Mol099341 746..757

Upon treatment with the pregnane X receptor (PXR) activator rifampicin (RIF), human hepatocellular carcinomaHepG2-derived ShP51 cells that stably express PXR showed epithelialmesenchymal transition (EMT)–like morphological changes and migration. Our recent DNA microarrays have identified hepatocyte nuclear factor (HNF) 4a and insulin-like growth factorbinding protein (IGFBP) 1 mRNAs to be downregulated and upregulated, respectively, in RIF-treated ShP51 cells, and these regulations were confirmed by the subsequent real-time polymerase chain reaction andWestern blot analyses. Using this cell system, we demonstrated here that the PXR-HNF4a-IGFBP1 pathway is an essential signal for PXR-induced morphological changes and migration. First, we characterized the molecular mechanism underlying the PXR-mediated repression of the HNF4a gene. Chromatin conformation capture and chromatin immunoprecipitation (ChIP) assays revealed that PXR activation by RIF disrupted enhancer-promoter communication and prompted deacetylation of histone H3 in the HNF4a P1 promoter. Cell-based reporter and ChIP assays showed that PXR targeted the distal enhancer of the HNF4a P1 promoter and stimulated dissociation of HNF3b from the distal enhancer. Subsequently, small interfering RNA knockdown of HNF4a connected PXR-mediated gene regulation with the PXRinduced cellular responses, showing that the knockdown resulted in the upregulation of IGFBP1 and EMT-like morphological changes without RIF treatment. Moreover, recombinant IGFBP1 augmented migration, whereas an anti-IGFBP1 antibody attenuated both PXR-induced morphological changes and migration in ShP51 cells. PXR indirectly activated the IGFBP1 gene by repressing the HNF4a gene, thus enabling upregulation of IGFBP1 to change the morphology of ShP51 cells and cause migration. These results provide new insights into PXR-mediated cellular responses toward xenobiotics including therapeutics.