Genetic disruption of all NO synthase isoforms enhances BMD and bone turnover in mice in vivo: Involvement of the renin-angiotensin system

Abstract

Introduction: NO is synthesized by three different NO synthase (NOS) isoforms, including neuronal (nNOS), inducible (iNOS) and endothelial NOS (eNOS). The roles of NO in bone metabolism have been extensively investigated in pharmacological studies and in studies with NOS isoform-deficient mice. However, because of the nonspecificity of agents and compensation among the NOS isoforms, the ultimate roles of endogenous NO are still poorly understood. To address this point, we successfully generated mice in which all three NOS genes are completely disrupted. In this study, we examined whether bone metabolism is abnormal in those mice. Materials and Methods: Experiments were performed in 12-wk-old male wildtype, singly nNOS-/-, iNOS-/-, and eNOS-/- and triply n/i/eNOS-/- mice. BMD was assessed by DXA. The kinetics of osteoblastic bone formation and those of osteoclastic bone resorption were evaluated by measurements of morphological and biochemical markers. Results: BMD was significantly higher only in the triply NOS -/- mice but not in any singly NOS-/- mice compared with the wildtype mice. Markers of osteoblastic bone formation, including bone formation rate, mineral apposition rate, and serum alkaline phosphatase concentration, were also significantly larger only in the triply NOS -/- mice compared with wildtype mice. Furthermore, markers of osteoclastic bone resorption, including osteoclast number, osteoclast surface, and urinary deoxypyridinoline excretion, were again significantly greater only in the triply NOS-/- mice. Importantly, the renin-angiotensin system in bone was significantly activated in the triply NOS-/- mice, and long-term oral treatment with an angiotensin II type 1 (AT1) receptor blocker normalized this pathological bone remodeling in those mice. Conclusions: These results provide the first direct evidence that genetic disruption of the whole NOS system enhances BMD and bone turnover in mice in vivo through the AT1 receptor pathway, showing the critical role of the endogenous NO/NOS system in maintaining bone homeostasis. © 2008 American Society for Bone and Mineral Research.