Processing/activation of CPP32-like proteases is involved in transforming growth factor beta(1)-induced apoptosis in rat hepatocytes

Apoptosis induced in rat hepatocytes by transforming growth factor beta(1) (TGF-beta(1)) was accompanied by the activation of interleukin-1 beta converting enzyme (ICE)-like proteases. Cell lysates were isolated at various times after TGF-beta(1) treatment and analyzed for ICE and CPP32-like activity, using N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (Ac-YVAD.AMC)and benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-3-trifluorome (Z-DEVD.AFC), respectively, CPP32-like but not ICE protease activity increased in a time dependent manner and preceded the onset of apoptosis. Kinetic studies in cell lysates indicated that more than one CPP32-like protease was being activated. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting of TGF-beta(1)-treated cells, which showed limited processing of CPP32 as shown by the appearance of the catalytically active p17 subunit. Loss of pro-Mch3 alpha was also observed but the catalytically active p19 subunit was not detected. Staurosporine, which induced a much greater level of hepatocyte apoptosis, produced a concomitant increase in CPP32/Mch3 alpha processing as shown by the appearance of the p17/p19 subunits and the corresponding increase in CPP32-like protease activity. Apoptosis, CPP32/Mch3 alpha processing and the increase in CPP32-like protease activity induced by TGF-beta(1) and staurosporine were abolished in hepatocytes pretreated with Z-Asp-Glu-Val-Asp (OMe) fluoromethylketone (Z-DEVD.FMK) or Z-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK). These peptide analogues were potent inhibitors of CPP32-like protease activity in lysates. Pretreatment of hepatocytes with cycloheximide also blocked TGF-beta(1)-induced apoptosis and the increase in CPP32-like activity. Unlike Z-VAD.FMK and Z-DEVD.FMK, cycloheximide did, not inhibit CPP32-like protease activity in cell lysates. Thus, cycloheximide may block apoptosis by inhibiting the synthesis of a protein, which is involved in the upstream events responsible for the activation of the CPP32-like protease activity. Our studies have identified two of the CPP32-like proteases, namely CPP32 and Mch3 alpha, which are activated during the execution phase of hepatocyte apoptosis.