REGULATION OF INTERLEUKIN-1 AND TUMOR-NECROSIS-FACTOR-ALPHA INDUCED GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE-EXPRESSION - POTENTIAL INVOLVEMENT OF ARACHIDONIC-ACID METABOLISM

Signal transduction pathways evoked by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) to stimulate expression of other cyt okines in mesenchymal cells are not clearly understood. Stimulation of the murine bone marrow stromal cell line +/+-1.LDA 11 with IL-1 (500 U/ml) in combination with TNF-alpha (500 U/ml) (IL-1 plus TNF-alpha) i nduced expression of c-jun mRNA as well as granulocyte-macrophage colo ny stimulating factor (GM-CSF) mRNA. We investigated the possibility t hat arachidonic acid metabolites, acting through protein kinase C (PKC ) and perhaps also through the PKC-responsive transcription factor c-j un/AP-1, may be responsible for regulating GM-CSF transcription in the se stromal cells. Expression of GM-CSF mRNA was preceded by IL-1 plus TNF-alpha induced arachidonate release (assayed using the H-3-derivati ve). Pretreatment of cells with the phospholipase A(2) inhibitor quina crine (20 mu M) inhibited accumulation of both c-jun and GM-CSF mRNA b ut had no influence on expression of other genes induced by IL-1 and T NF-alpha, including leukemia inhibitory factor (LIF). In addition, qui nacrine partially blocked IL-1 plus TNF-alpha induced H-3-arachidonic acid release from prelabeled stromal cells. Furthermore, exogenous ara chidonate (10 to 50 mu M) induced expression of c-jun. To investigate the role of arachidonate in GM-CSF transcription, we used a reporter v ector consisting of the murine GM-CSF promoter linked to firefly lucif erase. Transfection efficiency was monitored by assessing expression o f a constitutively active gene, RSV-beta galactosidase. In this system , quinacrine significantly inhibited IL-1 plus TNF-alpha induced GM-CS F transcription assayed with the reporter construct. Exogenous arachid onic acid alone (10 mu M) increased activity of GM-CSF reporter vector 1.5-fold over control. These results are consistent with the hypothes is that arachidonate metabolites are involved in the signaling pathway that leads to IL-1 plus TNF-alpha induced GM-CSF gene expression. Thu s, transcriptional activation of GM-CSF gene is mediated, in part, by the arachidonate cascade.