VITAMIN D RESISTANCE AND RELATED DISORDERS

维生素 D 抵抗和相关疾病

• 批准号: 6289849
• 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/6289849
• 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 thevitamin D receptor (VDR) to regulate calcium homeostasis, cellproliferation, differentiation, and various immunefunctions. Defects in the target actions of calcitriol thus have a broadspectrum of manifestations ranging from hyperreactivity to a lack of effects. Hereditary resistance to calcitriol (HVDRR) usuallyresults from a mutation in the VDR gene and manifests as rickets.A collaborative study with Constantine Stratakis, NICHD,investigating the mutations in a family with HVDRR lead to theisolation of a bacterial artificial chromosome containing the VDRgene, and physical mapping of the VDR gene by fluorescent in situhybridization and radiation hybrid analysis to chromosome12cen-q12, flanked by markers SHGC 30216 and SHGC 9798.Furthermore, we used skin fibroblasts from subjects with HVDRR toexplore mechanisms of calcitriol action. These mutant cells display abnormalities in many discrete steps of the receptoractivation pathway. We used these natural knockout cells tostudy the functional domains of VDR involved in the control ofreceptor localization. New technologies were developed to explorethe mechanisms of hormone uptake and VDR movement. We cloned andstably expressed fully functional GFP cimeras of VDR and RXR inkidney and osteoblast cells. We also developed photostable, biologically active red fluorescing derivatives of vitamin D.Confocal laser scanning microscopy was used for real-timesimultaneous recordings of calcitriol and VDR import into thenucleus. These studies showed that hormone binds VDR both inthe cytoplasm and in the nucleus, and indicated that calcitriolinduces translocation of cytoplasmic VDR into thenucleus, and suggested that translocating VDR carry the hormoneinto the nucleus. Binding of the translocating VDR tomicrotubules were demonstrated in vivo, with microscopy in livingcells, and in vitro, by copolymerization andcoimmunoprecipitation experiments. We identified a new targetsite for VDR, the centrosome. Our studies with cytoskeletoninhibitors indicated that microtubule minus-end movement may playa role in the hormone-induced centrosomal targeting of VDR, aswell as the transcriptional activity of VDR. Using site-direcetedmutagenesis, we found that the integrity of the activationfunction 2 domain of VDR is essential for the hormone- inducedtranslocation of VDR into the nucleus and into the centrosome.Coexpression experiments with wilde-type and heterodimerizationmutant RXR revealed a role for heterodimerizing RXR in thehormone-independent translocationof VDR. Another collaborative study with Dr. G.D.Roodmans laboratory (Univ Texas Health Sci Center, San Antonio,TX) explored the mechanisms causing calcitriolhypersensitivity of osteoclast precursors from patients withPagets disease. Binding studies with our green fluorescenttagged calcitriol revealed an increased VDR affinity forcalcitriol in osteoclasts from Pagets patients, which may beresponsible for the enhanced calcitriol sensitivity in Pagetsdisease. We will continue to use GFP chimeras of wilde-type andmutant VDR, RXR and coactivators to gain further understanding ofthe disorders of the vitamin D endocrine system. - vitamin D receptor, vitamin D resistance rickets, calcitriol, confocal microscopy, microtubules, translocation, VDR gene, GFP, analogs

维生素D的激素形式骨化三醇通过维生素D受体（VDR）来调节钙稳态、细胞增殖、分化和各种免疫功能。因此，骨化三醇靶向作用的缺陷具有从高反应性到缺乏作用的广泛表现。遗传性骨化三醇抗性（HVDRR）通常由VDR基因突变引起，表现为佝偻病。与NICHD的Constantine Stratakis合作研究了一个HVDRR家族的突变，导致分离出含有VDR基因的细菌人工染色体，并通过荧光原位杂交和辐射杂交分析将VDR基因定位到染色体12 cen-q12，两侧有SHGC 30216和SHGC 9798标记。此外，我们使用来自HVDRR受试者的皮肤成纤维细胞来探索骨化三醇的作用机制。这些突变细胞在受体激活途径的许多离散步骤中显示异常。我们使用这些天然敲除细胞来研究VDR参与受体定位控制的功能域。新的技术被开发来探索激素摄取和VDR运动的机制。我们克隆并在肾脏和成骨细胞中稳定表达VDR和RXR的全功能GFP嵌合体。我们还开发了光稳定的，生物活性的红色荧光衍生物的维生素D。共聚焦激光扫描显微镜用于实时同步记录骨化三醇和VDR输入到thenucleus。这些研究表明，激素在细胞质和细胞核中都能与VDR结合，并提示骨化三醇可诱导细胞质VDR向细胞核转位，从而推测转位的VDR可携带VDR进入细胞核。在活体细胞中用显微镜观察证实了VDR与微管的结合，在体外通过共聚和免疫共沉淀实验证实了VDR与微管的结合。我们发现了VDR的一个新靶点，中心体。我们对细胞凋亡抑制剂的研究表明，微管负端运动可能在维生素D诱导的VDR中心体靶向以及VDR的转录活性中起作用。利用定点突变技术，我们发现VDR激活功能2结构域的完整性对于激素诱导的VDR向细胞核和中心体的转位是必需的，野生型和异源二聚体化突变体RXR的共表达实验揭示了异源二聚体化RXR在VDR非依赖性转位中的作用。另一项与G.D.Roodmans博士实验室（德克萨斯大学健康科学中心，圣安东尼奥，TX）合作的研究探讨了佩吉特病患者破骨细胞前体骨化三醇超敏反应的机制。用我们的绿色荧光标记的骨化三醇进行的结合研究显示，Pagets患者破骨细胞中骨化三醇的VDR亲和力增加，这可能是Pagets病中骨化三醇敏感性增强的原因。我们将继续使用野生型和突变型VDR、RXR和辅激活因子的GFP嵌合体来进一步了解维生素D内分泌系统的疾病。- 维生素D受体，维生素D抵抗性佝偻病，骨化三醇，共聚焦显微镜，微管，易位，VDR基因，GFP，类似物