Processing/activation of CPP32-like proteases is involved in transforming growth factor beta1-induced apoptosis in rat hepatocytes

Hepatology. 1997 Jun;25(6):1516-26. doi: 10.1002/hep.510250634.

Abstract

Apoptosis induced in rat hepatocytes by transforming growth factor beta1 (TGF-beta1) was accompanied by the activation of interleukin-1beta converting enzyme (ICE)-like proteases. Cell lysates were isolated at various times after TGF-beta1 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-4-trifluoromethylcoumarin (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-beta1-treated cells, which showed limited processing of CPP32 as shown by the appearance of the catalytically active p17 subunit. Loss of pro-Mch3alpha 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/Mch3alpha processing as shown by the appearance of the p17/p19 subunits and the corresponding increase in CPP32-like protease activity. Apoptosis, CPP32/Mch3alpha processing and the increase in CPP32-like protease activity induced by TGF-beta1 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-beta1-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 Mch3alpha, which are activated during the execution phase of hepatocyte apoptosis.

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Apoptosis / physiology*
  • Caspase 3
  • Caspases*
  • Coumarins / chemistry
  • Coumarins / metabolism
  • Cycloheximide / pharmacology
  • Cysteine Endopeptidases / metabolism*
  • Endopeptidases / metabolism*
  • Enzyme Activation
  • Enzyme Induction
  • Kinetics
  • Liver / cytology
  • Liver / drug effects
  • Liver / physiology*
  • Male
  • Oligopeptides / chemistry
  • Oligopeptides / metabolism
  • Poly(ADP-ribose) Polymerases / chemistry
  • Poly(ADP-ribose) Polymerases / metabolism
  • Protein Processing, Post-Translational*
  • Protein Synthesis Inhibitors / pharmacology
  • Rats
  • Rats, Inbred F344
  • Transforming Growth Factor beta / pharmacology*

Substances

  • Coumarins
  • Oligopeptides
  • Protein Synthesis Inhibitors
  • Transforming Growth Factor beta
  • benzyloxycarbonyl-aspartyl-glutamyl-valyl-aspartyl-7-amino-4-trifluoromethylcoumarin
  • Cycloheximide
  • Poly(ADP-ribose) Polymerases
  • Endopeptidases
  • Casp3 protein, rat
  • Caspase 3
  • Caspases
  • Cysteine Endopeptidases