title: Cytokine-activated p42/p44 MAP kinase is involved in inducible nitric oxide synthase gene expression independent from NF-kappaB activation in vascular smooth muscle cells.
abstract: Recent evidence suggests the possible involvement of inducible nitric oxide synthase (iNOS) in the development and maintenance of hypertension in certain animal models. Inflammatory cytokines activate nuclear factor (NF)-kappaB, which plays a major role in transactivation of the inducible nitric oxide synthase (iNOS) gene. However, it remains unknown whether cytokine-mediated iNOS expression in vascular smooth muscle cells (VSMCs) requires signaling pathway(s) other than NF-kappaB activation. The purpose of this study was to determine whether the p42/p44 MAP kinase pathway is involved in cytokine-induced NF-kappaB activation and/or iNOS expression in cultured rat VSMCs. Nitrite/nitrate (NOx) production stimulated by interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha in VSMCs was markedly suppressed by inhibiting MAP kinase by pretreatment with a p42/p44 MAP kinase kinase (MAPKK)-1 inhibitor (PD98059) or by transfecting the dominant-interfering form of the nonphosphorylated MAPKK-1 expressing construct (MAPKK S222A). Inhibition of p42/p44 MAP kinase also antagonized the upregulation of iNOS mRNA and protein, as demonstrated by the quantitative RT-PCR method and Western blot analysis, respectively. Furthermore, rat iNOS promoter activity using an iNOS-luciferase construct stimulated by cytokines was inhibited by MAPKK-1 inhibition. However, kappaB-dependent transcription analysis revealed that cytokine-stimulated NF-kappaB activity was unaffected by MAP kinase inhibition. Western blot analysis using anti-IkappaB-alpha and anti-phospho-IkappaB-alpha antibodies showed that PD98059 had no effect on transient phosphorylation or degradation of IkappaB-alpha by cytokines. An electrophoretic mobility shift assay using synthetic oligonucleotide corresponding to the downstream NF-kappaB site of rat iNOS promoter as a probe showed that MAP kinase inhibition did not block cytokine-stimulated activation of NF-kappaB. These data suggest that the MAP kinase pathway is in part involved in cytokine-induced iNOS expression independent from NF-kappaB activation in rat VSMCs.
authors.affiliation: Department of Clinical and Molecular Endocrinology, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Doi
authors.forename: M
authors.initials: M
authors.lastname: Shichiri
authors.forename: K
authors.initials: K
authors.lastname: Katsuyama
authors.forename: F
authors.initials: F
authors.lastname: Marumo
authors.forename: Y
authors.initials: Y
authors.lastname: Hirata
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015870:Gene Expression / Q000502:physiology*; D005799:Genes, Dominant; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D016328:NF-kappa B / Q000502:physiology*; D009566:Nitrates / Q000037:antagonists & inhibitors; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000037:antagonists & inhibitors; D051381:Rats; D017208:Rats, Wistar
chemical_list: D016207:Cytokines; D004791:Enzyme Inhibitors; D005419:Flavonoids; D016328:NF-kappa B; D009566:Nitrates; D009573:Nitrites; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one