Interleukin-1 beta regulation of inducible nitric oxide synthase and cyclooxygenase-2 involves the p42/44 and p38 MAPK signaling pathways in cardiac myocytes

The genes encoding inducible nitric oxide synthase (iNOS) and cyclooxygenas e-2 (COX-2, also known as prostaglandin-endoperoxide synthase-2) are induce d in many types of cells in response to proinflammatory cytokines, We have previously shown that interleukin-1 beta (IL) stimulates iNOS and COX-2 mRN A in cardiac myocytes. Because IL has been shown to activate mitogen-activa ted protein kinase (MAPK) signaling pathways in many different cells, we te sted whether the p42/44 and p38 MAPK pathways were involved in IL stimulati on of iNOS and COX-2, using a specific inhibitor of p42/44 activation, PD98 059 (PD), and the p38 inhibitor SB205380 (SB). Nitrites were measured using the Griess reagent, prostaglandin PGE(2) by an enzyme immunoassay, iNOS an d COX-2 protein by Western blot analysis, and iNOS mRNA by Northern blot an alysis: Tested separately, the p38 kinase and MAPK inhibitors partially red uced IL stimulation of nitrite, iNOS protein, and iNOS mRNA; used together, they completely abolished the effect of IL. SE and PD inhibited IL-stimula ted COX-2 protein by 60% and 80%, respectively, and IL-stimulated COX-2 pro tein was totally prevented by the combination of inhibitors. PGE(2) product ion was inhibited more than 99% by either drug alone, suggesting a posttran slational effect on enzyme activity, To test whether this posttranslational effect involved the cytosolic phospholipase A(2) (cPLA(2)) isoform, Wester n blots were probed for cPLA(2) protein. Results indicated that IL stimulat ed cPLA(2) activity and synthesis, which was inhibited by SE but not PD, Th ese data indicate that (1) IL induction of iNOS synthesis depends on both t he p42/44 and p38 signaling pathways, acting primarily at the level of tran scriptional regulation; and (2) IL regulation of COX-2 synthesis involves t he p42/44 and p38 signaling pathways, with an additional level of regulatio n occurring posttranslationally, perhaps at the level of activation of the cPLA(2) isoform, which may be involved in intracellular signaling, as well as regulation of arachidonic acid release for COX-2 activity.