Abstract
Recently, it has been reported that calciumbinding proteins such as osteopontin and matrix Gla protein were expressed in calcified atherosclerotic lesion. In order to determine the role of osteopontin (OPN) in vascular calcification, we developed an in vitro model of vascular calcification by using bovine vascular smooth muscle cells (BVSMC) and demonstrated that alkaline phosphatase (ALP) plays an crucial role in BVSMC calcification and that the expression of OPN mRNA increases during this calcification. However, the mechanism by which OPN is induced in the development of vascular calcification still remains unknown. Therefore, we investigated the regulation of osteopontin expression during vascular calcification by using the above-mentioned model. Since β-glycerophosphate (β-GP) was used to induce calcification, we hypothesized that increased oncentrations of inorganic phosphate (Pi) resulting from the degradation of β-GP by ALP may modulate the expression of OPN in BVSMC. β-GP (3-10mM) dose-dependently increased the expression of OPN gene. Levamisole, a specific inhibitor of ALP, inhibited the induction of OPN gene by β-GP in a dose-dependent manner (10-5-10-3M). Furthermore, 2.5mM Pi restored the levels of OPN mRNA inhibited by levamisole, suggesting that Pi may directly increase OPN expression. Pi induced OPN mRNA in time-and dose-dependent manners. Since it is likely that calcium deposition resulting from raising the concentrations of Pi may induce OPN gene, we finally examined the effect of Pi on OPN expression under uncalcified condition [in the presence of etidronate (EHDP)]. Even in the presence of etidronate, OPN mRNA was upregulated by Pi. These data suggest that Pi may regulate OPN expression under calcifying microenvironment.
