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


CONCLUSIONS
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.
Department of Gastroenterology and Hepatology, University Medical Center Groningen, Groningen, University of Groningen, Groningen, The Netherlands.
Martijn O
MO
Hoeke
Department of Gastroenterology and Hepatology, University Medical Center Groningen, Groningen, University of Groningen, Groningen, The Netherlands.
Janette
J
Heegsma
Department of Gastroenterology and Hepatology, University Medical Center Groningen, Groningen, University of Groningen, Groningen, The Netherlands.
Mark
M
Hoekstra
Department of Gastroenterology and Hepatology, University Medical Center Groningen, Groningen, University of Groningen, Groningen, The Netherlands.
Han
H
Moshage
Department of Gastroenterology and Hepatology, University Medical Center Groningen, Groningen, University of Groningen, Groningen, The Netherlands.
Klaas Nico
KN
Faber
D001665:Binding Sites; D015153:Blotting, Western; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D047468:Immunoprecipitation; D055029:Inverted Repeat Sequences / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C096916:FXR1 protein, human; C082631:NR5A2 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; C099713:nuclear receptor subfamily 0, group B, member 2; D008156:Luciferases