Expression of the peripheral-type benzodiazepine receptor and apoptosis induction in hepatic stellate cells  Richard Fischer, Marcus Schmitt, Johannes G. Bode, Dieter Häussinger  Gastroenterology  Volume 120, Issue 5, Pages 1212-1226 (April 2001) DOI: 10.1053/gast.2001.23260 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 mRNA (A, C) and protein levels (B, C) of PBR, ANT 1, VDAC 1, and α-smooth muscle actin in HSCs (A–C) and PCs (B) in culture. Regulation of PBR expression by cytokines (D). Cells were harvested for RNA isolation after the time periods indicated and subjected to Northern blot analysis for PBR and GAPDH (A). For protein isolation, cells were harvested after the time period indicated and subjected to Western blot analysis using antibodies raised specifically against ANT 1, VDAC 1, PBR or α1-smooth muscle actin as a parameter of activation (B). PBR mRNA levels were analyzed by determination of density, and relative PBR mRNA levels (♦) are represented by the ratio PBR mRNA/GAPDH mRNA (C). The VDAC 1 (▴) and PBR protein level (●) measured on the day of culture of maximal intensity was arbitrarily set to 1.0, and the protein content found at other time points is expressed as a fraction thereof (C). For determination of PBR regulation by cytokines (D), HSCs cultured for 7 days were incubated for 24 hours in DMEM (0.5% FCS, 1% penicillin/streptomycin) with the substances indicated (IL-1β: 100 U/mL; IFNγ: 100 U/mL; IL-10: 250 ng/mL; M-CSF: 2 ng/mL). Data are given as means ± SEM (n = 3). SEM values were omitted when they were within the symbol size. Gastroenterology 2001 120, 1212-1226DOI: (10.1053/gast.2001.23260) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Subcellular localization of immunoreactive PBR, ANT 1 (A–C), and MnSOD (D–F) in HSCs after 8 days of culture and in vivo localization of immunoreactive PBR with GFAP in control (G–I) and CCl4-treated (J–L) rat liver. HSCs in culture for 8 days were immunostained with antibodies against PBR (second antibody FITC-conjugated, green), ANT 1, MnSOD, and GFAP (second antibody Cy3-conjugated, red, respectively) and studied by means of confocal laser scanning microscopy. Both PBR and ANT 1 as well as PBR and MnSOD colocalize (yellow) at day 8 of culture (A–C; D–F) and exhibit a punctate staining pattern, consistent with a mitochondrial localization. At day 1 and 21 of culture, HSCs stain positive for MnSOD and ANT 1, but not for PBR (not shown). In vivo liver injury was induced by CCl4 as described; injected vehicle served for control. Liver cryosections of rats treated for 14 days were PBR-immunostained (green; G–H, J–K). To detect HSCs, the slides were immunostained with GFAP (red; G+I, J+L). In control livers (G–I), PBR (green) was not detectable in GFAP-positive cells (red, arrow), but was prominent in surrounding hepatocytes. In CCl4-treated livers (J–L), GFAP-positive cells showed staining for PBR (arrows). Gastroenterology 2001 120, 1212-1226DOI: (10.1053/gast.2001.23260) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Induction of apoptosis by PK11195 and Ro5-4864 in HSCs cultured for 7 days and expression of DBI by HSCs. HSCs were kept in culture for 7 days, then exposed to potential inducers of apoptosis for 24 hours and apoptosis was determined by FITC-conjugated in situ nick end labeling reaction (TUNEL; A–E) or the FITC-conjugated annexin V assay (G–I). (A) Control; (B) PK11195 (100 μmol/L); (C) Ro5-4864 (100 μmol/L); (D) clonazepam (200 μmol/L); (E) CD95 ligand (50 ng/mL) plus enhancer (1 μg/mL). In A–E, cells were counterstained with TRITC-coupled phalloidin (red). TUNEL-positive cells exhibit a green nuclear staining. HSCs cultured for 7 days stain positive for DBI (F). They were immunostained with antibodies against DBI (second antibody FITC-conjugated, green); the nucleus was stained with propidium iodide (F). (G) control; (H) PK11195 (100 μmol/L); (I) Ro5-4864 (100 μmol/L). For G, H, and I, under each FITC-annexin V staining, the respective phase contrast images are given. Gastroenterology 2001 120, 1212-1226DOI: (10.1053/gast.2001.23260) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Caspase-3 activation by PK11195 in HSCs, but not in PCs. Incubation of HSCs for 24 hours with PK11195, but not with TGF-β1, leads to a dose-dependent and significant increase of caspase-3 activity. Positive control of caspase-3 activation by TGF-β1 is shown in PCs. PK11195 does not evoke an alteration of caspase-3 activity in PCs. Data are given as means ± SEM (n = 3). SEM values were omitted when they were within the symbol size. *Statistical significance compared with the corresponding control. Gastroenterology 2001 120, 1212-1226DOI: (10.1053/gast.2001.23260) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Concentration-dependence of PBR-ligand–induced apoptosis in HSCs cultured for 7 days. HSCs were cultured for 7 days and then exposed for 24 hours with PK11195 (●), Ro5-4864 (♦), or clonazepam (■), and the percentage of apoptotic cells was determined by the TUNEL reaction (A) or the annexin V-assay (B). Data are given as means ± SEM (n = 3). SEM values were omitted when they were within the symbol size. Gastroenterology 2001 120, 1212-1226DOI: (10.1053/gast.2001.23260) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of culture time on PK11195-induced apoptosis in HSCs. HSCs were cultured for the time periods indicated, and then exposed to PK11195 (100 μmol/L) for 24 hours. Thereafter, the percentage of apoptotic cells was determined by the TUNEL reaction (●) or the annexin V assay (♦). Data are given as means ± SEM (n = 3). SEM values were omitted when they were within the symbol size. Gastroenterology 2001 120, 1212-1226DOI: (10.1053/gast.2001.23260) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of PK11195 on the MMP in HSCs in culture for 7 days (A–C) and induction of apoptosis by CD95 ligand, but not by PK11195, in PCs (D–F). Mitochondrial membrane potential was measured in HSCs using the red fluorescence dye JC-1 (A–C) that stains only mitochondria with preserved membrane potential (A). Incubation of HSCs with PK11195 (100 μmol/L, B and C) induces a time-dependent disruption of the MMP first visible after 12 hours (B) and more pronounced after 16 hours (C). Under each condition, the corresponding phase contrast images are given. Induction of apoptosis in PCs is examined by annexin-V-FITC staining (green; D–F); phase contrast images are given under the corresponding condition. (D) Control; (E) 24 hours PK11195 (100 μmol/L); (F) 24 hours CD95 ligand (25 ng/mL) + enhancer protein (1 μg/mL). Gastroenterology 2001 120, 1212-1226DOI: (10.1053/gast.2001.23260) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 8 Time course of the PK11195 (100 μmol/L) effects on the collapse of MMP; ♦), phosphatidyl serine translocation (●), and nuclear DNA fragmentation (■) in HSCs 7 days in culture. HSCs in culture for 7 days were exposed to PK11195 (100 μmol/L) for the time period indicated and the MMP-disruption determined using JC-1 (♦). The phosphatidyl serine translocation was measured by Annexin-V FITC staining (●), and the nuclear DNA fragmentation examined with the TUNEL assay (■). Disruption of MMP precedes the phosphatidyl serine translocation, followed by the nuclear DNA fragmentation. Data are given as means ± SEM (n = 3). SEM values were omitted when they were within the symbol size. Gastroenterology 2001 120, 1212-1226DOI: (10.1053/gast.2001.23260) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 9 CD95 ligand–induced apoptosis in HSCs and liver PCs. HSCs cultured for 3 (♦), 7 (■), and 14 days (▴) and PC (●) cultured for 2 days were incubated for 24 hours with different concentrations of CD95 ligand and enhancer protein (1 μg/mL). The percentage of apoptotic cells was determined by annexin-V staining. CD95 ligand induced apoptosis in PC at concentration above 20 ng/mL, whereas HSCs were resistant to concentrations up to 100 ng/mL. Data are given as means ± SEM (n = 3). SEM values were omitted when they were within the symbol size. Gastroenterology 2001 120, 1212-1226DOI: (10.1053/gast.2001.23260) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 10 Lack of effect of endothelin-1 and CD95 ligand on PK11195-induced apoptosis in HSCs. HSCs were incubated for 24 hours with PK11195 with the concentrations indicated and the percentage of apoptotic cells determined by annexin-V staining. No significant difference is observed if HSCs are incubated only with PK11195 (●), or with PK11195 together with endothelin-1 (5 nmol/L; ♦), or CD95 ligand (100 ng/mL with enhancer protein, 1 μg/mL; ■). Data are given as means ± SEM (n = 3). SEM values were omitted when they were within the symbol size. Gastroenterology 2001 120, 1212-1226DOI: (10.1053/gast.2001.23260) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 11 Expression of Bcl-2, Bax, Bad, Bcl-XL, PTEN, PKB/Akt, phosphorylated PKB/Akt, and phosphorylated Bad in HSCs in culture (A); the effect of PK11195 on PKB/Akt, Bad, and Bcl-2 (B); and time course of PK11195-induced PKB/Akt dephosporylation (C). Levels of the indicated proteins in HSCs over culture time (A) and in HSCs cultured for 7 days and consecutively treated with PK11195 in DMEM (0.5% FCS/1% penicillin/streptomycin) for 20 hours (B) or for the time indicated (C) were examined using Western blot analysis with specific antibodies (for details, see Materials and Methods). Representative blots from 3 independent experiments are shown. Gastroenterology 2001 120, 1212-1226DOI: (10.1053/gast.2001.23260) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 12 Lack of effect of PK11195 on Bad expression and phosphorylation and Bcl-2 expression in liver PCs. Levels of the indicated proteins in PCs treated with PK11195 in DMEM (0.5% FCS/1% penicillin/streptomycin) for 20 hours were examined using Western blot analysis with specific antibodies (for details, see Materials and Methods). Representative blots from 3 independent experiments are shown. Gastroenterology 2001 120, 1212-1226DOI: (10.1053/gast.2001.23260) Copyright © 2001 American Gastroenterological Association Terms and Conditions