一种高效的26,27-六氟-1α,25-二羟基维生素D3对SV40转化的人胎儿成骨细胞钙化的影响。

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

作者信息

Miyahara T, Simoura T, Osahune N, Uchida Y, Sakuma T, Nemoto N, Kozakai A, Takamura T, Yamazaki R, Higuchi S, Chiba H, Iba K, Sawada N

机构信息

Department of Toxicology, Faculty of Pharmaceutical Sciences, Toyama Medical & Pharmaceutical University, 2630 Sugitani, Japan.

出版信息

Calcif Tissue Int. 2002 Jun;70(6):488-95. doi: 10.1007/s00223-001-1039-5. Epub 2002 May 20.

DOI:10.1007/s00223-001-1039-5

PMID:12016462

Abstract

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.

摘要

据报道，26,27-六氟-1α,25-二羟基维生素D3（F6-D3）在体内和体外生物系统中的效力比1α,25-二羟基维生素D3 [1,25(OH)2D3] 强5至10倍。然而，F6-D3对骨形成的影响尚未阐明。在本研究中，我们研究了F6-D3对SV40转染的人胎儿成骨细胞（SV-HFO）的影响，发现其在刺激钙化方面比1,25(OH)2D3强约100倍。F6-D3在增强碱性磷酸酶（ALP）、骨钙素（OCN）和骨桥蛋白（OPN）的mRNA表达方面也比1,25(OH)2D3有效约100倍。在存在10⁻⁸ M F6-D3和10⁻⁶ M 1,25(OH)2D3的情况下，钙化在第9天开始，并持续增加至第19天。ALP和OCN的mRNA表达在第4天达到最大值，此后下降。另一方面，当成骨细胞与低水平的[1β-³H]-F6-D3或[1β-³H]-1,25(OH)2D3一起孵育时，每个放射性峰均未被检测到。然而，当成骨细胞与放射性底物在酮康唑（一种CYP24的选择性抑制剂）存在下孵育时，每种底物均检测到一个明显的峰。这表明F6-D3以及1,25(OH)2D3均被CYP24代谢。成骨细胞与10⁻⁸ M [1β-³H]-F6-D3或10⁻⁸ M [1β-³H]-1,25(OH)2D3孵育4、9和14天。观察到一个小的1,25(OH)2D3峰，此后其水平下降。此外，当培养期延长时，出现了两个未知峰。就F6-D3而言，清楚地检测到F6-D3和26,27-六氟-23-氧代-1α,25(OH)2D3（23-氧代-F6）的峰，后者比前者高约4倍。两个峰均保留至第14天。根据细胞中的比放射性计算的未标记F6-D3和23-氧代-F6的量可能与1,25(OH)2D3及其代谢产物的量相似。F6-D3在刺激钙化方面的强活性可能是由于F6-D3在增强ALP、OCN和OPN的mRNA表达方面比1,25(OH)2D3强得多，并且细胞中积累的F6-D3和23-氧代-F6的量比1,25(OH)2D3及其代谢产物的量多得多。