[Phosphoproteins biosynthesis induced by 1,25(OH)2D3 in the rat calvarial osteoblasts].

Abstract

The present study attempts to explore the newly synthesized phosphoproteins secreted by osteoblast-like cells incubated with 1,25(OH)2D3. The phosphoproteins, which are non-collagenous proteins, may possess the ability to regulate bone mineral solubility. An osteoblast-enriched cell population isolated from 2 day-old rat calvaria by sequential enzymatic digestion was cultured in a defined medium containing dialized fetal calf serum protein (FCSP, 2 mg/ml) with 1, 5 and 10 x 10(-9)M 1,25(OH)2D3. At confluence, 32Pi (Na2H32PO4, NEX-011) was added for 24 hr. The medium proteins were precipitated by cold 10% TCA, dissolved in 15 mM Tris-HCl, pH 7.4 containing 7 M urea and chromatographed on hydroxyapatite columns (Bio-Rad, HTP). After stepwise elution with 6 mM, 0.1, 0.5 and 1.5 M Pi Hepes buffer pH 7.4 containing 3 M urea and 5 mM levamisole, the phosphoproteins were applied to 10% SDS-PAGE and autoradiographed. The 32Pi incorporated phosphoproteins of 75K, 66K, 58K, 42K, 38K, 24K, 22K, 19K, 15.5K, 13K and 3.5-10K molecular weight which were bound on a hydroxyapatite column were identified on autoradiograms of SDS-PAGE. The synthesis of 19K phosphoprotein was stable. However the synthesis of 75K, 66K, 38K and 15.5K phosphoproteins were increased by 1,25(OH)2D3. Therefore, 1,25(OH)2D3 induced phosphoproteins synthesized in rat calvarial osteoblasts, which can bind tightly on hydroxyapatite, may regulate the solubility of bone mineral and play a role in maintaining a blood/bone equilibrium.