Positive and negative regulation of a tumor necrosis factor response in melanoma cells.

Tumor necrosis factor (TNF) elicits a wide variety of responses in target cells by binding to cell surface receptors, but the signal transduced from these receptors in unclear. We examined the role of two different second messenger systems in the regulation of plasminogen activator inhibitor, type 2 (PAI-2) induction by TNF in SK-MEL-109 melanoma cells. Synthesis of PAI-2 and transcription of its mRNA could be induced by a protein kinase C (PKC) activator, phorbol myristate acetate. In addition, induction of PAI-2 synthesis by TNF was blocked by two PKC inhibitors, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride. The inhibitor of cyclic nucleotide-dependent protein kinases, N-[2-(methylamino)-ethyl]-5-isoquinoline sulfonamide dihydrochloride, was much less effective in decreasing PAI-2 synthesis. Staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride also inhibited both TNF- and phorbol myristate acetate-induced PAI-2 mRNA accumulation. We measured the binding of 3H-labeled phorbol dibutyrate to membrane and cytosol fractions of TNF-treated SK-MEL-109 cells and found a transient redistribution of 3H-labeled phorbol dibutyrate binding from cytosol to membrane fractions in response to TNF. In contrast to the positive regulation by PKC in promoting TNF-induced PAI-2 synthesis cAMP inhibited this response. Pretreatment of cells with agents that raise intracellular cAMP levels completely abolished TNF-induced PAI-2 synthesis. Addition of cAMP-elevating agents during TNF induction could also block PAI-2 synthesis. PAI-2 mRNA accumulation in response to TNF was inhibited, but not completely abolished, by cAMP-elevating agents, suggesting that cAMP also exerted its inhibitory effect at the translation level. The positive regulation of a TNF response by PKC and its negative modulation by cAMP may provide a means for intracellular coordination of signals from interacting extracellular factors in regulating TNF responses in different target cells.