Vitamin D Resistance And Related Disorders

维生素 D 抵抗及相关疾病

• 批准号: 7153417
• 负责人: JULIANNA 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/7153417
• 关键词: 1,25 dihydroxycholecalciferol; bone development; calcium disorder; calcium metabolism; genetically modified animals; heat shock proteins; histamine; homeostasis; hormone regulation /control mechanism; human tissue; intracellular transport; laboratory mouse; microscopy; molecular pathology; receptor expression; site directed mutagenesis; tissue /cell culture; vitamin D receptors; vitamin D resistant rickets

The hormonal form of vitamin D, calcitriol, acts through the vitamin D receptor (VDR) to regulate calcium homeostasis, maintenance of skeletal integrity, immune defense, hormone secretion, hair follicle cycling, and mammary gland development. The long-term objective of our studies is to elucidate the pathophysiology of diseases caused by excess or insufficient calcitriol actions. We apply modern imaging, biochemical, and genetic approaches to study the regulation of calcitriol metabolism and actions at the molecular level. Abnormal hormonal effects may result from insufficient or excess vitamin D supplementation or from abnormal VDR functions. One of the diseases caused by aberrant VDR functions is hereditary resistance to calcitriol (HVDDR). This disease is usually results from a mutation in the VDR gene and manifests as rickets. We have used skin fibroblasts from subjects with HVDDR to explore the molecular mechanisms of VDR activation. These fibroblasts either lack VDR or express mutant VDRs, which display abnormalities in discrete steps of the receptor activation pathway. We found that several mutations cause defects in VDR subcellular localization and dimerization with retinoid X receptor (RXR). Our first objective was to characterize these defects. We cloned functional fluorescent protein chimeras of VDR and RXR and explored the impact of VDR mutations on VDR functions by site-directed mutagenesis, dynamic microscopy experiments, and nuclear export assays. Microscopy showed that calcitriol and synthetic calcitriol analogues induce rapid receptor redistribution from the cytoplasm into the nucleus. Defects in DNA binding, hormone binding, coactivator binding, and dimerization selectively influenced intranuclear compartmentalization and nucleocytoplasmic trafficking of VDR. We identified sequences of the VDR and the RXR that define interactions with the nuclear import and export machinery and showed that the effect of mutations impairing VDR import or export correlated with the ability of VDR to regulate transcription of target genes. Recently we visualized changes in coactivator and corepressor intracellular trafficking in response to calcitriol. These studies lead to a better understanding of the growth regulatory effects of calcitriol. Our studies in rat osteosarcoma cells demonstrated that accelerated degradation of RXR and the expression of a dominant negative truncated RXR could cause resistance to the growth inhibitory effects of calcitriol. We then synthesized and purified calcitriol antagonists (BCA11 and BCA21), and found that similarly to VDR agonists they inhibited proliferation of cancer cell lines and the growth of human breast cancer xenografts in nude mice (US provisional patent No. 60/300,909; filed June 22, 2001; NIH reference No. E-213/01/0). Using GST pull-down experiments and microscopy, we found that both VDR agonists and antagonists induce the release of the corepressor NCoR from VDR, and this leads to the export of factors from the nucleus to the mitochondria, where they cause apoptosis. The presence or absence of these factors defines, at least in part, the sensitivity of cells to the growth inhibitory effects of vitamin D derivatives. Our second objective was to elucidate mechanisms that regulate the subcellular targeting and metabolism of the hormone, calcitriol. First, we studied subcellular targeting of calcitriol with microscopy. These experiments relied on using our biologically active fluorescing derivatives of calcitriol together with green fluorescent protein chimeras of heat shock proteins (hsp), which were generated by our collaborators Dr. Adams and colleagues at Cedar-Sinai Medical Center. We found that overexpression of hsp70 family of proteins increased cellular uptake of calcitriol. Moreover, mutational analysis demonstrated that members of the hsp70 family of proteins play important roles in calcitriol endocytosis, targeting to the endoplasmic reticulum and the mitochondria. Our ongoing studies explore the roles of Klotho protein, a protein that prevents several age-related diseases in mice, in the regulation of calcitriol targeting and metabolism. We generated GFP tagged Klotho and demonstrated that osteoblasts, which do not express Klotho, internalize Klotho protein from the cell culture media and this may be the mechanism by which Kloto influences mitochondrial conversion of vitamin D metabolites in osteoblasts. We continue to explore novel factors that regulate mitochondrial synthesis of calcitriol, such as histamine, and expand our findings to the level of the intact animal. In conclusion, we made major progress in all three areas of our research. Our findings lead to a better understanding of diseases associated with insufficient or excess calcitriol actions.

维生素 D 的激素形式骨化三醇通过维生素 D 受体 (VDR) 发挥作用，调节钙稳态、维持骨骼完整性、免疫防御、激素分泌、毛囊循环和乳腺发育。我们研究的长期目标是阐明骨化三醇作用过多或不足引起的疾病的病理生理学。我们应用现代成像、生化和遗传学方法在分子水平上研究骨化三醇代谢和作用的调节。维生素 D 补充不足或过量或 VDR 功能异常可能会导致荷尔蒙效应异常。 VDR 功能异常引起的疾病之一是骨化三醇遗传性耐药 (HVDDR)。这种疾病通常是由 VDR 基因突变引起的，表现为佝偻病。我们使用 HVDDR 受试者的皮肤成纤维细胞来探索 VDR 激活的分子机制。这些成纤维细胞要么缺乏VDR，要么表达突变的VDR，从而在受体激活途径的离散步骤中表现出异常。我们发现一些突变导致 VDR 亚细胞定位和与类视黄醇 X 受体 (RXR) 的二聚化缺陷。我们的首要目标是描述这些缺陷的特征。我们克隆了VDR和RXR的功能性荧光蛋白嵌合体，并通过定点诱变、动态显微镜实验和核输出测定探索了VDR突变对VDR功能的影响。显微镜显示骨化三醇和合成骨化三醇类似物诱导受体从细胞质快速重新分布到细胞核。 DNA 结合、激素结合、共激活剂结合和二聚化的缺陷选择性地影响 VDR 的核内区室化和核细胞质运输。我们鉴定了定义与核输入和输出机制相互作用的 VDR 和 RXR 序列，并表明损害 VDR 输入或输出的突变效应与 VDR 调节靶基因转录的能力相关。最近，我们观察了骨化三醇对细胞内共激活剂和辅阻抑剂运输的变化。这些研究使人们更好地了解骨化三醇的生长调节作用。我们对大鼠骨肉瘤细胞的研究表明，RXR 的加速降解和显性失活截短的 RXR 的表达可能会导致对骨化三醇的生长抑制作用的抵抗。然后我们合成并纯化了骨化三醇拮抗剂（BCA11和BCA21），发现与VDR激动剂类似，它们抑制癌细胞系的增殖和裸鼠中人乳腺癌异种移植物的生长（美国临时专利号60/300,909；2001年6月22日提交；NIH参考号E-213/01/0）。使用 GST 下拉实验和显微镜，我们发现 VDR 激动剂和拮抗剂都会诱导 VDR 释放辅阻遏物 NCoR，这导致因子从细胞核输出到线粒体，在线粒体中引起细胞凋亡。这些因素的存在或不存在至少部分地决定了细胞对维生素 D 衍生物的生长抑制作用的敏感性。 我们的第二个目标是阐明调节激素骨化三醇的亚细胞靶向和代谢的机制。首先，我们用显微镜研究了骨化三醇的亚细胞靶向。这些实验依赖于使用我们的生物活性荧光衍生物骨化三醇以及热休克蛋白 (hsp) 的绿色荧光蛋白嵌合体，这些嵌合体是由我们的合作者 Adams 博士和 Cedar-Sinai 医学中心的同事产生的。我们发现 hsp70 蛋白家族的过度表达增加了细胞对骨化三醇的摄取。此外，突变分析表明，hsp70 蛋白家族的成员在骨化三醇内吞作用中发挥重要作用，靶向内质网和线粒体。我们正在进行的研究探讨了 Klotho 蛋白（一种可预防小鼠多种与年龄相关的疾病的蛋白质）在骨化三醇靶向和代谢调节中的作用。我们生成了 GFP 标记的 Klotho，并证明不表达 Klotho 的成骨细胞会内化细胞培养基中的 Klotho 蛋白，这可能是 Kloto 影响成骨细胞中维生素 D 代谢物线粒体转化的机制。我们继续探索调节骨化三醇线粒体合成的新因子，例如组胺，并将我们的发现扩展到完整动物的水平。 总之，我们在研究的所有三个领域都取得了重大进展。我们的研究结果有助于更好地了解与骨化三醇作用不足或过量相关的疾病。