Inducible Nitric Oxide Synthase Mediates Bone Loss in Ovariectomized Mice

Abstract

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-1 beta, IL-6, and TNFalpha 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-alpha 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.

Negative Regulation of RANKL-induced Osteoclastic Differentiation in RAW264.7 Cells by Estrogen and Phytoestrogens

“…It has been established that in the presence of CSF-1 sufficient to maintain cell growth and survival, RANKL, via its tumor necrosis factor family receptor RANK, is sufficient to induce complete osteoclastic differentiation from hematopoietic precursors and that knock-out mice with defects in the RANKL system cannot form osteoclasts (26). It is also established that estrogen withdrawal causes rapid skeletal degradation, an effect that certainly involves other cells, including osteoblasts, but that has also been hypothesized to involve direct effects on osteoclast formation (3)(4)(5). This report demonstrates that phytoestrogens and ␤-estradiol directly reduce osteoclastic differentiation in the murine RAW264.7 cell model.…”

“…3). However, transgenic and knock-out mice with varying ER␣ and ER␤ expression established that estrogen effects on bone involve ER␣ and ER␤, which modulate signaling pathways involving Erk and nitric oxide and perform direct transcriptional activity (4). Estrogen responses in mesenchymal stem cell-derived bone-forming cells, osteoblasts, are extensively studied.…”

The role of nitric oxide in the mechanical repression of RANKL in bone stromal cells

“…We here have shown that nNOS is sensitive to strain up-regulation and NO generation in osteoprogenitor cells -essentially "taking over" for eNOS as the mechanically regulated NO synthase to preserve normal skeletal homeostasis. Finally, in mice deficient in the inflammatory NOS isoform, iNOS, although no bone abnormalities have been described under normal conditions, iNOS deficient mice are partially protected from ovariectomy [43] and inflammatory [44] induced bone loss. Taken together, this evidence reminds us that in the normal, NO synthase replete bone cell, nitric oxide regulation of skeletal remodeling is complex.…”

Beneficial Effects of Ajuga decumbens on Osteoporosis and Arthritis

“…15) Cuzzocrea et al suggest that iNOS is a potential target for therapy of postmenopausal osteoporosis in women. 24) We hypothesize that KE will be a useful agent in the treatment of osteoporosis and osteoarthritis because it has significant effects on the NO production and expression of iNOS in macrophages. We will investigate whether the anti-osteoporotic and anti-arthritic effect of KE are depressed by using the iNOS inhibitor.…”