Pike J W, Haussler M R
J Biol Chem. 1983 Jul 25;258(14):8554-60.
The uptake of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) by intact cells was investigated using the cultured embryonic 3T6 mouse fibroblast as a model. Suspended cells, incubated for 60-90 min in serum-containing culture medium supplemented with 1,25-(OH)2D3 (2 nM), maximally accumulate hormone which becomes bound to a typical vitamin D 3.3 S receptor protein. Incubation of cells with varying concentrations of 1,25-(OH)2D3 reveals the presence of 21,000 receptor molecules/3T6 cell, with an apparent uptake constant of 6-8 X 10(-10) M at 37 degrees C. This value contrasts with the equilibrium dissociation constant (Kd) for 1,25-(OH)2D3 binding of 6 X 10(-11) M as determined at 2 degrees C in disrupted cell cytosol. The distribution of unoccupied (R0) receptors is predominantly (greater than 85%) cytosolic in the hormone-deprived state (1,25-(OH)2D3 less than 0.05 nM), whereas exposure to 1,25-(OH)2D3 (2 nM) leads to almost complete nuclear localization of the occupied receptor at both 2 and 37 degrees C. This phenomenon was similarly supported through reconstitution of receptor and purified 3T6 nuclei in vitro in which binding also occurs at 2 degrees C. The majority (65%) of intact cell-formed receptor-nuclear complexes can be solubilized by micrococcal nuclease treatment, suggesting the participation of DNA in the acceptor binding site for the 1,25-(OH)2D3 receptor. Consistent with these data, DNA-binding of receptor also occurred in vitro at 2 degrees C and was a characteristic of both occupied (Rs) and unoccupied receptors. However, elution of the latter occurred at reduced ionic strength, implying that the hormone does physically alter the receptor protein. This binding was also sensitive to prior ethidium bromide saturation of DNA-cellulose, but not phosphocellulose. Although the biologic effects of the 1,25-(OH)2D3 hormone in 3T6 fibroblasts are as yet unknown, the present findings support previous work with 1,25-(OH)2D3 receptors and suggest that this cell represents a good model for the study of nuclear events associated with the molecular action of 1,25-(OH)2D3.
以培养的胚胎3T6小鼠成纤维细胞为模型，研究了完整细胞对1,25 - 二羟基维生素D3（1,25-(OH)2D3）的摄取。将悬浮细胞在补充有1,25-(OH)2D3（2 nM）的含血清培养基中孵育60 - 90分钟，激素会最大程度地积累，并与典型的维生素D 3.3 S受体蛋白结合。用不同浓度的1,25-(OH)2D3孵育细胞，发现每个3T6细胞存在21,000个受体分子，在37℃时的表观摄取常数为6 - 8×10(-10) M。该值与在2℃下破碎细胞胞质溶胶中测得的1,25-(OH)2D3结合的平衡解离常数（Kd）6×10(-11) M形成对比。在激素缺乏状态（1,25-(OH)2D3小于0.05 nM）下，未占据的（R0）受体主要（超过85%）分布在胞质溶胶中，而暴露于1,25-(OH)2D3（2 nM）会导致在2℃和37℃时被占据的受体几乎完全定位于细胞核。通过在体外将受体与纯化的3T6细胞核重组也同样支持了这一现象，在体外2℃时也会发生结合。大多数（约65%）完整细胞形成的受体 - 核复合物可通过微球菌核酸酶处理溶解，这表明DNA参与了1,25-(OH)2D3受体的受体结合位点。与这些数据一致，受体与DNA的结合在体外2℃时也会发生，并且是被占据的（Rs）和未被占据的受体的共同特征。然而，未被占据的受体在离子强度降低时会洗脱，这意味着激素确实会对受体蛋白产生物理改变。这种结合对DNA - 纤维素预先用溴化乙锭饱和敏感，但对磷酸纤维素不敏感。虽然1,25-(OH)2D3激素在3T6成纤维细胞中的生物学效应尚不清楚，但目前的研究结果支持了先前关于1,25-(OH)2D3受体的研究工作，并表明该细胞是研究与1,25-(OH)2D3分子作用相关的核事件的良好模型。
