Immunocytology with microwave-fixed fibroblasts shows 1 alpha,25-dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors.
Immunocytology with microwave-fixed fibroblasts shows 1 alpha,25-dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors.
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带有微波固定成纤维细胞的免疫细胞学显示1个α，25-二羟基维生素D3依赖性快速和雌激素依赖性维生素D受体的慢重组。
DOI：
10.1083/jcb.111.6.2385
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发表时间：
1990-12
期刊：
影响因子：
--
通讯作者：
Marx SJ
中科院分区：
文献类型：
--
作者：
Barsony J;Pike JW;DeLuca HF;Marx SJ
以前的研究没有证据表明维生素D受体(VDR)的区隔或亚细胞组织的调节。微波固定(9-15 S)和间接免疫检测系统的亲和素-生物素增强和藻红蛋白荧光团导致了足够的空间和时间分辨率，使这些过程能够分析。我们研究了来自正常人或患有四种不同类型遗传缺陷损害VDR功能的患者的培养成纤维细胞(突变细胞)。在缺乏1，25-二羟基维生素D3(骨化三醇)的情况下，VDR的分区受血清或雌激素的调节。在无血清和酚红培养的细胞中，VDR主要分布在胞浆内，但在加入血清或雌激素至少18h后，VDR主要分布在细胞核内(缓慢调节)。骨化三醇在部分VDR中启动了一个快速且多步骤的重组过程(快速调节)：在15-45 S内聚集，在30-45 S内沿原纤维排列，在45-90 S内聚集在核周，在1-3分钟内聚集在核内。我们发现骨化三醇在不同类型的培养细胞中对VDR的快速作用类似。这些连续的VDR模式变化显示出骨化三醇剂量依赖性和骨化三醇类似物对VDR的特异性。在突变的成纤维细胞中，骨化三醇后VDR模式的变化在选定的步骤中缺失或严重干扰。用小麦胚芽凝集素处理正常细胞，可以阻止蛋白质通过核孔的运输，也可以阻止依赖骨化三醇的VDR转位。我们的结论是，微波固定后的免疫细胞学为调节VDR的组织和定位提供了证据。
Prior studies have given no evidence for regulation of vitamin D receptor (VDR) compartmentalization or subcellular organization. Microwave fixation (9-15 s) and an indirect immunodetection system of avidin-biotin enhancement and phycoerythrin fluorophore resulted in sufficient spatial and temporal resolution to allow analysis of these processes. We studied cultured fibroblasts from normals or from patients with four different types of hereditary defect compromising VDR function (mutant cells). Compartmentalization of VDRs in the absence of 1,25-dihydroxyvitamin D3 (calcitriol) was regulated by serum or estrogen. VDRs were mainly cytoplasmic in cells cultured without serum and phenol red, but VDRs were mainly intranuclear after addition of serum or an estrogen to cells for at least 18 h (slow regulation). Calcitriol initiated a rapid and multistep process (rapid regulation) of reorganization in a portion of VDRs: clumping within 15-45 s, alignment of clumps along fibrils within 30-45 s, perinuclear accumulation of clumps within 45-90 s, and intranuclear accumulation of clumps within 1-3 min. We found similar rapid effects of calcitriol on VDRs in various other types of cultured cells. These sequential VDR pattern changes showed calcitriol dose dependency and calcitriol analogue specificity characteristic for the VDR. In mutant fibroblasts VDR pattern changes after calcitriol were absent or severely disturbed at selected steps. Treatment of normal cells with wheat germ agglutinin, which blocks protein transport through nuclear pores, also blocked calcitriol-dependent translocation of VDRs. We conclude that immunocytology after microwave fixation provides evidence for regulation of VDR organization and localization.