Volume 120, Issue 7, Pages 1810-1817 (June 2001) The bile acid–activated phosphatidylinositol 3-kinase pathway inhibits Fas apoptosis upstream of bid in rodent hepatocytes  Yasuhiro Takikawa, Hideyuki Miyoshi, Christian Rust, Patricia Roberts, Richard Siegel, Pijus K. Mandal, Randal E. Millikan, Gregory J. Gores  Gastroenterology  Volume 120, Issue 7, Pages 1810-1817 (June 2001) DOI: 10.1053/gast.2001.24835 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 GCDC, but not TCDC, treatment of hepatocytes results in release of cytochrome c from mitochondria. (A) Mouse hepatocytes were isolated and cultured as described in Materials and Methods. After 24 hours in culture,mouse hepatocytes were treated with the diluent (control), which was media (Dulbecco's modified Eagle medium [DMEM]), 50 μmol/L TCDC, 50 μmol/L TCDC plus 250 nmol/L wortmannin, or GCDC for 6 hours. The cells were lysed and a cytosolic fraction obtained as described in Materials and Methods. Western blot analysis of the cytosolic fraction was performed for cytochrome c using anti-cytochrome c mouse monoclonal antibodies. The immunoblot shown is 1 blot representative of 5 similar experiments. (B) McNtcp.24 cells were transfected with cytochrome c-GFP as described in Materials and Methods. Twenty-four hours after transfection, the cells were treated with media, TCDC, TCDC plus wortmannin, or GCDC as described (in panel A) for 4 hours. Confocal microscopy was performed to determine the cellular localization of cytochrome c-GFP. The confocal images shown are representative of 4 similar experiments. (C) McNtcp 24 cells were cotransfected with cytochrome c-GFP and a constitutively active PI 3-K. Twenty-four hours after transfection, the cells were treated with media (control), 50 μmol/L GCDC, and 50 μmol/L GCDC plus 250 nmol/L wortmannin for 4 hours and observed by confocal microscopy (control). Similarly, McNtcp 24 cells were also cotransfected with cytochrome c-GFP plus a DN-PI 3-K and observed after 4 hours of treatment with TCDC. The confocal images shown are representative of 4 similar experiments. Collectively, these data show that TCDC activation of PI 3-K inhibits cytochrome c release. Gastroenterology 2001 120, 1810-1817DOI: (10.1053/gast.2001.24835) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 TCDC, as well as GCDC, induces Fas aggregation. Mouse hepatocytes were isolated and cultured as described in Materials and Methods. After 24 hours in culture, mouse hepatocytes were treated with diluent (control), which was media (DMEM), 50 μmol/L GCDC, or 50 μmol/L TCDC for 1 hour. Cell lysates were then prepared and used for immunoprecipitation using anti-Fas antibody (Jo2 antibody) under limited antibody conditions (0.5 μg/mL) or excess antibody conditions (5 μg/mL). Western blot analysis of the immunoprecipitate was performed using a Fas-specific goat polyclonal antibody. The immunoblot shown is representative of 6 similar experiments. Gastroenterology 2001 120, 1810-1817DOI: (10.1053/gast.2001.24835) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Both GCDC and TCDC treatment of hepatocytes are associated with translocation of FADD and caspase 8 to the plasma membrane. (A) Mouse hepatocytes were isolated and cultured as described in Materials and Methods. After 24 hours in culture, mouse hepatocytes were treated with the diluent (control), which was media (DMEM), 50 μmol/L TCDC, or GCDC for 1 hour. Mouse hepatocytes were lysed and a membrane fraction obtained as described in Materials and Methods. Western blot analysis of the membrane fraction was performed using anti-Fas goat polyclonal antibody and anti-caspase 8 rabbit polyclonal antibody. The immunoblot shown is representative of 3 similar experiments. (B) McNtcp.24 cells were transfected with DN-FADD-GFP or C360S-caspase-GFP as described in Materials and Methods. Twenty-four hours after transfection, cells were treated with media (control), GCDC, or TCDC for 4 hours and then visualized by confocal microscopy. The confocal images shown are representative of 5 similar experiments. Gastroenterology 2001 120, 1810-1817DOI: (10.1053/gast.2001.24835) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 TCDC attenuates caspase 8–like activity. (A) McNtcp.24 cells were treated with diluent control, which was media (DMEM), GCDC (50 μmol/L), or GCDC plus IETD-fmk (50 μmol/L) for 2 hours. The cells were then mounted on the microscope stage. Following addition of IETD-CarMAC (25 μmol/L), intracellular fluorescence was measured over 30 minutes using excitation and emission wavelengths of 380 and 520 nm, respectively. Increases in caspase 8–like activity are expressed as fluorescence increases per minute in each cell. The data are the mean and standard error of 5 separate experiments. (B) Cells were treated with GCDC, mounted on the microscope stage, and incubated in the presence of 25 μmol/L IETD-CarMac as described in panel A. After 30 minutes, the media was exchanged with probe-free media and fluorescence measured over an additional 20 minutes. Next, 20 μmol/L digitonin was added to the buffer and fluorescence measured after 5 minutes. The data depicted are from 1 experiment, which is representative of 3 separate experiments. (C) Representative photomicrographs of intracellular fluorescence are shown. Note the minimal fluorescence at 0 and 30 in a control cell, and 0 minute in a GCDC-treated cell. Fluorescence was extremely bright, however, and diffuse in a GCDC-treated cell incubated with 25 μmol/L IETD-CarMac for 30 minutes. (D) McNtcp.24 cells were treated with diluent (control), 50 μmol/L TCDC, 50 μmol/L GCDC, and 50 μmol/L TCDC plus 250 nmol/L wortmannin for 2 hours. After this period of incubation, the cells were mounted on the stage of an inverted fluorescent microsope and 25 μmol/L IETD-CarMAC was added. The fluorescence was imaged over 30 minutes. Caspase 8–like activity in GCDC and TCDC plus wortmannin-treated cells is significantly greater than control, wortmannin, and TCDC-treated cells, P < 0.001. The data represent the mean and SD of 5 separate experiments. Gastroenterology 2001 120, 1810-1817DOI: (10.1053/gast.2001.24835) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 TCDC inhibits Bid translocation to mitochondria by a PI 3-K–dependent mechanism. McNtcp 24 cells were transfected with Bid-GFP as described in Materials and Methods. Twenty-four hours after transfection, the cells were treated with diluent (control), which was media (DMEM), 50 μmol/L TCDC, 50 μmol/L GCDC, and 50 μmol/L TCDC plus 250 nmol/L wortmannin for 1 hour. Mitochondria were stained with 50 nmol/L TMRM 30 minutes before the confocal microscopy. Bid-GFP images are shown in the left column, TMRM in the middle column, and overlay images of GFP and TMRM fluorescence are shown in the images in the right column. Bid-GFP translocation to mitochondria occurred in cells treated with GCDC or TCDC plus the PI 3-K inhibitor wortmannin. The confocal images shown are representative of 5 similar experiments. Gastroenterology 2001 120, 1810-1817DOI: (10.1053/gast.2001.24835) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 The TCDC/PI 3-K survival signal inhibits apoptosis by blocking caspase 8 activation and Bid translocation to mitochondria. The principal findings of this study are summarized in this diagram. Following TCDC treatment of hepatocytes, Fas undergoes aggregation with recruitment of FADD and procaspase 8 to the receptor complex. However, the simultaneous activation of the PI 3-K survival pathway prevents caspase activation and Bid translocation to the mitochondria blocking the apoptosis cascade and permitting cell survival. The corresponding figures supporting the mechanistic steps identified in the diagram are listed on each arrow. Steps inhibited by the TCDC/PI 3-kinase survival pathway are represented by dotted arrows. Gastroenterology 2001 120, 1810-1817DOI: (10.1053/gast.2001.24835) Copyright © 2001 American Gastroenterological Association Terms and Conditions