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


      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.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Atherosclerosis
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Nathan C.
        Nitric oxide as a secretory product of mammalian cells.
        FASEB J. 1992; 6: 3051-3064
        • Moncada S.
        • Higgs A.
        The l-arginine-nitric oxide pathway.
        N. Engl. J. Med. 1993; 329: 2002-2012
        • Forstermann U.
        • Kleinert H.
        Nitric oxide synthase: expression and expressional control of the three isoforms.
        Naunyn Schmiedebergs Arch. Pharmacol. 1995; 352: 351-364
        • Kanno K.
        • Hirata Y.
        • Imai T.
        • Marumo F.
        Induction of nitric oxide synthase gene by interleukin in vascular smooth muscle cells.
        Hypertension. 1993; 22: 34-39
        • Cornwell T.L.
        • Arnold E.
        • Boerth N.J.
        • Lincoln T.M.
        Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP.
        Am. J. Physiol. 1994; 267: C1405-1413
        • Iwashina M.
        • Shichiri M.
        • Marumo F.
        • Hirata Y.
        Transfection of inducible nitric oxide synthase gene causes apoptosis in vascular smooth muscle cells.
        Circulation. 1998; 98: 1212-1218
        • Xie Q.W.
        • Whisnant R.
        • Nathan C.
        Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon γ and bacterial lipopolysaccharide.
        J. Exp. Med. 1993; 177: 1779-1784
        • Lowenstein C.
        • Alley E.
        • Raval P.
        • Snowman A.
        • Snyder S.
        • Russell S.
        • Murphy W.
        Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon γ and lipopolysaccharide.
        Proc. Natl. Acad. Soc. USA. 1993; 90: 9730-9734
        • Eberhardt W.
        • Kunz D.
        • Hummel R.
        • Pfeilschifter J.
        Molecular cloning of the rat inducible nitric oxide synthase gene promoter.
        Biochem. Biophys. Res. Commun. 1996; 223: 752-756
        • Barnes P.J.
        • Karin M.
        Nuclear factor-κβ: a pivotal transcription factor in chronic inflammatory diseases.
        N. Engl. J. Med. 1997; 336: 1066-1071
        • Mercurio F.
        • Zhu H.
        • Murray B.W.
        • Shevchenko A.
        • Bennett B.L.
        • Li J.
        • Young D.B.
        • Barbosa M.
        • Mann M.
        • Manning A.
        • Rao A.
        IKK-1 and IKK-2: cytokine-activated IκB kinases essential for NF-κB activation.
        Science. 1997; 278: 860-866
        • DiDonato J.A.
        • Hayakawa M.
        • Rothwarf D.M.
        • Zandi E.
        • Karin M.
        A cytokine-responsive IκB kinase that activates the transcription factor NF-κB.
        Nature. 1997; 388: 548-554
        • Katsuyama K.
        • Shichiri M.
        • Marumo F.
        • Hirata Y.
        NO inhibits cytokine-induced iNOS expression and NF-κB activation by interfering with phosphorylation and degradation of IκB-α.
        Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1796-1802
        • Katsuyama K.
        • Shichiri M.
        • Marumo F.
        • Hirata Y.
        Role of nuclear factor-κB activation in cytokine- and sphingomyelinase-stimulated inducible nitric oxide synthase gene expression in vascular smooth muscle cells.
        Endocrinology. 1998; 139: 4506-4512
        • Katsuyama K.
        • Shichiri M.
        • Kato H.
        • Imai T.
        • Marumo F.
        • Hirata Y.
        Differential inhibitory actions by glucocorticoid and aspirin on cytokine-induced nitric oxide production in vascular smooth muscle cells.
        Endocrinology. 1999; 140: 2183-2190
        • Kitamura Y.
        • Takahashi H.
        • Nomura Y.
        • Taniguchi T.
        Possible involvement of Janus kinase Jak2 in interferon-γ induction of nitric oxide synthase in rat glial cells.
        Eur. J. Pharmacol. 1996; 306: 297-306
        • Hellendall R.P.
        • Ting J.P.
        Differential regulation of cytokine-induced major histocompatibility complex class II expression and nitric oxide release in rat microglia and astrocytes by effectors of tyrosine kinase, protein kinase C, and cAMP.
        J. Neuroimmunol. 1997; 74: 19-29
        • Lee B.S.
        • Kang H.S.
        • Pyun K.H.
        • Choi I.
        Roles of tyrosine kinases in the regulation of nitric oxide synthesis in murine liver cells: modulation of NF-κB activity by tyrosine kinases.
        Hepatology. 1997; 25: 913-919
        • Kleinert H.
        • Wallerath T.
        • Fritz G.
        • Ihrig-Biedert I.
        • Rodriguez-Pascual F.
        • Geller D.A.
        • Forstermann U.
        Cytokine induction of NO synthase II in human DLD-1 cells: roles of the JAK-STAT, AP-1 and NF-κB-signaling pathways.
        Br. J. Pharmacol. 1998; 125: 193-201
        • Corbett J.A.
        • Kwon G.
        • Marino M.H.
        • Rodi C.P.
        • Sullivan P.M.
        • Turk J.
        • McDaniel M.L.
        Tyrosine kinase inhibitors prevent cytokine-induced expression of iNOS and COX-2 by human islets.
        Am. J. Physiol. 1996; 270: C1581-1587
        • Nakashima O.
        • Terada Y.
        • Inoshita S.
        • Kuwahara M.
        • Sasaki S.
        • Marumo F.
        Inducible nitric oxide synthase can be induced in the absence of active nuclear factor-κB in rat mesangial cells: involvement of the Janus kinase 2 signaling pathway.
        J. Am. Soc. Nephrol. 1999; 10: 721-729
        • Joly G.A.
        • Ayres M.
        • Kilbourn R.G.
        Potent inhibition of inducible nitric oxide synthase by geldanamycin, a tyrosine kinase inhibitor, in endothelial, smooth muscle cells, and in rat aorta.
        FEBS Lett. 1997; 403: 40-44
        • Hirata Y.
        • Yoshimi H.
        • Takaichi S.
        • Yanagisawa M.
        • Masaki T.
        Binding and receptor down-regulation of a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells.
        FEBS Lett. 1988; 239: 13-17
        • Kanno K.
        • Hirata Y.
        • Emori T.
        • Ohta K.
        • Eguchi S.
        • Imai T.
        • Marumo F.
        l-Arginine infusion induces hypotension and increases diuresis/natriuresis with concomitant increased urinary excretion of nitrite/nitrate and cyclic GMP in human.
        Clin. Exp. Pharmacol. Physiol. 1992; 19: 619-625
        • Shichiri M.
        • Kato H.
        • Doi M.
        • Marumo F.
        • Hirata Y.
        Induction of max by adrenomedullin and CGRP antagonizes endothelial apoptosis.
        Mol. Endocrinol. 1999; 13: 1353-1363
        • Shichiri M.
        • Yokokura M.
        • Marumo F.
        • Hirata Y.
        Endothelin-1 inhibits apoptosis of vascular smooth muscle cells induced by nitric oxide and serum deprivation via MAP kinase pathway.
        Arterioscler. Thromb. Vasc. Biol. 2000; 20: 989-997
        • Shichiri M.
        • Hirata Y.
        Antiangiogenesis signals by endostatin.
        FASEB J. 2001; 15: 1044-1053
        • Meydan N.
        • Grunberger T.
        • Dadi H.
        • Shahar M.
        • Arpaia E.
        • Lapidot Z.
        • Leeder J.S.
        • Freedman M.
        • Cohen A.
        • Gazit A.
        • Levitzki A.
        • Roifman C.M.
        Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor.
        Nature. 1996; 379: 645-648
        • Xie Q.W.
        • Kashiwabara Y.
        • Nathan C.
        Role of transcription factor NF-κB/Rel in induction of nitric oxide synthase.
        J. Biol. Chem. 1994; 269: 4705-4708
        • Doi M.
        • Shichiri M.
        • Katsuyama K.
        • Marumo F.
        • Hirata Y.
        Cytokine-activated p42/p44 MAP kinase is involved in inducible nitric oxide synthase gene expression independent from NF-κB activation in vascular smooth muscle cells.
        Hypertens. Res. 2000; 23: 643-649
        • Marks-Konczalik J.
        • Chu S.C.
        • Moss J.
        Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor κB-binding sites.
        J. Biol. Chem. 1998; 273: 22201-22208
        • Kleinert H.
        • Euchenhofer C.
        • Ihrig B.I.
        • Forstermann U.
        Glucocorticoids inhibit the induction of nitric oxide synthase II by down-regulating cytokine-induced activity of transcription factor nuclear factor-κB.
        Mol. Pharmacol. 1996; 49: 15-21
        • Yoza B.K.
        • Hu J.Y.Q.
        • McCall C.E.
        Protein-tyrosine kinase activation is required for lipopolysaccharide induction of interleukin β and NF-κB activation but not NF-κB nuclear translocation.
        J. Biol. Chem. 1996; 271: 18306-18309
        • Levitzki A.
        • Gazit A.
        Tyrosine kinase inhibition: an approach to drug development.
        Science. 1995; 267: 1782-1788
        • Bach E.A.
        • Tanner J.W.
        • Marsters S.
        • Ashkenazi A.
        • Aguet M.
        • Shaw A.S.
        • Schreiber R.D.
        Ligand-induced assembly and activation of the interferon γ receptor in intact cells.
        Mol. Cell. Biol. 1996; 16: 3214-3221
        • Bates M.E.
        • Bertics P.J.
        • Busse W.W.
        IL-5 activates a 45-kilodalton mitogen-activated protein (MAP) kinase and Jak-2 tyrosine kinase in human eosinophils.
        J. Immunol. 1996; 156: 711-718
        • Yu C.R.
        • Lin J.X.
        • Fink D.W.
        • Akira S.
        • Bloom E.T.
        • Yamauchi A.
        Differential utilization of Janus kinase-signal transducer activator of transcription signaling pathways in the stimulation of human natural killer cells by IL-2, IL-12, and IFN-α.
        J. Immunol. 1996; 157: 126-137
        • Zhang Y.
        • Turkson J.
        • Carter S.C.
        • Smithgall T.
        • Levitzki A.
        • Kraker A.
        • Krolewski J.J.
        • Medveczky P.
        • Jove R.
        Activation of Stat3 in v-Src-transformed fibroblasts requires cooperation of Jak1 kinase activity.
        J. Biol. Chem. 2000; 275: 24935-24944
        • Kamijo R.
        • Harada H.
        • Matsuyama T.
        • Bosland M.
        • Gerecitano J.
        • Shapiro D.
        • Le J.
        • Koh S.I.
        • Kimura T.
        • Green S.J.
        • Mak T.W.
        • Taniguchi T.
        • Vilcek J.
        Requirement for transcription factor IRF-1 in NO synthase induction in macrophages.
        Science. 1994; 263: 1612-1615