models of cholestasis demonstrating congruence between the
in vitro and in vivo observations (6). Thus, the mechanisms of bile acid-induced hepatocyte apoptosis are of clinical and scientific importance.
Apoptosis may occur by two fundamental pathways: (i) the death receptor
or extrinsic pathway; and (ii) the mitochondrial or intrinsic pathway.
Bile acids induce hepatocyte apoptosis in a short term in
vitro paradigm by a Fas death receptor-dependent mechanism (3). Hepatocyte apoptosis was also decreased in the lpr bile
duct-ligated mouse, which has minimal Fas expression (6). Although
hepatocyte apoptosis was diminished in this model of extrahepatic
cholestasis, the rate of apoptosis increased over time suggesting a
Fas-independent mechanism of apoptosis was also occurring in these
animals (6). The mechanism responsible for this delayed,
Fas-independent pathway of bile salt-induced apoptosis was not
elucidated in this study. This delayed apoptosis may also be death
receptor mediated as hepatocytes express tumor necrosis factor receptor
R1, tumor necrosis factor-related apoptosis inducing ligand
(TRAIL)1 receptor 1 (TRAIL-R1, also referred to as death receptor-4 (DR4)), and TRAIL
receptor 2 (TRAIL-R2, also called death receptor 5 (DR5)/killer/TRICK2). All these death receptors signal apoptosis
by recruiting the cytoplasmic adapter protein FADD (Fas-associated
death domain) to an oligomerized receptor complex (7-11). FADD in turn
facilitates the binding and activation of procaspase 8, an initiator
caspase (10), which then catalyzes a series of proteolytic events that
contribute to the process recognized biochemically and morphologically
as apoptosis.
Bile acids have also been postulated to cause direct mitochondrial
cytotoxicity, and the Fas-independent apoptosis could also occur via
the mitochondrial pathway (12, 13). Indeed, bile acids will induce
mitochondrial generation of reactive oxygen species, mitochondrial
membrane potential depolarization, and the mitochondrial permeability
transition (4, 14). The biochemical hallmark of the mitochondrial
pathway of apoptosis is cytochrome c release from the
intermembrane space into the cytosol (13, 15). Cytosolic cytochrome
c allosterically facilitates binding of apoptosis activating
factor-1 with procaspase 9. This multimeric protein complex facilitates
activation of caspase 9 which then initiates a caspase cascade causing
cell death by apoptosis. Mitochondrial dysfunction can also occur in
death receptor-mediated apoptosis, especially so called "Type II
cells" such as hepatocytes (12). Mitochondrial cytochrome
c release is FADD/caspase 8-dependent during
death receptor-mediated apoptosis of type II cells, whereas in the
classic mitochondrial pathway of apoptosis, mitochondrial dysfunction
is independent of FADD/caspase-8 activation (13). Thus, inhibition of
FADD/caspase 8 signaling can help differentiate death
receptor-associated versus direct mitochondrial dysfunction in type II cells such as hepatocytes.
The overall objective of this study was to examine the mechanisms
contributing to Fas-independent apoptosis during bile acid cytotoxicity. To address this objective, we formulated the following questions. (i) Do toxic bile acids induce apoptosis in Fas-deficient cells? (ii) Does cytochrome c release occur in this model of
apoptosis? (iii) Is mitochondrial cytochrome c
release-dependent or -independent of the FADD/caspase 8 activation process and (iv) does bile acid modulate expression of death
receptors or their cognate ligands? To address these questions, we
generated a human cell line expressing the human bile acid transporter
to induce bile acid-mediated apoptosis. We choose the hydrophobic bile
acid glycochenodeoxycholate (GCDC) as the toxic bile salt for these
studies because it is a primary bile salt that accumulates
intrahepatically during cholestasis and is a potent inducer of
hepatocyte apoptosis (1, 16).
Generation of HuH-BAT Cell Lines and Culture
HuH-7 cells, a human hepatocellular carcinoma cell line, were
stably transfected with the sodium-dependent taurocholate
co-transporting polypeptide (Ntcp). An expression vector for the human
Ntcp (pcDNA3-Ntcp) was a generous gift from Peter Maier, Zurich,
Switzerland. HuH-7 cells were cultured until 30% subconfluent in
Eagle's minimum essential medium containing 10% fetal bovine
serum. Transfection with pcDNA3-Ntcp was performed using
LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD). Stably
transfected clones were selected in medium containing 1200 mg/ml G418.
Individual clones were subcloned and screened for bile acid transport
measurement as previously described (17). Established clones (HuH-BAT
for HuH-Bile Acid Transporting) were grown in Eagle's minimum
essential medium supplemented with 10% fetal bovine serum, 10% bovine
calf serum, 100,000 units/liter penicillin, 100 mg/liter streptomycin,
100 mg/liter gentamycin, and 200 mg/liter G418.
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 (16).
Plasmids and Transfection
Plasmids for dominant-negative (DN)-FADD-green fluorescent
protein (GFP) (pcDNA3-GFP- Subcellular Fractionation
Cytosolic extracts for the immunoblot assay of cytochrome
c were obtained as described by Leist et al.
(20). Briefly, at the desired time points, the culture medium was
exchanged with premeabilization 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
permeabilization buffer was removed and centrifuged for 10 min at
13,000 × g. Supernatants representing the cytosolic
extract were employed for the immunoblot analysis.
Measurement of Caspase-8-like Activity
Cytosolic extracts for the enzyme assay were prepared by the
method as described (17) with minor modification. In brief, cells were
homogenized in hypotonic buffer (25 mM HEPES, 5 mM MgCl2, 1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin, 2 µg/ml leupeptin, pH 7.5), and centrifuged for 10 min at 1,000 × g. Caspase-8-like activity was measured by adding 50 µl of
cytosol to 450 µl of assay buffer containing 25 mM HEPES (pH 7.5), 10 mM dithiothreitol, 0.1% CHAPS, 0.5 mM phenylmethylsulfonyl fluoride, 100 units/ml aprotinin,
and 20 µM IETD-AFC. Fluorescence was monitored using a
fluorometer (model LS50, PerkinElmer Life Sciences, Norwalk, CT) as
described previously (3, 17).
Reverse Transcriptase (RT)-PCR
Death Receptors--
Expressions of Fas, TNF receptor 1, DR4,
DR5, and DR6 in HuH-BAT cell were evaluated using RT-PCR. Total RNA was
extracted from the cells (3). cDNA was prepared using an oligo(dT)
primer and Moloney leukemia virus reverse transcriptase as
previously described in detail (3). Primers used in this experiments
were: Fas, 5'-GCGAAAGCCCATTTTTCTTCC-3' and 5'-ATTTATTGCCACTGTTTCAGG-3'; TNF receptor 1, 5'-TGTGTCTCCTGTAGTAAC TG-3' and
5'-ACGAATTCCTTCCAGCGCAA-3'; DR4, 5'-CAGAACGTCCTGGAGCCTGTAAC-3'
and 5'-ATGTCCATTGCCTGATTCTTTGTG-3'; DR5, 5'-GGGAAGAAGATTCTCCTGAGATGT
G-3' and 5'-ACATTGTCCTCAGCCCCAGGTCG-3'; DR6, 5'-ACAGAAGGCCTCGAATCTCA-3'
and 5'-TGCATTCTCGGTCAGTCAAG-3'; glyceraldehyde-3-phosphate
dehydrogenase, 5'-ACCACAGTCCATGCCATCAC-3' and
5'-TCCACCACCCTGTTGCTGTA-3'. For TRAIL receptor 2, primers flanking a
putatively retained intronic sequence were designed to yield two PCR
products differing by 87 base pairs in length according to the presence
of the intron (21).
Death Ligands
For the quantitation of TRAIL-R2/DR5 mRNA, real time PCR was
performed with the Roche LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) using SYBR green as the fluorophore (Molecular Probes, Eugene, OR). After electophoresis in 1% low melting
temperature agarose gel, gels containing the expected base pairs PCR
product bands were cut and the products were eluted into Tris-HCl using DNA elution kit (Qiagen, Valencia, CA). The TRAIL-R2/DR5 standards were
prepared at the concentrations of 109, 108,
107, 106, 105, 104, and
103 copies/µl. The linear relationship between copy
number and cycle number was then determined. The standard curve was
used to calculate the copy number in the experimental sample.
Reporter Gene Assay
HuH-BAT cells cultured in 6-well plates were co-transfected with
20 ng of TK-Renilla-CMV and 1 µg of pGL2-full, pGL2 SV-half, pGL2-SV-SmaI, or pGL2. Twelve hours after the transfection, cells were
incubated with GCDC or media (control) for 8 h and then cell lysates were prepared as previously described (22). Both firefly and
Renilla luciferase activities were quantitated using the dual luciferase reporter assay system (Promega, Madison, WI) according to
the manufacturer's instructions.
Immunoblot Analysis
Samples were resolved by 14% SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose membrane, and blotted
with appropriate primary antibodies at dilution of 1:1,000.
Peroxidase-conjugated secondary antibodies
(BIOSOURCE International, Camarillo, CA) were
incubated at a dilution of 1:2,000. Bound antibody was visualized using
chemiluminescent substrate (ECL; Amersham Pharmacia Biotech, Arlington
Heights, IL) and exposed to Kodak X-Omat film. Primary antibodies:
rabbit anti-DR4, goat anti-DR5, rabbit anti-DR6, and rabbit anti-FLIP
were obtained from Alexis. Goat anti-TNF-receptor 1, goat anti-TRAIL,
and goat anti-actin were obtained from Santa Cruz Biotechnology Inc.
(Santa Cruz, CA). Rabbit anti-DR5 was obtained from R&D systems Co.
Mouse anti-cytochrome c was obtained from Phrmingen, San
Diego, CA.
Cross-linking and Immunoprecipitation
Cells were treated with 2 mM
3,3'-dithiobis(succinimidylpropionate) (Pierce Chemical Co., Rockford,
IL), a cleavable cross-linker, for 20 min at 4 °C. After the
reaction was quenched with 50 mM Tris-HCl for 10 min at
4 °C, cells were washed with PBS. Cells were lysed for 30 min on ice
with lysis buffer (1% Triton X-100, 150 mM NaCl, 10%
gycerol, 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) and centrifuged at
15,000 × g for 15 min at 4 °C. Immunoprecipitation
was performed by incubating 2 mg of supernatant protein in 1 ml of
lysis buffer containing goat anti-DR5 antibody (Alexis Co., Lausen,
Switzerland) and 40 ml of protein-Sepharose 4B (Zymed
Laboratories Inc. Laboratories, San Francisco, CA) at 4 °C
overnight with agitation. Polypeptides were eluted by boiling for 5 min
in 2 × Laemmli sample buffer. For an analysis of DR5
oligomerization, immunoprecipitation was performed with excess and
limited amounts of antibody as described previously (3).
Immunocytochemistry
Cells were cultured in 35-mm dishes on collagen-coated glass
coverslips. For immunocytochemistry, the media was aspirated and the
cells were rinsed with phosphate-buffered saline (pH 7.2). Next, the
cells were fixed with freshly prepared 3% paraformaldehyde in PBS (pH
6.9) for 20 min at 37 °C. After the fixation, the cells were washed
three times with PBS (pH 7.2), and then incubated in PBS containing
0.1% Triton X-100 for 2 min. The dishes were rinsed in PBS, and the
cells next incubated with the primary antisera for 2 h at
37 °C. Primary antisera were rabbit anti-DR4 (Alexis, 1:300
dilution) and goat anti-DR5 (Alexis, 1:300 dilution). After three
washes with PBS, the cells were incubated with tetramethylrhodamine methyl ester-conjugated secondary antibodies (Molecular Probes Inc.,
Eugene OR), 10 µg/ml, for 45 min at 37 °C. Cells were washed again, and the coverslips mounted on glass slides and viewed by laser
scanning confocal microscopy (Axiovert 100 M-LSM 510, Carl Zeiss Inc.,
Thornwood, NY) using excitation and emission wavelengths of 555 and 580 nm, respectively.
Materials and Reagents
DAPI was from Molecular Probes Inc. (Eugene, OR). IETD-CHO and
IETD-AFC was obtained from Enzyme Systems Products (Livermore, CA).
GCDC was obtained from Sigma.
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 ANOVA for repeated measures and a post-hoc Bonferroni test to correct for
multiple comparisons.
GCDC Induces Apoptosis in the Fas-deficient HuH-BAT Cell
Line--
Because bile acid-mediated apoptosis requires transport of
bile acids into cells (3), we first developed a bile salt transporting cell line. The Fas-deficient p53 mutant, human hepatoma cell line, HuH-7, was stably transfected with the sodium-dependent
bile acid transporter (HuH-BAT). Expression of the transport protein
was verified by immunoblot analysis, and efficient
sodium-dependent bile acid uptake was confirmed by
performing uptake studies with radiolabeled taurocholate (Fig.
1A). GCDC-induced HuH-BAT cell apoptosis was concentration- and time-dependent (Fig. 1,
Band C). Maximal apoptosis was observed at 12 h at a
GCDC concentration of 200 µM. GCDC did not induce
apoptosis in the parent non-bile salt transporting HuH-7 cells.
Chenodeoxycholate (CDC) and taurochenodeoxycholate (TCDC) were also
examined for their ability to induce apoptosis in this cell line.
Following incubation with 200 µM CDC or TCDC for 12 h, HuH-BAT cell apotosis was 43.1 ± 7.9 and 23.9 ± 3.7%, respectively (data not shown). As has been reported in other studies (23), ursodeoxycholate (100 µM) did not induce apoptosis
but did inhibit GCDC-mediated apoptosis by 38% (data not shown). This latter observation helps establish the validity of this model for
studying bile acid apoptosis. Taken together, these results suggest
GCDC and other bile acids can induce a delayed apoptosis in the absence
of Fas by a mechanism requiring bile acid transport into the cell.
GCDC-mediated Cytochrome c Release and Apoptosis Is FADD and
Caspase-8 Dependent--
Mitochondrial release of cytochrome
c into the cytosol was observed in this model of
Fas-independent, bile acid-mediated apoptosis (Fig.
2). The mitochondrial release of
cytochrome c was time dependent and maximal at 4 h; the
occurrence of cytochrome c release preceded the morphologic
changes of apoptosis. Next, the effect of inhibiting the FADD/caspase
8-activation process on mitochondrial cytochrome c release
was examined to determine if a known death receptor was involved.
Transfection of HuH-BAT cells with a dominant negative FADD-GFP
construct attenuated GCDC-mediated cytochrome c release (Fig. 2A). GCDC associated cytochrome c release
was also prevented by transfection with CrmA (a small pox protein which
potently inhibits caspase 8 and caspase 1) or using the selective
tetrapeptide caspase 8 inhibitor IETD-CHO (Fig. 2B). All
three approaches of inhibiting the FADD/caspase-8 activation pathway
also markedly attenuated GCDC cytotoxicity (Fig.
3, A-C). Transfection with dominant negative FADD-GFP also inhibited GCDC-associated increases in
caspase 8-like activity demonstrating caspase 8 activation was
FADD-dependent (Fig. 3D). Collectively, these
observations support a death receptor pathway in GCDC-mediated
apoptosis despite the absence of Fas expression.
HuH-BAT Cells Express Multiple Death Receptors, but Only the Death
Ligand TRAIL--
Because GCDC-induced apoptosis of HuH-BAT cells
appeared to be death receptor-mediated, we profiled the mRNA
expression of known human death receptors and ligands in this cell line
by RT-PCR (Fig. 4). As reported by others
these cells do not express Fas (24). However, mRNA expression for
TNF-R1, TRAIL-R1/DR4, -R2/DR5, and death receptor-6 (DR6) was observed.
These cells also express TRAIL but not TNF- GCDC Enhances TRAIL-R2/DR5 Expression--
Because there was
constitutive expression of TRAIL and TRAIL-R1/DR4 and -R2/DR5 without
apoptosis, we reasoned that GCDC induced apoptosis by increasing
expression of the ligand or its receptors. Indeed, TRAIL-R2/DR5 but not
TRAIL-R1/DR4 or TRAIL increased following GCDC treatment of HuH-BAT
cells (Fig. 5). TRAIL-R2/DR5 was observed as two bands of 49 and 43 kDa
by polyacrylamide gel electrophoresis. This observation is consistent
with published data demonstrating two splice variants for TRAIL-R2/DR5
(21, 25). Both TRAIL-R2/DR5 isoforms increased following GCDC treatment
of the cells suggesting enhanced transcription was occurring as opposed
to alternations in mRNA splicing. To further examine this concept,
we evaluated TRAIL-R2/DR5 mRNA levels by quantitative real time PCR
(Fig. 6). GCDC treatment of HuH-BAT cells
increased TRAIL-R2/DR5 mRNA expression 10-fold. The increased
expression of TRAIL-R2/DR5 mRNA and protein by GCDC suggested this
death receptor may contribute to GCDC-mediated apoptosis.
To determine if GCDC increased TRAIL-R2/DR5 expression by enhancing
transcription, we next performed luciferase reporter gene assays (Fig.
7). The full genomic fragment, pGL2-full,
demonstrated basal transactivation of TRAIL-R2/DR5 in this p53 mutated
cell line. Moreover, GCDC enhanced this p53-independent transactivation of pGL2-full. The pGL2-full construct contains three high homology lesions to the p53 consensus DNA-binding sequence (18) as indicated in
Fig. 7 (BS1, BS2, and BS3). A previous study demonstrated that BS2 is
the most responsive to p53-dependent transactivation of the
TRAIL-R2/DR5 gene (18). In that study, pGL2-SV-SmaI, which contains the BS2 site, demonstrated transactivation following transfection of wild-type p53 (18). Interestingly, the transcriptional activation by GCDC was observed in both pGL2 and pGL2-SV half, but not
in pGL2-SV-SmaI. Indeed GCDC increased expression of the pGL2-SV-half
~3-fold. These results suggest GCDC increases TRAIL-R2/DR5 gene
expression, in part, by enhancing transcription.
GCDC Sensitizes HuH-BAT Cells to TRAIL--
We reasoned that if
GCDC increases TRAIL-R2/DR5 expression, it should sensitize the cells
to exogenous TRAIL. HuH-BAT cells were treated with GCDC (200 µM), FLAG-tagged TRAIL plus an anti-FLAG antibody to
induce ligand aggregation, or both GCDC plus TRAIL (Fig.
8A). Indeed, apoptosis in the
presence of GCDC plus TRAIL was greater than the sum of apoptosis in
GCDC-treated and TRAIL-exposed cells. These data demonstrate that GCDC
sensitizes cells to TRAIL presumably by up-regulating the receptor for
TRAIL-R2/DR5. Recently, the expression of FLICE inhibitory protein
(FLIP) has been implicated as an inhibitor of TRAIL-induced apoptosis
(26-29). Therefore, we next evaluated cellular expression levels of
FLIP in GCDC-treated cells (Fig. 8B). However, the
expression of FLIP was unchanged during GCDC exposure suggesting that
the sensitization to TRAIL-induced apoptosis by GCDC is not mediated by
down-regulation of FLIP, but rather by increased expression of
TRAIL-R2/DR5.
GCDC-induced Apoptosis Is Associated with TRAIL-R2/DR5, but Not
TRAIL-R1 Oligomerization--
As assessed by crystal structure
analysis, TRAIL and its cognate receptors have been shown to
oligomerize, a process necessary for transducing the death signal (8,
30, 31). Therefore, we determined if TRAIL-R2/DR5 oligomerization
occurs in GCDC-mediated apoptosis. We employed the technique of limited
antibody immunoprecipitation following treatment of the cells with a
chemical cross-linker to assess TRAIL-R2/DR5 and -R1/DR4
oligomerization. This technique has been used by us and others to
assess Fas receptor oligomerization (3, 32, 33), and is based on the
concept that oligomerized, cross-linked proteins are more efficiently
immunoprecipitated that monomers (3). Following GCDC treatment of
HuH-BAT cells for 60 min, we observed more efficient
immunoprecipitation of TRAIL-R2/DR5 than in untreated cells under
limiting antibody conditions (Fig.
9A). In contrast, TRAIL-R1 did
not appear to be oligomerized by GCDC treatment as assessed by this
assay (Fig. 9A). In addition, aggregation of TRAIL-R2/DR5
receptors was also observed using immunocytochemistry (Fig.
9B). The cellular distribution of TRAIL-R2/DR5 receptors was
diffused in untreated cells but became punctate in GCDC-treated cells
consistent with aggregation or capping of the receptor. In contrast,
TRAIL-R1/DR5 immunofluorescence remained diffuse following exposure to
GCDC (Fig. 9B). These observations are consistent with
activation of TRAIL-R2/DR5 by cytotoxic bile acids.
The principal findings of this study relate to the cellular
mechanisms of Fas-independent bile salt-mediated apoptosis. The results
demonstrate that GCDC-mediated apoptosis in a Fas-deficient cell line:
(i) is FADD and caspase 8 dependent; (ii) is associated with increased
TRAIL-R2/DR5 mRNA and protein expression without a change in FLIP
protein levels; and (iii) involves oligomerization of TRAIL-R2/DR5 but
not TRAIL-R1. The results provide new information suggesting bile acid
cytotoxicity, in the absence of Fas expression, is mediated by TRAIL
and its cognate receptor TRAIL-R2/DR5. Each of these findings will be
discussed below.
Deoxycholate (100 µM) has previously been shown to induce
apoptosis of HuH-7 cells (23). In contrast, we did not observe apoptosis by GCDC in this cell line in the absence of Ntcp. It is
widely accepted that unconjugated bile acids such as deoxycholate either diffuse across plasma membranes or are transported by a family
of transport polypeptides referred to as organic anion transporters
(34). In contrast, conjugated bile acids such as GCDC are predominantly
transported into cells by the Ntcp (34). In our experience, bile acid
transport into the cell is required to render GCDC apoptotic (35).
Likely, the differences between the previous study and our current
results relates to the bile acid employed, GCDC versus DCA,
and the different modes of transport into the cell for these two bile acids.
Previous in vitro experiments demonstrated delayed
hepatocyte apoptosis in the bile duct-ligated lpr mouse. This
observation suggested that, in addition to activating a Fas (6), bile
acids may also activate additional apoptotic pathways. However, lpr mice are not completely Fas deficient and do express minute amounts of
Fas making the above interpretation of the in vivo data
problematic. Therefore, to determine if bile acids may also activate
other apoptotic pathways, we determined if bile acid apoptosis occurred in a Fas-deficient human hepatoma cell line stably transfected with a
sodium-dependent bile acid transporter, HuH-BAT cells. This
cell line underwent apoptosis despite the absence of Fas. These
data suggest cytotoxic bile acids can activate multiple apoptotic processes.
Several observations implicate a death receptor pathway and not direct
mitochondrial cytotoxicity for bile acid-mediated apoptosis despite the absence of Fas expression. Apoptosis by GCDC was inhibited by transfection with a dominant negative FADD implying a necessary role
for FADD in the apoptotic cascade. Likewise, inhibition of caspase 8 by
transfection with CrmA or use of a selective, tetrapeptide inhibitor
also attenuated apoptosis. Although cytochrome c release was
observed following bile acid treatment, it was also prevented by
dominant negative FADD transfection and caspase 8 inhibition (Fig. 2).
These data are also consistent with a recent report showing that a
caspase 8 inhibitor also blocked mitochondrial generation of reactive
oxygen species in bile acid-treated hepatocytes (36). Thus, death
receptor-mediated signaling appears to be responsible for mitochondrial
dysfunction during bile acid cytotoxicity.
Death receptor-mediated bile acid cytotoxicity in the Fas-deficient
cell line appears to be TRAIL-mediated. Although TRAIL and TRAIL-R1/DR4
and -R2/DR5 are constitutively expressed by the cells, the cytotoxic
bile acid GCDC sensitized the cells to TRAIL cytotoxicity. This
sensitization was associated with an increased expression of
TRAIL-R2/DR5 mRNA and protein. Sensitization to TRAIL cytotoxicity
by up-regulating TRAIL-R2/DR5 has also been observed in p53
overexpression-induced apoptosis (28). These data suggest that enhanced
expression of TRAIL-R2/DR5 is a common mechanism for apoptosis
induction by injurious agents. In addition to increasing TRAIL-R2/DR5
expression, bile acids may also sensitize the cells to TRAIL killing by
altering affinity of binding proteins for the receptor complex or by
enhancing the density of death receptors in plasma membrane
microdomains. Both inhibitor and enhancing proteins for death receptor
signaling have been identified (37, 38), and it is possible that bile
acids bind to these cytoplasmic proteins influencing their activities.
Aggregation of plasma membrane receptors and their signaling in
cholesterol-rich lipid rafts has also been described (39), and bile
acids which are known to insert into lipid bilayers may increase the
density of the death receptors in lipid rafts promoting their
aggregation. The mechanisms by which bile acids sensitize cells
TRAIL-R2/DR5 apoptosis maybe multifactorial and will require further
experimental definition.
The potential hepatotoxicity of TRAIL has been controversial (40).
Recently, data have emerged suggesting that TRAIL in a reduced state
with high zinc binding is not hepatotoxic while more oxidized and less
zinc containing ligands may be hepatotoxic (40). Our data suggest the
context of the hepatocyte may also be important in determining if TRAIL
will be hepatotoxic. In cholestasis with impairment of bile acid
secretion, the retention of bile acids within the hepatocyte may
sensitize the cell to TRAIL-mediated apoptosis by enhancing
TRAIL-R2/DR5 expression. This information will be important in
selecting patients for anti-tumor therapy with this ligand as is
currently being considered.
We identified enhanced transcription for TRAIL-R2/DR5 in HuH-BAT cells
following GCDC exposure. Both p53-dependent and
-independent mechanisms for TRAIL-R2/DR5 induction have been identified
(41, 42). HuH-BAT cells are derived from the HuH-7 cell line which is
known to have a cysteine for tyrosine mutation at codon 220 (A:T Collectively, our previous and current studies demonstrate the
importance of death receptor-mediated apoptosis in bile salt cytotoxicity. These studies suggest that death receptor-mediated apoptosis should also be important in liver injury during cholestasis. However, further in vivo studies are required to evaluate
this concept. Appropriate studies employing cholestatic models of liver injury in the Bid knockout mouse or CrmA (a potent inhibitor of caspase
8) expressing transgenic animals would be useful to determine the
in vivo relevance of our in vitro observations.
Such further studies would not only provide a more complete picture of
the role of death receptors in cholestatic liver injury, but also help
evaluate BID and caspase 8 as respective therapeutic targets for
reducing liver injury during cholestasis.
The secretarial assistance of Sara Erickson
is gratefully acknowledged. We thank Harald Wajant, Stuttgart,
Germany, for providing pcDNA3-GFP- *
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.
¶
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, August 15, 2001, DOI 10.174/jbc.M105300200
The abbreviations used are:
TRAIL, tumor
necrosis factor-related apoptotis inducing ligand;
AFC, 7-amino-4-trifluoromethylcoumarin;
DAPI, 4',6-diamidino-2-phenylindole
dihydrochloride;
DR, death receptor;
FADD, Fas-associated death domain
protein;
GCDC, glycochenodeoxycholic acid;
FLIP, FLICE inhibitory
protein;
Ntcp, sodium-dependent taurocholate
co-transporting polypeptide;
PBS, phosphate-buffered saline;
RT-PCR, reverse transcriptase-polymerase chain reaction;
TRAIL-R, tumor
necrosis factor-related apoptotis inducing ligand-receptor;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
TNF, tumor necrosis factor;
CDC, chenodeoxycholate;
GFP, green fluorescent
protein;
TCDC, taurochenodeoxycholate..
The Bile Acid Glycochenodeoxycholate Induces TRAIL-Receptor
2/DR5 Expression and Apoptosis*
,,,,¶
Division of Gastroenterology and Hepatology,
Mayo Medical School, Clinic, and Foundation, Rochester, Minnesota 55905 and the § Laboratory of Molecular Oncology and Cell Cycle
Regulation, Howard Hughes Medical Institute, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania 19104
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
expression by these cells. GCDC treatment increased
expression of TRAIL-R2/DR5 mRNA and protein 10-fold while
expression of TRAIL-R1 was unchanged. Furthermore, aggregation of
TRAIL-R2/DR5, but not TRAIL-R1/DR4 was observed following GCDC
treatment of the cells. Induction of TRAIL-R2/DR5 expression and
apoptosis by bile acids provides new insights into the mechanisms of
hepatocyte apoptosis and the regulation of TRAIL-R2/DR5 expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FADD) (3) and CrmA (pCl-CrmA) (3) have
been previously described. The expression vector pc-DNA3-GFP-FADD encodes a polypeptide that contains GFP fused to the COOH-terminal death domain. This construct acts as a dominant-negative inhibitor because it lacks the NH2-terminal death effector domain of
FADD (11). The pGL2-full, pGL2 SV-half, pGL2-SV-SmaI, and pGL2 gene reporter constructs for TRAIL-R2/DR5 have been previously described (18). The TK-Renilla-CMV plasmid was purchased from Promega (Madison,
WI) and used to normalize for transfection efficiency in luciferase
assays. HuH-BAT cells were transiently transfected using LipofectAMINE
as previously described by us (19). In brief, cells grown in 3.5-cm
dishes were transfected by adding 1 ml of Opti-MEM I containing 6 µg
of LipofectAMINE (Life Technologies, Inc.), 6 µl of LipofectAMINE
Plus reagent (Life Technologies, Inc.), and each plasmid:
pcDNA3-GFP-FADD (0.5 µg) and pCl-CrmA (0.45 µg). The cells were
incubated in the above mixture for 3 h at 37 °C in a 5%
CO2, 95% air incubator. After this incubation, 1 ml
of Eagle's minimum essential medium containing 20% fetal bovine serum
was added to the transfection medium in each culture dish. Twenty-four
hours later, the medium was aspirated and replaced with 2 ml of
Eagle's minimum essential medium containing 10% fetal bovine serum.
The transfection of CrmA was performed by co-transfection with the GFP
expression plasmid (pE-GFP-N1, 0.15 µg/dish) to identify the
transfected cells under fluorescent microscopy. The transfection efficiency was ~60-70% for all plasmids as estimated by the
percentage of cells expressing GFP was visualized by fluorescence microscopy.
Expression of Fas ligand (FasL), TNF-, TRAIL
were evaluated using the following primers: FasL,
5'-ATGCAGCAGCCCTTCAATTACC-3' and 5'-CCAGTAGTGCAGTAGCTCATC-3';
TNF-, 5'-CAGAGGGAAGAGTTCCCCCAG-3' and 5'-CCTTGGTCTGGTAGGAGACG-3';
TRAIL, 5'-AGACCTGCGTGCTGATCGTG-3' and 5'-TTATTTTGCGGCCCAGAGCC-3'. Total
cellular RNA was isolated using Trizol reagent (Life Technologies,
Inc.). After reverse transcription as previously described (3),
the cDNA product was amplified by PCR with Taq DNA
polymerase using standard protocols (3). The amplified products (8 µl) were separated on 1% agarose gels, stained with ethidium
bromide, and photographed using ultraviolet illumination.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
GCDC-induces apoptosis in HuH-BAT cells.
A, the Fas-deficient human hepatoma cell line,
HuH7, was stably transfected with the sodium-dependent bile
salt transporter (Ntcp) generating the HuH-bile acid transporting
(HuH-BAT) cell line. Ntcp expression was verified by immunoblot
analysis (inset). Kinetic analysis demonstrated linear,
sodium-dependent bile acid uptake in the transfected but
not the parent cell line. Bile acid uptake was assessed using
radiolabeled taurocholate. B, apoptosis was observed in the
bile acid transporting HuH-BAT cell line but not in the parent HuH7
cells after 12 h of incubation with GCDC (50-200
µM). Apoptosis was quantitated using DAPI and fluorescent
microscopy. C, HuH-BAT cells were incubated in the presence
of GCDC (200 µM) or diluent (media) for 12 h and
apoptosis quantitated over time. All data were expressed as mean ± S.D. from three individual experiments.
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Fig. 2.
GCDC-induced mitochondrial cytochrome
c is FADD and caspase 8-dependent.
A, immunoblot analysis of cytosolic extracts from HuH-BAT
cells was performed using anti-cytochrome c mouse monoclonal
antibody after treatment with media (control) and GCDC (200 µM). The peak cytochrome c release was
observed after 4 h of incubation. B, HuH-BAT
cells in 10-cm plates were transfected with dominant negative (DN)-FADD
or CrmA expressing plasmids (5 µg of DNA/each plate). Transfected or
untransfected cells were incubated with 200 µM GCDC for
4 h. For the far right panel, untransfected cells were
treated with the selective caspase 8 inhibitor IETD-CHO (50 µM).
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Fig. 3.
FADD and caspase 8 mediate GCDC-induced
apoptosis in HuH-BAT cells. A, HuH-BAT cells in 3.5-cm
dishes were transfected with DN-FADD-GFP expressing plasmid DNA (0.5 µg of DNA/dish). Equivalent amounts of GFP expressing plasmid was
used as a control for the DN-FADD-GFP transfection. Cells were used for
experiments 48 h after transfection. Apoptosis was assessed after
treatment with GCDC (200 µM, 12 h) or media using
DAPI loading and fluorescent microscopy. Apoptotic cells are expressed
as a percentage of total GFP-expressing cells. p < 0.01 for GCDC versus control, DN-FADD, or DN-FADD plus GCDC
by ANOVA. B, HuH-BAT cells in 3.5-cm dishes were
co-transfected with CrmA (0.45 µg of DNA/dish) and GFP (0.15 µg of
DNA/dish). Equivalent amounts of GFP expressing plasmid was used as a
control for co-transfection. Cells were used for experiments 48 h
after transfection. The number of apoptotic cells are expressed as a
percentage of total GFP-expressing cells. p < 0.01 for
GCDC versus control, CrmA, or CrmA plus GCDC by ANOVA.
C, GCDC-induced apoptosis was evaluated in the presence or
absence of the selective tetrapeptide caspase-8 inhibitor, IETD-CHO, 50 µM. p < 0.01 for GCDC versus
control, IETD or IETD plus GCDC by ANOVA. D, caspase-8
activity was measured in cell lysates using the fluorogenic substrate,
IETD-AMC. p < 0.01 for GCDC versus control,
DN-FADD, or DN-FADD plus GCDC by ANOVA. Data were expressed as
mean ± S.D. from three (A, B, and C) or
four (D) individual experiments.
or Fas ligand. To
confirm this mRNA expression profile at the protein level,
immunoblot analysis was performed (Fig.
5). Protein expression for all mRNA
transcripts was observed. Although multiple death receptors are
expressed in these cells, the only death ligand detected was TRAIL.
Thus, these observations suggest TRAIL and one of its cognate receptors
contributes to GCDC cytotoxicity in the absence of Fas.
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Fig. 4.
HuH-BAT cells express multiple death
receptors and ligands. Reverse transcriptase-PCR analysis of death
receptors and their ligands. HuH-BAT cells were cultured for 4 h
in the presence or absence of 200 µM GCDC. Total RNA was
isolated from HuH-7 cells (lane 2), HuH-BAT cells
(lane 3), and GCDC-treated HuH-BAT cells (lane
4), and subsequent RT-PCR was performed using Fas, TNF receptor 1, TRAIL receptor 1, TRAIL receptor 2, DR6, Fas ligand, TRAIL (ligand),
TNF- , and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). Lane 1 is a positive control for each
PCR. SW480 cells, a human colon cancer cell line, were used as a
positive control for Fas, TNF receptor 1, TRAIL receptor 1, TRAIL
receptor 2, and DR6. LNCaP cells, a human prostate cancer cell line,
were used as a positive control for Fas ligand and TNF-. Col6(7)
cells, a human melanoma cells, were used as a positive control for
TRAIL.
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Fig. 5.
GCDC increases DR5, but DR4 protein
expression. HuH-BAT cells were incubated in the presence of GCDC
(200 µM) or media (Control). Cells were lysed
after 1, 2, 4, and 8 h incubation, and equivalent amounts of
protein were immunoblotted with anti DR4, anti-DR5, anti-TNF-R1, and
anti-DR6 antisera (Panel A). Immunblot analysis was also
performed using anti-TRAIL antisera (Panel B). Immunblot
with anti-actin antisera was performed as a control for protein
loading.
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Fig. 6.
GCDC up-regulates DR5 mRNA. HuH-BAT
cells were incubated for 0, 1, 2, 4, and 6 h in the presence of
GCDC (200 µM). Total cellular RNA was isolated and
reverse transcription was performed. cDNA from reverse
transcription were amplified in the PCR reaction mixture in the
presence of cyber green. Cyber green fluorescence was continuously
monitored using the LightCycler as described under "Experimental
Procedures." A, the standard cDNA samples containing
109, 108, 107, 106,
105, 104, and 103 copies of PCR
products were amplified using the primer for DR5, and the cycle number
of each fluorescence curve during the initial amplification phase was
determined (left panel). The cycle numbers and the copy
numbers were plotted (right panel) to generate a standard
curve. B, the fluorescence intensities of cDNA samples
derived from HuH-BAT cells were monitored, and the copy number of each
sample was calculated using the equation obtained from the left panel
of A. Equivalent loading of cDNA samples was
evaluated by performing the same quantitative real time PCR using
primers for 18 S RNA.
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Fig. 7.
GCDC activates DR5 promoter activity.
A, schematic description of plasmids used in luciferase
reporter assay. B, HuH-BAT cells were co-transfected with 20 ng of TK-Renilla-CMV and 1 µg of indicated reporter plasmids. Twelve
hours after the transfection, cells were incubated with GCDC or media
(control) for 8 h. Both firefly and Renilla luciferase activities
were quantitated and data were expressed as the ratio of firefly
luciferase activity/Renilla luciferase activity. All data were
expressed as mean ± S.D. from four individual experiments.
SV, SV40 basal promoter.
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Fig. 8.
GCDC sensitizes cells to apoptosis by
exogenous TRAIL. A, HuH-BAT cells were cultured in
96-well plates. FLAG-tagged TRAIL (400 ng/ml) and anti-FLAG antibody M2
(2 µg/ml) were added to the culture media. Anti-FLAG antibody causes
cross-linking of TRAIL leading to a maximum activity of TRAIL. Cells
were incubated with FLAG-TRAIL plus M2 in the presence or absence of
GCDC (200 µM) for 12 h. Apoptosis was evaluated by
DAPI staining and fluorescent microscopy as described under
"Experimental Procedures." Data were expressed as mean ± S.D.
from three individual experiments. B, HuH-BAT cells were
incubated in the presence of GCDC (200 µM) or media
(Control). Cells were lysed after 1, 2, 4, and 8 h
incubation, and equivalent amounts of protein were immunoblotted with
anti-FLIP antisera. Immunblot for with anti-actin antisera was
performed as a control for protein loading.
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Fig. 9.
GCDC treatment of HuH-BAT cells results in
DR5, but not DR4 aggregation. A, untreated (Control)
and GCDC (200 µM, 4 h)-treated HuH-BAT cells were
incubated with the cleavable cross-linking agent
3,3'-dithiobis(succinimidylpropionate) and lysed for
immunoprecipitation using limiting (1 µg/ml) or excess (10 µg/ml)
amounts of anti-DR5 or DR4 antisera. Western blot analysis of the
immunoprecipitate was performed as described under "Experimental
Procedures." B, control and GCDC (200 µM,
4 h)-treated cells were were fixed and innunocytochemistry
performed with anti-DR4/TRAIL-R1 and anti-DR5/TRAIL-R2 antisera.
Immunofluorescence was granular and cytoplasmic for DR4 in both treated
and untreated cell. Although DR5 immunofluorescence was granular in
untreated cells it became punctate consistent with receptor aggregation
in treated cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G:C) (43). Mutations in this region of the gene are known to disrupt
p53 function (43). Because the HuH-BAT cells have a mutant p53, bile
acid induction of TRAIL-R2/DR5 would appear to be p53 independent. Bile
acids have been shown to modulate gene expression by binding to the
nuclear hormone receptor FXR (44-46). In addition, bile acids can also
activate NF-
B (22), a transcription factor, and the JNK signal
transduction cascade which can result in transcriptionally active AP1
complexes (47). The role of these different pathways in regulating
TRAIL-R2/DR5 induction by bile acids will require further study. These
studies may, however, be important for many fields of biology. For
example, induction of TRAIL-R2/DR5 may promote apoptosis of cancer
cells by chemotherapeutic agents. If bile acid activation of the FXR nuclear hormone receptor is shown to induce TRAIL-R2/DR5, agonists for
this receptor (e.g. GW4064) could be employed in the
treatment of cancer (48).
ACKNOWLEDGEMENTS
FADD.
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Maloney, P. R.,
Willson, T. M.,
and Kliewer, S. A.
(2000)
Mol. Cell
6,
517-526[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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