A highly potent 26,27-Hexafluoro-1a,25-dihydroxyvitamin D3 on calcification in SV40-transformed human fetal...

26,27-hexafluoro-1a,25-dihydroxyvitamin D3 (F6-D3) has been reported to be 5-10 times more potent than 1a,25-dihydroxyvitamin D3[1,25(OH)2D3] in biological systems in vivo and in vitro. However, the effect of F6-D3 on bone formation has yet to be clarified. In the present study, we investigated the effect of F6-D3 on SV40-transfected human fetal osteoblastic cells (SV-HFO) and found it to be about 100 times greater than that of 1,25(OH)2D3 in stimulating calcification. F6-D3 was also about 100 times more effective than 1,25(OH)2D3 in enhancing the expression of mRNA for alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In the presence of 10?8 M F6-D3 and 10?6 M 1,25(OH)2D3, the calcification began on day 9 and increased up to day 19. Expression of mRNA for ALP and OCN reached a maximum on day 4 and thereafter declined. On the other hand, when osteoblastic cells were incubated with a low level of [1b-3H]-F6-D3- or [1b-3H]-1,25(OH)2D3, each radioactive peak could not be detected. However, on the incubation of osteoblastic cells and radioactive substrate in the presence of ketoconazole, a selective inhibitor of CYP24, a clear peak for each substrate was detected. This suggested that F6-D3 as well as 1,25(OH)2D3 is metabolized by CYP24. Osteoblastic cells were incubated with 10?8 M[1b-3H]-F6-D3 or 10?8 M[1b-3H]-1,25(OH)2D3 for 4, 9, and 14 days. A small peak of 1,25(OH)2D3 was observed and thereafter its level decreased. In addition, two unknown peaks increased when the culture period was extended. In the case of F6-D3, peaks of F6-D3 and 26,27-hexafluoro-23-oxo-1a,25(OH)2D3(23-oxo-F6) were clearly detected, the latter being about 4 times higher than the former. Both peaks was retained up to day 14. The amount of unlabeled F6-D3 and 23-oxo-F6 calculated from the specific radioactivity in the cells may be similar to the amount of 1,25(OH)2D3 and its metabolites. The strong activity of F6-D3 in stimulating calcification may be due to the fact that F6-D3 is much more potent than 1,25(OH)2D3 in enhancing the expression of mRNA for ALP, OCN, and OPN and that the amount of F6-D3 and 23-oxo-F6 accumulated in the cells is much greater than that of 1,25(OH)2D3 and its metabolite.