Distinct involvement of NF-kappa B and p38 mitogen-activated protein kinase pathways in serum deprivation-mediated stimulation of inducible nitric oxide synthase and its inhibition by 4-hydroxynonenal

Cytokine-induced expression of inducible nitric oxide synthase (iNOS) and c oncomitant production of nitric oxide (NO) involve activation of mitogen-ac tivated protein (MAP) kinases and are in most cases mediated by the transcr iption factor NF-KB. We investigated the role of p38 MAP kinase activation and I kappaB phosphorylation in iNOS expression in a novel iNOS-inducing mo del in mouse macrophages. Deprivation of serum from the culture medium of R AW 264.7 cells up-regulated iNOS and NO production, which were inhibited by 4-hydroxy-2-nonenal (HNE), a component of oxidatively modified low-density lipoprotein (oxLDL). Serum withdrawal induced phosphorylation of Akt, I ka ppaB, and p38 MAP kinase. Pretreatment with the potent P13 kinase inhibitor wortmannin, the NF-KB inhibitor PDTC or the specific p38 MAP kinase inhibi tor SB203580 each partially attenuated the induction of iNOS and NO product ion, demonstrating that both p38 activation and I kappaB phosphorylation ar e required for iNOS expression. SB203580, however, did not prevent the phos phorylation of Akt and I kappaB, suggesting that the p38 MAP kinase signal contributes to iNOS gene expression through an I kappaB-phosphorylation-ind ependent pathway. HNE, which markedly inhibited iNOS expression and NO prod uction, prevented the serum withdrawal-triggered I kappaB phosphorylation b ut not that of Akt or p38 MAP kinase. A high concentration of HNE stimulate d dephosphorylation of I kappaB but promoted activation of p38 MAP kinase. Taken together, those results suggest that NF-kappaB and p38 MAP kinase lie in separate signal pathways for serum deprivation-stimulated iNOS expressi on and NO production. HNE selectively suppresses the former pathway, target ing a site downstream of Akt. (C) 2001 Wiley-Liss, Inc.