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, 103 (1), 137-45

Toxic Bile Salts Induce Rodent Hepatocyte Apoptosis via Direct Activation of Fas

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Toxic Bile Salts Induce Rodent Hepatocyte Apoptosis via Direct Activation of Fas

W A Faubion et al. J Clin Invest.

Abstract

Cholestatic liver injury appears to result from the induction of hepatocyte apoptosis by toxic bile salts such as glycochenodeoxycholate (GCDC). Previous studies from this laboratory indicate that cathepsin B is a downstream effector protease during the hepatocyte apoptotic process. Because caspases can initiate apoptosis, the present studies were undertaken to determine the role of caspases in cathepsin B activation. Immunoblotting of GCDC-treated McNtcp.24 hepatoma cells demonstrated cleavage of poly(ADP-ribose) polymerase and lamin B1 to fragments that indicate activation of effector caspases. Transfection with CrmA, an inhibitor of caspase 8, prevented GCDC-induced cathepsin B activation and apoptosis. Consistent with these results, an increase in caspase 8-like activity was observed in GCDC-treated cells. Examination of the mechanism of GCDC-induced caspase 8 activation revealed that dominant-negative FADD inhibited apoptosis and that hepatocytes isolated from Fas-deficient lymphoproliferative mice were resistant to GCDC-induced apoptosis. After GCDC treatment, immunoprecipitation experiments demonstrated Fas oligomerization, and confocal microscopy demonstrated DeltaFADD-GFP (Fas-associated death domain-green fluorescent protein, aggregation in the absence of detectable Fas ligand mRNA. Collectively, these data suggest that GCDC-induced hepatocyte apoptosis involves ligand-independent oligomerization of Fas, recruitment of FADD, activation of caspase 8, and subsequent activation of effector proteases, including downstream caspases and cathepsin B.

Figures

Figure 1
Figure 1
Cathepsin B activity increases during GCDC-induced apoptosis of McNtcp.24 cells, and cathepsin B inhibitors reduce apoptosis. Cells were incubated in medium alone or with 50 μM GCDC in the absence or presence of 100 μM FA-fmk (throughout the experiment) or 0.1 μM CA-074-Me (pretreatment for 10 min before adding GCDC). Cathepsin B activity (a) was measured in cells after 2 h of incubation using the fluorogenic substrate VLK-CMAC as described in Methods. P < 0.01 for GCDC vs. control, GCDC plus FA-fmk, or GCDC plus CA-074-Me by ANOVA. Apoptosis (b) was quantitated after 2 h of incubation as described in Methods. P < 0.01 for GCDC vs. control, GCDC plus FA-fmk, or GCDC plus CA-074-Me by ANOVA. GCDC, glycochenodeoxycholate.
Figure 2
Figure 2
Immunoblot analysis shows PARP and lamin B1 cleavage. McNtpc.24 cells were treated with GCDC for the indicated length of time. HL-60 cells with and without etoposide appear on the right as positive and negative controls, respectively. The appearance of 89-kDa and 45-kDa cleavage products of PARP (top) and lamin B1 (bottom), respectively, are characteristic of caspase-mediated cleavage. PARP, poly(ADP-ribose) polymerase.
Figure 3
Figure 3
CrmA expression prevents cathepsin B activation and apoptosis in McNtcp.24 cells treated with GCDC. In contrast, inhibition of cathepsin B activity does not prevent the increase in caspase 8 activity. Forty-eight hours after transfection with an expression vector for CrmA or the empty vector, McNtcp.24 cells were treated with 50 μM GCDC or the diluent. After 2 h of incubation, apoptosis (a) was quantitated as described in Fig. 1. P < 0.01 for GCDC vs. control, CrmA, or GCDC plus CrmA. The inset shows an immunoblot demonstrating CrmA expression in transfected cells. Cathepsin B activity (b) was measured after 2 h of incubation using the fluorogenic substrate VLK-CMAC. P < 0.01 for GCDC vs. control, CrmA, or GCDC plus CrmA by ANOVA. Caspase 8 activity (c) was assessed in lysates prepared from McNtcp.24 cells incubated in medium alone or with 50 μM GCDC at 37°C in the presence of 100 μM FA-fmk or 0.1 μM CA-074-Me as described in Fig. 1. P = NS for GCDC vs. GCDC plus FA-fmk or GCDC plus CA-074-Me. P < 0.01 for all GCDC-treated cells compared with control. NS, not significant.
Figure 4
Figure 4
The increased cleavage of IETD-AFC in cytosol from GCDC-treated McNtcp.24 cells is diminished by prior transfection with CrmA. Untransfected McNtcp.24 cells were incubated in the absence (control; open squares) or presence of 50 μM GCDC (solid squares and solid circles). Cells were transfected with CrmA for 48 h before incubation in the presence of GCDC (solid circles). Cytosol was prepared at selected time points and assayed for caspase 8–like protease activity using the fluorogenic substrate IETD-AFC.
Figure 5
Figure 5
MC159 or DN FADD–GFP inhibits apoptosis in GCDC-treated McNtcp.24 cells. Forty-eight hours after transfection with plasmid encoding MC159 (a) or DN FADD–GFP (b), McNtcp.24 cells were treated with 50 μM GCDC or diluent. At the indicated time points, cells were stained with DAPI and examined by microscopy. In the top panel, P < 0.01 for GCDC vs. control, vector (empty plasmid), MC159, or MC159 plus GCDC by ANOVA; P = NS between GCDC vs. vector plus GCDC. In the bottom panel, P < 0.01 for GFP plus GCDC vs. GFP, DN FADD–GFP, or DN FADD–GFP plus GCDC by ANOVA. DAPI, 4′,6-diamidino-2-phenylindole; DN, dominant negative; FADD, Fas-associated death domain; GFP, green flourescent protein.
Figure 6
Figure 6
Hepatocytes isolated from the Fas-deficient lpr mouse are resistant to GCDC-induced apoptosis. Hepatocytes isolated from lpr and wild-type (Wt; MRLMPJ+/+) mice were cultured in medium for 8 h before addition of diluent or 50 μM GCDC for another 4 h. At 0, 2, and 4 h of incubation, apoptosis was quantitated morphologically (a) as described in Fig. 1. Representative photomicrographs of MRL and lpr mouse hepatocytes treated with 50 μM GCDC for 2 h before DAPI staining (b). Note the chromatin margination/condensation and nuclear fragmentation in MRL hepatocytes and their relative absence in the lpr hepatocytes.
Figure 7
Figure 7
McNtcp.24 cells express Fas receptor but not FasL mRNA. After McNtcp.24 cells were cultured for 2 h in the presence or absence of 50 μM GCDC, total RNA was isolated for subsequent RT-PCR using Fas, FasL, and GAPDH. RNA from ileum served as a positive control for both Fas receptor and FasL. The identities of the PCR products were verified by DNA sequencing. FasL, Fas ligand; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcriptase.
Figure 8
Figure 8
GCDC treatment of primary mouse hepatocytes cells results in Fas receptor oligomerization. Untreated and GCDC (50 μM)-treated cells were incubated with the cleavable cross-linking reagent 3,3′-dithio-bis(sulfosuccinimidyl proprionate) and lysed for immunoprecipitation using limiting (0.5 μg/ml) or excess (5 μg/ml) amounts of anti-Fas antibody. Western blot analysis of the immunoprecipitate was performed as described in Methods.
Figure 9
Figure 9
Intracellular localization of DN FADD–GFP in McNtcp.24 cells. Forty-eight hours after transfection with DN FADD–GFP, cells were either untreated (control) or treated with 10 ng/ml FasL, 50 μM tauro ursodeoxycholate (TUDC), 50 μM GCDC, 1 μM staurosporine (SP), or 50 μM glycodeoxycholate (GDC) for 4 h. GFP fluorescence was imaged by laser scanning confocal microscopy.

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