Human FXR Regulates SHP Expression through Direct Binding to an LRH-1 Binding Site, Independent of an IR-1 and LRH-1

BackgroundFarnesoid 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.MethodsA 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.ResultshSHP 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.ConclusionFXR/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.背景：法尼醇X受体/类视黄醇X受体α（FXR/RXRα）是肝脏与肠道内胆汁酸合成及转运的核心转录调控因子。FXR可被胆汁酸激活，而RXRα则由维生素A衍生物9-顺式视黄酸（9cRA）激活。值得注意的是，9cRA会抑制FXR/RXRα与其应答元件——反向重复序列1（IR-1）——的结合。尽管如此，大多数FXR/RXRα靶基因在两种配体共同存在时表达量达到峰值，其中包括小异二聚体伴侣（SHP）。本研究重新探讨了FXR/RXRα对人源SHP的调控作用。 方法：本研究通过分析一段579 bp的人源SHP启动子元件，定位受FXR/鹅脱氧胆酸（CDCA）以及RXRα/9cRA调控的应答元件。将构建的人源SHP启动子质粒转染至表达FXR/RXRα的DLD-1、HEK293以及HepG2细胞中，并用CDCA、GW4064（合成型FXR配体）以及/或9cRA处理细胞，随后对其进行分析。通过体外下拉实验分析FXR与DNA的相互作用。 结果：缺失此前已鉴定的IR-1（−291/−279）区域的人源SHP启动子元件，仍可在很大程度上保留FXR/CDCA介导的激活效应，但无法对9cRA产生应答。FXR介导的人源SHP启动子激活主要依赖于−122/−69区域。下拉实验结果显示，FXR可直接结合−122/−69序列，而该结合可被−78/−70位点处的肝受体同源物1（LRH-1）结合位点的定点突变所阻断。即便在包含IR-1的人源SHP启动子背景中，上述突变也会显著削弱FXR/CDCA介导的激活效应。LRH-1并不会增强FXR/RXRα介导的人源SHP启动子活性激活。 结论：FXR/CDCA激活的SHP表达主要通过直接结合LRH-1结合位点介导，该位点不受LRH-1调控，且无法对9cRA产生应答。9cRA诱导的SHP表达则需要与直接重复序列2（DR-2）及DR-4重叠的IR-1区域。本研究首次证实，FXR与RXRα激动剂具有协同刺激但相互独立的调控作用。