RANKL Stimulates Inducible Nitric-oxide Synthase Expression and Nitric Oxide Production in Developing Osteoclasts AN AUTOCRINE NEGATIVE FEEDBACK MECHANISM TRIGGERED BY RANKL-INDUCED INTERFERON-β VIA NF-κB THAT RESTRAINS OSTEOCLASTOGENESIS AND BONE RESORPTION

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-κB ligand (RANKL) differentiation, we found that inducible nitric-oxide synthase (iNOS) expression and NO generation were stimulated by interferon (IFN)-γ or lipopolysaccharide, but not by interleukin-1 or tumor necrosis factor-α. Surprisingly, iNOS expression and NO release were also triggered by RANKL. This response was time- and dose-dependent, required NF-κB 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-β is also a RANKL-induced autocrine negative feedback inhibitor that limits osteoclastogenesis, we investigated whether IFN-β is involved in this novel RANKL/iNOS/NO autoregulatory pathway. IFN-β was induced by RANKL and stimulated iNOS expression and NO release, and a neutralizing antibody to IFN-β inhibited iNOS/NO elevation in response to RANKL, thereby enhancing osteoclast formation. Thus, RANKL-induced IFN-β 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.