Regulatory Mechanisms of Renal Vitamin D Activation and Degradation

项目摘要/摘要 1，25-二羟基维生素D(1，25(OH)2D3)在肾脏中由Cyp27b1合成，并在所有靶点失活 组织通过细胞色素P24A1。这两个基因在肾脏中受到许多激素的相互调节，这些激素 维持适当的生理水平的1，25(OH)2D3循环。尽管它们很重要， 然而，促进Cyp27b1在肾脏或肾脏中表达的分子细节知之甚少。 细胞色素P24A1在这个组织和其他地方。这表明我们对维生素的理解存在着巨大的缺陷。 D系统，这一缺口在各种矿物质代谢疾病中尤为突出。在最近的研究中，我们已经 确定小鼠Cyp27b1基因座的关键基因组区域，该区域介导PTH、FGF23对Cyp27b1的调节 和1，25(OH)2D3，这是一种只存在于肾脏的模块。删除此模块的组件将减少 Cyp27b1在肾脏中的表达，但对非肾脏靶细胞中Cyp27b1的表达没有影响 (NRTCs)。鉴于这些发现，我们建议进行以下分子研究。目标1：描述 小鼠肾脏中介导Cyp27b1的内分泌模块及其单独成分 PTH、1，25(OH)2D3和FGF23在体内的表达和调节我们将使用CHIP-SEQ分析和 体内CRISPR/Cas9缺失研究以进一步表征我们在 肾脏的功能是调节Cyp27b1的表达。我们还将定义独立的 Cyp27b1基因中的一个模块，在NRTCs中介导炎症信号的活动。这些 研究旨在促进我们对支持新陈代谢的分子机制的理解 维生素D的激活目标2：确定控制CYP24A1表达的调节子模块的特征 体内甲状旁腺素、1，25(OH)2D3和FGF23对肾脏的调节作用。肾脏中的CYP24A1在肾脏中发挥作用 在调节血液中1，25(OH)2D3和其他维生素D代谢物水平方面的协调作用。我们将使用 目的1中概述的方法，以阐明该基因下调的基因组机制 PTH，并被FGF23和1，25(OH)2D3上调。对老鼠的突变分析将使我们能够定义 增强子簇以及转录因子和共调节子的作用部位 这些活动。目标3：利用饮食操作来测试和确认已开发的调节假说 在Cyp27b1受损的与疾病相关的小鼠品系中。为了达到这个目的，我们计划测试一个假设 模型，该模型解释了在删除 肾脏特有的调节模块。我们将使用饮食控制钙、磷和维生素D代谢物来测试 我们的假设。

PROJECT SUMMARY/ABSTRACT 1,25-Dihydroxyvitamin D (1,25(OH)2D3) is synthesized in the kidney by Cyp27b1 and inactivated in all target tissues via Cyp24a1. The two genes are reciprocally regulated in the kidney by a number of hormones that serve to maintain appropriate physiologic levels of circulating 1,25(OH)2D3. In spite of their importance, however, little is known of the molecular details that facilitate expression of Cyp27b1 in the kidney or of Cyp24a1 in this tissue and elsewhere. This represents an enormous deficit in our understanding of the vitamin D system, a gap that is highlighted in a variety of diseases of mineral metabolism. In recent studies, we have identified key genomic regions in the Cyp27b1 locus in mice that mediate Cyp27b1 regulation by PTH, FGF23 and 1,25(OH)2D3, a module that is present only in the kidney. Deletion of components of this module reduce Cyp27b1 expression in the kidney but have no effect on Cyp27b1 expression in non-renal target cells (NRTCs). In view of these findings, we propose the following molecular studies. Aim 1: Characterize the endocrine module and its individual components in the mouse kidney that mediates Cyp27b1 expression and regulation by PTH, 1,25(OH)2D3 and FGF23 in vivo. We will use ChIP-seq analysis and CRISPR/Cas9 deletion studies in vivo to further characterize a regulatory module we have discovered in the kidney that functions to regulate the expression of Cyp27b1. We will also define the location of an independent module in the Cyp27b1 gene locus that mediates the actions of inflammatory signaling in NRTCs. These studies are designed to advance our understanding of the molecular mechanisms that underpin the metabolic activation of vitamin D. Aim 2: Characterize the regulatory sub-modules that control Cyp24a1 expression and regulation by PTH, 1,25(OH)2D3 and FGF23 in the kidney in vivo. Cyp24a1 in the kidney plays a coordinating role in regulating blood levels of 1,25(OH)2D3 and other vitamin D metabolites. We will use the methods outlined in Aim 1 to elucidate the genomic mechanisms through which this gene is downregulated by PTH and upregulated by both FGF23 and 1,25(OH)2D3. Mutational analysis in mice will allow us to define the clusters of enhancers and the sites of action of transcription factors and comodulators that are responsible for these activities. Aim 3: Utilize dietary manipulation to test and confirm regulatory hypotheses developed in Cyp27b1-compromised mouse strains relevant to disease. In this aim, we plan to test a hypothetical model that explains the complex phenotypes that have emerged following deletion of components of the kidney-specific regulatory module. We will use dietary manipulation of Ca, Pi, and vitamin D metabolites to test our hypotheses.