This Article Full Text Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cuzzocrea, S. Articles by Maggi, A. Search for Related Content PubMed PubMed Citation Articles by Cuzzocrea, S. Articles by Maggi, A.

Inducible Nitric Oxide Synthase Mediates Bone Loss in Ovariectomized Mice

Institute of Pharmacology, University of Messina (S.C., L.D., T.G., R.D.P., A.P.C.), Department of Biomorphology, University of Messina School of Medicine (E.M., D.P.), and Department of Obstetrical and Gynecological Sciences, University of Messina (Z.R.), 98100 Messina, Italy; Center of Excellence on Neurodegenerative Diseases and Center for Pharmacology and Biotechnology, University of Milan (E.V., A.M.), 20129 Milan, Italy; and Department of Rheumatology, University Hospital Nijmegen (F.A.J.V.d.L.), 6500 HB, Nijmegen, The Netherlands

Address all correspondence and requests for reprints to: Salvatore Cuzzocrea, Ph.D., Institute of Pharmacology, School of Medicine, University of Messina, Torre Biologica-Policlinico Universitario Via C. Valeria-Gazzi, 98100 Messina, Italy. E-mail: salvator{at}unime.it; or to: Prof. Adriana Maggi, Center MPL, Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Balzaretti 9, 20129 Milan, Italy. E-mail: adriana.maggi{at}unimi.it.

Several clinical studies have shown that bone loss may be attributed to osteoclast recruitment induced by mediators of inflammation. In different experimental paradigms we have recently demonstrated that estrogen exhibits antiinflammatory activity by preventing the induction of inducible nitric oxide synthase (iNOS) and other components of the inflammatory reaction. To verify whether this could explain the estrogen-dependent blockade of osteoporosis, we investigated the effect of ovariectomy in mice in which iNOS activity had been blunted by genetic or pharmacological manipulation. The consequences of iNOS blockade were evaluated initially on bone formation and resorption by histomorphometric analysis. The proximal tibiae of mice with iNOS genotypes revealed that 32 d after ovariectomy bone volume and bone formation rate were significantly decreased, and osteoclast surface was increased. Conversely, in iNOS knockout (iNOSKO) and wild-type (WT) mice treated with a specific inhibitor of iNOS, N-iminoethyl-L-lysine, ovariectomy did not result in bone depletion. In WT mice, ovariectomy also affected bone formation, as shown by a decreased mineral apposition rate. Also in this case, iNOS inactivation prevented the effect of ovariectomy. Immunocytochemical analysis showed that after ovariectomy iNOS protein accumulates in chondrocytes, and a significant increase in nitrotyrosine and poly(ADP-ribose) synthetase staining was observed in the femur metaphyses. The increase in nitrotyrosine and poly(ADP-ribose) synthetase formation induced by ovariectomy was significantly reduced in sections from iNOSKO mice. These data indicate that in WT mice the observed induction of iNOS has functional relevance, because it leads to overproduction of nitric oxide and accumulation of highly reactive molecules, triggering a local inflammatory reaction. These inflammatory foci attract cytokines, well known actors in the mechanism of osteoclastogenesis. In iNOSKO mice the measurements of IL-1ß, IL-6, and TNF plasma levels showed that ovariectomy fails to elicit the increase observed in WT animals and suggests that iNOS plays a primary role in the protective effects of estrogens. To further support this hypothesis, we show that estradiol-dependent activation of estrogen receptor- blocks phorbol 12-acetate 13-myristate-induced transcription of iNOS promoter in transfected cells, thus demonstrating that the promoter of iNOS is under estrogen negative control.

Our findings point to a key role of iNOS in mediating the negative effects of estrogen depletion on bones and provide a novel mechanistic explanation for the effects of menopause in osteoporosis and possibly also in other diseases in which the inflammatory component is elevated.

This article has been cited by other articles:

				H. Zheng, X. Yu, P. Collin-Osdoby, and P. Osdoby RANKL Stimulates Inducible Nitric-oxide Synthase Expression and Nitric Oxide Production in Developing Osteoclasts: AN AUTOCRINE NEGATIVE FEEDBACK MECHANISM TRIGGERED BY RANKL-INDUCED INTERFERON-beta VIA NF-{kappa}B THAT RESTRAINS OSTEOCLASTOGENESIS AND BONE RESORPTION J. Biol. Chem., June 9, 2006; 281(23): 15809 - 15820. [Abstract] [Full Text] [PDF]

				C. Zaragoza, E. Lopez-Rivera, C. Garcia-Rama, M. Saura, A. Martinez-Ruiz, T. R. Lizarbe, F. Martin-de-Lara, and S. Lamas Cbfa-1 mediates nitric oxide regulation of MMP-13 in osteoblasts. J. Cell Sci., May 1, 2006; 119(Pt 9): 1896 - 1902. [Abstract] [Full Text] [PDF]

				E. Vegeto, S. Belcredito, S. Ghisletti, C. Meda, S. Etteri, and A. Maggi The Endogenous Estrogen Status Regulates Microglia Reactivity in Animal Models of Neuroinflammation Endocrinology, May 1, 2006; 147(5): 2263 - 2272. [Abstract] [Full Text] [PDF]

				V. Garcia Palacios, L. J. Robinson, C. W. Borysenko, T. Lehmann, S. E. Kalla, and H. C. Blair Negative Regulation of RANKL-induced Osteoclastic Differentiation in RAW264.7 Cells by Estrogen and Phytoestrogens J. Biol. Chem., April 8, 2005; 280(14): 13720 - 13727. [Abstract] [Full Text] [PDF]

				R. J. van't Hof, J. MacPhee, H. Libouban, M. H. Helfrich, and S. H. Ralston Regulation of Bone Mass and Bone Turnover by Neuronal Nitric Oxide Synthase Endocrinology, November 1, 2004; 145(11): 5068 - 5074. [Abstract] [Full Text] [PDF]

				X. Fan, E. Roy, L. Zhu, T. C. Murphy, C. Ackert-Bicknell, C. M. Hart, C. Rosen, M. S. Nanes, and J. Rubin Nitric Oxide Regulates Receptor Activator of Nuclear Factor-{kappa}B Ligand and Osteoprotegerin Expression in Bone Marrow Stromal Cells Endocrinology, February 1, 2004; 145(2): 751 - 759. [Abstract] [Full Text] [PDF]