Crosstalk between estrogen and bile acid signaling pathway

描述（由申请人提供）：除了在正常性和生殖功能的发育和维持中的众所周知的作用外，雌激素在非经典内分泌器官系统（如心血管系统和肝脏）中发挥广泛的生物学作用。胆汁酸是胆固醇的代谢产物，已被鉴定为核受体法尼醇X受体（FXR）的一类信号分子。胆汁酸对FXR的激活调节了大量涉及胆汁酸、胆固醇、脂质和葡萄糖稳态维持的FXR靶基因。胆汁酸盐输出泵（BSEP）是胆汁酸通过激活FXR而进行正性调节的一种重要的胆汁酸外排器，在维持肝脏和胆汁胆汁中胆汁酸稳态中起着重要作用。在胆汁酸水平失衡引起的疾病中，妊娠肝内胆汁淤积症（ICP）和胆石病都与性激素雌激素有关。虽然多种危险因素与这些疾病有关，但研究表明雌激素在诱发这些疾病中起着关键作用。雌激素诱导的胆汁淤积和胆结石病均已在动物模型中重现。然而，我们对潜在机制的理解还远未完成。我们的初步研究发现，雌激素在体内外均能抑制人BSEP的表达，且这种抑制作用是由雌激素受体1（ER 1）通过与FXR的物理相互作用介导的，表明雌激素/ER 1和胆汁酸/FXR信号通路之间存在交叉作用。待检验的中心假设是，雌激素下调BSEP表达是通过一种新的非经典反式阻遏途径介导的，这是ER 1和FXR之间的直接相互作用，因此是ICP和胆石病的常见风险因素。提出了两个具体的目标来检验这一假设。具体目标1是通过以下三种方法确定雌激素介导的BSEP表达抑制的机制：（1）在体外和体内确定ER 1和FXR之间的物理相互作用;（2）确定雌激素对FXR和ER 1辅助调节因子向BSEP启动子的募集改变;（3）绘制ER 1中与FXR直接相互作用的区域。具体目标2是通过以下两种方法在体内研究雌激素介导的BSEP抑制、肝和胆的病理后果：（1）用活体成像技术测定整个妊娠期间小鼠体内人BSEP反式激活的动力学;和（2）雌激素介导的BSEP抑制在雌激素诱导的胆汁淤积和胆石病中的病因作用将在WT和ER 1中进行研究（-/-）小鼠。在完成这项研究后，我们将通过ER 1和FXR之间的直接相互作用，牢固地建立一种新的非经典雌激素反式抑制途径，揭示了雌激素/ER 1和胆汁酸/FXR信号通路之间的这种串扰的机理，并建立了BSEP抑制在雌激素诱导的胆汁淤积和胆石病中的共同病因作用。这些发现将开辟一个新的领域，通过研究雌激素对各种FXR靶基因的影响，促进我们对雌激素相关的各种生物学和病理学活动的理解。同样重要的是，这些发现将促进我们对雌激素诱导的ICP和胆石病的机制和发病机制的理解。研究结果将首次在这两种不同但相关的疾病之间建立共同的病因学联系，最后，研究结果将为开发治疗药物提供分子基础，通过调节BSEP表达和ER 1/FXR信号通路来治疗或预防这些疾病。

DESCRIPTION (provided by applicant): In addition to the well-known roles in development and maintenance of normal sexual and reproductive function, estrogen exerts a vast range of biological effects in nonclassical endocrine organ systems, such as cardiovascular system and liver. Bile acids are metabolites of cholesterol and have been identified as a class of signaling molecules for nuclear receptor farnesoid X receptor (FXR). Activation of FXR by bile acids regulates a myriad of FXR target genes involving in maintenance of bile acids, cholesterol, lipids and glucose homeostasis. Bile acid salt export pump (BSEP) positively regulated by bile acids through activating FXR plays a critical role in maintaining bile acid homeostasis in the liver and bile as a canalicular bile acid effluxer. Among diseases resulted from imbalance of bile acid levels, intrahepatic cholestasis of pregnancy (ICP) and gallstone disease are both associated with sex hormone estrogen. Although multiple risk factors are linked to those disorders, studies have demonstrated that estrogen plays a key role in the induction of the diseases. Both estrogen-induced cholestasis and gallstone disease have been reproduced in animal model. However, our understanding on the underlying mechanisms is far from complete. In our preliminary study, we found that estrogen repressed human BSEP expression in vitro and in vivo, and the repression was mediated by estrogen receptor 1 (ER1) through physically interacting with FXR, indicating a crosstalk between estrogen/ER1 and bile acids/FXR signaling pathway. The central hypothesis to be tested is that down-regulation of BSEP expression by estrogen is mediated through a novel nonclassical transrepression pathway, a direct interaction between ER1 and FXR and as a consequence is a common risk factor for ICP and gallstone disease. Two specific aims are proposed to test the hypothesis. Specific Aim 1 is to determine the mechanisms for estrogen-mediated repression of BSEP expression by the following three approaches: (1) the physical interaction between ER1 and FXR will be firmly established in vitro and in vivo; (2) altered recruitment of FXR and ER1 coregulators to the BSEP promoter by estrogen will be determined; and (3) the region in ER1 directly interacting with FXR will be mapped. Specific Aim 2 is to investigate estrogen-mediated BSEP repression, hepatic and biliary pathological consequences in vivo with the following two approaches: (1) the dynamics of human BSEP transactivation in vivo in mice during the entire pregnancy will be determined with the Living Imaging technology; and (2) the etiological role of estrogen-mediated BSEP repression in estrogen-induced cholestasis and gallstone disease will be investigated in wt and ER1(-/-) mice. Upon completion of the proposed study, we will have firmly established a novel nonclassical estrogen transrepression pathway through a direct interaction between ER1 and FXR, revealed mechanistic insights into such crosstalk between the estrogen/ER1 and bile acids/FXR signaling pathway, and established a common etiological role of BSEP repression in estrogen-induced cholestasis and gallstone disease. The findings will open a new field to advance our understanding on the diverse biological and pathological activities associated with estrogens through investigating estrogen's effects on various FXR target genes. Equally significant, the findings will advance our understanding on the mechanisms and pathogenesis of estrogen-induced ICP and gallstone disease. For the first time, the results will establish a common etiological link between the two distinct but related diseases, and finally, the findings will provide a molecular basis for developing therapeutic agents to treat or prevent those disorders by modulating BSEP expression and ER1/FXR signaling pathways.