TRANSCRIPTIONAL REGULATION OF BILE SALT EXPORT PUMP BY ENDOBIOTICS AND XENOBIOTI

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 胆固醇转化为胆汁酸，然后分泌成胆汁，是消除过量胆固醇的主要途径。遗传学和生化研究已经证实，胆盐输出泵(BSEP)负责分泌。内源性和外源性BSEP功能或表达受损可导致胆汁淤积性肝损伤。另一方面，内源性和外源性促进BSEP的表达对胆汁淤积和高胆固醇血症有治疗作用。阐明BSEP表达调控的分子机制将具有生理、毒理学和治疗意义。该项目的长期目标是阐明BSEP表达受内生生物和外源生物通过不同但功能相关的途径协调调节的分子机制。这个试点项目的具体目的是：特定的目标#1确定是否通过c-jun/c-fos激活途径实现低脂血症性谷固酮诱导BSEP的表达，以及特定的目标#2确定核受体肝脏同源受体1(LRH-1)是否在转录上调节BSEP的表达。针对特定的目标#1，提出了两种实验方法：(A)超电泳迁移率改变分析将在体外确定c-jun/c-fos是否与BSEP启动子中的谷固酮反应元件(Gure)结合；(B)染色质免疫沉淀将在体内确定c-jun/c-fos是否被招募到Gure。针对特定的目的#2，提出了两种实验方法：(A)LRH-1的表达将被RNAi特异性沉默，并将确定LRH-1对BSEP表达的影响；(B)将对BSEP启动子中潜在的LRH-1结合位点进行突变分析，以确定LRH-1的功能位点(S)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Conversion of cholesterol to bile acids and subsequent secretion into bile represents a major pathway in the elimination of excessive cholesterol. Genetic and biochemical studies have established that the bile salt export pump (BSEP) is responsible for the secretion. Impairment of BSEP function or expression by endobiotics and xenobiotics lead to cholestatic liver injury. On the other hand, enhancement of BSEP expression by endobiotics and xenobiotics has therapeutic effect on cholestasis and hypercholesterolemia. Elucidation of the molecular mechanism for the regulation of BSEP expression will have physiological, toxicological and therapeutic significance. The long-term objective of the proposed project is to elucidate the molecular mechanism by which the expression of BSEP is coordinately regulated by endobiotics and xenobiotics through distinct but functionally related pathways. The specific aims of this pilot project are: specific aim #1 to determine whether hypolipidemic guggulsterone-mediated induction of BSEP expression is achieved through the c-Jun/c-fos activation pathway and specific aim #2 to determine whether nuclear receptor liver homology receptor 1 (LRH-1) transcriptionally regulates BSEP expression. Two experimental approaches are proposed for specific aim #1: (a) super electrophoretic mobility shift assay will be performed to determine whether c-Jun/c-fos binds to the guggulsterone responsive element (GuRE) in the BSEP promoter in vitro; (b) chromatin immunoprecipitation will be performed to determine whether c-Jun/c-fos is recruited to the GuRE in vivo. Two experimental approaches are proposed for specific aim #2: (a) LRH-1 expression will be specifically silenced by RNAi and the effect of LRH-1 knockdown on BSEP expression will be determined; (b) mutational analysis of the potential LRH-1 binding sites in the BSEP promoter will be performed to identify the functional LRH-1 site(s).