Abstract
Nitric oxide (NO) is a multifunctional signaling molecule and a key vasculoprotective and potential osteoprotective factor. NO regulates normal bone remodeling and pathological bone loss in part through affecting the recruitment, formation, and activity of bone-resorbing osteoclasts. Using murine RAW 264.7 and primary bone marrow cells or osteoclasts formed from them by receptor activator of NF-kappaB ligand (RANKL) differentiation, we found that inducible nitric-oxide synthase (iNOS) expression and NO generation were stimulated by interferon (IFN)-gamma or lipopolysaccharide, but not by interleukin-1 or tumor necrosis factor-alpha. Surprisingly, iNOS expression and NO release were also triggered by RANKL. This response was time- and dose-dependent, required NF-kappaB activation and new protein synthesis, and was specifically blocked by the RANKL decoy receptor osteoprotegerin. Preventing RANKL-induced NO (via iNOS-selective inhibition or use of marrow cells from iNOS-/- mice) increased osteoclast formation and bone pit resorption, indicating that such NO normally restrains RANKL-mediated osteoclastogenesis. Additional studies suggested that RANKL-induced NO inhibition of osteoclast formation does not occur via NO activation of a cGMP pathway. Because IFN-beta is also a RANKL-induced autocrine negative feedback inhibitor that limits osteoclastogenesis, we investigated whether IFN-beta is involved in this novel RANKL/iNOS/NO autoregulatory pathway. IFN-beta was induced by RANKL and stimulated iNOS expression and NO release, and a neutralizing antibody to IFN-beta inhibited iNOS/NO elevation in response to RANKL, thereby enhancing osteoclast formation. Thus, RANKL-induced IFN-beta triggers iNOS/NO as an important negative feedback signal during osteoclastogenesis. Specifically targeting this novel autoregulatory pathway may provide new therapeutic approaches to combat various osteolytic bone diseases.
Highlights
Normal bone remodeling requires a homeostatic balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts (OCs).3 Excessive OC bone resorption leads to bone loss in many skeletal pathologies such as rheumatoid arthritis, periodontal disease, postmenopausal osteoporosis, implant osteolysis, and tumor-associated bone loss [1]
Cytokines and Other Inflammatory Stimuli Differentially Regulate inducible nitric-oxide synthase (iNOS) mRNA Expression and Nitric oxide (NO) Production in Murine RAW Cells and RAW-OCs—Previously, we showed that inflammatory stimuli such as tumor necrosis factor (TNF)-␣, IL-1, IFN-␥, and LPS significantly increase NO production in avian OC-like cells, but not in authentic isolated avian OCs, whereas protein kinase C activation by phorbol 12-myristate 13-acetate (PMA) or intracellular calcium elevation by the ionophore A23187 stimulates NO production in authentic avian OCs and inhibits their bone pit resorption [12]
The iNOS/NO system is up-regulated by certain inflammatory signals in murine RAW-OCs and their precursor cells, and this regulation differs in some respects from that seen in avian OCs and their bone marrow precursor cells
Summary
Normal bone remodeling requires a homeostatic balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts (OCs).3 Excessive OC bone resorption leads to bone loss in many skeletal pathologies such as rheumatoid arthritis, periodontal disease, postmenopausal osteoporosis, implant osteolysis, and tumor-associated bone loss [1].
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