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

Martijn O. Hoeke, Janette Heegsma, Mark Hoekstra, Albert Moshage, Klaas Nico Faber*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

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

Methods: A 579-bp hSHP promoter element was analyzed to locate FXR/chenodeoxycholic acid (CDCA)-and RXR alpha/9cRA-responsive elements. hSHP promoter constructs were analyzed in FXR/RXR alpha-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/RXR alpha-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 RXR alpha agonists.

Original languageEnglish
Article numbere88011
Number of pages10
JournalPLoS ONE
Volume9
Issue number2
DOIs
Publication statusPublished - 3-Feb-2014

Keywords

  • ORPHAN NUCLEAR RECEPTOR
  • FARNESOID-X-RECEPTOR
  • SALT EXPORT PUMP
  • CHOLESTEROL 7-ALPHA-HYDROXYLASE GENE
  • BILE-ACID BIOSYNTHESIS
  • LIVER RECEPTOR
  • FEEDBACK-REGULATION
  • MOLECULAR-BASIS
  • DNA-BINDING
  • ACTIVATION

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