Jääskeläinen T, Itkonen A, Mäenpää P H
Department of Biochemistry and Biotechnology, University of Kuopio, Findland.
Eur J Biochem. 1995 Mar 1;228(2):222-8.
We have studied the role of retinoid X receptor alpha (RXR alpha) in vitamin D receptor (VDR) responsive interactions using nuclear extracts from human osteoblast-like MG-63 osteosarcoma cells and its specific response element from human osteocalcin gene (OC-VDRE). An RXR alpha-specific antibody was not capable of recognizing the two VDR responsive complexes formed with the OC-VDRE. Addition of in-vitro-produced RXR alpha to the binding reaction resulted in decreased binding of the two VDR-responsive interactions and, simultaneously, formation of a new complex, which was identified with RXR alpha- and VDR-specific antibodies. A similarly migrating RXR alpha- and VDR-responsive complex was also formed when baculovirus-expressed VDR was used with the in-vitro-produced RXR alpha in the absence of a nuclear extract or when VDRE from mouse osteopontin gene (OP-VDRE) was used as a binding site. Characterization of DNA binding properties for this VDR-RXR alpha complex revealed that both half sites of OC-VDRE are required for DNA binding. These results indicate that RXR alpha is probably not the physiological accessory factor in the MG-63 osteosarcoma cells needed for the VDR-VDRE interactions within the human osteocalcin gene promoter, although it is capable of dimerizing with recombinant VDR and the native VDR from these cells and although these dimers are capable of binding in vitro to the OC-VDRE.
我们使用来自人成骨细胞样MG-63骨肉瘤细胞的核提取物及其来自人骨钙素基因的特异性反应元件（OC-VDRE），研究了视黄酸X受体α（RXRα）在维生素D受体（VDR）反应性相互作用中的作用。一种RXRα特异性抗体无法识别与OC-VDRE形成的两种VDR反应性复合物。在结合反应中加入体外产生的RXRα导致两种VDR反应性相互作用的结合减少，同时形成一种新的复合物，该复合物可用RXRα和VDR特异性抗体鉴定。当在没有核提取物的情况下将杆状病毒表达的VDR与体外产生的RXRα一起使用，或者当使用来自小鼠骨桥蛋白基因的VDRE（OP-VDRE）作为结合位点时，也会形成一种迁移情况类似的RXRα和VDR反应性复合物。对这种VDR-RXRα复合物的DNA结合特性进行表征发现，OC-VDRE的两个半位点都是DNA结合所必需的。这些结果表明，RXRα可能不是人骨钙素基因启动子内VDR-VDRE相互作用所需的MG-63骨肉瘤细胞中的生理辅助因子，尽管它能够与重组VDR以及这些细胞中的天然VDR二聚化，并且尽管这些二聚体能够在体外与OC-VDRE结合。
