Volume 129, Issue 1, Pages 269-284 (July 2005) Bid Is Upstream of Lysosome-Mediated Caspase 2 Activation in Tumor Necrosis Factor α–Induced Hepatocyte Apoptosis  M. Eugenia Guicciardi, Steven F. Bronk, Nathan W. Werneburg, Xiao-Ming Yin, Gregory J. Gores  Gastroenterology  Volume 129, Issue 1, Pages 269-284 (July 2005) DOI: 10.1053/j.gastro.2005.05.022 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Bid is activated upstream of cathepsin B during TNF-α–induced hepatocyte apoptosis and is required for lysosomal permeabilization. (A) Immunoblot analysis of Bax, Bak, Bid, Bcl-XL, and Mcl-1 on untreated isolated hepatocytes from WT and Ctsb−/− mice showed that the 2 phenotypes express comparable cellular levels of these proteins. (B) Isolated hepatocytes from WT and Ctsb−/− mice were incubated with TNF-α (28 ng/mL) and AcD (0.2 μg/mL) for up to 6 hours, and mitochondrial fractions were obtained as described in Materials and Methods. Aliquots of 50 μg of mitochondrial proteins were analyzed by immunoblot for Bid and cytochrome c oxidase (COX) as a measure of protein loading. The t-Bid was readily detectable in both WT and Ctsb−/− mitochondria. (C) Isolated hepatocytes from WT and Bid−/− mice were incubated with TNF-α/AcD for 2 hours, and cytosolic cathepsin B activity was measured as described in Materials and Methods. A significant increase in cytosolic cathepsin B activity was detectable in WT, but not in Bid−/−, cells (#x002A;P < .05, WT vs Bid−/−). (D) Isolated hepatocytes from WT and Bid−/− mice were loaded with LysoTracker Red, treated with TNF-α/AcD for 2 hours, and imaged by confocal microscopy as described in Materials and Methods. Lysosomal permeabilization (as indicated by LysoTracker Red release) was significantly impaired in Bid−/− cells. Cont, control. Gastroenterology 2005 129, 269-284DOI: (10.1053/j.gastro.2005.05.022) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Bid is activated upstream of cathepsin B during TNF-α–induced hepatocyte apoptosis and is required for lysosomal permeabilization. (A) Immunoblot analysis of Bax, Bak, Bid, Bcl-XL, and Mcl-1 on untreated isolated hepatocytes from WT and Ctsb−/− mice showed that the 2 phenotypes express comparable cellular levels of these proteins. (B) Isolated hepatocytes from WT and Ctsb−/− mice were incubated with TNF-α (28 ng/mL) and AcD (0.2 μg/mL) for up to 6 hours, and mitochondrial fractions were obtained as described in Materials and Methods. Aliquots of 50 μg of mitochondrial proteins were analyzed by immunoblot for Bid and cytochrome c oxidase (COX) as a measure of protein loading. The t-Bid was readily detectable in both WT and Ctsb−/− mitochondria. (C) Isolated hepatocytes from WT and Bid−/− mice were incubated with TNF-α/AcD for 2 hours, and cytosolic cathepsin B activity was measured as described in Materials and Methods. A significant increase in cytosolic cathepsin B activity was detectable in WT, but not in Bid−/−, cells (#x002A;P < .05, WT vs Bid−/−). (D) Isolated hepatocytes from WT and Bid−/− mice were loaded with LysoTracker Red, treated with TNF-α/AcD for 2 hours, and imaged by confocal microscopy as described in Materials and Methods. Lysosomal permeabilization (as indicated by LysoTracker Red release) was significantly impaired in Bid−/− cells. Cont, control. Gastroenterology 2005 129, 269-284DOI: (10.1053/j.gastro.2005.05.022) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Caspase 2 is activated during TNF-α–induced hepatocyte apoptosis. (A) Isolated hepatocytes from WT and Ctsb−/− mice were incubated in the absence (cont) or presence of TNF-α/AcD for 4 hours, and caspase 2 catalytic activity was measured at the single-cell level as described in Materials and Methods. Caspase 2 activity was significantly increased in WT hepatocytes, but not in Ctsb−/− hepatocytes (#x002A;P < .01, WT vs Ctsb−/−). (B) Procaspase 2 processing was analyzed by immunoblot in cell lysates from McArdle7777 cells after 4 hours of treatment with TNF-α/AcD in the presence or absence of the specific cathepsin B inhibitor R-3032 (20 μmol/L). A representative immunoblot is shown. Equal protein loading was verified by immunoblot for actin. TNF-α–induced processing of procaspase 2 was impaired by the inhibition of cathepsin B, as quantitated by densitometric analysis from 3 separate experiments (70% processed procaspase 2 vs 35% after TNF-α treatment in the absence or presence of R-3032). (C) Caspase 2 expression in McArdle7777 was silenced by siRNA as described in Materials and Methods. Transfection with caspase 2 siRNA efficiently inhibited caspase 2 expression but did not affect the expression of other caspases, such as caspase 3, 7, 8, and 9. (D) Apoptosis was quantitated by morphological criteria in McArdle7777 cells transfected with caspase 2 siRNA (Casp-2) after 4 hours of treatment with TNF-α/AcD. Untransfected McArdle7777 cells (−) and cells mock-transfected with scrambled siRNA (Scr.) were subjected to the same treatment and used as controls. Transfection with caspase 2 siRNA significantly attenuated apoptosis (#x002A;P < .01). Gastroenterology 2005 129, 269-284DOI: (10.1053/j.gastro.2005.05.022) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Caspase 2 activation contributes to mitochondrial dysfunction in TNF-α–induced hepatocyte apoptosis. Untransfected McArdle7777 cells (−) and cells transfected either with scrambled siRNA (Scr.) or with caspase 2 siRNA (Casp-2) were treated with TNF-α/AcD for 6 hours. (A) Mitochondrial membrane potential (Δφ) was measured as described in Materials and Methods. Transfection with caspase 2 siRNA prevented TNF-α–induced loss of Δφ. (B) Cytochrome c release was measured at the indicated time points by immunohistochemical analysis in caspase 2 or untransfected McArdle7777 cells. (C) Quantitation of cells showing diffuse fluorescence, indicative of release of cytochrome c into the cytosol, was performed in a minimum of 5 high-power microscopic fields per each time point and group. Transfection with caspase 2 siRNA significantly reduced and delayed cytochrome c release. Cont, control; MRH, McArdle7777. Gastroenterology 2005 129, 269-284DOI: (10.1053/j.gastro.2005.05.022) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Caspase 2 deficiency attenuates TNF-α–induced apoptosis in murine hepatocytes. Isolated hepatocytes from WT and caspase 2 knockout (C2−/−) mice were incubated with TNF-α/AcD for 8 hours in the presence or absence of the cathepsin B inhibitor R-3032 or the pan-caspase inhibitor Q-VD-OPh (5 μmol/L). (A) Apoptosis was quantitated by morphological criteria after DAPI staining. The data are expressed as -fold increase over the control value, which was arbitrarily set to 1, and represent the mean ± SE of 6 samples from 2 separate isolations. (B) Caspase-3/-7 activity was measured as described in Materials and Methods. The data are expressed as -fold increase of rhodamine fluorescent units (RFLUs) over the control value, which was arbitrarily set to 1, and represent the mean ± SE of 6 samples from 2 separate isolations. Apoptosis was quantitated after 24 hours of treatment with TNF-α/AcD in the presence or absence of cathepsin B or caspase 2 inhibition in primary mouse hepatocytes (C) and McArdle7777 cells (D). The data are representative of at least 3 separate experiments performed in triplicate and are expressed as mean ± SE. Inhibition of cathepsin B or caspase 2 reduces TNF-induced apoptosis in murine cells. Cont, control. Gastroenterology 2005 129, 269-284DOI: (10.1053/j.gastro.2005.05.022) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Bid, cathepsin B, and caspase 2 are part of the same TNF-α–activated signaling pathway that acts upstream of the mitochondria. (A) Isolated hepatocytes from WT, Bid−/−, and Ctsb−/− mice were incubated with or without TNF-α/AcD in the presence or absence of the caspase 2 inhibitor z-VDVAD-fmk (5 μmol/L) for 8 hours, and apoptosis was quantitated by morphological criteria after DAPI staining. Bid−/− and Ctsb−/− mouse hepatocytes were significantly more resistant to TNF-α–induced apoptosis compared with wild-type hepatocytes (#x002A;P < .05; #x002A;#x002A;P < .001). Simultaneous inhibition of caspase 2 reduced apoptosis to levels comparable to those of untreated cells. (B) Cytochrome c release was measured at the indicated time points by immunoblot analysis in cytosolic extracts from WT, Bid−/−, and Ctsb−/− mouse hepatocytes. Release of cytochrome c was greatly reduced in the absence of Bid or cathepsin B and in the presence of the caspase 2 inhibitor. Gastroenterology 2005 129, 269-284DOI: (10.1053/j.gastro.2005.05.022) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Bax activation during TNF-α/AcD–induced hepatocyte apoptosis is cathepsin B independent. (A) McArdle7777 cells were incubated in the absence (cont) or presence (TNF-α) of TNF-α/AcD for 4 hours; an experimental group was also treated with TNF-α/AcD in the presence of the cathepsin B inhibitor R-3032 (TNF-α + R-3032). Active Bax was immunoprecipitated from cell lysates of each group by using a monoclonal antibody for detection of conformationally changed Bax (6A7). Active Bax was not detectable in controls, but it was readily identified in TNF-α–treated cells, both in the presence and absence of the cathepsin B inhibitor R-3032. (B) WT and Ctsb−/− mouse hepatocytes were incubated with TNF-α/AcD for up to 6 hours, and mitochondrial fractions (50 μg) were analyzed by immunoblot for Bax. Prolonged incubation with TNF-α resulted in Bax translocation to the mitochondria in both phenotypes with similar kinetics. (C) Mitochondria from McArdle7777 cells incubated in the absence (control) or presence (TNF-α) of TNF-α and R-3032 (TNF-α + R-3032) for 4 hours were subjected to alkali extraction to produce alkali-sensitive (AS) and alkali-resistant (AR) mitochondrial membrane fractions. Aliquots of 20 μg of both the fractions, together with aliquots of total mitochondrial extracts (M), were analyzed by immunoblot for Bax. Incubation with TNF-α resulted in deep insertion of Bax into the outer mitochondrial membrane, as indicated by the appearance of Bax in the AR fraction (lane 6). Bax insertion in the mitochondrial membrane was not cathepsin B dependent (lane 9). Gastroenterology 2005 129, 269-284DOI: (10.1053/j.gastro.2005.05.022) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Model of TNF-α–activated apoptotic signaling pathways. Engagement of TNF-R1 by TNF-α leads to activation of caspase 8 and cleavage of Bid. Bid, in turn, contributes to both lysosomal permeabilization with consequent release of cathepsin B into the cytosol and mitochondrial permeabilization, likely by facilitating Bax insertion onto the mitochondrial membrane. Cytosolic cathepsin B activates caspase 2, thus resulting in amplification of mitochondria permeabilization with the release of apoptogenic factors such as cytochrome c and activation of a caspase cascade. Activation of parallel pathways may account for the partial permeabilization of mitochondria in the absence of either Bid or cathepsin B. Gastroenterology 2005 129, 269-284DOI: (10.1053/j.gastro.2005.05.022) Copyright © 2005 American Gastroenterological Association Terms and Conditions