Matsue M, Kageyama R, Denhardt D T, Noda M
Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-Ku, Japan.
Bone. 1997 Apr;20(4):329-34. doi: 10.1016/s8756-3282(97)00005-7.
The active form of vitamin D, 1,25(OH)2 vitamin D3 (D3), is a potent modulator of osteoblastic function. In this study, we examined, the expression of a negative-type basic helix-loop-helix transcription factor, HES-1, in osteoblastic cells and the regulation of its expression by D3. We found that HES-1 is expressed as a 1.7 kb mRNA in rat osteoblastic osteosarcoma ROS17/2.8 cells. Treatment with D3 suppressed HES-1 mRNA levels by about 50%. This suppression was observed within 24 h and lasted for at least 48 h. The suppressive effect was dose-dependent starting at 10(-9) mol/L and saturated at 10(-8) mol/L. The vitamin D3 suppression of HES-1 mRNA level was blocked by actinomycin D as well as cycloheximide, suggesting the involvement of transcriptional control, which requires new protein synthesis. Proteins in the crude nuclear extracts prepared from ROS17/2.8 cells bound to the N-box sequence (CACNAG). To examine the function of HES-1 in osteoblasts, HES-1 was overexpressed in ROS17/2.8 cells. Overexpression of HES-1 suppressed the vitamin D-dependent upregulation of osteopontin gene expression in these cells. Vitamin D suppression of HES-1 gene expression was also observed in normal rat calvaria-derived osteoblast-enriched cells. These results indicate that HES-1 is expressed in osteoblastic cells and is involved in vitamin D3 regulation of osteoblastic gene expression.
维生素D的活性形式，1,25(OH)2维生素D3（D3），是成骨细胞功能的有效调节剂。在本研究中，我们检测了负性碱性螺旋-环-螺旋转录因子HES-1在成骨细胞中的表达及其受D3的调控情况。我们发现HES-1在大鼠成骨肉瘤ROS17/2.8细胞中以1.7 kb的mRNA形式表达。用D3处理可使HES-1 mRNA水平降低约50%。这种抑制在24小时内即可观察到，并持续至少48小时。抑制作用从10(-9)mol/L开始呈剂量依赖性，在10(-8)mol/L时达到饱和。放线菌素D和环己酰亚胺均可阻断维生素D3对HES-1 mRNA水平的抑制作用，提示其涉及转录调控，且需要新蛋白质合成。从ROS17/2.8细胞制备的粗核提取物中的蛋白质与N-box序列（CACNAG）结合。为了研究HES-1在成骨细胞中的功能，我们在ROS17/2.8细胞中过表达了HES-1。HES-1的过表达抑制了这些细胞中骨桥蛋白基因表达依赖维生素D的上调。在正常大鼠颅骨来源的富含成骨细胞的细胞中也观察到了维生素D对HES-1基因表达的抑制作用。这些结果表明，HES-1在成骨细胞中表达，并参与维生素D3对成骨细胞基因表达的调控。
