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

项目总结 慢性肾脏病(CKD)患者经历了广泛的骨骼和矿物质代谢 异常，包括骨量丢失和成纤维细胞生长因子23(FGF23)的过量产生 骨细胞。血清FGF23水平在CKD病程早期升高，且高水平独立相关 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骼异常和降低FGF23水平方面，但该方法受到穷人的限制 了解起作用的分子机制。在这项提案的初步数据中，我们表明 核受体，肝细胞核因子4α(hNF4α)与骨骼特异通路有机械联系 在FGF23过量的动物模型中控制FGF23的产生。我们还发现FGF23启动子包含 预测的HNF4反应元件(HNF4-RE)以及HNF4α在成骨细胞和骨细胞中表达。在……里面 新的初步数据显示，hNF4α刺激成骨细胞增殖，且成骨细胞特异性 Hnf4α的缺失导致成骨细胞和小鼠的FGF23过多，动物的骨量减少。我们也 结果表明，在已建立的进行性慢性肾脏病模型的骨骼中，HNF4α的表达被抑制了98%。 Col4a3KO小鼠。这些观察结果支持了HNF4α的一个重要的新角色。在本提案中，我们将测试 假设慢性肾脏病中HNF4α缺乏会导致FGF23过多和骨骼异常。在Aim1我们 将通过评估HNF4α在体外的增殖、活性和分化来确定其在骨骼中的作用 过表达或缺乏HNF4α的成骨细胞。我们将通过rna来鉴定骨hnf4α转录组。 携带HNF4α特异性缺失小鼠的测序和骨表型分析 成骨细胞/骨细胞(HNF4、α、Oc-Cko)。为了研究hnf4α是否介导成骨反应，我们将 将WT和HNF4αOc-Cko小鼠暴露于分解代谢和合成代谢的PTH挑战。在AIM2中，我们将研究HNF4α 通过检测HNF4α是否直接与HNF4-RE结合，作为骨组织中FGF23产生的分子抑制因子 在FGF23启动子上抑制FGF23转录。我们还将确定成纤维细胞生长因子23对HNF4α抑制的反应 在FGF23产量低的小鼠中。最后，在Aim3中，我们将检验以下假设：在慢性肾脏病中，HNF4α降低 激活刺激FGF23转录。我们将确定慢性肾脏病期间HNF4α表达何时下降 进展以及HNF4α是否对磷酸盐有反应。我们将展示这种药物的治疗潜力 通过将Col4a3KO与新生成的含有靶向骨骼的小鼠杂交来靶向HNF4α通路 HNF4α(hNF4αOc-CTG)的过表达以及对FGF23水平和骨量的校正 Col4a3KO/HnF4αOc-CTG复合小鼠。该项目将有助于对分子调控的新见解。 骨和FGF23在健康和慢性肾脏病中的作用，并支持我们开发新的治疗方法的最终目标 以改善慢性肾脏病的预后。

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.