Requirement of calcium and phosphate ions in expression of sodium-dependent vitamin C transporter 2 and osteopontin in MC3T3-E1 osteoblastic cells

Abstract

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 μM) 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.