Knockdown of ATP8B1 expression leads to specific downregulation of the bile acid sensor FXR in HepG2 cells: effect of the FXR agonist GW4064

Pilar Martínez-Fernández, Loreto Hierro, Paloma Jara, Luis Alvarez
2009 American Journal of Physiology - Gastrointestinal and Liver Physiology  
Martínez-Fernández P, Hierro L, Jara P, Alvarez L. Knockdown of ATP8B1 expression leads to specific downregulation of the bile acid sensor FXR in HepG2 cells: effect of the FXR agonist GW4064. Farnesoid X receptor (FXR) is a bile acid-sensing nuclear receptor that controls bile acid homeostasis. It has been suggested that downregulation of FXR contributes to the pathogenesis of an inherited disorder of bile secretion caused by mutations in ATP8B1. We have investigated the relationship between
more » ... P8B1 knockdown and FXR downregulation in the human hepatoblastoma cell line HepG2. Transfection of HepG2 cells with ATP8B1 small interfering RNA (siRNA) duplexes led to a 60% reduction in the endogenous levels of ATP8B1 mRNA and protein and a concomitant decrease in FXR mRNA and protein content, as well as in FXR phosphorylation. This decrease was accompanied by a marked reduction in mRNA levels of a subset of FXR targets, such as bile salt export pump (ABCB11), small heterodimer partner, and uridine 5Ј-diphosphate-glucuronosyltransferase. ATP8B1 inhibition specifically targeted FXR since mRNA expression of other prominent nuclear receptors, such as pregnane X receptor and constitutive androstane receptor, or liver-enriched transcription factors, such as hepatocyte nuclear factor 1␣ (HNF-1␣) and HNF-4␣, was not altered. The expression of other key genes involved in bile acid synthesis, detoxification, and transport also remained unchanged upon ATP8B1 knockdown. Supporting the specificity of the effect, siRNA-mediated silencing of ABCB11, whose defect is associated with another inherited disorder of bile secretion, did not affect FXR expression. Treatment with the synthetic FXR agonist GW4064 was able to partially neutralize ATP8B1 siRNA-mediated FXR downregulation and fully counteract inhibition of FXR target genes. Collectively these findings indicate that ATP8B1 knockdown specifically downregulates FXR, and this action can be circumvented by treatment with FXR agonists. farnesoid X receptor; ABCB11; nuclear receptors; progressive familial intrahepatic cholestasis FARNESOID X RECEPTOR (FXR; NRIH4) is a nuclear receptor that functions as a ligand-activated transcription factor. Bile salts such as chenodeoxycholate, cholate, deoxycholate, and their conjugates are its physiological ligands (39). To modulate target gene transcription, it also usually requires heterodimerization with retinoid X receptor (RXR; NR2B1) (12). FXR accounts for pleiotropic effects in the enterohepatic system, but, so far, its most well-defined action is the regulation of bile acid homeostasis (18, 21) . In the liver, and in response to elevated concentrations of bile acids, FXR coordinately induces the expression of genes involved in bile acid detoxification (SULT2A1, UGT2B4), sinusoidal efflux (organic
doi:10.1152/ajpgi.90371.2008 pmid:19228886 fatcat:awycjgpv3nbkrmwefqncttiywa