TRANSCRIPTIONAL REGULATION OF BILE SALT EXPORT PUMP BY ENDOBIOTICS AND XENOBIOTI

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。胆盐输出泵(BSEP)似乎负责胆汁酸的分泌。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。阐明BSEP表达调控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检验核心假设，即BSEP的表达是由内生生物和外源生物通过不同但功能相关的机制协调调节的。这一试验项目的具体目的包括：(1)确定核受体FXR在氧固醇22(R)-OHC调节BSEP表达中的反式激活作用；(2)确定氧固醇22(R)-OHC与另外两种FXR激活剂CDCA和Guggulsterone的相互作用模式。第一个特定目的是为了验证这样一个假设，即氧固醇诱导的BSEP表达上调是通过激活核受体FXR来实现的。提出了两种方法来验证这一假设：(A)将构建显性负FXR突变体，并测试它们抑制22(R)-OHC上调BSEP表达的能力；(B)BSEP启动子中的FXR结合位点IR-1将发生突变，其调节报告表达的能力将被测试以响应氧固醇22(R)-OHC。第二个特定目的是验证BSEP的表达受FXR激活剂Oxsterol 22(R)-OHC、胆汁酸CDCA和异种谷固酮协调调控的假设。建议用两种方法来验证这一假设：(1)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)转BSEP启动子记者的Huh 7细胞将单独或联合使用22(R)-OHC、CDCA和Guggulsterone，然后测量报告活性。

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.