The role of protein kinase C isozymes in TNF-alpha-induced cytotoxicity to a rat intestinal epithelial cell line.
Tumor necrosis factor (TNF)-alpha can induce cytotoxicity and apoptosis in a number of cell types and has been implicated in the regulation of many inflammatory processes. It has been suggested that protein kinase C (PKC) is one of the intracellular mediators of the actions of TNF-alpha. In the present study, the role of PKC isoforms in TNF-alpha-mediated cytotoxicity and apoptosis in intestinal cells was investigated using the rat epithelial cell line, IEC-18. Cells were incubated with TNF-alpha in the presence or absence of the transcription inhibitor actinomycin D (AMD). The extent of cell damage was enhanced when AMD was added to incubation medium, suggesting that new protein synthesis plays a role in the cytotoxic action of TNF. TNF-alpha also induced the translocation of PKC-alpha, -delta, and -epsilon from cytosol to the membrane fraction of the intestinal cells. Furthermore, the cytotoxic and apoptotic effects of TNF were reduced by pretreating the cells with the PKC-epsilon translocation inhibitor, PKC-epsilonV1-2. In contrast, although cells incubated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) also displayed an increase in cell injury, the extent of cytotoxicity and apoptosis was not enhanced by AMD. Furthermore, PMA-induced cell damage was reduced by rottlerin, a PKC-delta inhibitor. Caspase-3, an enzyme implicated in the mediation of apoptosis, was activated in cells in response to either TNF-alpha or PMA stimulation, and its effects on this activity were reduced by selective inhibition of PKC-epsilon and -delta, respectively. Furthermore, inhibition of caspase-3 activity reduced apoptosis. These data suggest that activation of selective PKC isoforms mediate the effects of TNF-alpha on intestinal epithelial cell injury.
Department of Physiology, University of Western Ontario, London, Ontario, Canada N6A 5C1.
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Chang
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Tepperman
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D053938:DNA Fragmentation; D003609:Dactinomycin / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects*; D019085:Fluorescent Antibody Technique, Direct; D006801:Humans; D015151:Immunoblotting; D007422:Intestines / Q000166:cytology*; D007527:Isoenzymes / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D007527:Isoenzymes; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate