Osteoporosis and Molecular Targets of Vitamin D

描述(由申请人提供)：维生素D和钙缺乏是与年龄相关的骨丢失和增加骨折风险的危险因素。肠道钙吸收减少(这与肠道中1，25(OH)2D3的两个主要分子靶标TRPV6和Calbindin-D9k的表达减少有关)，加上24(OH)酶对1，25(OH)2D3分解代谢的增加，导致年龄相关性骨丢失。虽然1，25(OH)2D3的主要作用是肠道钙吸收，但我们对维生素D诱导钙转运的机制还知之甚少。此外，维生素D缺乏导致骨质疏松症的机制尚不清楚。这项探索性研究的统一假设是，1，25(OH)2D3介导的肠道钙吸收的基本机制仍未确定，维生素D受体(VDR)介导的维持钙稳态的事件涉及一系列辅因子，包括与表观遗传调节相关的辅因子，并且这一过程随着年龄的增长而恶化。为了在第一个特定目的中检验这一假设，我们建议检查Calbindin和TRPV6之间的功能相互关系。在使用双KO小鼠的初步研究中，我们发现在没有Calbindin-D9k和TRPV6的情况下，低钙或1，25(OH)2D3反应的肠道钙吸收效率最低(与WT和单个KO小鼠相比)，这表明TRPV6和Calbindin-D9k(共同定位于肠道)共同作用影响钙吸收。我们假设Calbindin-D9k不是钙扩散的促进剂(从而挑战了维生素D介导的跨细胞钙吸收的传统模型)，而是通过TRPV6调节钙内流。这些研究(使用共聚焦显微镜、共IP、GST下拉试验和电生理学)将使我们能够首次了解Calbindin-D9k和TRPV6之间的功能相互关系，从而了解它们在1，25(OH)2D3调控的肠道钙转运中的作用。这些研究的发现可能会导致开发药理工具来增强肠道钙吸收，从而帮助在衰老过程中保持钙的反应性。在具体目标2中，我们建议研究1，25(OH)2D3维持钙稳态的机制，以及随年龄增长而发生的维生素D调节的变化。除了靶蛋白的功能意义外，了解1，25(OH)2D3控制钙稳态的分子机制也很重要。表观基因组控制是一个新的和快速发展的领域，我的实验室已经获得了第一个证据，证明特定的蛋白甲基转移酶与p160辅活化子GRIP1在调控1，25(OH)2D3靶基因表达方面存在协同作用。我们将研究CARM1甲基转移酶和协同辅助因子在包括TRPV6和24(OH)ase在内的关键的1，25(OH)2D3靶基因调控中的作用，以及VDR、VDR相关因子的功能和VDR功能的表观遗传调节如何随年龄变化。此外，我们将通过芯片序列分析肾脏和肠道中VDR调控基因(新的和已知的)中1，25(OH)2D3响应的组蛋白甲基化模式和调控辅助因子的共现以及随年龄发生的变化。VDR共激活子是1，25(OH)2D3作用的主要调节子。了解1，25(OH)2D3对VDR和辅活化子的募集变化和表观遗传学变化可能是了解钙稳态失调和对维生素D的反应随年龄变化的关键。长期目标是确定维生素D作用的分子途径，以揭示维持钙平衡的新治疗策略。确定最有效的维生素D类似物，通过转录和表观遗传机制防止或逆转钙稳态的恶化，可能对如何治疗骨病，特别是骨质疏松症和骨量减少具有长期的影响。这种应用适合于R21，因为它“涉及相当大的风险，但可能导致突破，可能对生物医学或临床研究领域产生重大影响”。

DESCRIPTION (provided by applicant): Vitamin D and calcium deficiencies are risk factors for age-related bone loss and increased fracture risk. Decreased intestinal calcium absorption (which correlates with decreased expression of TRPV6 and calbindin-D9k, the two major molecular targets of 1,25(OH)2D3 in the intestine) combined with an increase in the catabolism of 1,25(OH)2D3 by 24(OH)ase contribute to age related bone loss. Although a principal action of 1,25(OH)2D3 is intestinal calcium absorption, we are far from understanding the mechanisms involved in vitamin D induced transport of calcium. In addition, the mechanisms by which inadequate vitamin D status contributes to osteoporosis are not yet known. The unifying hypothesis of this exploratory study is that basic mechanisms involved in 1,25(OH)2D3 mediated intestinal calcium absorption remain to be defined, that a complex of cofactors, including those associated with epigenetic regulation, are involved in vitamin D receptor (VDR) mediated events involved in maintaining calcium homeostasis and that there is a deterioration in this process with aging. To test this hypothesis in the first specific aim we propose to examine the functional interrelationship between calbindin and TRPV6. In initial studies using double KO mice we found that intestinal calcium absorption in response to low calcium or 1,25(OH)2D3 is least efficient in the absence of both calbindin-D9k and TRPV6 (compared to WT and single KO mice), suggesting that TRPV6 and calbindin-D9k (which are co-localized in the intestine) act together to affect calcium absorption. We hypothesize that calbindin-D9k is not a facilitator of calcium diffusion (thus challenging the traditional model of vitamin D mediated transcellular calcium absorption) but rather a modulator of calcium influx via TRPV6. Studies are proposed (using confocal microscopy, co-ip., GST pull down assays and electrophysiology) that will enable us to understand for the first time the functional interrelationship between calbindin-D9k and TRPV6 and thus their role in 1,25(OH)2D3 regulated intestinal calcium transport. Findings from these studies may lead to the development of pharmacological tools to enhance intestinal calcium absorption and thus help to maintain calcium responsiveness during aging. In specific aim 2 we propose to examine mechanisms by which 1,25(OH)2D3 acts to maintain calcium homeostasis and changes in vitamin D regulation that occur with aging . Besides the functional significance of target proteins it is also important to understand the molecular mechanisms by which 1,25(OH)2D3 controls calcium homeostasis. Epigenomic control is a new and rapidly evolving field and my lab has obtained the first evidence that specific protein methyltransferases cooperate with the p160 coactivator GRIP1 in regulating 1,25(OH)2D3 target gene expression. We will examine the role of CARM1 methyltransferase and cooperating cofactors in the regulation of key 1,25(OH)2D3 target genes including TRPV6 and 24(OH)ase and how the function of VDR, VDR associated factors and epigenetic regulation of VDR function are altered with age. In addition we will analyze by ChIP seq genome wide changes in histone methylation patterns and co-occurrence of regulatory cofactors in response to 1,25(OH)2D3 in VDR regulated genes (novel as well as known) in kidney and intestine and changes that occur with aging. VDR coactivators are master regulators of 1,25(OH)2D3 action. Understanding altered recruitment by 1,25(OH)2D3 of VDR and coactivators and epigenetic changes may be the key to understanding dysregulation of calcium homeostasis and altered responsiveness to vitamin D that occurs with aging. The long term goal is to define molecular pathways of vitamin D action in order to reveal new therapeutic strategies to sustain calcium balance. Identifying the most effective vitamin D analog to prevent or reverse deterioration of calcium homeostasis through transcriptional and epigenetic mechanisms could have long range implications for how bone diseases, particularly osteoporosis and osteopenia, are treated. This application is appropriate for an R21 since it "involves considerable risk but may lead to a breakthrough that could have a major impact on a field of biomedical or clinical research".