p38 but not p44/42 mitogen-activated protein kinase is required for nitricoxide synthase induction mediated by lipopolysaccharide in RAW 264.7 macrophages

Protein kinase C (PKC)-alpha, -beta 1, and -delta are known to be involved in the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. The role of mitogen-activated protein kinases (MAPK) p4 4/42 and p38 in the LPS effect was studied further. LPS-mediated NO release and the inducible form of NO synthase expression were inhibited by the p38 inhibitor, SE 203580, but not by the MARK kinase inhibitor, PD 98059. Ten- minute treatment of cells with LPS resulted in the activation of p44/42 MAR K, p38, and c-Jun NH2-terminal kinase. Marked or slight activation, respect ively, of p44/42 MAPK or p38 was also seen after 10-min treatment with 12-O - tetradecanoylphorbol-13-acetate, but c-Jun NH2-terminal kinase activation did not occur. Tyrosine kinase inhibitor,genestein, attenuated the LPS-ind uced activation of both p44/42 MARK and p38, whereas the PKC inhibitors, Ro 31-8220 and calphostin C, or long-term treatment with 12-O-tetradecanoy-ph orbol- 13-acetate resulted in inhibition of p44/42 MARK activation, but had only a slight effect on p38 activation, indicating that LPS-mediated PKC a ctivation resulted in the activation of p44/42 MAPK. Nuclear factor-kappa B (NF-kappa B)-specific DNA-protein-binding activity in the nuclear extracts was enhanced by 10-min, 1-h, or 24-h treatment with LPS. Analysis of the p roteins involved in NF-kappa B binding showed translocation of p65 from the cytosol to the nucleus after 10-min treatment with LPS. The onset of NF-ka ppa B activation correlated with the cytosolic degradation of both inhibito ry proteins of NF-kappa B, I kappa B-alpha and I kappa B-beta. I kappa B-al pha. was resynthesized rapidly after loss (1-h LPS treatment), whereas I ka ppa B-beta levels were not restored until after 24-h treatment. SE 203580 b ut not PD 98059 inhibited the LPS-induced stimulation of NF-kappa B DNA-pro tein binding. Thus, activation of p38 but not p44/42 MARK by LPS resulted i n the stimulation of NF-kappa B-specific DNA-protein binding and the subseq uent expression of inducible form of NO synthase and NO release in RAW 264. 7 macrophages.