IMMUNOCYTOCHEMICAL LOCALIZATION OF THE 1,25-DIHYDROXYVITAMIN-D3 RECEPTOR IN TARGET-CELLS
IMMUNOCYTOCHEMICAL LOCALIZATION OF THE 1,25-DIHYDROXYVITAMIN-D3 RECEPTOR IN TARGET-CELLS
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DOI：
10.1210/endo-122-4-1224
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发表时间：
1988-04-01
期刊：
影响因子：
4.8
通讯作者：
DEMPSTER, DW
中科院分区：
文献类型：
--
作者：
CLEMENS, TL;GARRETT, KP;DEMPSTER, DW
我们使用一种针对纯化禽1,25-二羟维生素D3受体的单克隆抗体（9A7），开发了一种免疫细胞化学技术，用于在固定组织和培养细胞中可视化蛋白质。在Bouin' s固定的鸡肠中，1,25-二羟维生素D3受体样免疫反应性主要定位于上皮细胞的细胞核，并且在隐窝中比在绒毛细胞中更丰富。受体染色在肝细胞中较低或无法检测到，但在肝窦内壁的细胞核中存在。在大鼠脑中，受体样免疫反应性丰富且分布广泛，但并不总是与维生素d依赖性钙结合蛋白的存在一致；含有丰富钙结合蛋白的小脑浦肯野细胞缺乏1,25-二羟基维生素D3受体。在分解的大鼠骨细胞中，包括破骨细胞在内的单个核细胞中存在受体免疫反应性。两个独立的成骨细胞样大鼠骨肉瘤细胞克隆，在先前的研究中显示为受体阳性（17/2.8）或阴性（24.1），其核免疫反应性与配体结合决定的受体水平完全一致。在受体阳性的3T6成纤维细胞中，通过10-7 M 1,25-二羟维生素D3处理48小时，可以观察到激素诱导的受体上调现象。我们的研究证明了免疫细胞化学方法在靶组织中观察1,25-二羟维生素D3受体的可行性，并表明它主要是一个相对未被占据和完全激活状态的核蛋白。此外，维生素d依赖性钙结合并不是1,25-二羟基维生素D3作用的普遍标志。相反，我们的观察表明1,25-二羟基维生素D3受体的表达可能与细胞分化状态有关。
We have used a monoclonal antibody (9A7) against the purified avian 1,25-dihydroxyvitamin D3 receptor to develop an immunocytochemical technique for visualization of the protein in fixed tissues and cultured cells. In Bouin''s-fixed, chick intestine, 1,25-dihydroxyvitamin D3 receptor-like immunoreactivity was localized mainly in nuclei of epithelial cells and was more abundant in the crypt than in the villar cells. Receptor staining was low or undetectable in liver hepatocytes but was present in nuclei of cells lining the hepatic sinusoids. In rat brain, receptor-like immunoreactivity was abundant and widely distributed, but did not always coincide with the presence of vitamin D-dependent calcium-binding protein; 1,25-dihydroxyvitamin D3 receptor was absent from cerebellar Purkinje cells that contained abundant calcium-binding protein. In disaggregated rat bone cells, receptor immunoreactivity was present in mononuclear cells including osteoclasts. Two separate clones of osteoblast-like, rat osteosarcoma cells, shown in previous studies to be either receptor positive (17/2.8) or negative (24.1), demonstrated nuclear immunoreactivity in exact concordance with receptor levels as determined by ligand binding. The phenomenon of hormone-induced up-regulation of receptor was visualized in receptor-positive 3T6 fibroblasts by demonstration of markedly enhanced nuclear reactivity in cells treated with 10-7 M 1,25-dihydroxyvitamin D3 for 48 h. Our studies demonstrate the feasibility of the immunocytochemical approach to visualize the 1,25-dihydroxyvitamin D3 receptor in target tissues and show that it is predominantly a nuclear protein in the relatively unoccupied and fully activated states. Moreover, the vitamin D-dependent calcium binding is not a universal marker for 1,25-dihydroxyvitamin D3 action. Rather, our observations suggest that the expression of the 1,25-dihydroxyvitamin D3 receptor may be connected with the state of cellular differentiation.