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Regulatory Mechanisms of Renal Vitamin D Activation and Degradation

肾脏维生素 D 激活和降解的调节机制

基本信息

批准号：
10373006
负责人：
J WESLEY PIKE
金额：
$ 39.27万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-11 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10373006
关键词：
Blood Bone Diseases CRISPR/Cas technology CYP27B1 gene Cells ChIP-seq Chemicals Chronic Kidney Failure Complex Dihydroxycholecalciferols Disease Distal Endocrine Enhancers Experimental Models Gene Expression Genes Genetic Transcription Genomic Segment Genomics Homeostasis Hormonal Hormones Human Individual Inflammatory Intestines Introns Kidney Location Maintenance Mediating Medicine Metabolic Activation Metabolism Methods Minerals Modeling Modification Molecular Mouse Strains Mus Mutation Analysis Nature Phenotype Physiological Play Production Property Regulation Renal function Research Role Signal Transduction Site Skeleton System Testing Tissues Vitamin D cell type combat design dietary dietary manipulation genomic locus hormone regulation in vivo novel novel strategies promoter response selective expression transcription factor virtual

项目摘要

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.

项目概要/摘要 1,25-二羟基维生素 D (1,25(OH)2D3) 在肾脏中由 Cyp27b1 合成，并在所有目标中失活通过 Cyp24a1 的组织。这两个基因在肾脏中受到多种激素的相互调节，用于维持循环 1,25(OH)2D3 的适当生理水平。尽管它们很重要，然而，我们对促进 Cyp27b1 在肾脏或细胞中表达的分子细节知之甚少。该组织和其他地方的 Cyp24a1。这表明我们对维生素的理解存在巨大缺陷 D系统的缺口在多种矿物质代谢疾病中凸显。在最近的研究中，我们有确定了小鼠 Cyp27b1 基因座中通过 PTH、FGF23 介导 Cyp27b1 调节的关键基因组区域 1,25(OH)2D3，一种仅存在于肾脏中的模块。删除该模块的组件reduce Cyp27b1 在肾脏中表达，但对非肾脏靶细胞中 Cyp27b1 表达没有影响（NRTC）。鉴于这些发现，我们提出以下分子研究。目标 1：表征小鼠肾脏中介导 Cyp27b1 的内分泌模块及其各个成分 PTH、1,25(OH)2D3 和 FGF23 在体内的表达和调节。我们将使用 ChIP-seq 分析体内 CRISPR/Cas9 缺失研究进一步表征了我们在肾的功能是调节 Cyp27b1 的表达。我们还将定义一个独立的位置 Cyp27b1 基因位点中介导 NRTC 炎症信号传导作用的模块。这些研究旨在增进我们对代谢基础分子机制的理解维生素 D 的激活。目标 2：表征控制 Cyp24a1 表达的调节子模块以及体内肾脏中 PTH、1,25(OH)2D3 和 FGF23 的调节。 Cyp24a1 在肾脏中发挥着在调节 1,25(OH)2D3 和其他维生素 D 代谢物的血液水平中发挥协调作用。我们将使用目标 1 中概述的方法阐明了该基因被下调的基因组机制 PTH 并受 FGF23 和 1,25(OH)2D3 的上调。小鼠的突变分析将使我们能够定义增强子簇以及负责转录因子和共调节子的作用位点这些活动。目标 3：利用饮食控制来测试和确认所制定的监管假设在与疾病相关的 Cyp27b1 受损小鼠品系中。为了这个目标，我们计划测试一个假设的该模型解释了删除组件后出现的复杂表型肾脏特异性调节模块。我们将使用钙、磷和维生素 D 代谢物的饮食控制来测试我们的假设。