Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905
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ABSTRACT |
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Cholangiocytes are the target of a group of liver diseases termed the cholangiopathies that include conditions characterized by periductal inflammation and cholangiocyte apoptosis. Because inflammation is associated with oxidative stress, we developed the hypothesis that cholangiocytes exposed to oxidative stress will be depleted of endogenous cytoprotective molecules, leading to cholangiocyte apoptosis. To begin to test this hypothesis, we explored the relationships among glutathione (GSH) depletion, expression of Bcl-2 (a protooncogene that inhibits apoptosis), and apoptosis in a nonmalignant human cholangiocyte cell line. Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using buthionine sulfoximine (BSO). Bcl-2 expression was assessed by quantitative immunoblot analysis, and apoptosis quantified by fluorescence microscopy using the DNA binding dye 4',6'-diamidino-2-phenylindole. Bcl-2 message was assessed by RNase protection assay, and Bcl-2 protein synthesis and half-life by pulse-chase analysis. Exposure of human cholangiocytes in culture to BSO reduced GSH levels by 93 ± 3% (P < 0.01). In addition, treatment of cholangiocytes with BSO reduced Bcl-2 levels by 87 ± 2% (P < 0.01) and was associated with a time-dependent increase in the number of cells undergoing apoptosis; ~11 ± 1% of cultured cells demonstrated morphological changes of apoptosis by 72 h compared with 1.5 ± 0.1% in untreated cholangiocytes (P < 0.01). Maintenance of GSH levels by addition of glutathione ethyl ester in the presence of BSO blocked the BSO-associated increase in apoptosis in BSO-treated cholangiocytes and also prevented the decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady-state levels of bcl-2 mRNA or Bcl-2 protein synthesis. However, Bcl-2 protein half-life decreased 57% in BSO-treated vs. untreated cells. Our results using a human cholangiocyte cell line demonstrate that reduction in the cellular levels of an antioxidant such as GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis, an observation that may be important in the pathogenesis of the inflammatory cholangiopathies.
cholangiopathies; ductopenia; glutathione ethyl ester
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INTRODUCTION |
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THE INFLAMMATORY cholangiopathies are a group of liver diseases in which the bile duct epithelial cell, or cholangiocyte, is the target of a variety of pathological insults. In several of these conditions, cholangiocyte death by apoptosis has been demonstrated (4, 5, 25, 28, 37). Thus, although cholangiocyte apoptosis is known to occur in these conditions, the mechanism of programmed cell death is unknown.
The pathophysiology of inflammatory cholangiopathies is likely
multifactorial. In the inflammatory cholangiopathies, chronic inflammation could potentially cause cholangiocyte apoptosis by at
least three mechanisms: 1) through
cytotoxic T-lymphocyte induction of apoptosis via the Fas receptor-Fas
ligand system, or the perforin-granzyme B system (23, 24, 27);
2) through cytokines that are known to cause apoptosis, such as tumor necrosis factor- (TNF-
) and interferon-
(7, 32, 33, 49); and
3) through oxidative stress, perhaps
by decreasing glutathione (GSH), the major antioxidant in hepatic
epithelia (11, 21, 26). In particular, oxidative stress is
a common component of inflammation because of the generation of toxic
oxygen species by local inflammatory cells (i.e., monocytes and
neutrophils) and promotion of radical formation by cytokines (i.e.,
TNF; 15, 31, 38, 43, 46, 51). Although much has been learned about the
mechanisms of cytotoxic T-cell- and cytokine-induced apoptosis, the
mechanisms of oxidative stress-mediated apoptosis remain poorly
defined. Because the mechanisms of oxidative stress-induced apoptosis
appear to be germane to the inflammatory component of the
cholangiopathies, a better understanding of cholangiocyte apoptosis
resulting from oxidative stress would be of clinical and scientific
importance.
Oxidative stress is characterized by the generation of numerous toxic
oxygen species, including the superoxide anion
(O2), H2O2,
· OH, and singlet oxygen (47). The main defense against oxidative stress is lipid soluble antioxidants, e.g., vitamin E,
-carotene, etc., and the water soluble antioxidant GSH. Oxidative stress is usually accompanied by cellular GSH depletion (50). Large
amounts of GSH are secreted into bile (almost one-half of the GSH
released by the adult liver is secreted into bile), raising the
possibility that biliary and cholangiocyte GSH is an important defense
mechanism for these cells. Indeed, liver tissue GSH levels are markedly
reduced in one of the human inflammatory cholangiopathies, primary
biliary cirrhosis (14). Of relevance, the cellular GSH status has been
found to be important in modulating apoptosis in other cell types (3,
26, 44, 52). The importance of GSH depletion in promoting apoptosis has
been amply demonstrated in that apoptosis is prevented by replenishing
intracellular GSH concentrations in several models of apoptosis (3, 8,
20, 44, 52). However, GSH depletion alone may not be sufficient to
induce apoptosis. Changes in GSH appear to affect apoptosis by
regulating protein expression of members of the Bcl-2 family of
proteins (20, 35, 48).
The cellular threshold for apoptosis is highly regulated by a growing number of the Bcl-2 family of proteins (39-41). Multiple mammalian members have been reported to date, including Bcl-2, Bax, Bcl-xL, Bcl-xS, Bak, Bad, Mcl-1, and Bfl-1 (22, 42). Except for Bad, these proteins are integral membrane proteins localized predominantly to the endoplasmic reticulum, nuclear, and outer mitochondrial membranes. Members of this family can be antiapoptotic (Bcl-2, Bcl-xL, Mcl-1, Bfl-1) and proapoptotic (Bcl-xS, Bax, Bad, Bak). In cholangiocytes, only the expression of Bcl-2, Bax, and Bcl-xL has been documented to date (19). Bcl-2, a 25-kDa protein, and Bcl-xL, a 30-kDa protein, are potent repressors of apoptosis. These proteins are thought to inhibit apoptosis by one of several possible mechanisms, including 1) functioning as ion channels in the mitochondrial membrane and preventing the mitochondrial membrane permeability transition and 2) functioning as adapter molecules sequestering proapoptotic molecules from executing the apoptotic machinery (13, 18, 40). Of special importance in understanding the link between GSH depletion and apoptosis are the observations demonstrating loss of GSH, Bcl-2 depletion, and apoptosis (8, 20, 35, 48). For example, induction of apoptosis by the prooxidant nitric oxide (NO) is associated with GSH depletion and loss of Bcl-2 and an increase in Bax (20). These studies suggest that GSH depletion induces apoptosis by altering expression of the Bcl-2 family member proteins.
Based on the above-mentioned concepts, we developed the general hypothesis that GSH depletion in cholangiocytes alters the expression of Bcl-2 family members resulting in changes in cholangiocyte apoptosis. Thus the specific aims of this study were to address the following questions: 1) does GSH depletion induce cholangiocyte apoptosis, 2) does GSH depletion alter the expression of Bcl-2, Bax, and Bcl-xL protein, and 3) what is the mechanism by which GSH depletion results in altered expression of this family of proteins.
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EXPERIMENTAL MODEL AND ASSAYS |
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Cell culture and GSH depletion.
A human cholangiocyte cell line (H69), immortalized with the simian
virus 40 (SV40) large T antigen, was obtained from Dr. Doug Jefferson
(Tufts University, Boston, MA) and cultured as previously described
(17). Although immortal, this cell line is nonmalignant;
in vitro, it does not display anchorage independent growth and in vivo
it does not produce tumors in nude mice (19). Cultured cells were
treated with buthionine sulfoximine (BSO, 0.05 mM) to induce GSH
depletion. BSO causes GSH depletion by directly blocking
-glutamylcysteine synthetase, the enzyme that catalyzes the
rate-limiting step in GSH synthesis. BSO was added to the medium of
70% confluent cultured cells every 24 h for 3 days. The total number
of cells at 3 days was not significantly reduced in the BSO treatment
group (29 ± 3.3 × 104/plate)
compared with controls (34 ± 2.2 × 104/plate;
P > 0.05). Thus cell proliferation
occurred in the treated and untreated cultures, and apoptosis did not
appear to occur merely as a result of cell cycle arrest. In some
experiments, beauvericin (25 µM), a potassium ionophore, was used to
experimentally induce apoptosis as previously described by us (37). To
test whether BSO treatment of H69 cells would enhance apoptosis,
beauvericin was added to cell cultures for 2 h after treatment with BSO
for 3 days. To test whether a GSH precursor would maintain cellular GSH
levels, in some experiments glutathione ethyl ester (GSHee, 3 mM) was
added to the medium every 8 h for 3 days.
GSH measurements. GSH levels were measured using monochlorobimane employing a methodological adaptation previously described by us (16). Briefly, at desired intervals, culture medium was removed and cells were incubated in the presence of 166 µmol/l of monochlorobimane in 4 ml of HEPES-buffered saline for 30 min at 37°C. The medium was then aspirated and replaced with a hypotonic solution (HEPES-buffered saline and distilled water, 1:1 dilution) containing 1% Triton to induce cell detachment from culture flasks. The cell lysate was removed from flasks, and protein was determined using the Bio-Rad assay (9). The cell lysate was then extracted with an equal volume of methylene chloride to remove unreacted monochlorobimane. Fluorescence was quantitated in the aqueous phase using excitation and emission wavelengths of 398 and 488 nm, respectively, in a fluorometer (model 450; Sequoia-Turner, Mountain View, CA). Standard curves were prepared by incubating GSH in the presence of glutathione S-transferase (6 U/ml) for 30 min.
Morphological assessment of apoptosis. Fluorescence microscopy and the nuclear binding dye 4',6'-diamidino-2-phenylindole were used to quantitate nuclear morphological changes indicative of cell death by apoptosis in cultured cells, as previously described by us in detail (37).
Immunoblot analysis for Bcl-2, Bax, and Bcl-xL. Cell lysates were prepared from cultured H69 cells, and immunoblot analysis was performed as described previously by us in detail (19, 37). The primary antibody for detection of Bcl-2 was a monoclonal mouse anti-human Bcl-2 oncoprotein (Dako, Carpenteria, CA) prepared in a 1:1,000 dilution. The secondary antibody was a goat anti-mouse human IgG peroxidase conjugate antibody prepared in a 1:3,000 dilution (Biosource International, Camarillo, CA). For determination of Bax and Bcl-xL, the nylon transfer membrane was incubated with 1:2,000 dilution of a polyclonal rabbit anti-Bax and anti-Bcl-xL antibody, respectively (Santa Cruz Biotechnology, Santa Cruz, CA); the secondary antibody for both was a goat anti-rabbit used at a 1:7,000 dilution (Biosource International). Immunoreactive areas were analyzed using an imaging densitometer (model GS-700) and the Molecular Analyst Software (Bio-Rad Laboratories, Hercules, CA).
Ribonuclease protection assay for Bcl-2.
The assays were performed using the RPA II kit (Ambion, Austin, TX) and
10 µg of total RNA extracted from H69 cells as previously described
by us (1). Briefly, an antisense
32P-labeled riboprobe was
transcribed from the pCRII-bcl-2 using [-32P]UTP and
T7 RNA polymerase. The primary RNA
transcript was purified by excision from a 5% acrylamide-8 M urea
denaturing gel and subsequently eluted into a solution of 0.5 M
ammonium acetate, 1 mM EDTA, and 0.1% SDS at 37°C. The antisense
was hybridized with total RNA from the cells at 45°C for 12 h. The
unhybridized RNA chains were digested by a mixture of RNase
A/T1 (150-200 U/ml). The
protected hybrid (504 bp) was resolved in a 5% acrylamide-8 M urea gel
and detected after exposure to X-ray film (Kodak, Rochester, NY) for 24 h at
70°C. Glyceraldehyde-3-phosphate dehydrogenase
(Clontech Laboratories, Palo Alto, CA) was employed as a housekeeping
gene.
Determination of Bcl-2 protein synthesis and half-life. Synthesis and half-life of Bcl-2 protein were determined as described by Merino et al. (30) and Blagosklonny et al. (6). Briefly, cells were labeled with 0.2 mCi/ml [35S]methionine-cysteine (Trans35S-label, ICN Radiochemicals) for 4 h. For synthesis experiments, labeling was performed in untreated cells or after 72 h of incubation with BSO. In the half-life experiments, for chasing out the radiolabeled L-[35S]methionine-cysteine, cells were washed with medium containing excess methionine and incubated in this same medium with or without BSO for 5-24 h. Immunoprecipitation was carried out by incubating 3 × 106 cells from each sample in 0.1 ml of lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid, 1 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.23 U/ml aprotinin, 10 µM leupeptin, 1 mM benzamidine) for 1 h at 4°C. Cell lysates were transferred to a fresh tube containing 40 µl of 1% Triton X-100 solution-protein A-Sepharose (1:1, vol/vol; Sigma) that had been preabsorbed with 2 µl of rabbit serum. After rotation overnight at 4°C, the supernatant was transferred to a fresh tube and 2 µl of rabbit anti-human Bcl-2 antiserum were added. After rotation at 4°C for 2 h, 40 µl of 1% Triton X-100 solution-protein A-Sepharose (1:1, vol/vol) were added and the samples were rotated for an additional 2 h at 4°C. Finally, immune complexes on protein A-Sepharose were recovered by centrifugation at 16,000 g for 1 min, and the pellet was washed four times with 1 ml each of 1% Triton X-100 lysis buffer before resuspending the protein A-Sepharose beads in 50 µl of Laemmli sample buffer, heating at 85°C for 10 min, and subjecting the samples to SDS-PAGE (12% gels). Gels were fixed, enhanced (Enlightening, NEN, Boston, MA), dried, and exposed to X-ray film (BIOMAX MR, Kodak, Rochester, NY). Radioactive areas were analyzed using an imaging densitometer (model GS-700; Bio-Rad Laboratories) and the Molecular Analyst Software (Bio-Rad Laboratories).
Plasmid construct for Bcl-2 expression. A human cDNA containing the full open reading frame of Bcl-2 (1 kb) in a parent vector pCEP4 (10.4 kb) expressing the hygromycin-resistance gene was kindly provided by Dr. Jennifer Pietenpol (Vanderbilt Cancer Center, Nashville, TN). Plasmid DNAs were transformed into competent cells (Inra F' competent cells - One Shot Kit; Invitrogen, Carlsbad, CA). The resulting colonies were purified with Wizard Plusl maxipreps DNA purification system (Promega). Plasmid DNAs used for transfection were further purified by ethanol precipitation.
Establishing a stable H69 cell line overexpressing Bcl-2. DNA transfections were performed when cells were ~40% confluent. The cells were transfected by the addition of 20 µg of the DNA plasmid to each 100-mm petri dish. Serum-free medium containing a 1:9 ratio of DNA-single cationic lipid (Perfect Transfection Kit; Invitrogen, San Diego, CA) was used, and cells were incubated in the mixture for 8 h. Next, transfected cells were cultured in serum-supplemented medium for 36 h. After recovery from transfection, cells were grown in selective medium containing 200 µg/ml of hygromycin B (GIBCO, Gaithersburg, MD). The antibiotic was added every 3 days. A culture dish with nontransfected H69 cells treated with hygromycin B was used as a control for hygromycin selection. By 7 days there were no cells present in this dish. After 3 wk of transfection, resistant clones were isolated using cloning cylinders (Bellco Glass, Vineland, NJ) and transferred for expansion and analysis. Bcl-2 overexpression was assessed by Immunoblot Analysis. Stable transformants were grown in medium containing 100 µg/ml of hygromycin B.
Materials. Monochlorobimane was purchased from Molecular Probes (Eugene, OR). All other reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated.
Statistics. All values are expressed as means ± SE from at least three different experiments. Statistical differences between means were calculated by Student's t-test, and results were considered statistically different at P < 0.05 level.
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RESULTS |
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Is GSH depletion associated with enhanced cholangiocyte apoptosis? GSH levels in untreated cultured cholangiocytes were stable over 3 days and averaged 16.3 ± 0.6 nmol/mg of protein (Fig. 1A), a value similar to cellular GSH concentrations previously reported for freshly isolated rat cholangiocyte suspensions (34). After incubation of cultured cholangiocytes in BSO (0.05 mM), cellular GSH levels decreased by 93% (1 ± 0.3 nmol/mg of protein) after 24 h and remained at that same level for the next 48 h (Fig. 1A). Exposure of cultured cholangiocytes to BSO (0.05 mM) was also associated with a time-dependent increase in the number of cells undergoing apoptosis, with 11 ± 1.6% of cultured cells demonstrating morphological changes of apoptosis by 3 days (Fig. 1B). The percentage of apoptosis in untreated cells was stable over 72 h and averaged 1.6 ± 0.1% (Fig. 1B). Cell necrosis was not observed with these concentrations of BSO as assessed by trypan blue exclusion.
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Does maintenance of intracellular GSH levels prevent apoptosis in BSO-treated cholangiocytes? To determine whether GSH depletion or BSO itself was inducing cholangiocyte apoptosis, we used GSHee to maintain intracellular GSH concentration in the presence of BSO. First, we demonstrated that addition of GSHee could maintain cellular GSH levels in cholangiocytes. GSHee (3 mM) was added to the culture medium every 8 h for 3 days in addition to BSO (0.05 mM) every 24 h. Administration of GSHee to BSO-treated cholangiocytes maintained cellular GSH levels similar to control values (Fig. 2A). The effect of this maintenance of cellular GSH levels on the extent of BSO-induced apoptosis is shown in Fig. 2B; addition of GSHee decreased significantly the percentage of cells undergoing apoptosis to 2.6 ± 0.1% compared with 10.8 ± 0.7% in cells treated with BSO alone (P < 0.01). These data are consistent with the interpretation that BSO induces cholangiocyte apoptosis via GSH depletion.
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Is BSO treatment of cultured cholangiocytes associated with a decrease in Bcl-2 protein? Having demonstrated that GSH depletion in cholangiocytes is associated with increased apoptosis, we addressed the possible mechanisms associated with these events. We measured Bcl-2, Bax, and Bcl-xL protein expression in BSO-treated cholangiocytes by quantitative immunoblot analysis. BSO treatment decreased the amount of the antiapoptotic protein Bcl-2 by 50% of initial values at 24 and 48 h, and by 87% of the initial value at 72 h, as assessed by densitometry; in contrast, there was no change in the expression of Bax and Bcl-xL protein (Fig. 3). Thus GSH depletion results in a specific loss of cellular Bcl-2 without alterations in at least two other members of this protein family.
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Does maintenance of GSH levels by GSHee in BSO-treated cholangiocytes prevent the decrease in Bcl-2 protein? To demonstrate a direct cause-and-effect relationship between GSH depletion and decreased Bcl-2 protein expression, we asked whether maintenance of GSH by GSHee in BSO-treated cholangiocytes would prevent the decrease in Bcl-2 protein. GSHee (3 mM) was added to culture medium every 8 h for 3 days and BSO (0.05 mM) every 24 h. Bcl-2 protein was assessed by quantitative immunoblot analysis. Administration of GSHee to BSO-treated cholangiocytes maintained cellular GSH levels similar to control (Fig. 2A), and it also prevented the decrease in Bcl-2 protein (Fig. 4). These data demonstrate that the loss of cellular Bcl-2 observed during treatment with BSO is mediated by GSH depletion.
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What is the mechanism by which GSH depletion results in altered expression of Bcl-2 protein? Conceptually, GSH depletion could cause loss of cellular Bcl-2 by altering transcription, translation, or the half-life of the protein. To determine whether the decrease of the anti-apoptotic Bcl-2 protein was due to altered levels of bcl-2 mRNA, we assessed the steady-state levels of bcl-2 mRNA by the RNase protection assay. We observed no change in the steady-state levels of bcl-2 mRNA in BSO-treated cells compared with controls at 24, 48, and 72 h (Fig. 5). Thus, despite a nearly 90% decrease in the amount of Bcl-2 protein, there was no change in bcl-2 mRNA. To explain a BSO-induced decrease in Bcl-2 protein without a concomitant change in message, we examined for a posttranscriptional alteration of Bcl-2 at the level of translation or at the level of degradation by assessing Bcl-2 protein synthesis and half-life using [35S]methionine pulse-chase analysis. We observed no change in Bcl-2 synthesis in BSO-treated cells (1.68 ± 0.28 arbitrary densitometry units) compared with controls (1.76 ± 0.26 arbitrary densitometry units; P > 0.05). The half-life of Bcl-2 in control cholangiocytes was 23 h. Our calculated half-life is consistent with the value reported for Bcl-2 in a human myeloid leukemia cell line (6). In contrast, Bcl-2 half-life was reduced by ~57% (10 h) in BSO-treated cholangiocytes (Fig. 6). Thus the decrease in Bcl-2 protein after GSH depletion appears to be due to accelerated rate of Bcl-2 protein degradation.
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Is a decrease in Bcl-2 necessary for BSO-induced apoptosis? To ascertain whether a loss of cellular Bcl-2 is necessary for BSO-induced apoptosis, we established a stable H69 cell line overexpressing Bcl-2. As assessed by immunoblot analysis, this stably transfected cell line demonstrated a 25-fold increase in Bcl-2 protein content compared with untransfected cells (Fig. 7A). GSH levels decreased to similar values in BSO-treated untransfected cells and BSO-treated transfected cells. In contrast, the Bcl-2 overexpressing cell line was markedly resistant to BSO-induced apoptosis (Fig. 7B). Indeed, BSO-induced apoptosis was 15.4 ± 0.6% in wildtype cells but was only 2.2 ± 0.8% in Bcl-2-transfected H69 cells (P < 0.01). These data suggest that the loss of cellular Bcl-2 observed during GSH depletion contributes to the observed cholangiocyte apoptosis.
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DISCUSSION |
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The major findings reported here relate to the cellular mechanisms regulating apoptosis in cholangiocytes. Using a human immortalized but nonmalignant cholangiocyte cell line, we made the novel observations that experimentally induced reduction in endogenous GSH is associated with 1) increased apoptosis; 2) decreased Bcl-2 protein, an anti-apoptotic molecule, due to increased Bcl-2 degradation; and 3) no change in Bax and Bcl-xL protein. These results support our hypothesis that GSH depletion alters the expression of Bcl-2 and induces cholangiocyte apoptosis. Our data indicate that GSH participates in apoptosis in cholangiocytes and that depletion of this molecule by either oxidation, decreased synthesis, extrusion, or decreased uptake may be critical in predisposing cholangiocytes to apoptotic cell death.
Many models exist to induce GSH depletion. Depletion of GSH may be induced by using GSH synthesis inhibitors (i.e., BSO), GSH-S-transferase-dependent thiol alkylating agents (e.g., diethyl maleate, phorone, etc.), peroxides (e.g., tert-butyl hydroperoxide, H2O2, etc.), GSSG reductase inhibitors (i.e., bis-chloronitrosourea), and nonenzymatic GSH reacting agents (i.e., diazene and diamide; 29, 36, 45). We choose to use the GSH synthesis inhibitor BSO because of its specificity and because the block in GSH synthesis can be overcome by supplying the cell with GSHee (2, 29, 53). Indeed, depletion of GSH by oxidizing agents, such as t-butyl hydroperoxide, is temporary because the oxidized glutathione (GSSG) formed is rapidly reduced by GSSG reductase. Alkylating compounds that react with the thiol group of GSH such as diethyl maleate have a variety of additional effects: 1) its metabolite, maleate, affects certain enzymes and inhibits protein synthesis (29) and 2) the decrease in GSH is short-lived and actually leads to an increase in GSH levels through the increased synthesis of GSH that follows the absence of feedback inhibition by GSH on its own synthesis (12). Thus, although GSH depletion induced by BSO is profound and not oxidant driven, we chose this model because, to address the questions of interest to us, it clearly had advantages over other approaches, as itemized previously. Because intracellular cholangiocyte GSH concentrations have not been measured during bile duct inflammation, it is simply not possible to confidently comment on the physiological or pathophysiological relevance of the GSH depletion observed in our model. However, we note that this model also provides original information regarding the fate of Bcl-2 in altered redox states of the cell.
For these studies, we employed the H69 cell line, a human cholangiocyte cell line immortalized with the SV40 large T antigen because nonmalignant human primary cell cultures are difficult to obtain on a consistent basis and have a limited survival in culture. The H69 cells retain their cholangiocyte phenotype and, although transformed, are nonmalignant (i.e., do not demonstrate anchorage independent growth or grow in nude mice) (19). Furthermore, this cell line allowed us to establish cells stably overexpressing Bcl-2, a technique that would not have been feasible with primary cell cultures. Finally, these cells undergo beauvericin-induced apoptosis at similar concentrations and over the same time course as normal rat cholangiocytes. Thus the results obtained with the H69 cells should be pertinent to nontransformed cholangiocytes.
Although the rates of apoptosis may appear modest in the BSO-treated cells (11% at 72 h), in the context of organ physiology even small amounts of observed apoptosis may be highly significant. For example, Schulte-Hermann and colleagues (10) have calculated that even a 3% rate of unopposed hepatocyte apoptosis observed in a liver biopsy would result in a 25% reduction in liver volume in 72 h. Furthermore, GSH depletion and the associated loss of Bcl-2 can certainly predispose the cell to apoptosis by other proapoptotic stimuli as we directly demonstrated using beauvericin. Thus the rates of apoptosis by themselves could potentially lead to ductopenia over time and/or predispose to further apoptosis by proapoptotic stimuli.
Intracellular GSH depletion has been reported to induce or enhance apoptosis in several different systems, including polymorphonuclear leukocytes, thymocytes, lymphocytes, and hepatocytes (3, 26, 44, 52). In our experiments, a role for GSH depletion in the induction of apoptosis was suggested by the observation that maintenance of GSH levels by GSHee prevented apoptosis. Indeed, inhibition of apoptosis by reversing GSH depletion with the use of different GSH precursors has been reported in several cell systems (3, 8, 20, 44, 52). To begin to address the possible mechanisms associated with apoptosis in experimentally induced GSH depletion, we measured Bcl-2, Bax, and Bcl-xL protein. BSO treatment significantly decreased the amount of the anti-apoptotic Bcl-2 protein, without affecting Bax and Bcl-xL protein. We also observed that the maintenance of GSH levels by GSHee prevented both cholangiocyte apoptosis and the decrease in Bcl-2 protein. These data reinforce a cause-and-effect relationship between GSH depletion and decreased Bcl-2 protein; they also strengthen the specificity of BSO. Several in vitro studies have suggested an association between alterations in GSH and Bcl-2 levels and susceptibility for apoptosis. For example, apoptosis induced in neurons by glutamate or N-methyl-D-aspartate (NMDA) is associated with a decrease in both GSH levels and the anti-apoptotic Bcl-2 protein (35). In this system Flupirtine, a NMDA receptor antagonist, completely abolished the NMDA-induced reductions in GSH and Bcl-2 and decreased apoptotic cell death. Also, NO-induced apoptosis in adenocarcinoma and hepatocellular carcinoma cell lines (HT-29 and Hep 3B, respectively) is associated with a decrease in intracellular GSH, a decrease in the expression of Bcl-2, and an increase in Bax (20). In this same experiment, addition of N-acetylcysteine increased GSH and Bcl-2 protein expression and prevented apoptosis. Finally, in a model of HIV-induced apoptosis in lymphocytes, overexpression of HIV protease was associated with decreased intracellular GSH and decreased Bcl-2 protein expression (48). Thus, although GSH depletion induced by a variety of perturbations is associated with diminished Bcl-2 and increased apoptosis, the mechanism by which GSH depletion-repletion alters Bcl-2 protein remains unclear.
Expression of Bcl-2 is regulated at both transcriptional and posttranscriptional levels. To determine the mechanism of Bcl-2 downregulation in BSO-induced apoptosis in cholangiocytes, we assessed the steady-state levels of Bcl-2 message by RNase protection assay. Despite a substantial decrease in the amount of Bcl-2 protein, we observed no change in the steady-state levels of Bcl-2 message, a finding suggesting a posttranscriptional process at either the level of translation or degradation. Indeed, we were able to demonstrate for the first time that the half-life of Bcl-2 is markedly decreased in GSH-depleted cholangiocytes. These results suggest that Bcl-2 protein degradation is regulated by the redox state of the cell. Interestingly, lymphocyte GSH depletion and proteolysis of Bcl-2 have been described but not mechanistically linked in apoptosis (48). Our work suggests that an oxidized redox status promotes activation of a Bcl-2 degradation pathway as a mechanism of apoptosis.
Our work has potential pathophysiological importance, since little is known about regulatory mechanisms of apoptosis in cholangiocytes. The data support a role for GSH as a cytoprotective molecule involved in maintaining the balance between proliferation and cell death by apoptosis in the biliary tree. This work provides the framework for the following general hypothesis: the biliary concentration and/or metabolism of cytoprotective biliary constituents, such as GSH, are modified in the cholangiopathies resulting in loss of Bcl-2, accelerated apoptosis, and ultimately ductopenia. Further investigations using in vivo models will ultimately be required to test this hypothesis.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-24031 (N. F. LaRusso) and DK-41876 (G. J. Gores) and by the Mayo Foundation.
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FOOTNOTES |
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Address for reprint requests: N. F. LaRusso, Center for Basic Research in Digestive Diseases, Mayo Clinic, 200 First St., SW, Rochester, MN 55905.
Received 31 December 1997; accepted in final form 8 June 1998.
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