Altered AP-1/Ref-1 redox pathway and reduced proliferative response in iNOS-deficient vascular smooth muscle cells

Abstract

We previously reported that injury-induced medial vascular smooth muscle cell (VSMC) proliferation and neointima formation in carotid arteries of inducible nitric oxide synthase knockout (iNOS KO) mice were significantly reduced compared with wild type (WT). However, the molecular pathway underlying such differences is not known. In this in vitro study, we discovered that the AP-1/Ref-1/thioredoxin signaling pathway is altered in aortic VSMC from iNOS KO mice, which leads to reduced growth response when compared with aortic VSMC from WT mice. After equal initial seeding, the cell number after 7 days in serum medium was less in iNOS KO cells compared with WT VSMC (1.2-0.6-10⁵ vs 3.2-1.1-10⁵; p < 0.05). Significantly more iNOS KO cells remained in the G0/G1 phase compared with WT cells after 24-h serum treatment (82.6-13.7% vs 62.3-14.6%; p < 0.05) by cell-cycle analysis. Nuclear PCNA expression was also less in the iNOS KO cells, which was not affected by exogenous NO or superoxide. Superoxide generation after 24-h serum stimulation was less in the iNOS KO cells compared with WT cells. After 30-min serum stimulation, AP-1 DNA binding was reduced and a lack of increase in nuclear c-Jun protein was observed in iNOS KO VSMC. RT-PCR analysis confirmed a lack of inducible c-Jun mRNA after serum stimulation in the KO cells. In addition, KO cells had less nuclear reducing factor-1 (Ref-1) and serum-inducible thioredoxin protein expression. Reduced proliferative response of iNOS KO VSMC to serum treatment is associated with altered AP-1/Ref-1/thioredoxin pathway activation.

Keywords

AP-1 iNOS KO proliferation Ref-1 thioredoxin VSMC

References

Hansson GK , Geng YJ , Holm J , Hardhammar P , Wennmalm A , Jennische E. Arterial smooth muscle cells express nitric oxide synthase in response to endothelial injury. J Exp Med 1994; 180: 733-738.

Arthur JF , Yin ZL , Young HM , Dusting GJ. Induction of nitric oxide synthase in the neointima induced by a periarterial collar in rabbits. Arterioscler Thromb Vasc Biol 1997; 17: 737-740.

Yan Z , Hansson GK. Overexpression of inducible nitric oxide synthase by neointimal smooth muscle cells. Circ Res 1998; 82: 21-29.

Chyu KY , Dimayuga P , Zhu J et al. Decreased neointimal thickening after arterial wall injury in inducible nitric oxide synthase knockout mice. Circ Res 1999; 85: 1192-1198.

Tolbert T , Thompson JA , Bouchard P , Oparil S. Estrogen-induced vasoprotection is independent of inducible nitric oxide synthase expression: evidence from the mouse carotid artery ligation model. Circulation 2001; 104: 2740-2745.

Yamasaki K , Edington HD , McClosky C et al. Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer. J Clin Invest 1998; 101: 967-971.

Rai RM , Lee FY , Rosen A et al. Impaired liver regeneration in inducible nitric oxide synthase deficient mice. Proc Natl Acad Sci USA 1998; 95: 13 829-13 834.

Stallmeyer B , Kampfer H , Kolb N , Pfeilschifter J , Frank S. The function of nitric oxide in wound repair: inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization. J Invest Dermatol 1999; 113: 1090-1098.

Zhu DY , Liu SH , Sun HS , Lu YM. Expression of inducible nitric oxide synthase after focal cerebral ischemia stimulates neurogenesis in the adult rodent dentate gyrus. J Neurosci 2003; 23: 223-229.

10.

Xia Y , Roman LJ , Masters BS , Zweier JL. Induciblenitric-oxide synthase generates superoxide from the reductase domain. J Biol Chem 1998; 273: 22635-22639.

11.

Nakamura H , Nakamura K , Yodoi J. Redox regulation of cellular activation. Annu Rev Immunol 1997; 15: 351-369.

12.

Souza HP , Souza LC , Anastacio VM et al. Vascular oxidant stress early after balloon injury: evidence for increased NAD(P)H oxidoreductase activity. Free Radic Biol Med 2000; 28: 1232-1242.

13.

Ryder K , Nathans D. Induction of protooncogene c-jun by serum growth factors. Proc Natl Acad Sci USA 1988; 85: 8464-8467.

14.

Xanthoudakis S , Curran T. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity. EMBO J 1992; 11: 653-665.

15.

Hirota K , Matsui M , Iwata S , Nishiyama A , Mori K , Yodoi J. AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc Natl Acad Sci USA 1997; 94: 3633-3638.

16.

Diamond DA , Parsian A , Hunt CR et al. Redox factor-1 (Ref-1) mediates the activation of AP-1 in HeLa and NIH 3T3 cells in response to heat shock. J Biol Chem 1999; 274: 16959-16964.

17.

Holmgren A. Thioredoxin. Annu Rev Biochem 1985; 54: 237-271.

18.

Li PF , Dietz R , von HR. Differential effect of hydrogen peroxide and superoxide anion on apoptosis and proliferation of vascular smooth muscle cells. Circulation 1997; 96: 3602-3609.

19.

Jang M , Pezzuto JM. Effects of resveratrol on 12-O-tetradecanoylphorbol-13-acetate-induced oxidative events and gene expression in mouse skin. Cancer Lett 1998; 134: 81-89.

20.

Hassid A , Arabshahi H , Bourcier T , Dhaunsi GS , Matthews C. Nitric oxide selectively amplifies FGF-2-induced mitogenesis in primary rat aortic smooth muscle cells. Am J Physiol 1994; 267: H1040-H1048.

21.

Tarpey MM , Fridovich I. Methods of detection of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite. Circ Res 2001; 89: 224-236.

22.

Johnson RS , van Lingen B , Papaioannou VE , Spiegelman BM. A null mutation at the c-jun locus causes embryonic lethality and retarded cell growth in culture. Genes Dev 1993; 7: 1309-1317.

23.

Wisdom R , Johnson RS , Moore C. c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms. EMBO J 1999; 18: 188-197.

24.

Zingarelli B , Hake PW , Yang Z , O’Connor M , Denenberg A , Wong HR. Absence of inducible nitric oxide synthase modulates early reperfusion-induced NF-kappaB and AP-1 activation and enhances myocardial damage. FASEB J 2002; 16: 327-342.

25.

Angel P , Hattori K , Smeal T , Karin M. Thejunproto-oncogeneis positively autoregulated by its product, Jun/AP-1. Cell 1988; 55: 875-885.

26.

Xanthoudakis S , Miao GG , Curran T. The redox and DNA-repair activities of Ref-1 are encoded by nonoverlapping domains. Proc Natl Acad Sci USA 1994; 91: 23-27.

27.

Tell G , Zecca A , Pellizzari L et al. An ‘environment to nucleus’ signaling system operates in B lymphocytes: redox status modulates BSAP/Pax-5 activation through Ref-1 nuclear translocation. Nucleic Acids Res 2000; 28: 1099-1105.

28.

Kakolyris S , Kaklamanis L , Giatromanolaki A et al. Expression and subcellular localization of human AP endonuclease 1 (HAP1/Ref-1) protein: a basis for its role in human disease. Histopathology 1998; 33: 561-569.

29.

Seki S , Akiyama K , Watanabe S , Hatsushika M , Ikeda S , Tsutsui K. cDNA and deduced amino acid sequence of a mouse DNA repair enzyme (APEX nuclease) with significant homology to Escherichia coli exonuclease III. J Biol Chem 1991; 266: 20 797-27 802.

30.

Takagi Y , Gon Y , Todaka T et al. Expression of thioredoxin is enhanced in atherosclerotic plaques and during neointima formation in rat arteries. Lab Invest 1998; 78: 957-966.

31.

Suschek CV , Schnorr O , Hemmrich K et al. Critical role of L-arginine in endothelial cell survival during oxidative stress. Circulation 2003; 107: 2607-2614.