Role of NF- kappaB binding sites in the regulation of inducible nitric oxide synthase by tyrosine kinase

ABSTRACT

In macrophages, lipopolysaccharide (LPS) alone or in combination with interferon- gamma (IFN- gamma) has been shown to release a nitric oxide (NO) through the increase of the transcription of the inducible nitric oxide synthase (iNOS) gene. To investigate the exact intracellular signaling pathway of the regulation of iNOS gene transcription by LPS plus IFN- gamma, the effects of protein tyrosine kinase (PTK) inhibitor and protein kinase C (PKC) inhibitors on NO production, iNOS mRNA expression, nuclear factor- kappaB (NF- kappaB) binding activity and the promoter activity of iNOS gene containing two NF- kappaB sites have been examined in a mouse macrophage RAW 264.7 cells. LPS or IFN- gamma, stimulated NO production, and their effect was enhanced synergistically by mixture of LPS and IFN- gamma. The PTK inhibitor such as tyrphostin reduced LPS plus IFN- gamma-induced NO production, iNOS mRNA expression and NF- kappaB binding activity. In contrast, PKC inhibitors such as H-7, Ro-318220 and staurosporine did not show any effect on them. In addition, transfection of RAW 264.7 cells with iNOS promoter linked to a CAT reporter gene revealed that tyrphostin inhibited the iNOS promoter activity through the NF- kappaB binding site, whereas PKC inhibitors did not. Taken together, these suggest that PTK, but not PKC pathway, is involved in the regulation of the iNOS gene transcription through the NF- kappaB sites of iNOS promoter in RAW 264.7 macrophages by LPS plus IFN- gamma.