人 FXR 通过直接结合 LRH-1 结合位点而不是通过 IR-1 和 LRH-1 调节 SHP 的表达。

Human FXR regulates SHP expression through direct binding to an LRH-1 binding site, independent of an IR-1 and LRH-1.

机构信息

Department of Gastroenterology and Hepatology, University Medical Center Groningen, Groningen, University of Groningen, Groningen, The Netherlands.

出版信息

PLoS One. 2014 Feb 3;9(2):e88011. doi: 10.1371/journal.pone.0088011. eCollection 2014.

DOI:10.1371/journal.pone.0088011

PMID:24498423

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3912179/

Abstract

BACKGROUND

Farnesoid X receptor/retinoid X receptor-alpha (FXR/RXRα) is the master transcriptional regulator of bile salt synthesis and transport in liver and intestine. FXR is activated by bile acids, RXRα by the vitamin A-derivative 9-cis retinoic acid (9cRA). Remarkably, 9cRA inhibits binding of FXR/RXRα to its response element, an inverted repeat-1 (IR-1). Still, most FXR/RXRα target genes are maximally expressed in the presence of both ligands, including the small heterodimer partner (SHP). Here, we revisited the FXR/RXRα-mediated regulation of human SHP.

METHODS

A 579-bp hSHP promoter element was analyzed to locate FXR/chenodeoxycholic acid (CDCA)- and RXRα/9cRA-responsive elements. hSHP promoter constructs were analyzed in FXR/RXRα-transfected DLD-1, HEK293 and HepG2 cells exposed to CDCA, GW4064 (synthetic FXR ligand) and/or 9cRA. FXR-DNA interactions were analyzed by in vitro pull down assays.

RESULTS

hSHP promoter elements lacking the previously identified IR-1 (-291/-279) largely maintained their activation by FXR/CDCA, but were unresponsive to 9cRA. FXR-mediated activation of the hSHP promoter was primarily dependent on the -122/-69 region. Pull down assays revealed a direct binding of FXR to the -122/-69 sequence, which was abrogated by site-specific mutations in a binding site for the liver receptor homolog-1 (LRH-1) at -78/-70. These mutations strongly impaired the FXR/CDCA-mediated activation, even in the context of a hSHP promoter containing the IR-1. LRH-1 did not increase FXR/RXRα-mediated activation of hSHP promoter activity.

CONCLUSION

FXR/CDCA-activated expression of SHP is primarily mediated through direct binding to an LRH-1 binding site, which is not modulated by LRH-1 and unresponsive to 9cRA. 9cRA-induced expression of SHP requires the IR-1 that overlaps with a direct repeat-2 (DR-2) and DR-4. This establishes for the first time a co-stimulatory, but independent, action of FXR and RXRα agonists.

摘要

背景

法尼醇 X 受体/视黄醇 X 受体-α（FXR/RXRα）是肝脏和肠道胆汁盐合成和转运的主要转录调节因子。FXR 被胆汁酸激活，RXRα 被维生素 A 衍生物 9-顺式视黄酸（9cRA）激活。值得注意的是，9cRA 抑制 FXR/RXRα 与其反应元件（反向重复 1 [IR-1]）的结合。尽管如此，大多数 FXR/RXRα 靶基因在两种配体存在的情况下表达最大，包括小异二聚体伴侣（SHP）。在这里，我们重新研究了 FXR/RXRα 介导的人 SHP 调节。

方法

分析了 579bp 的 hSHP 启动子元件，以定位 FXR/鹅去氧胆酸（CDCA）和 RXRα/9cRA 反应元件。在暴露于 CDCA、GW4064（合成 FXR 配体）和/或 9cRA 的转染 DLD-1、HEK293 和 HepG2 细胞中分析了 hSHP 启动子构建体。通过体外拉下测定分析了 FXR-DNA 相互作用。

结果

缺乏先前鉴定的 IR-1（-291/-279）的 hSHP 启动子元件在很大程度上保持了其对 FXR/CDCA 的激活，但对 9cRA 无反应。FXR 介导的 hSHP 启动子的激活主要依赖于-122/-69 区域。拉下测定显示 FXR 与-122/-69 序列直接结合，该结合在-78/-70 处的肝受体同源物-1（LRH-1）结合位点的特异性突变中被阻断。这些突变强烈削弱了 FXR/CDCA 介导的激活，即使在包含 IR-1 的 hSHP 启动子的情况下也是如此。LRH-1 并未增加 FXR/RXRα 介导的 hSHP 启动子活性的激活。