Distinct involvement of NF-kappaB 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 concomitant production of nitric oxide (NO) involve activation of mitogen-activated protein (MAP) kinases and are in most cases mediated by the transcription factor NF-kappaB. We investigated the role of p38 MAP kinase activation and IkappaB phosphorylation in iNOS expression in a novel iNOS-inducing model in mouse macrophages. Deprivation of serum from the culture medium of RAW 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, IkappaB, and p38 MAP kinase. Pretreatment with the potent PI3 kinase inhibitor wortmannin, the NF-kappaB inhibitor PDTC or the specific p38 MAP kinase inhibitor SB203580 each partially attenuated the induction of iNOS and NO production, demonstrating that both p38 activation and IkappaB phosphorylation are required for iNOS expression. SB203580, however, did not prevent the phosphorylation of Akt and IkappaB, suggesting that the p38 MAP kinase signal contributes to iNOS gene expression through an IkappaB-phosphorylation-independent pathway. HNE, which markedly inhibited iNOS expression and NO production, prevented the serum withdrawal-triggered IkappaB phosphorylation but not that of Akt or p38 MAP kinase. A high concentration of HNE stimulated dephosphorylation of IkappaB but promoted activation of p38 MAP kinase. Taken together, these results suggest that NF-kappaB and p38 MAP kinase lie in separate signal pathways for serum deprivation-stimulated iNOS expression and NO production. HNE selectively suppresses the former pathway, targeting a site downstream of Akt. Copyright 2001 Wiley-Liss, Inc.