Hypertension Research
Online ISSN : 1348-4214
Print ISSN : 0916-9636
ISSN-L : 0916-9636
Cytokine-Activated p42/p44 MAP Kinase Is Involved in Inducible Nitric Oxide Synthase Gene Expression Independent from NF-xB Activation in Vascular Smooth Muscle Cells
Masaru DOIMasayoshi SHICHIRIKoichi KATSUYAMAFumiaki MARUMOYukio HIRATA
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2000 Volume 23 Issue 6 Pages 659-667

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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)-xB, 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-xB activation. The purpose of this study was to determine whether the p42/p44 MAP kinase pathway is involved in cytokine-induced NF-xB activation and/or iNOS expression in cultured rat VSMCs. Nitrite/nitrate (NOx) production stimulated by interleukin (IL)-1β or tumor necrosis factor (TNF)-α 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, xB-dependent transcription analysis revealed that cytokine-stimulated NF-xB activity was unaffected by MAP kinase inhibition. Western blot analysis using anti-IxB-α and anti-phospho-IxB-α antibodies showed that PD98059 had no effect on transient phosphorylation or degradation of IxB-α by cytokines. An electrophoretic mobility shift assay using synthetic oligonucleotide corresponding to the downstream NF-xB site of rat iNOS promoter as a probe showed that MAP kinase inhibition did not block cytokine-stimulated activation of NF-xB. These data suggest that the MAP kinase pathway is in part involved in cytokine-induced iNOS expression independent from NF-xB activation in rat VSMCs. (Hypertens Res 2000; 23: 659-667)

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