Identification of osteoblast cell-surface antigen regulating calcification as insulin receptor-related receptor, the alkali-sensor: A novel modulator of bone metabolism.

BACKGROUND AND AIMS: The regulatory mechanism of calcification mediated by osteoblasts is ambiguous. Previously, we had found a unique cell-surface A7 antigen recognized by an osteoblast-specific monoclonal antibody, which is specifically expressed in osteoblasts in bone tissues and is involved in the regulation of calcification. This study aimed to identify the molecular entity of the A7 antigen, which is a possible regulator of calcification, expressed on the surface of osteoblasts. METHODS: Immunoprecipitated A7 antigen was analyzed using SDS-PAGE and mass spectrometry followed by Mascot analysis. Gene expression of the candidate molecule was confirmed by quantitative RT-PCR analysis. Calcification was estimated by using Alizarin Red staining of primary osteoblasts obtained from the calvariae of newborn rats. Immunofluorescence detection of osteoblast-specific antigens was performed using cryosections of the ankle joints obtained from rats with adjuvant-induced arthritis. RESULTS AND DISCUSSION: Proteomics analysis of the A7 antigen strongly suggested it as the insulin receptor-related receptor (IRR), which is an alkali-sensor. IRR mRNA expression was confirmed in rat calvaria-derived primary osteoblasts. Immunoprecipitation and immunoblotting analyses further confirmed the A7 antigen as IRR. Calvaria-derived primary osteoblasts briefly exposed to an alkaline environment showed remarkable stimulation of calcification, involving the IRR-mediated activation of extracellular signal-regulated kinases. Furthermore, immunohistochemical analysis of A7 antigen localization at pathological bone destruction sites showed a possible direct contact between active osteoclasts and osteoblasts expressing the A7 antigen, IRR, the alkali-sensor. CONCLUSIONS: A unique osteoblasts surface antigen regulating calcification was identified to be an IRR, the alkali-sensor expressed on the surface of osteoblasts. IRR- expressing osteoblasts were localized in contact with active osteoclasts at pathological bone destruction sites. The current study provides basic evidence that could pave the way for the development of novel medicines targeting the IRR to cure pathological bone diseases.