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

Abstract

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.

Introduction

Nitric oxide (NO) is synthesized from l-arginine by the catalytic action of three distinct isozymes of NO synthase (NOS), and appears to play diverse physiological roles, including vasodilation, neurotransmission, and mediation of immune responses [1], [2], [3]. Unlike two Ca²⁺-dependent constitutive isozymes of NOS dominantly expressed in neuron (nNOS) and endothelium (eNOS), a cytokine-inducible isozyme (iNOS) produces large amounts of NO in response to bacterial lipopolysaccharides (LPS) and certain inflammatory cytokines in a variety of cells. Stimulation with LPS, interleukin (IL)-1β or tumor necrosis factor (TNF)-α causes augmented iNOS expression and subsequent massive NO production in vascular smooth muscle cells (VSMCs) [4]. High-output NO derived from iNOS in VSMCs profoundly relaxes vascular tonus, inhibits cell proliferation, and induces apoptosis of VSMCs [5], [6]. Therefore, overexpression of the iNOS gene in VSMCs has been implicated in the pathogenesis of endotoxin shock as well as vascular remodeling and atherosclerosis.

The promoter region of murine iNOS gene contains several potential cis-elements for the binding of transcription factors, including IFN-γ responsive element (γ-IRE), γ-activated site (GAS), IFN-α-stimulated response element (ISRE), AP-1 sites, nuclear factor-kappa B (NF-κB) sites and so forth [7], [8], [9]. NF-κB activation is essential for the cytokine-induced expression of iNOS gene in a variety of cell types including VSMCs [10]. NF-κB is a heterodimeric complex usually consisting of p50/p65 subunit which associates with a cytoplasmic inhibitor (IκB-α) to form an inactive ternary complex [10]. Activation of NF-κB by cytokines requires proteasome-mediated degradation of IκB-α after phosphorylation by serine/threonine protein kinases, termed IκB kinases (IKK) [11], [12], and polyubiquination, thereby rendering NF-κB heterodimer to translocate into the nucleus and bind to the κB sites of the genes. We have recently shown that proteasome inhibitors suppress the cytokine-induced iNOS expression by blocking IκB-α degradation and NF-κB activation in rat VSMCs [13], [14], [15]. Neutral sphingomyelinase also induces iNOS expression, but distinct from the proteasome-mediated IκB-α degradation pathway, suggesting the existence of yet unspecified pathways in mediation of iNOS expression [14].

Recent accumulating lines of evidence suggest that protein tyrosine kinases (PTK) may be involved in the cytokine-induced iNOS expression in several cell types. For example, interferon-γ (IFN-γ) in combination with LPS and/or other cytokines stimulated NO production and iNOS mRNA expression, whose effects were blocked by PTK inhibitors (genistein, herbimycin A, tyrphostin) in murine macrophage and rat glial cells [16], rat astrocytes and microglia [17], murine embryonic liver cells [18], human colon cancer cells [19], human pancreatic islets [20] and rat mesangial cells [21]. However, these PTK inhibitors failed to affect NO production stimulated by IL-1β alone or LPS plus cytokines in rat VSMCs and murine endothelial cells [22]. The reason(s) for the apparent discrepancy among the inhibitory effects of PTK inhibitors on the cytokine-induced iNOS expression in various cell types thus far reported remains unknown.

These observations led us to study: (1) whether PTK(s) is/are involved in IL-1β- and TNF-α-induced iNOS expression in rat VSMCs, and if so; (2) to delineate the relationship of PTK(s) to NF-κB activation; and (3) to characterize PTK(s) responsible for the cytokine-induced iNOS expression.