Yoichi Negishi

Establishment of permanent cell lines exhibiting vitamin D-dependent expression of β-galactosidase activity

The active hormonal form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), has been described as a principal mediator of skeletal homeostasis. Treatment of rat osteosarcoma (ROS)17/2.8, an osteoblast-like cell line, with 1alpha,25(OH)2D3 results in a ligand-dependent increase in transcription of the bone-specific osteocalcin gene. We isolated permanent cell lines that were established by transfecting ROS 17/2.8 cells with plasmids consisting of the human osteocalcin gene promoter containing the vitamin D responsive element linked to a bacterial beta-galactosidase gene. In one of many cell lines, especially in clone NK-31, 1alpha,25(OH)2D3 strongly stimulated beta-galactosidase activity. Reverse transcription-polymerase chain reaction analysis also showed endogenous osteocalcin gene expression and beta-galactosidase gene expression in clone NK-31 cells, which paralleled the increase in beta-galactosidase activity. Using a synthetic analogue of 1alpha,25(OH)2D3, 24,24-difluoro-1alpha,25-dihydroxyvitamin D3, we found that the levels of this activity and these gene expressions were nearly parallel to those of 1alpha,25(OH)2D3. 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 at high doses (concentration: 10(-7) M) also induced beta-galactosidase activity in clone NK-31. These cell lines, harboring the plasmid-carrying beta-galactosidase gene under the control of the osteocalcin gene promoter, may contribute to studies on the regulation by 1alpha,25(OH)2D3 or to the development of synthetic analogues of 1alpha,25(OH)2D3.