Cancellation of the calcification in cultured osteoblasts by CLEC-2

This study aims to investigate the effect of CLEC-2 on calcification in cultured mouse osteoblasts. In the RT-PCR and cell ELISA analysis, it was confirmed that osteoblasts express podoplanin, osteopontin, osteocalcin and sclerostin in culture, and that expressions of osteopontin and osteocalcin increased in calcification medium. The expression of podopla-nin, osteopontin, osteocalcin and sclerostin did not change in osteoblasts with CLEC-2, indicating that CLEC-2 does not af-fect the expression of these bone proteins in osteoblasts. However, the amounts of calcified nodules and alkaline phos-phatase activity were significantly suppressed in cultured osteoblasts by CLEC-2. The quantitative analysis showed that both the calcified nodule amount and alkaline phosphatase activity decreased with CLEC-2 while there was no influence in the cell viability with CLEC-2. Further, the expression of RUNX2 was observed in cytoplasm and in nucleus of cultured mouse osteoblasts while the expression decreased with CLEC-2. In Matrigel-based three-dimensional culture a significant cell process elongation of osteoblasts was observed and the elongation was strongly suppressed with CLEC-2. Considering these, CLEC-2 may have an ability to cancel the calcification of osteoblasts by blocking the maturation of osteoblast via interaction with CLEC-2 receptor podoplanin without any involvements of bone-associated protein production.