FAS-MEDIATED APOPTOSIS IN MOUSE HEPATOCYTES INVOLVES THE PROCESSING AND ACTIVATION OF CASPASES

The mechanism of Fas antigen-induced hepatocyte apoptosis was investig ated. Using a monoclonal antibody directed against the Fas antigen, ap optosis was induced in freshly isolated murine hepatocytes within 90 m inutes of antibody addition as assessed by plasma membrane bleb format ion, chromatin condensation, and DNA fragmentation. Pretreatment of th e cells with the caspase inhibitors, N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), benzyl oxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone ( Z-VAD-FMK), or Z-Asp-2,6-dichlorobenzuyloxymethylketon inhibited anti- fas-mediated apoptosis. Likewise, the serine protease inhibitors, N-to syl-L-phenyl chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (D Cl), prevented apoptosis, whereas N-tosyl-L-lysine chloromethyl ketone (TLCK), Ac-Leu-Leu-L-norleucinal, Ac-Leu-Leu-L-methional, and ns-epox ysuccinyl-L-leucylamido-(4-guanidino)butane were without effect. Exami nation of CED-3/caspase-3-related caspases revealed that pro-caspases- 3 (CPP32) and -7 (Mch-3 alpha) were rapidly processed after Fas antige n stimulation. Caspase-7 was further cleaved to form the catalytically active subunits. In contrast, the p17 subunit of caspase-3 was not de tected, indicating slow formation or rapid degradation. The activation of CED-3-related caspases was further confirmed by an increase in the rate of Z-DEVD-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC) hydroly sis that was sensitive to Ac-DEVD-CHO and was inhibited by pretreatmen t of the cells with TPCK but not by DCI. In contrast, no increase in t he rates of hydrolysis of Z-YVAD-AFC, a substrate for caspase-1, was d etected. Investigation of the in situ proteolytic cleavage of the CED- 3 related caspases substrate, poly(ADP-ribose) polymerase, revealed th at this protein was not degraded in hepatocytes undergoing Fas-mediate d apoptosis. Taken together, our results show that processing of caspa ses, in particular, caspases-7 and -3, occurs during Fas-induced apopt osis of mouse hepatocytes and suggest a role of these proteases as wel l as serine protease(s) in the apoptotic response.