Cytokine-activated Jak-2 is involved in inducible nitric oxide synthase expression independent from NF-κB activation in vascular smooth muscle cells

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Inflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, activate nuclear factor-kappa B (NF-κB) which transactivates inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs). However, it remains obscure whether cytokine-mediated iNOS expression in VSMCs requires signaling pathway(s) other than NF-κB activation. The present study was designed to elucidate whether protein tyrosine kinases (PTKs) are involved in the cytokine-induced NF-κB activation and iNOS expression in cultured rat VSMCs. Both IL-1β and TNF-α stimulated NF-κB activity, iNOS mRNA and protein expression with massive nitrite/nitrate (NOx) production in rat VSMCs. PTK inhibitors (genistein, herbimycin A) dose-dependently inhibited the cytokine-stimulated NOx production and iNOS mRNA expression. However, neither genistein nor herbimycin A affected the cytokine-stimulated phosphorylation and degradation of IκB-α, or NF-κB activation, whereas they completely blocked the cytokine-stimulated iNOS transcriptional activity. Tyrphostin B42 (AG490), a Jak-2 tyrosine kinase inhibitor, similarly blocked the cytokine-induced NOx production, iNOS expression and its promoter activity without affecting NF-κB-dependent transcription. Transfection of a dominant-negative Jak-2 mutant antagonized the cytokine-induced NOx production and iNOS expression, while wild-type Jak-2 expressing construct was without effect. These data indicate that the cytokine-induced iNOS expression involves activation of Jak-2 signaling pathway independent from NF-κB activation in rat VSMCs.