p38 MAPK-mediated transcriptional activation of inducible nitric-oxide synthase in glial cells. Roles of nuclear factors, nuclear factor kappa B, cAMP response element-binding protein, CCAAT/enhancer-binding protein-beta, and activating transcription factor-2.

Previous studies have shown that mitogen-activated protein kinase (MAPK) cascades signal the induction of inducible nitric-oxide synthase (iNOS) in glial cells (Bhat, N. R., Zhang, P., Lee, J. C., and Hogan E. L. (1998) J. Neurosci. 18, 1633-1641; Bhat, N. R., Zhang, P., and Bhat, A. N. (1999) J. Neurochem. 72, 472-478). This study further investigates the role of p38 MAPK in the transcriptional activation of the iNOS gene by transient transfection with constitutively active upstream kinases in the pathway (i.e. MAPK kinase 3 (MKK3b(E)) and MAPK kinase 6 (MKK6b(E)). Expression in C-6 glial cells of either MKK3b(E) or MKK6b(E) resulted in an induction of the activity of a cotransfected rat iNOS promoter-reporter (iNOS-luciferase (Luc)) gene and an enhancement of cytokine-induced expression of iNOS mRNA, both of which were inhibitable by the p38 MAPK inhibitor SB203580. The MKK constructs also induced cAMP response element-mediated (CRE-Luc) and nuclear factor kappa B-dependent (nuclear factor kappa B-Luc) transcriptional activities. Transfection with dominant negative (dn) forms of CRE-binding protein (CREB) and CCAAT/enhancer-binding protein (C/EBP), the two CRE-binding transcription factors targeted by the p38 MAPK pathway, resulted in opposite effects; dnCREB enhanced and dnC/EBP inhibited iNOS-Luc parallel to their effects on CRE-Luc. In addition, the induction, by MKK3b(E) and MKK6b(E), of iNOS promoter activity was enhanced by a wild-type activating transcription factor (ATF-2), whereas a phosphorylation-defective form of ATF-2 had a suppressive effect. The results of these molecular studies provide evidence for an important role for the p38 MAPK pathway in the transcriptional activation of the iNOS gene in rat glial cells involving the transcription factors nuclear factor kappa B, C/EBP, and ATF-2.