VITAMIN D RESISTANCE AND RELATED DISORDERS

维生素 D 抵抗和相关疾病

• 批准号: 6162053
• 负责人: J BARSONY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6162053
• 关键词: 1,25 dihydroxycholecalciferol; clinical research; confocal scanning microscopy; cyclic GMP; endoplasmic reticulum; fibroblasts; genetic disorder; green fluorescent proteins; human subject; microtubules; receptor coupling; receptor expression; vitamin D receptors; vitamin D resistant rickets

The hormonal form of vitamin D, calcitriol, acts through the vitamin D receptor (VDR) to regulate important functions such as calcium homeostasis, cell proliferation, differentiation, and immune functions. Defects in the target actions of calcitriol thus have a broad spectrum of manifestations ranging from hyperreactivity to a lack of effects. Hereditary resistance to calcitriol usually results from a mutation in the VDR gene and manifests as rickets. We used skin fibroblasts from subjects with this rare hereditary disease to explore mechanisms of calcitriol action. These mutant cells display abnormalities in many discrete steps of the receptor activation pathway. They have been used to establish that certain receptor mutations compromise many receptor functions but allow other functions to be retained normally. We used these natural "knockout" cells to show that a nongenomic action of calcitriol to raise intracellular cGMP level is mediated through the VDR. To study VDR localization in living cells, we developed the first biologically active fluorescent ligand for calcitriol: 38-BODIPY-calcitriol. This labeled hormone made possible the use of confocal microscopy to show that VDR reside both in the cytoplasm and the nucleus, and that calcitriol exposure induces translocation of cytoplasmic VDR into the nucleus. We identified a docking site for VDR in the endoplasmic reticulum, bound to calreticulin. Experiments in living cells also showed that VDR associates with microtubules during translocation, and this finding was confirmed by in vitro copolymerization studies with adenovirus expressed VDR and purified tubulin. Fluorescent labeling techniques proved useful in elucidating the mechanisms of VDR activation and led us to understand better abnormalities in the vitamin D effector system. A transcriptionally active chimeric protein was prepared in Dr. Hager's laboratory, containing the green fluorescent protein and the glucocorticoid receptor (GFP-GR). Transient expression of GFP-GR in mouse adenocarcinoma cells allowed us to study receptor motion and intranuclear targeting. We confirmed that unliganded GR resides in the cytoplasm and that hormone addition initiates a cytoplasm to nucleus translocation. We detected distinct subnuclear localizations of agonist (dexamethasone) and antagonist (RU486) activated receptors. After dexamethasone, GFP-GR accumulated in a discrete series of foci, while after RU486, GFP-GR remained in a reticular pattern, not forming bright foci. Another adenocarcinoma cell line (3134) was used to define the nature of these foci. These cells carry a Aminichromosome@ containing 800 GR binding sites in a head-to-tail tandem repeat. Agonist exposure caused GFP-GR accumulation along this array. A mutant GFP-GR, with altered DNA-binding failed to localize to this array and failed to activate transcription of this Aminichromosome. We prepared green fluorescent protein VDR chimeras to study translocation and intranuclear distribution of VDR. Transient expression of this GFP-VDR in mouse adenocarcinoma and in COS-7 cells showed that the chimeric protein is competent for hormone dependent transactivation and confirmed that hormone induces translocation of cytoplasmic VDR into the nucleus. Green fluorescent protein chimeras of mutant VDR variants are being used to gain further understanding of the mechanisms of vitamin D resistant rickets.

维生素D的激素形式，骨化三醇，通过维生素D 维生素D受体（VDR）调节重要功能，如钙 稳态、细胞增殖、分化和免疫功能。 因此，骨化三醇靶向作用的缺陷具有广谱性 从反应过度到缺乏效果 对骨化三醇的遗传性耐药性通常是由以下基因突变引起的： VDR基因并表现为佝偻病我们使用皮肤成纤维细胞， 患有这种罕见遗传性疾病的受试者，以探索 骨化三醇作用。这些突变细胞在许多方面显示出异常， 受体活化途径的离散步骤。池使用 为了确定某些受体突变损害了许多受体， 功能，但允许其他功能正常保留。我们使用 这些天然的“敲除”细胞表明， 骨化三醇升高细胞内cGMP水平是通过 VDR。为了研究VDR在活细胞中的定位，我们开发了第一个 骨化三醇的生物活性荧光配体： 38-BODIPY-骨化三醇。这种标记的激素使得使用 共聚焦显微镜显示VDR既存在于细胞质中， 细胞核，并且钙三醇暴露会诱导细胞核的移位 细胞质VDR进入细胞核。我们找到了VDR的对接点 与钙网蛋白结合实验 活细胞也表明，VDR与微管相关， 易位，这一发现得到了证实， 与腺病毒表达的VDR和纯化的聚合研究 微管蛋白荧光标记技术被证明是有用的， VDR激活的机制，使我们更好地了解 维生素D效应系统异常 具有转录 活性嵌合蛋白在Hager博士的实验室制备， 含有绿色荧光蛋白和糖皮质激素受体， （GFP-GR）。GFP-GR在小鼠腺癌细胞中的瞬时表达 使我们能够研究受体运动和核内靶向。我们 证实了未配体的GR存在于细胞质中， 添加引发细胞质到细胞核的易位。我们检测到 激动剂（地塞米松）的不同亚核定位， 拮抗剂（RU 486）激活受体。地塞米松后，GFP-GR GFP-GR在一系列离散的病灶中积累，而在RU 486后，GFP-GR 仍保持网状模式，不形成明亮的病灶。 另一 使用腺癌细胞系（3134）来确定这些细胞的性质。 焦点这些细胞携带含有800 GR结合的氨基染色体 头-尾串联重复序列中的位点。激动剂暴露导致GFP-GR 沿着沿着这个阵列的累积。一种DNA结合改变的突变型GFP-GR 未能定位到该阵列，也未能激活转录 这个氨基染色体。我们制备了绿色荧光蛋白VDR 嵌合体来研究VDR的易位和核内分布。 这种GFP-VDR在小鼠腺癌中的瞬时表达和在小鼠淋巴结转移中的瞬时表达。 COS-7细胞显示，嵌合蛋白是激素的感受态 依赖的反式激活，并证实激素诱导 细胞质VDR易位到细胞核中。绿色荧光 突变VDR变体的蛋白质嵌合体被用于获得进一步的 了解维生素D抵抗性佝偻病的机制。