核受体FXR调控内源性雌激素II相代谢的作用机制研究

The homeostasis of endogenous estrogen in human affects the organism development and disease occurrence. Based on our previously proved facts that activation of FXR receptor could significantly down-regulate the expression of UGT2B7 and SULT1E1 (both could metabolize estradiol), increase the estradiol levels in female mice, and reduce the formation of estradiol-related phase II metabolites (including glucuronides and sulfates), we innovatively propose the scientific issue that FXR receptor regulate the phase II metabolism and disposition of estrogen through influencing the expression of estrogen-metabolizing related UGT (or SULT) isozymes and efflux transporters. First, we will systematically evaluate the glucuronidation and sulfation of estrogen (including estradiol, estrone and estriol) by several approaches, including reaction phenotyping, enzyme kinetic analysis, chemical inhibition assays and metabolic activity correlation analysis. Meanwhile, we will establish the HeLa cell lines overexpressing the UGT (or SULT) enzymes which could not be obtained commercially to determine their contribution to the metabolism of estrogen. Based on the engineered HeLa-UGT/SULT cell models above, the contribution and significance of efflux transporters (BCRP and MRPs) to the disposition of estrogen-related metabolites were performed through chemical inhibition experiments (Ko143 and MK571) and gene manipulation methods (overexpressing or lower expressing of a single efflux transporter). Besides, overexpressing or lower expressing of β-glucuronidase (or steroid sulfatase) were tested to confirm the roles of de-glucuronidation (or de-sulfation) in efflux transporters regulating the phase II metabolism of estrogen. In addition, vesicular transport assays and sandwich cultured human hepatocytes models were both applied to reveal the action mechanisms of (P-gp, BCRP and MRPs) in regulating cellular glucuronidation or sulfation of estrogen. Furthermore, we will investigate the regulation effects of FXR receptor towards estrogen-metabolizing related UGT (or SULT) enzymes and efflux transporters in HepG2 and Caco-2 cell lines, and elucidate the molecular mechanism of gene regulation by chemical intervention (GW4064 and CDCA) and gene manipulation (luciferase reporter gene assays; electrophoretic mobility shift assays, EMSA; chromatin immunoprecipitation assay, ChIP; co-immunoprecipitation, Co-IP; etc.). Moreover, we will validate the regulatory effects of FXR receptor on the expression of estrogen-metabolizing related UGT (or SULT) enzymes and efflux transporters in FXR-/- mice, and build the communication network relationship in FXR receptor regulating the phase II metabolism and disposition of estrogen. This project is of great significance in revealing the crucial role of FXR receptor in the homeostasis of endogenous estrogen in human, and also propose a new idea for clinical intervention of estrogen imbalance-related diseases.

雌激素的稳态平衡影响着机体的发育与疾病的发生发展。本项目根据前期发现激活FXR受体可显著下调UGT2B7和SULT1E1（二者均代谢雌二醇）表达，提升小鼠雌二醇水平，降低雌二醇II相代谢物形成的事实，创新性地提出FXR通过UGT/SULT酶-外排转运蛋白代谢网络调控机体雌激素II相代谢的科学问题。首先通过UGT/SULT重组酶体系和高表达UGT/SULT酶的HeLa细胞模型，筛选代谢雌激素的亚型酶；基于上述HeLa细胞模型、囊泡转运模型和“三明治”人肝细胞模型等，评价外排转运蛋白对雌激素II相代谢的调控机制；在FXR-/-小鼠及肝肠细胞水平，研究FXR对UGT/SULT酶与外排转运蛋白的调控效应，通过化学干预与基因操纵，阐明其调控的分子机制，沟通FXR调控雌激素II相代谢的网络关系。本项目对于揭示FXR在雌激素稳态平衡中的关键作用具有重要意义，将为雌激素失衡相关疾病的临床干预提供新思路。

FXR激动剂的使用可以显著提高小鼠体内雌二醇水平、降低其II相代谢物的形成，发挥调节机体雌激素水平作用。但是，其调控作用机制尚不明确。本课题主要完成了以下工作：①优化雌二醇、雌酮、雌三醇及其葡萄糖醛酸结合物和磺酸结合物的UHPLC/MS-MS分析条件；结合体外酶孵育体系，明确雌激素在肝肠微粒体和肝S9 fraction中的代谢特征；UGT1A1、UGT1A10、UGT2B7和SULT1E1、SULT1A1为介导雌激素II相代谢失活的关键亚型酶；完成上述亚型酶的孵育体系优化，获得适用于表征五种关键酶代谢活性的体外评价体系；②采用UGT1A1高表达的稳转HeLa细胞模型，以雌二醇为底物，结合化学抑制剂法（Ko143和MK571）和基因沉默法（shRNA质粒）发现，外排转运蛋白BCRP和MRPs不同程度地参与雌二醇葡萄糖醛酸苷的外排；③FXR受体激动剂（GW4064和CDCA）可以显著影响雌激素II相代谢酶的mRNA表达；采用质粒瞬时转染法（FXR质粒或si_FXR）发现，高表达或低表达FXR受体，可以显著改变雌激素II相代谢关键酶的mRNA表达，在蛋白表达水平，对SULT1E1表现显著差异；④采用双荧光素酶报告基因，EMSA和CHIP等实验发现，FXR受体可以通过调控中间转录因子CAR受体，与SULT1E1启动子区域的位点（-948~-933 bp）结合，启动转录调控作用；再者，FXR受体也可以通过抑制PGC1α和HNF4α的结合来调节SULT1E1的表达；⑤此外，FXR受体还可以直接与雌激素I相代谢关键酶CYP1A1（-478-488bp）和CYP1B1（-1460-1475bp）的启动子区域结合，发挥转录调控作用，进而影响雌激素I相代谢酶的活性。本研究一定程度上揭示了FXR受体调控雌激素代谢，维持机体雌激素稳态平衡的关键作用，为雌激素失衡相关疾病的临床干预提供了科学依据。