Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota 55905
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ABSTRACT |
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Toxic bile salts induce hepatocyte apoptosis by a Fas-dependent, Fas ligand-independent mechanism. To account for this observation, we formulated the hypothesis that toxic bile salts induce apoptosis by effecting translocation of cytoplasmic Fas to the cell surface, resulting in transduction of Fas death signals. In McNtcp.24 cells the majority of Fas was cytoplasmic, as assessed by cell fractionation and immunofluorescence studies. However, cell surface Fas increased sixfold after treatment with the toxic bile salt glycochenodeoxycholate (GCDC) in the absence of increased Fas protein expression. Moreover, in cells transfected with Fas-green fluorescence protein, cell surface fluorescence also increased in GCDC-treated cells, directly demonstrating Fas translocation to the plasma membrane. Both brefeldin A, a Golgi-disrupting agent, and nocodazole, a microtubule inhibitor, prevented the GCDC-induced increase in cell surface Fas and apoptosis. In conclusion, toxic bile salts appear to induce apoptosis by promoting cytoplasmic transport of Fas to the cell surface by a Golgi- and microtubule-dependent pathway.
brefeldin A; Fas-green fluorescence protein; JO-2 antisera; nocodazole; vesicle trafficking
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INTRODUCTION |
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CHOLESTASIS, DEFINED AS AN impairment in bile formation, is a feature of many chronic human liver diseases (28). The impairment in bile formation results in the retention of normal bile constituents within the liver, including high concentrations of bile salts (11). The retention and accumulation of bile salts within the liver during cholestasis is thought to exacerbate and promote liver injury (17, 23). Indeed, the pathophysiological importance of bile salt-induced liver injury is demonstrated in children with subtype 2 of the progressive familial intrahepatic cholestasis syndromes (26). These children have mutations in the cannalicular transport protein for bile salt secretion into bile and develop progressive liver disease because of the inability to excrete bile salts from the hepatocyte (26). The cellular and molecular mechanisms of bile salt-induced hepatocyte injury are, therefore, of clinical and scientific importance.
We have recently demonstrated that toxic bile salts induce hepatocyte apoptosis by a mechanism dependent on the Fas death receptor (10). The Fas receptor is a member of a growing family of death receptors that when aggregated or oligomerized signal a caspase protease-mediated death cascade culminating in apoptosis (9). Although Fas receptor activation is usually triggered by engaging the Fas ligand, in our studies the toxic bile salt glycochenodeoxycholate (GCDC) induced Fas oligomerization in the absence of Fas ligand. Furthermore, hepatocyte apoptosis was also observed in the bile duct-ligated, Fas ligand-deficient gld mouse (20). These observations suggest that a ligand-independent mechanism of Fas-mediated liver injury occurs during cholestasis. This is a potential new mechanism for cholestatic and toxin-induced liver injury.
Fas receptor oligomerization independent of ligand has been observed when Fas is overexpressed, when keratinocytes are exposed to ultraviolet light, and during drug-induced apoptosis by ganciclovir (1, 4, 21). Ganciclovir-induced apoptosis is mediated by a redistribution of cytoplasmic Fas to the cell surface (4). These observations suggest that one mechanism for the post-transcriptional regulation of Fas receptors is to sequester this death receptor within intracellular pools. The receptors can then be shuttled to the plasma membrane, presumably by a vesicular transport pathway, to initiate cell death signals. An increase in the cell surface density of Fas receptors likely promotes their aggregation, causing apoptosis. On the basis of this information, we formulated the hypothesis that bile salt-mediated, Fas-dependent apoptosis is caused by trafficking of preexisting cytoplasmic Fas receptors to the plasma membrane. To test this hypothesis, we sought to answer the following specific questions: 1) What is the cell surface vs. cytoplasmic distribution of Fas? 2) Does an increase in cell surface Fas occur during exposure to toxic bile salts? 3) Does inhibition of Fas translocation attenuate bile salt-mediated apoptosis? and 4) Are bile salt-treated hepatocytes more sensitive to Fas-stimulated apoptosis? We chose GCDC as the toxic bile salt for these studies because it is a primary bile salt whose concentrations increase during cholestasis.
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EXPERIMENTAL PROCEDURES |
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Cell culture. McNtcp.24 cells, a hepatocyte-derived cell line that stably expresses the sodium-dependent taurocholate-cotransporting polypeptide and efficiently transports bile salts, were cultured in DMEM containing 10% fetal bovine serum and 10% calf serum (22). We have previously shown that this cell line undergoes bile salt-mediated apoptosis with the same kinetics and at the same concentrations as primary rat hepatocytes (22). We have also demonstrated that this cell line does not express Fas ligand as assessed by PCR (10). For the cell fractionation experiments, mouse hepatocytes were employed. Mouse hepatocytes were isolated and cultured as previously described (10). p53 knockout mice were obtained from Jackson Laboratories (Bar Harbor, ME).
Immunofluorescence. Quantitative immunofluorescence was performed using a digitized fluorescence microscopy system employing a Zeiss Axiovert 35M microscope (Carl Zeiss, Thornwood, NY) and the Metafluor imaging system (Universal Imaging, West Chester PA), which has been previously described in detail (2). Briefly, McNtcp.24 cells were cultured on collagen-coated glass coverslips in 30-mm dishes. The cells were fixed in formaldehyde (3% formaldehyde, 0.1 M PIPES, 1.0 mM EGTA, and 1.0 mM MgSO4, pH 7.2) for 30 min, blocked for 30 min (blocking buffer 3 mM KH2PO4, 7 mM K2HPO4, 5% goat serum, 5% glycerol, and 0.04% NaN3, pH 7.2), and incubated with the polyclonal rabbit anti-Fas antibody at 1:100 dilution in phosphate buffer (3 mM KH2PO4 and 7 mM K2HPO4, pH 7.2). For measurements of total cellular Fas, cells were permeabilized by incubation in PBS containing 0.1% saponin for 30 min at room temperature. For measurements of both cell surface and total cellular Fas, cells were then washed with phosphate buffer and incubated with Alexa 488 goat anti-rabbit antisera at a dilution of 1:2,000 in phosphate buffer for 30 min, washed with phosphate buffer and then distilled water, air dried, and mounted in Permount. Individual cells were imaged at fluorescein wavelengths. Cellular fluorescence was quantitated by multiplying the average fluorescence intensity in the cell by the number of pixels above background using a threshold of zero.
Fractionation of mouse hepatocytes. Mouse hepatocytes were fractionated into plasma membrane and cytoplasmic fractions using a modification of the method of Berman et al. (6). Cells (0.5 × 106/ml with 10 ml/dish) were cultured on 100-mm polystyrene plates overnight. Cultured cells were scraped off the plates using a cell lifter in PBS (137 mM NaCl, 2 mM KCl, 8 mM Na2HPO4, and 1 mM KH2PO4), collected by centrifugation (50 g for 2 min), and homogenized in SIE buffer (0.25 M sucrose, 3.0 mM imidazole, 0.1% ethanol) at 4°C. The pellet was homogenized in a Teflon glass Potter-Elvehjem homogenizer (Curtin Matheson Scientific, Houston, TX) with a Wheaton Teflon pestle for 10 strokes and then centrifuged at 150 g for 10 min. The pellet was then homogenized in a Dounce homogenizer by hand with a tight-fitting pestle for 25 strokes, and the previous supernatant from the motorized homogenization and the current homogenate from the hand homogenization were centrifuged at 150 g for 10 min twice, discarding the pellet each time. The resultant supernatant was centrifuged at 2,000 g for 20 min. The pellet was resuspended in SIE buffer and layered atop a discontinuous sucrose gradient consisting of 45%, 41.5%, and 37% sucrose in 0.5 mM CaCl2 and 5.0 mM Tris · HCl. This was centrifuged in a Beckman sw-28 rotor at 90,000 g for 2 h. The resultant band (plasma membrane fraction) at the 37%-41.5% interface was removed, diluted with PBS, and centrifuged in a Beckman TI-70 rotor at 250,000 g for 1 h. The resultant pellet consisting of purified plasma membrane sheets was brought up in PBS.
Immunoblot analysis for Fas receptor in cell fractions. After SDS-PAGE electrophoresis of cell proteins using a 12% polyacrylamide precast minigels (Bio-Rad, Hercules, CA), proteins were transferred to a Nitrobind nitrocellulose membrane (Micron Separation, Westboro, MA) by electroblotting. The membrane was then rinsed briefly with 20 mM Tris-0.5 M NaCl, pH 7.0 (TBS), blocked with 5% wt/vol skim milk in TBS-0.05% Tween 20, pH 7.0 (TTBS) for 30 min at room temperature to prevent nonspecific binding, and then incubated for 60 min with a 1:1,000 dilution of polyclonal rabbit anti-Fas antisera. Membranes were washed three times in TTBS for 10 min each at room temperature and then incubated for 60 min at room temperature with a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. After three washes in TTBS for 10 min each at room temperature followed by a single wash in TBS for 10 min at room temperature, the blots were developed with the enhanced chemiluminescent substrate (Amersham, Arlington Heights, IL) and exposed to Kodak Biomax film.
Fas-GFP and confocal microscopy. Primers containing additional restriction site sequences (Bgl II in the sense and Sac II in the antisense) were made from the murine Fas sequence obtained from GenBank (accession no. E05335). The sense primer for Fas was 5'-AGATCTATGCTGTGGATCTGGGCT-3', and the reverse primer was 5'-CCGCGGCTCCAGACATTGTCCTTC-3'. Mouse liver cDNA was amplified using PCR buffer (20 mM Tris, pH 8.4, and 50 mM KCl), 1.5 mM MgCl, 200 µM dNTPs, 0.4 µM sense primer, 0.4 µM reverse primer, and 2.5 U Taq polymerase. Samples were heated to 94°C for 2 min and then amplified using the MJ Research (Watertown, MA) PTC 200 Peltier thermal cycler for 30 cycles with the following times: 94°C for 1 min, 59.8°C for 1 min, and 72°C for 1 min. The samples were then placed at 72°C for 10 min. The PCR product was then cloned into Promega's pGEM T-vector following the manufacturer's instructions. The PCR product was cut out of the PCR vector with Bgl II and Sac II restriction enzymes and subcloned into Clontech's pEGFP-N1 vector that had been cut with the same enzymes. All PCR products were confirmed as the appropriate product by sequencing using dye terminator technology. McNtcp.24 cells were transfected as previously described with the Fas-green fluorescence protein (GFP) construct (16). GFP fluorescence was imaged by confocal microscopy as previously described in detail (10, 22).
Quantification of apoptosis. We quantitated apoptosis as previously described in detail (10) by assessing the nuclear changes of apoptosis using the nuclear binding dye 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) and fluorescence microscopy.
Bile acid uptake. Steady-state bile acid uptake by McNtcp.24 cells was quantitated as we have previously described in detail (31). Tritiated taurocholate (New England Nuclear, Boston, MA) was employed for these studies.
Statistical analysis. All data represent at least three experiments using cells, tissue, or extract from a minimum of three separate isolations and are expressed as means ± SE unless otherwise indicated. Differences between groups were compared using an ANOVA for repeated measures and a post hoc Bonferroni test for multiple comparisons. All statistical analyses were performed with the statistical software package InStat from GraphPAD (San Diego, CA).
Materials. DMEM media was purchased from Bio Whittaker (Walkersville, MD). Anti-Fas polyclonal antibody M-20 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit horseradish peroxidase was obtained from BioSource International (Camarillo, CA). Goat anti-rabbit Alexa 488 was obtained from Molecular Probes (Eugene, OR). Bovine serum and fetal calf serum were purchased from Summit Biotechnology (Ft. Collins, CO). Enhanced chemiluminescence Western blot detection kit was obtained from Amersham Pharmacia (Piscataway, NJ). JO-2 antisera was purchased from PharMingen (San Diego, CA). All other chemicals were purchased from Sigma (St. Louis, MO). Brefeldin A was in a stock solution of 5 mg/ml in DMSO and nocodazole 4 mM in dH2O.
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RESULTS |
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What is the cytoplasmic vs. plasma membrane distribution of Fas?
The cellular distribution of Fas, cytoplasmic vs. plasma membrane, was
assessed using cell fractionation studies in mouse hepatocytes and
quantitative fluorescence microscopy in McNtcp.24 cells (Fig.
1). Cell fractionation studies (Fig.
1A) demonstrated that the majority of total cellular Fas in
mouse hepatocytes was cytoplasmic and not associated with the plasma
membrane (~71% of Fas was cytoplasmic vs. 29% in the plasma
membrane). Because of the limitations of cell fractionation studies,
including contamination of fractions, redistribution of proteins during
the fractionation procedure, and proteolysis, we confirmed these
results using quantitative fluorescence microscopy (Fig. 1B).
Fas-associated immunofluorescence was measured in permeabilized cells
(total cell Fas) and nonpermeabilized cells (cell surface Fas). These
studies also demonstrated that Fas was predominantly cytoplasmic (54%
in the cytoplasm). Thus in McNtcp.24 cells and mouse hepatocytes, the
majority of Fas is sequestered within intracellular compartments.
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Does an increase in cell surface Fas occur during GCDC treatment?
We next determined whether GCDC-mediated apoptosis was associated with
an increase in cell surface Fas. The increase in cell surface Fas was
initially determined by cell fractionation studies (Fig. 1). An
approximately sixfold increase in plasma membrane Fas was identified
after treatment of McNtcp.24 cells with 50 µM GCDC for 1 h. We chose
to confirm this observation by combining cell immunofluorescence with
quantitative fluorescence microscopy in which we could visualize the
cells' surface fluorescence directly (Fig.
2). Indeed, a sixfold increase in cell
surface fluorescence was also observed using this approach. The
increase in cell surface Fas could not be explained by an increase in
Fas protein expression; immunoblot analysis of whole cell lysates
following treatment with GCDC for 4 h identified no significant
increase in total cellular Fas compared with untreated cells (Fig.
3). Furthermore, actinomycin D (200 µM),
an inhibitor of transcription, did not block the increase in cell
surface Fas (data not shown).
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Does inhibition of Fas translocation attenuate bile salt-mediated
apoptosis?
Fas has been reported to be shuttled to the cell surface in fibroblasts
by a protein secretory pathway involving the Golgi apparatus (5).
Brefeldin A blocks Golgi-dependent protein secretion (7). Therefore, we
postulated that brefeldin A would block the GCDC-mediated increase in
cell surface Fas and apoptosis. Incubation of McNtcp.24 cells with
brefeldin A (10 µg/ml) plus GCDC (50 µM) prevented the increase in
cell surface Fas (Fig. 5). Moreover,
brefeldin A also blocked GCDC-induced apoptosis (Fig. 5).
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What is the mechanism for bile acid-associated increase in cell
surface trafficking of Fas?
Although p53 has been suggested to mediate Fas trafficking (5), bile
acid-associated Fas trafficking pathway was not p53 dependent (Fig.
7). Indeed, rates of GCDC-mediated
apoptosis and increases in Fas-associated immunofluorescence were
virtually identical in hepatocytes obtained from wild-type and p53
knockout mice.
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Does GCDC render cells more sensitive to apoptosis by the JO-2 Fas
agonistic antisera?
We reasoned that by increasing cell surface expression of Fas,
GCDC-treated cells may become more susceptible to apoptosis induced by
Fas agonists. Indeed, apoptosis by the JO-2 Fas agonistic antisera was
significantly increased in GCDC-treated cells (Fig. 9). The ability of GCDC to sensitize
hepatocytes to apoptosis by Fas stimulation was inhibited by both
brefeldin A and nocodazole (Fig. 9), an observation suggesting that Fas
trafficking from the cell interior to the surface was necessary for
GCDC sensitization of hepatocytes to Fas-stimulated apoptosis. These
data demonstrate that the increased cell surface expression of Fas
observed in GCDC-treated cells is functional in regard to transducing
death signals.
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DISCUSSION |
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The principal findings of this study relate to the cellular mechanisms of Fas-dependent, Fas ligand-independent apoptosis by toxic bile salts. The results demonstrate that 1) Fas is predominantly intracellular in hepatocytes; 2) GCDC cytotoxicity is associated with an increase in cell surface Fas; 3) inhibition of protein trafficking with brefeldin A, nocodazole, or chelerythrine attenuates the increase in cell surface Fas and apoptosis during treatment with GCDC; and 4) and GCDC-treated hepatocytes are sensitized to cell death by Fas agonistic antisera. Toxic bile salts appear to effect a Fas-dependent apoptotic cascade by a process requiring an increase in the cell surface density of this death receptor.
Because of the propensity for the death domains of death receptors to spontaneously associate (3), the density of cell death receptors on the plasma membrane must be tightly regulated by the cell. One post-transcriptional mechanism for regulating the plasma membrane density of surface receptors is to sequester receptors within intracellular pools. The receptors can then be shuttled to the plasma membrane by appropriate stimuli in a regulated fashion. Consistent with these concepts, the death receptors Fas and tumor necrosis factor receptor-1 can be largely intracellular, sequestered within the Golgi complex (5, 14). Recently, it has been demonstrated that ganciclovir- and transcription-independent, p53-mediated apoptosis are mediated by a redistribution of cytoplasmic Fas to the cell surface (5). In our current study, we found an increase in cell surface Fas during treatment by toxic concentrations of GCDC. The increase in cell surface Fas was blocked by the protein secretion inhibitor brefeldin A and the microtubule poison nocodazole. These data suggest that bile salts are able to effect shuttling of Fas death receptors from intracellular stores utilizing a classic protein secretion pathway.
Activation of PKC appears to contribute to the shuttling of intracellular Fas to the plasma membrane. Indeed, we have shown that the PKC inhibitor chelerythrine blocks the increase in cell surface Fas and bile salt-induced apoptosis. These observations mechanistically link our previously published results showing attenuation of bile salt cytotoxicity by PKC inhibitors to our current observations demonstrating the role of Fas in bile salt-mediated apoptosis (15). However, we cannot exclude additional mechanisms of bile salt stimulation of Fas trafficking to the plasma membrane. Bile salts may also perturb Golgi signaling. Morphological studies employing radiolabeled bile salts, a fluorine-containing bile salt analog, and fluorescently labeled glycocholate have all demonstrated bile salt localization with the Golgi apparatus (8, 18, 27). Association of toxic bile salts with the Golgi network may potentially initiate intracellular stress signals, resulting in the vesicular trafficking of Fas to the plasma membrane.
We have previously demonstrated Fas oligomerization independent of Fas
ligand by GCDC treatment of hepatocytes (10). The results of this study
suggest that an increase in cell surface Fas is required for this
oligomerization event. However, it is unclear whether an increase in
cell surface density of Fas receptors alone is sufficient for their
spontaneous oligomerization or if other additional processes are
involved in this model of toxin-induced apoptosis. Interestingly, a
protein inhibitor of tumor necrosis factor receptor-1 oligomerization
has been identified and referred to as silencer of death domain (SODD)
(13). Because of the similarities in signaling by death
domain-containing receptors, it is likely that a similar protein may
exist for Fas. Bile salts could also potentially facilitate Fas
oligomerization by binding to such inhibitory proteins and disabling
their binding to Fas. Toxic bile salts may also facilitate binding of
Fas-associated death domain protein (FADD) or the recently described
FADD-like interleukin-1-converting enzyme (FLICE)-associated huge
protein (FLASH) to plasma membrane-associated Fas (12).
Direct measurements of bile salts on binding of the death-inducing
signaling complex proteins to the Fas receptor will be needed to
address these questions. Finally, we cannot exclude the existence of an
undiscovered Fas-binding ligand in this process.
Our observations may have relevance to the clinical observation that colchicine, an agent that depolymerizes microtubules, is salutary in human cholestatic liver diseases (29). Perhaps colchicine is beneficial by preventing Fas from translocation to the plasma membrane by inhibiting microtubule transport in these liver diseases. We were unable to directly test colchicine in our in vitro model because it unexpectedly induced cytotoxicity in the McNtcp.24 cell line. However, our data with nocodazole, also a microtubule-depolymerizing agent, support this concept.
In summary, the data in the current study suggest that toxic bile salts cause cell death, in part, by effecting translocation of Fas from the cytoplasm to the cell surface. The shuttling of Fas and the resulting apoptosis can be inhibited by the Golgi-disrupting agent brefeldin A and the microtubule poison nocodazole. By inference, a Golgi-associated and microtubule-dependent pathway appears to be involved in the trafficking of Fas to the cell surface during bile salt cytotoxicity. The increase in density of cell surface Fas receptors promotes their oligomerization, initiating a caspase-dependent death-signaling pathway (10). These results provide a new model for toxin-induced liver injury, namely a Fas-dependent but Fas ligand-independent mechanism for liver injury. The implications of these results for potential therapy of cholestatic liver diseases are currently under investigation.
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ACKNOWLEDGEMENTS |
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The secretarial assistance of Sara Erickson is gratefully acknowledged.
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FOOTNOTES |
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This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-41876), the Gainey Foundation, St. Paul, MN, and by the Mayo Foundation, Rochester, MN.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. J. Gores, Mayo Medical School, Clinic, and Foundation, 200 First St. SW, Rochester, Minnesota 55905 (E-mail: gores.gregory{at}mayo.edu).
Received 21 September 1999; accepted in final form 22 January 2000.
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