TRANSCRIPTIONAL REGULATION OF BILE SALT EXPORT PUMP BY ENDOBIOTICS AND XENOBIOTI

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定是研究者机构。胆汁盐输出泵（BSEP）似乎负责胆汁酸的分泌。内源性和外源性物质对BSEP功能或表达的损害导致胆汁淤积性肝损伤。阐明调控BSEP表达的分子机制将具有治疗、毒理学和药学意义。拟议项目的长期目标是测试BSEP的表达通过不同但功能相关的机制由内源性和外源性物质协调调节的中心假设。本试验项目的具体目的包括：（1）确定核受体FXR在氧化甾醇22（R）-OHC调节BSEP表达中的反式激活作用;（2）确定氧化甾醇22（R）-OHC与其他两种FXR激活剂CDCA和guggulsterone的相互作用模式。第一个具体目的是为了检验这一假设，即通过激活核受体FXR来实现氧化甾醇诱导的BSEP表达上调。提出了两种方法来检验这一假设：（a）构建显性负性FXR突变体，并检测其抑制22（R）-OHC上调BSEP表达的能力;（B）突变BSEP启动子中的FXR结合位点IR-1，并检测其响应氧固醇22（R）-OHC调节报告基因表达的能力。第二个具体目的是检验BSEP表达受FXR激活剂氧固醇22（R）-OHC、胆汁酸CDCA和异生物质guggulsterone协调调节的假设。本文提出了两种方法来验证这一假设：（1）用22（R）-OHC、CDCA和guggulsterone单独或联合处理人原代肝细胞，然后测量BSEP mRNA水平;（2）用22（R）-OHC、CDCA和guggulsterone单独或组合处理用BSEP启动子报道基因转染的Huh 7细胞，随后测量报告子活性。

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. The bile salt export pump (BSEP) appears to be responsible for the secretion of bile acids. Impairment of BSEP function or expression by endobiotics and xenobiotics lead to cholestatic liver injury. Elucidation of molecular mechanisms for regulated expression of BSEP will have therapeutic, toxicological, and pharmaceutical significance. The long-term goal of the proposed project is to test the central hypothesis that expression of BSEP is coordinately regulated by endobiotics and xenobiotics through distinct but functionally related mechanisms. The specific aims of this pilot project include: (1) To determine the transactivative role of nuclear receptor FXR in the regulated expression of BSEP by oxysterol 22(R)-OHC; and (2) to determine the interactive modes of oxysterol 22(R)-OHC with the other two FXR activators CDCA and guggulsterone. The first specific aim is designed to test the hypothesis that oxysterol-induced up-regulation of BSEP expression is achieved through activation of nuclear receptor FXR. Two approaches are proposed to test this hypothesis: (a) dominant negative FXR mutants will be constructed and their ability to suppress the up-regulated expression of BSEP by 22(R)-OHC will be tested; and (b) the FXR binding site IR-1 in the BSEP promoter will be mutated and its ability to regulate reporter expression will be tested in response to oxysterol 22(R)-OHC. The second specific aim is designed to test the hypothesis that BSEP expression is coordinately regulated by FXR activators oxysterol 22(R)-OHC, bile acid CDCA, and xenobiotic guggulsterone. Two approaches are proposed to tested the hypothesis: (1) human primary hepatocytes will be treated with 22(R)-OHC, CDCA, and guggulsterone individually or in a combination of the two, followed by measurement of BSEP mRNA levels; (2) Huh 7 cells transfected with BSEP promoter reporters will be treated with 22(R)-OHC, CDCA, and guggulsterone individually or in a combination, followed by measurement of reporter activity.