Arachidonic acid increases c-fos and Egr-1 mRNA in 3T3 fibroblasts by formation of prostaglandin E2 and activation of protein kinase C.

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Studying Swiss 3T3 fibroblasts, we report that arachidonic acid strongly stimulates mRNA levels of the growth-associated immediate early genes c-fos and Egr-1. Structurally related compounds like arachidonic acid methyl ester, arachidonyl alcohol, or eicosatetraynoic acid are ineffective, indicating a specific role of free unesterified arachidonic acid or an arachidonic acid metabolite in c-fos and Egr-1 mRNA accumulation. Blocking the conversion of arachidonic acid to prostaglandins by inhibiting cyclooxygenase abolishes arachidonic acid-induced accumulation of c-fos and Egr-1 mRNA. Inhibition of the lipoxygenase or cytochrome P-450 epoxygenase pathways has no significant effect on arachidonic acid-induced c-fos and Egr-1 mRNA levels, indicating that prostaglandin synthesis is necessary for the increase in c-fos and Egr-1 mRNA. Reversed phase high performance liquid chromatography revealed prostaglandin E2 (PGE2) as the major arachidonic acid metabolite in Swiss 3T3 fibroblasts. When added to the cells, PGE2 stimulates c-fos and Egr-1 mRNA levels to the same degree as arachidonic acid. Also, the inhibition of arachidonic acid-stimulated c-fos and Egr-1 mRNA accumulation by indomethacin is reversed by PGE2. Contrary to reports that PGE2 caused an increase in cAMP levels in Swiss 3T3 fibroblasts, we found that arachidonic acid and PGE2 only minimally increase cAMP levels as compared with untreated cells. In contrast, inhibition of protein kinase C by calphostin C and chelerythrine or down-regulation with phorbol 12-myristate 13-acetate drastically reduces PGE2 and arachidonic acid-induced c-fos and Egr-1 mRNA levels. These data indicate that arachidonic acid exerts its stimulatory effect on c-fos and Egr-1 mRNA via synthesis of PGE2 and subsequent activation of protein kinase C, probably through a PGE2 receptor coupled to phospholipase C.