Osteoporosis and Molecular Targets of Vitamin D

描述（由申请人提供）：维生素D和钙缺乏是年龄相关性骨质流失和骨折风险增加的风险因素。肠钙吸收减少（与TRPV 6和钙结合蛋白-D9 k（肠中1，25（OH）2D 3的两个主要分子靶点）的表达减少相关）与24（OH）酶对1，25（OH）2D 3的催化作用增加相结合，导致年龄相关的骨丢失。虽然1，25（OH）2D 3的主要作用是肠道钙吸收，但我们远未了解维生素D诱导钙转运的机制。此外，维生素D不足导致骨质疏松症的机制尚不清楚。该探索性研究的统一假设是，1，25（OH）2D 3介导的肠钙吸收的基本机制仍有待确定，辅因子复合物（包括与表观遗传调节相关的辅因子）参与维生素D受体（VDR）介导的维持钙稳态的事件，并且该过程随年龄增长而恶化。为了在第一个具体目标中验证这一假设，我们建议检查钙结合蛋白和TRPV 6之间的功能相互关系。在使用双KO小鼠的初始研究中，我们发现响应于低钙或1，25（OH）2D 3的肠钙吸收在钙结合蛋白-D9 k和TRPV 6两者不存在的情况下效率最低（与WT和单KO小鼠相比），这表明TRPV 6和钙结合蛋白-D9 k（共定位于肠中）一起作用以影响钙吸收。我们假设钙结合蛋白-D9 k不是钙扩散的促进剂（从而挑战维生素D介导的跨细胞钙吸收的传统模型），而是通过TRPV 6的钙内流的调节剂。提出了研究（使用共聚焦显微镜，co-ip.，GST下拉测定和电生理学），这将使我们能够首次了解钙结合蛋白-D9 k和TRPV 6之间的功能相互关系，从而了解它们在1，25（OH）2D 3调节的肠道钙转运中的作用。这些研究的结果可能会导致药理学工具的发展，以提高肠道钙的吸收，从而有助于保持钙的反应在衰老过程中。在具体目标2中，我们建议检查1，25（OH）2D 3用于维持钙稳态和维生素D调节随衰老发生的变化的机制。除了靶蛋白的功能意义外，了解1，25（OH）2D 3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迅速发展的领域，我的实验室已经获得了第一个证据，即特定的蛋白质甲基转移酶与p160共激活因子GRIP 1在调节1，25（OH）2D 3靶基因的表达。我们将研究CARM 1甲基转移酶和协同辅因子在调节关键的1，25（OH）2D 3靶基因（包括TRPV 6和24（OH）ase）中的作用，以及VDR功能、VDR相关因子和VDR功能的表观遗传调节如何随年龄变化。此外，我们将通过ChIP seq分析组蛋白甲基化模式的全基因组变化和肾脏和肠道中VDR调节基因（新的和已知的）中响应1，25（OH）2D 3的调节辅因子的共同出现，以及随着衰老发生的变化。VDR共活化剂是1，25（OH）2D 3作用的主要调节剂。了解VDR的1，25（OH）2D 3和辅激活因子以及表观遗传变化的改变可能是了解钙稳态失调和对维生素D的反应性改变的关键。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，以防止或逆转恶化的钙稳态通过转录和表观遗传机制可能有长期的影响，如何治疗骨疾病，特别是骨质疏松症和骨质减少症。该应用程序适用于R21，因为它“涉及相当大的风险，但可能导致可能对生物医学或临床研究领域产生重大影响的突破”。

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".