Yoon K G, Rutledge S J, Buenaga R F, Rodan G A
Department of Bone Biology and Osteoporosis Research, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Biochemistry. 1988 Nov 15;27(23):8521-6. doi: 10.1021/bi00423a003.
Osteocalcin is an abundant noncollagenous protein in bone, and its synthesis is stimulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In this study, the rat osteocalcin gene was isolated, sequenced, and found to be a single-copy gene that is highly conserved between human and rat. Northern blot analysis of RNAs from a number of rat tissues revealed osteocalcin mRNA only in calvariae, consistent with bone-specific expression of osteocalcin. In order to investigate promoter activity and its modulation by 1,25(OH)2D3, plasmids containing the osteocalcin promoter region linked to the reporter enzyme bacterial chloramphenicol acetyltransferase (CAT) were used to transfect rat osteosarcoma ROS 17/2.8 cells, which express osteocalcin endogenously, and UMR 106 cells, which lack osteocalcin expression. Transfected ROS 17/2.8 cells exhibited a higher basal CAT activity than UMR 106 cells. Moreover, 1,25(OH)2D3 stimulated the CAT expression 5-10-fold only in ROS 17/2.8 cells and not in UMR 106 cells. By use of unidirectional deletion analysis, a domain strongly responsive to 1,25(OH)2D3 was identified between bases -1035 and -871 upstream from the site of transcription initiation, while a weakly responsive region was found further downstream.
骨钙素是骨骼中一种丰富的非胶原蛋白，其合成受1,25 - 二羟维生素D3 [1,25(OH)2D3]刺激。在本研究中，大鼠骨钙素基因被分离、测序，发现是一个单拷贝基因，在人和大鼠之间高度保守。对多种大鼠组织的RNA进行Northern印迹分析，结果显示仅在颅盖骨中存在骨钙素mRNA，这与骨钙素的骨特异性表达一致。为了研究启动子活性及其受1,25(OH)2D3的调控情况，将含有与报告酶细菌氯霉素乙酰转移酶（CAT）相连的骨钙素启动子区域的质粒用于转染内源性表达骨钙素的大鼠骨肉瘤ROS 17/2.8细胞以及缺乏骨钙素表达的UMR 106细胞。转染后的ROS 17/2.8细胞表现出比UMR 106细胞更高的基础CAT活性。此外，1,25(OH)2D3仅在ROS 17/2.8细胞中使CAT表达增加5 - 10倍，而在UMR 106细胞中则无此作用。通过单向缺失分析，在转录起始位点上游 - 1035至 - 871碱基之间鉴定出一个对1,25(OH)2D3有强烈反应的结构域，而在更下游发现了一个弱反应区域。
