Processing/activation of CPP32-like proteases is involved in transforming growth factor β1-induced apoptosis in rat hepatocytes

Salmaan H. Inayat-Hussain, Carole Couet, Gerald M. Cohen, Kelvin Cain

    Research output: Contribution to journalArticle

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    Abstract

    Apoptosis induced in rat hepatocytes by transforming growth factor β1 (TGF-β1) was accompanied by the activation of interleukin-1β converting enzyme (ICE)dike proteases. Cell lysates were isolated at various times after TGF-β1 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-β1-treated cells, which showed limited processing of CPP32 as shown by the appearance of the catalytically active p17 subunit. Loss of pro-Mch3α 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/Mch3α processing as shown by the appearance of the p17/p19 subunits and the corresponding increase in CPP32-like protease activity. Apoptosis, CPP32/Mch3α processing and the increase in CPP32-like protease activity induced by TGF-β1 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-β1-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 Mch3α, which are activated during the execution phase of hepatocyte apoptosis.

    Original languageEnglish
    Pages (from-to)1516-1526
    Number of pages11
    JournalHepatology
    Volume25
    Issue number6
    DOIs
    Publication statusPublished - 1997

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    Transforming Growth Factors
    Hepatocytes
    Peptide Hydrolases
    Apoptosis
    Caspase 1
    Cycloheximide
    Staurosporine
    Hepatocyte Growth Factor
    Sodium Dodecyl Sulfate
    Polyacrylamide Gel Electrophoresis
    Western Blotting
    Peptides

    ASJC Scopus subject areas

    • Hepatology

    Cite this

    Processing/activation of CPP32-like proteases is involved in transforming growth factor β1-induced apoptosis in rat hepatocytes. / Inayat-Hussain, Salmaan H.; Couet, Carole; Cohen, Gerald M.; Cain, Kelvin.

    In: Hepatology, Vol. 25, No. 6, 1997, p. 1516-1526.

    Research output: Contribution to journalArticle

    Inayat-Hussain, Salmaan H. ; Couet, Carole ; Cohen, Gerald M. ; Cain, Kelvin. / Processing/activation of CPP32-like proteases is involved in transforming growth factor β1-induced apoptosis in rat hepatocytes. In: Hepatology. 1997 ; Vol. 25, No. 6. pp. 1516-1526.
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