Effect of 1,25(OH) 2D 3 on its receptor mRNA levels and osteocalcin synthesis in human osteosarcoma cells

Abstract

Hormone-dependent accumulation of specific binding sites for 1,25(OH) 2D 3 and changes in human 1,25-dihydroxyvitamin D receptor (hVDR) mRNA levels were examined in cell lines (MG-63, SaOs-2 and U2-Os) derived from human bone. Osteocalcin synthesis and secretion as well as alkaline phosphatase activity were also characterized as biochemical markers of the osteoblastic phenotype. Specific binding sites for 1,25(OH) 2D 3 were quantified by incubating cultured intact cells with [ 3H]1,25(OH) 2D 3 at 37° C. Based on the uptake of 1,25(OH) 2D 3, there were about 3000 to 4000 receptor molecules per cell with apparent dissociation constants varying between 0.02 to 0.03 nM. The binding was saturated with 1,25(OH) 2D 3 in 3 to 6 h after the hormone addition and further exposure to the hormone resulted in an upregulation of the bindings sites. The levels were elevated by as little as 10 to 200 pM 1,25(OH) 2D 3, and maximal binding was achieved with 0.2–0.7 nM 1,25(OH) 2D 3. Treatment with 1,25(OH) 2D 3 also resulted in a clear increase (about 3-fold) in hVDR mRNA by 24 h in all three cell lines. The increase in hVDR mRNA level was time- and dose-dependent. MG-63 cells responded with 2- and 15-fold increases, respectively, in intracellular and secreted levels of osteocalcin after the 1,25(OH) 2D 3-treatment. In dot-blot hybridization assay, MG-63 cells expressed osteocalcin mRNA which was inducible with 1,25(OH) 2D 3 while, in SaOs-2 and U2-Os cells, osteocalcin mRNA was not detected under the same circumstances. Also, no secretion of osteocalcin was detected in SaOs-2 and U2-Os cells with or without addition of 1,25(OH) 2D 3.