Vitamin D Resistance And Related Disorders

维生素 D 抵抗及相关疾病

• 批准号: 7337522
• 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/7337522
• 关键词: Vitamin; Resistance; Related; Disorders

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. We recently found that VDR export is essential for the effect of calcitriol to inhibit cell growth. On the other hand, accelerated degradation of RXR and the expression of a dominant negative truncated RXR could cause resistance to the growth inhibitory effects of calcitriol in many cancer cell types. Our recent collaborative studies showed that VDR import rate changes contribute to the sensitivity of the enterocyte-like Caco-2 cells to synthetic calcitriol derivatives. Interestingly, not only VDR agonists, but also VDR antagonists display antiproliferative effects in cancer models. We synthesized and purified calcitriol antagonists (BCA11 and BCA21), and found that 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. Using the red fluorescing calcitriol and the green fluorescing VDR and reverse photobleaching experiments, we visualized the nuclear import and export of the liganded VDR. 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. Our ongoing studies at Georgetown University in collaboration with Dr. Verbalis recently demonstrated that chronic hyponatremia causes osteoporosis in rats, and elucidated the effects of hyponatremia on osteoclast formation and resorptive activities. 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）起作用以调节钙稳态、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量或不足引起的疾病的病理生理学。我们应用现代影像学、生物化学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。异常的激素效应可能是由于维生素D补充不足或过量或VDR功能异常。 VDR功能异常引起的疾病之一是遗传性骨化三醇抗性（HVDDR）。这种疾病通常是由VDR基因突变引起的，表现为佝偻病。我们已经使用来自HVDDR受试者的皮肤成纤维细胞来探索VDR激活的分子机制。这些成纤维细胞缺乏VDR或表达突变型VDR，其在受体活化途径的离散步骤中显示异常。我们发现几个突变导致VDR亚细胞定位缺陷和与类维生素A X受体（RXR）的二聚化。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讨了VDR突变对VDR功能的影响。显微镜检查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中。DNA结合、激素结合、辅激活因子结合和二聚化的缺陷选择性地影响VDR的核内区室化和核质运输。我们确定了VDR和RXR的序列，它们定义了与核输入和输出机制的相互作用，并表明损害VDR输入或输出的突变的影响与VDR调节靶基因转录的能力相关。最近，我们观察到骨化三醇对辅激活子和辅阻遏子细胞内运输的影响。我们最近发现VDR输出对于骨化三醇抑制细胞生长的作用是必不可少的。另一方面，RXR的加速降解和显性负性截短RXR的表达可能导致许多癌细胞类型对骨化三醇的生长抑制作用产生抗性。我们最近的合作研究表明，VDR输入速率的变化有助于肠细胞样Caco-2细胞对合成骨化三醇衍生物的敏感性。有趣的是，不仅VDR激动剂，而且VDR拮抗剂在癌症模型中显示抗增殖作用。我们合成并纯化了骨化三醇拮抗剂（BCA 11和BCA 21），发现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激动剂和拮抗剂都诱导从VDR释放辅阻遏物NCoR，这导致因子从细胞核输出到线粒体，在那里它们引起凋亡。这些因子的存在或不存在至少部分地限定了细胞对维生素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骨化三醇的生物活性荧光衍生物。利用红色荧光钙三醇和绿色荧光VDR以及反向光漂白实验，我们可视化了配体VDR的核输入和输出。我们正在进行的研究探索Klotho蛋白的作用，Klotho蛋白是一种预防小鼠几种年龄相关疾病的蛋白质，在调节骨化三醇靶向和代谢中的作用。我们产生了GFP标记的Klotho，并证明了不表达Klotho的成骨细胞从细胞培养基中内化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。 最近，我们在乔治敦大学与Verbalis博士合作进行的研究表明，慢性低钠血症导致大鼠骨质疏松症，并阐明了低钠血症对破骨细胞形成和吸收活动的影响。总之，我们在所有三个研究领域都取得了重大进展。我们的研究结果导致更好地了解与骨化三醇作用不足或过量相关的疾病。

项目成果

Degradation of RXRs influences sensitivity of rat osteosarcoma cells to the antiproliferative effects of calcitriol.

• DOI: 10.1210/mend.16.5.0821
• 发表时间: 2002-05
• 期刊: Molecular endocrinology
• 作者: K. Prüfer;C. Schröder;K. Hegyi;J. Bársony

The human vitamin D receptor gene (VDR) is localized to region 12cen-q12 by fluorescent in situ hybridization and radiation hybrid mapping: genetic and physical VDR map.

通过荧光原位杂交和辐射杂交作图：遗传和物理 VDR 图谱，将人类维生素 D 受体基因 (VDR) 定位于 12cen-q12 区域。

• DOI: 10.1359/jbmr.1999.14.7.1163
• 发表时间: 1999
• 期刊: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research
• 作者: Taymans,SE;Pack,S;Pak,E;Orban,Z;Barsony,J;Zhuang,Z;Stratakis,CA
• 通讯作者: Stratakis,CA