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，并在所有靶标中灭活通过CYP24A1组织。这两个基因在肾脏在肾脏中受到相互调节用于维持1,25（OH）2d3的循环的适当生理水平。尽管很重要，但是，几乎不知道促进肾脏或肾脏表达的分子细节该组织和其他地方的CYP24A1。这代表了我们对维生素的理解的巨大赤字 D系统，一种在各种矿物质代谢疾病中突出显示的差距。在最近的研究中，我们有在小鼠中鉴定了CYP27B1基因座中的关键基因组区域，这些小鼠介导PTH介导CYP27B1调节的关键基因组区域还有1,25（OH）2d3，仅在肾脏中存在的模块。删除该模块的组件减少肾脏中的CYP27B1表达，但对非肾脏靶细胞中的CYP27B1表达没有影响（NRTC）。鉴于这些发现，我们提出了以下分子研究。目标1：特征内分泌模块及其在介导CYP27B1的小鼠肾脏中的单个成分 PTH的表达和调节，体内1,25（OH）2d3和FGF23。我们将使用Chip-seq分析和 crispr/cas9删除研究的体内研究，以进一步表征我们在功能调节CYP27B1表达的肾脏。我们还将定义独立的位置 CYP27B1基因基因座中介导NRTC中炎症信号的作用的模块。这些研究旨在促进我们对代谢基础的分子机制的理解维生素D的激活D AIM 2：表征控制CYP24A1表达的调节子模块 PTH的调节，体内肾脏中的1,25（OH）2d3和FGF23。肾脏中的CYP24A1播放协调在调节1,25（OH）2d3和其他维生素D代谢物的血液水平中的作用。我们将使用 AIM 1中概述的方法阐明了该基因被下调的基因组机制 PTH并由FGF23和1,25（OH）2d3上调。小鼠的突变分析将使我们能够定义增强剂的集群以及负责转录因子和共同调节剂的作用部位这些活动。目标3：利用饮食操纵来测试和确认发展的调节假设在与疾病相关的CYP27B1症状小鼠菌株中。在此目标中，我们计划测试一个假设的解释了在删除组件后出现的复杂表型的模型肾脏特异性调节模块。我们将使用CA，PI和维生素D代谢物的饮食操纵来测试我们的假设。