Abstract
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.
Highlights
PAI- and transcription of its mRNA could be induced by a protein kinase C (PKC) activator, phorbol myristate acetate
Positive Regulation of PAI- Induction by Tumor necrosis factor (TNF)-To study the signal transduction pathway of the TNF receptor, we monitored a specific response previously described in SKMEL-109 melanoma cells: the synthesis of PAI- that is readily observed by SDS-PAGE [15]
The results described in this report point to PKC as a mediator of a TNF response, the induction of PAI- in SK-MEL109 melanoma cells
Summary
Material-Recombinant human TNF wasa gift of Dr T. Assay-TNF was iodinated to a specific activity of >lOO Ci/g [17] and binding assays were carried out with cells in suspension as previously described [14]. Deionized glyoxal was added to 20% and RNA was denatured for 5 min at 65 ‘C and fractionated by electrophoresis in 1% agarose gels. Confluent SK-MEL-109 cells in 175-cm’ flasks were placed in medium with 1% serum for 24 h, treated with TNF in serum-free medium, washed with ice-cold PBS, scraped off with a rubber policeman in a small volume of PBS, and collected by centrifugation. The protein concentration was determined with the bicinchoninic acid protein assay (Pierce Chemical Co.), and 100-200 .ug of protein from each fraction was aliquoted into microtubes containing an equal volume of 2 x binding medium Specific binding for each time point was determined by subtracting nonspecific binding in triplicate assays
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