The involvement of protein kinase C, calcium, and 5-lipoxygenase in the production of tumor necrosis factor by a cloned interleukin-3 dependent cell line with natural cytotoxic activity.
The cloned interleukin-3 dependent cell line, M1-A5 was studied to determine whether protein kinase C, calcium mobilization, and 5-lipoxygenase activity were involved in the signal transduction pathways required for the production of TNF. TNF release was stimulated by 10 ng/ml phorbol myristate acetate (PMA), 2 microM calcium ionophore A23187, and 1 microgram/ml lipopolysaccharide (LPS) with synergism seen between PMA and A23187. All signals were blocked by phloretin and the PMA signal was blocked by H-7, both drugs acting as protein kinase C inhibitors. Desensitization of protein kinase C by PMA (1 microgram/ml for 24 h) provided evidence that both PMA- and LPS-stimulated TNF production were protein kinase C-dependent while A23187-stimulated TNF production was not. Both the calcium chelator, EGTA, and the intracellular calcium antagonist, TMB-8, inhibited TNF production stimulated by all agents, indicating that TNF stimulation by all agents was calcium dependent. Finally, the 5-lipoxygenase inhibitors, ketoconazole and L-656,224, but not the cyclo-oxygenase inhibitor ASA, inhibited TNF stimulated by all agents. These findings indicate that, although TNF production by M1-A5 cells can be stimulated either by a calcium/protein kinase C- or by a calcium-dependent signal, there is a convergence of signals at the level of 5-lipoxygenase activation.
MacIntyre
J P
JP
Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.
Pope
B L
BL
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002999:Clone Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D004533:Egtazic Acid / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D016859:Lipoxygenase Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D002121:Calcium Channel Blockers; D007377:Interleukin-3; D016859:Lipoxygenase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D004533:Egtazic Acid; D001094:Arachidonate 5-Lipoxygenase; D011493:Protein Kinase C; D002118:Calcium