Vitamin D Resistance And Related Disorders

维生素 D 抵抗及相关疾病

• 批准号: 6543040
• 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/6543040
• 关键词: 1,25 dihydroxycholecalciferol; adipose tissue; analog; athymic mouse; binding proteins; breast neoplasms; calcium; cell differentiation; cell proliferation; estrogen receptors; fibroblasts; gene expression; green fluorescent proteins; homeostasis; hormone regulation /control mechanism; human tissue; intracellular transport; neoplastic cell; neoplastic growth; proteasome; protein degradation; 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, cell proliferation, differentiation, and various immune functions. Defects in calcitriol actions thus have a broad spectrum of manifestations ranging from a lack of effects to hyperactivity. Lack of hormone effects may result from insufficient vitamin D supplementation or from defects in VDR functions. Hereditary resistance to calcitriol (HVDDR) usually results from a mutation in the VDR gene and manifests as rickets. We have used skin fibroblasts from subjects with HVDDR to explore mechanisms of calcitriol action. These mutant cells display abnormalities in many discrete steps of the receptor activation pathway, including defects in nuclear hormone uptake and in VDR subcellular targeting. We developed new technologies to explore these abnormalities at the cellular level. We cloned functional GFP chimeras of VDR and stably expressed them in kidney and osteoblast cells. The naturally occurring mutations in the VDR were then generated by site-directed mutagenesis and expressed in kidney-derived cell lines such as 293, CV-1, and COS-7 cells. Microscopy showed that calcitriol and synthetic calcitriol analogues induce rapid receptor redistribution from the cytoplasm into the nucleus. Defects in DNA binding, hormone binding, and dimerization selectively influenced subcellular trafficking of VDR. Calcitriol antagonists (BCA11 and BCA21), which we synthesized and purified recently, also inhibited VDR subcellular trafficking. During the characterization of these antagonists we realized that they are highly potent inhibitors of cancer cell proliferation (US provisional patent No. 60/300,909; filed June 22, 2001; NIH reference No. E-213/01/0). Studies in cultured cancer cell lines (breast, melanoma, glioma, prostate, colon, and osteosarcoma) showed that they counteract the growth stimulatory effect of calcitriol, which is induced by physiological hormone concentration. Both BCA11 and BCA21 still acted as calcitriol to inhibit proliferation at high, pharmacological concentrations. BCA11 not only inhibited breast cancer growth, but also induced differentiation. BCA11 was as potent as the commonly used estrogen antagonist tamoxifen in inhibiting the growth of human breast cancer xenografts in nude mice without causing any noticeable side effects. BCA11 was also effective to inhibit the growth of estrogen receptor negative breast cancers. Because VDR is expressed in 80% of breast cancers while only 40% express estrogen receptors, antagonists of VDR could have important potential for drug development. Hormone resistance, however, may develop after prolonged treatment and further studies are needed to overcome this problem. Accelerated degradation of VDR or degradation of its heterodimerization partner, RXR, also influence calcitriol effects. We found that the antiproliferative effect of calcitriol is more sensitive to changes in RXR degradation than other VDR functions. Such an accelerated proteasomal degradation of RXR causes calcitriol resistance in rat osteosarcoma cells (ROS). Stable expression of YFP-RXR in ROS cells restored the responsiveness to the antiproliferative effects of calcitriol and retinoids, as did treatments with proteasomal inhibitors. We started to explore calcitriol storage and release in the adipose tissue. Ongoing studies are directed at understanding uptake mechanisms, hormonal regulation and physiological importance of calcitriol release from the adipose tissue. We continue to use GFP chimeras of wild-type and mutant VDR and intracellular vitamin D binding proteins together with our fluorescent labeled calcitriol to gain further understanding of the disorders of the vitamin D endocrine system.

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、细胞增殖、分化和各种免疫功能。因此，骨化三醇作用的缺陷具有广泛的表现，从缺乏作用到过度活跃。缺乏激素效应可能是由于维生素D补充不足或VDR功能缺陷所致。遗传性耐骨化三醇（HVDDR）通常是由于VDR基因突变引起的，表现为佝偻病。我们使用了HVDDR患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活化途径的许多离散步骤中显示异常，包括核激素摄取和VDR亚细胞靶向的缺陷。我们开发了新技术来探索细胞水平上的这些异常。我们克隆了VDR的功能性GFP嵌合体，并在肾脏和成骨细胞中稳定表达。然后通过定点诱变产生VDR中天然存在的突变，并在肾脏来源的细胞系如293、CV-1和COS-7细胞中表达。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、激素结合和二聚化的缺陷选择性地影响VDR的亚细胞运输。我们最近合成和纯化的骨化三醇拮抗剂（BCA 11和BCA 21）也抑制VDR亚细胞运输。 在这些拮抗剂的表征过程中，我们认识到它们是癌细胞增殖的高效抑制剂（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）。对培养的癌细胞系（乳腺癌、黑色素瘤、神经胶质瘤、前列腺癌、结肠癌和骨肉瘤）的研究表明，它们抵消了骨化三醇的生长刺激作用，骨化三醇的生长刺激作用是由生理激素浓度诱导的。BCA 11和BCA 21在高药理学浓度下仍充当骨化三醇以抑制增殖。BCA 11不仅抑制乳腺癌生长，而且诱导分化。BCA 11在抑制裸鼠人乳腺癌异种移植瘤生长方面与常用的雌激素拮抗剂他莫昔芬一样有效，而不会引起任何明显的副作用。BCA 11也有效抑制雌激素受体阴性乳腺癌的生长。由于80%的乳腺癌表达VDR，而只有40%的乳腺癌表达雌激素受体，因此VDR拮抗剂可能在药物开发中具有重要潜力。然而，激素抵抗可能在长期治疗后发展，需要进一步研究来克服这个问题。 VDR的加速降解或其异源二聚化伴侣RXR的降解也影响骨化三醇的作用。我们发现骨化三醇的抗增殖作用对RXR降解的变化比其他VDR功能更敏感。RXR的这种加速的蛋白酶体降解导致大鼠骨肉瘤细胞（ROS）中的骨化三醇抗性。ROS细胞中YFP-RXR的稳定表达恢复了对骨化三醇和类维生素A抗增殖作用的反应性，蛋白酶体抑制剂治疗也是如此。 我们开始探索骨化三醇在脂肪组织中的储存和释放。正在进行的研究旨在了解从脂肪组织释放骨化三醇的摄取机制、激素调节和生理重要性。我们继续使用野生型和突变型VDR和细胞内维生素D结合蛋白的GFP嵌合体以及我们的荧光标记的骨化三醇，以进一步了解维生素D内分泌系统的疾病。