Requirement of Calcium and Phosphate Ions in Expression of Sodium-dependent Vitamin C Transporter 2 and Osteopontin in MC3T3-E1 Osteoblastic Cells

Osteoclasts dissolve mineralized bone matrix at bone resorption sites and release large amounts of calcium (Ca(2+)) and phosphate (PO(4)(3-)) ions into the extracellular fluid. However, the exact nature of Ca(2+) and PO(4)(3-) on osteoblasts remains unclear. We proposed that Ca(2+) and PO(4)(3-) ions are required for the expression of sodium-dependent vitamin C transporter (SVCT) 2 and a differentiation marker, osteopontin (OPN), in osteoblasts as a response to the osteoclastic degradation. Results from Northern blotting indicated that a deficiency of Ca(2+) or PO(4)(3-) inhibited both SVCT2 and OPN expression in a time-dependent manner, whereas elevated Ca(2+) (1 to 4 mM) or PO(4)(3-) (1 to 4 mM) dose-dependently induced SVCT2, OPN expression and OPN promoter activity. In addition, the L-type calcium channel blocker, nifedipine (5 to 20 micro M) and the phosphate transporter inhibitor, foscarnet (0.15 to 0.6 mM), dose-dependently abolished Ca(2+)- and PO(4)(3-)-induced SVCT2, OPN expression and OPN promoter activity. Furthermore, the results from L-ascorbic acid uptake assay and Western blotting indicated that the stimulatory effect of Ca(2+) and PO(4)(3-) on functional SVCT2 protein expression. These findings suggested that Ca(2+) and PO(4)(3-) regulate osteoblastic phenotype by entering into cells to stimulate SVCT2 and OPN expression.