The molecular property of Ninjurin1 and its role in bone development

Abstract

Osteoclasts (OCs) are bone-resorbing cells that originate from hematopoietic stem cells and develop through the fusion of mononuclear myeloid precursors. Dysregulation of OC development causes skeletal disorders such as osteopetrosis, osteoporosis, and rheumatoid arthritis. Although the molecular mechanisms underlying osteoclastogenesis have been well established, the means by which OCs maintain their survival during OC development remain unknown. In the present study, it was found that Ninjurin1 (Ninj1), a cell surface protein, supports OC development by enhancing the survival of prefusion OCs (preOCs). Ninj1 expression was dynamically regulated during osteoclastogenesis. In addition, Ninj1-/- mice exhibit mild osteopetrosis owing to impaired OC development. However, typical markers of OC differentiation such as Nfatc1, c-Fos, integrinβ3, Oscar, and Calcr were unaffected by Ninj1 deficiency. Furthermore, other important events of OC development such as transmigration, fusion, and actin ring formation were also not impaired by the absence of Ninj1. Instead, Ninj1 deficiency increased Caspase-9-dependent intrinsic apoptotic cell death in preOCs. Furthermore, overexpression of Ninj1 enhanced the survival of mouse macrophage/preOC RAW264.7 cells in osteoclastogenic culture, which suggests that Ninj1 is important for the survival of preOCs. Finally, analysis of publicly available microarray datasets revealed a potent correlation between high NINJ1 expression and bone disorders in humans. Regarding to Ninj1 molecular property, it was revealed that Ninj1 assembles into a homomeric protein complex composed of two to six monomeric Ninj1 molecules, and the intracellular region of Ninj1 encompassing Leu101 to Ala110 is important for Ninj1 assembly. Furthermore, Ninj1 is an N-glycosylated protein, and the N-glycosylation enhances homomer formation and regulates protein stability and plasma membrane sorting. These findings suggest that Ninj1 plays an important role in bone homeostasis by enhancing the survival of preOCs and might represent a potent therapeutic target for destructive bone disorders. Moreover, targeting of N-glycosylation in Ninj1 might be a regulatory strategy for modulation of Ninj1 function(s).