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

Abstract

<div><p>Background</p><p>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 <i>SHP</i>.</p><p>Methods</p><p>A 579-bp <i>hSHP</i> promoter element was analyzed to locate FXR/chenodeoxycholic acid (CDCA)- and RXRα/9cRA-responsive elements. <i>hSHP</i> 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 <i>in vitro</i> pull down assays.</p><p>Results</p><p><i>hSHP</i> 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 <i>hSHP</i> 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 <i>hSHP</i> promoter containing the IR-1. LRH-1 did not increase FXR/RXRα-mediated activation of <i>hSHP</i> promoter activity.</p><p>Conclusion</p><p>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.</p></div