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Positive and negative regulation of a tumor necrosis factor response in melanoma cells
1Department of Biological Sciences, State University of New York, Albany 12222.
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.
Insights
Tumor necrosis factor (TNF) signaling in melanoma cells involves protein kinase C (PKC) activation and cyclic adenosine monophosphate (cAMP) inhibition to regulate plasminogen activator inhibitor type 2 (PAI-2) production.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Cancer research
Background:
- Tumor necrosis factor (TNF) triggers cellular responses via surface receptors, but downstream signaling remains unclear.
- Understanding TNF-induced signaling is crucial for targeted cancer therapies.
- Plasminogen activator inhibitor type 2 (PAI-2) is implicated in melanoma cell behavior.
Purpose of the Study:
- To investigate the roles of protein kinase C (PKC) and cyclic adenosine monophosphate (cAMP) in TNF-induced PAI-2 production in SK-MEL-109 melanoma cells.
- To elucidate the signaling mechanisms by which TNF regulates PAI-2 synthesis and mRNA expression.
Main Methods:
- Utilized PKC activators (phorbol myristate acetate) and inhibitors (staurosporine, HCL) to assess PAI-2 induction.
- Measured PAI-2 mRNA accumulation and protein synthesis in response to TNF and modulators.
- Investigated the effect of cAMP-elevating agents on TNF-induced PAI-2 responses.
- Analyzed the translocation of phorbol dibutyrate binding in TNF-treated cells.
Main Results:
- PKC activation by phorbol myristate acetate induced PAI-2 synthesis and mRNA expression.
- PKC inhibitors blocked TNF-induced PAI-2 synthesis and mRNA accumulation.
- TNF treatment caused a transient redistribution of phorbol dibutyrate binding from cytosol to membrane fractions.
- cAMP-elevating agents inhibited TNF-induced PAI-2 synthesis and mRNA accumulation, suggesting effects at both transcriptional and translational levels.
Conclusions:
- PKC positively regulates TNF-induced PAI-2 production in melanoma cells.
- cAMP negatively modulates TNF-induced PAI-2 production, acting at both mRNA and protein levels.
- The interplay between PKC and cAMP signaling provides a mechanism for coordinating cellular responses to TNF.