Tumor necrosis factor - α - induced activation of protein kinase c isoforms is mediated by phospholipid second messengers and not by calcium in human non-pigmented ciliary epithelial cells

Purpose. To determine the effect of tumor necrosis factor-α (TNF-α) on the activation of protein kinase C isoforms in SV-40 transformed human non-pigmented epithelial (HNPE) cells. Methods. HNPE cells were used for PKC translocation assays and inlracellular calcium ([Ca²⁺]_i) measurements. Incubation times ranged from 1 - 60 minutes. Control cells were untreated throughout the incubation period. Western blotting was performed using monoclonal/polyclonal antibodies against PKCa, PKC6, PKCE and PKC; for cytosolic (CS) and plasma membrane (PM) fractions of HNPE cells. Intracellular calcium measurements were made using a fura-2 sensitive imaging system. Results. In HNPE cells, [Ca;'], levels did not increase following treatments with TNF-a or phorbol esters (PMA). However, TNF-a and PMA, were both able to translocate PKCa from CS to PM within 10-30 minutes. PMA did not effectively induce the translocation of other PKC isoforms within 30 minutes of incubation. PKC6 isoform translocated within 30 minutes of incubation with TNF-a. TNF-a, however, had no effect on PKCe and PKC; isoforms. Ch-Ceramide and D-609 (an inhibitor of phospholipases C and D), prevented the translocation of all four PKC isoforms in the presence of PMA or TNF-a. Conclusions. PMA is a potent translocator of the classical PKC (cPKC) isoforms in HNPE cells. TNF-a activates cPKCs and novo PKCs in a time dependent manner possibly by increasing the diacylglycerol pool and not by the mobilization of intracellular calcium.