The role of protein kinase C isozymes in TNF-alpha-induced cytotoxicity toa 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 in flammatory processes. It has been suggested that protein kinase C (PKC) is one of the intracellular mediators of the actions of TNF-alpha. In the pres ent study, the role of PKC isoforms in TNF-alpha -mediated cytotoxicity and apoptosis in intestinal cells was investigated using the rat epithelial ce ll line, IEC-18. Cells were incubated with TNF-alpha in the presence or abs ence of the transcription inhibitor actinomycin D (AMD). The extent of cell damage was enhanced when AMD was added to incubation medium, suggesting th at new protein synthesis plays a role in the cytotoxic action of TNF. TNF-a lpha 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 cell s with the PKC-epsilon translocation inhibitor, PKC-epsilon V1-2. In contra st, although cells incubated with the phorbol ester phorbol 12-myristate 13 -acetate (PMA) also displayed an increase in cell injury, the extent of cyt otoxicity and apoptosis was not enhanced by AMD. Furthermore, PMA-induced c ell 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 ac tivity were reduced by selective inhibition of PKC-epsilon and -delta, resp ectively. Furthermore, inhibition of caspase-3 activity reduced apoptosis. These data suggest that activation of selective PKC isoforms mediate the ef fects of TNF-alpha on intestinal epithelial cell injury.