Role of HNF4a in the regulation of FGF23 in health and disease

项目摘要 慢性肾脏病（CKD）患者的骨和矿物质代谢范围广泛 异常，包括骨量丢失和成纤维细胞生长因子23（FGF 23）的过量产生， 骨细胞血清FGF 23水平在CKD病程早期升高，高水平是独立相关的 CKD进展、心血管疾病和死亡的风险更大。这些发现激发了人们的兴趣 在设计治疗方法以减少骨异常和降低FGF 23水平方面， 对分子机制的理解。在这个建议的初步数据，我们表明， 核受体，肝细胞核因子4 α（HNF 4 α）与骨特异性途径机制相关 在FGF 23过量的动物模型中控制FGF 23的产生。我们还表明，FGF 23启动子含有 预测的HNF 4反应元件（HNF 4-RE），并且Hnf 4 α在成骨细胞和骨细胞中表达。在 新的初步数据表明，HNF 4 α刺激成骨细胞增殖，成骨细胞特异性 Hnf 4 α的缺失导致成骨细胞和小鼠中FGF 23过量，并导致动物中骨量降低。我们也 显示Hnf 4 α表达在已建立的进行性CKD模型的骨中被抑制了98%， Col 4a 3 KO小鼠。这些观察结果支持HNF 4 α的重要新作用。在本提案中，我们将测试 假设CKD中HNF 4 α缺乏导致过量FGF 23和骨异常。在aim 1中， 通过体外评估HNF 4 α的增殖、活性和分化， 成骨细胞过度表达或缺乏HNF 4 α。我们将通过RNA技术来表征骨HNF 4 α转录组， 对携带Hnf 4 α特异性缺失的小鼠进行测序并完成骨表型分析， 成骨细胞/骨细胞（Hnf 4 αOc-cKO）。为了研究HNF 4 α是否介导成骨反应，我们将 将WT和Hnf 4 αOc-cKO小鼠暴露于分解代谢和合成代谢PTH激发。在目标2中，我们将研究HNF 4 α 通过检测HNF 4 α是否直接与HNF 4-RE结合， 作用于FGF 23启动子以抑制FGF 23转录。我们还将确定FGF 23对HNF 4 α抑制的反应 在低FGF 23产生的小鼠中。最后，在Aim 3中，我们将检验以下假设：在CKD中，HNF 4 α降低， 活化刺激FGF 23转录。我们将确定CKD期间HNF 4 α表达何时降低， HNF 4 α是否对磷酸盐有反应。我们将展示 通过将Col 4a 3 KO与新产生的含有骨靶向的小鼠杂交来靶向HNF 4 α通路 Hnf 4 α过表达（HNF 4 αOc-cTg），并通过评估FGF 23水平和骨量的校正， Col 4a 3 KO/Hnf 4 αOc-cTg复合小鼠。该项目将有助于对分子调控的新见解， 骨和FGF 23在健康和CKD中的作用，并支持我们开发新的治疗方法的最终目标 改善CKD的预后。

PROJECT SUMMARY Patients with chronic kidney disease (CKD) experience a wide-range of bone and mineral metabolism abnormalities, including bone mass loss and excess production of fibroblast growth factor 23 (FGF23) by osteocytes. Serum FGF23 levels rise early in the course of CKD, and high levels are independently associated with greater risks of CKD progression, cardiovascular disease and death. These findings have stimulated interest in designing therapies to reduce bone abnormalities and lower FGF23 levels, but the approach is limited by poor understanding of the molecular mechanisms at play. In preliminary data of this proposal, we show that the nuclear receptor, hepatocyte nuclear factor 4 alpha (HNF4α) is mechanistically linked to bone specific pathways controlling FGF23 production in animal models of FGF23 excess. We also show that FGF23 promoter contains a predicted HNF4 response element (HNF4-RE) and that Hnf4α is expressed in osteoblasts and osteocytes. In new preliminary data, we show that HNF4α stimulates osteoblast proliferation and that osteoblast specific deletion of Hnf4α leads to FGF23 excess in osteoblasts and mice, and to low bone mass in animals. We also show that Hnf4α expression is repressed by 98% in bones from an established model of progressive CKD, the Col4a3KO mouse. These observations support an important new role for HNF4α. In this proposal, we will test the hypothesis that HNF4α deficiency in CKD contributes to excess FGF23 and bone abnormalities. In Aim1 we will establish the role of HNF4α in bone, by assessing in vitro the proliferation, activity and differentiation of osteoblasts that overexpress or lack HNF4α. We will characterize the bone HNF4α transcriptome by RNA sequencing and complete the bone phenotype analysis of mice carrying the specific deletion of Hnf4α in osteoblasts/osteocytes (Hnf4αOc-cKO). To investigate whether HNF4α mediates the osteogenic response, we will expose WT and Hnf4αOc-cKO mice to catabolic and anabolic PTH challenges. In Aim2 we will investigate HNF4α as a molecular suppressor of FGF23 production in bone, by testing whether HNF4α directly binds to HNF4-RE on FGF23 promoter to inhibit FGF23 transcription. We will also determine FGF23 response to HNF4α inhibition in mice with low FGF23 production. Finally, in Aim3 we will test the hypothesis that in CKD, decreased HNF4α activation stimulates FGF23 transcription. We will determine when HNF4α expression decreases during CKD progression and whether HNF4α responds to phosphate. We will demonstrate the therapeutic potential of targeting HNF4α pathways by crossing Col4a3KO to newly generated mice containing a bone targeted overexpression of Hnf4α (HNF4αOc-cTg) and by assessing correction of FGF23 levels and bone mass in Col4a3KO/Hnf4αOc-cTg compound mice. The project will contribute to new insights into the molecular regulation of bone and FGF23 in health and in CKD, and support our ultimate goal of developing novel therapeutic approaches to improve outcomes in CKD.