Abstract
Previous studies show that feedback inhibition of bile acid production by bile acids is mediated by multiple mechanisms, including activation of pregnane X receptor (PXR). Consistent with these studies, the antibiotic rifampicin, a ligand for human PXR, reduces hepatic bile acid levels in cholestasis patients. To delineate the mechanisms underlying PXR-mediated suppression of bile acid biosynthesis, we examined the functional cross-talk between human PXR and HNF-4, a key hepatic activator of genes involved in bile acid biosynthesis including the cholesterol 7-alpha hydroxylase (CYP7A1) and sterol 12-alpha hydroxylase (CYP8B1) genes. Treatment with rifampicin resulted in repression of endogenous human CYP7A1 expression in HepG2 cells that was reversed by PXR small interfering RNA. The coactivator PGC-1 enhanced transcriptional activity of HNF-4, and this enhancement was suppressed by rifampicin-activated PXR. Endogenous PGC-1 from mouse liver extracts bound to PXR, and recombinant PGC-1 directly interacted with both PXR and HNF-4 in vitro. Rifampicin-dependent interaction of PXR with PGC-1 was shown in cells by coimmunoprecipitation, and intranuclear localization studies using confocal microscopy provided further evidence for this interaction. In chromatin immunoprecipitation studies, rifampicin treatment did not inhibit HNF-4 binding to the native promoters of CYP7A1 and CYP8B1 but resulted in dissociation of PGC-1 and concomitant gene repression. Most interestingly, these rifampicin effects were also observed in the phosphoenolpyruvate carboxykinase gene that contains a functional HNF-4-binding site and is central to hepatic gluconeogenesis. Our study suggests that ligand-activated PXR interferes with HNF-4 signaling by targeting the common coactivator PGC-1, which underlies physiologically relevant inhibitory cross-talk between drug metabolism and cholesterol/glucose metabolism.
MeSH terms
-
Animals
-
Bile Acids and Salts / metabolism
-
Binding Sites
-
Blotting, Western
-
COS Cells
-
Cell Line
-
Cell Nucleus / metabolism
-
Cholesterol / metabolism*
-
Cholesterol 7-alpha-Hydroxylase / metabolism
-
Chromatin / chemistry
-
Chromatin / metabolism
-
Cytochrome P-450 CYP3A
-
Cytochrome P-450 Enzyme System / metabolism
-
DNA-Binding Proteins / chemistry*
-
DNA-Binding Proteins / metabolism
-
Enzyme Inhibitors / pharmacology
-
Gene Expression Regulation
-
Genes, Reporter
-
Genetic Vectors
-
Glucose / metabolism*
-
Glutathione / metabolism
-
Glutathione Transferase / metabolism
-
Green Fluorescent Proteins / chemistry
-
Heat-Shock Proteins
-
Hepatocyte Nuclear Factor 4
-
Humans
-
Immunoprecipitation
-
Ligands
-
Liver / metabolism*
-
Mice
-
Oligonucleotides / chemistry
-
Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha
-
Phosphoenolpyruvate Carboxykinase (GTP) / metabolism
-
Phosphoproteins / chemistry*
-
Phosphoproteins / metabolism
-
Plasmids / metabolism
-
Pregnane X Receptor
-
Promoter Regions, Genetic
-
Protein Binding
-
RNA, Messenger / metabolism
-
RNA, Small Interfering / metabolism
-
Receptors, Cytoplasmic and Nuclear / chemistry*
-
Receptors, Cytoplasmic and Nuclear / metabolism
-
Receptors, Steroid / chemistry*
-
Recombinant Proteins / chemistry
-
Reverse Transcriptase Polymerase Chain Reaction
-
Rifampin / pharmacology
-
Sepharose / chemistry
-
Signal Transduction
-
Steroid 12-alpha-Hydroxylase / metabolism
-
Transcription Factors / chemistry*
-
Transcription Factors / metabolism
-
Transcription, Genetic
-
Transcriptional Activation
-
Transfection
Substances
-
Bile Acids and Salts
-
Chromatin
-
DNA-Binding Proteins
-
Enzyme Inhibitors
-
Heat-Shock Proteins
-
Hepatocyte Nuclear Factor 4
-
Ligands
-
Oligonucleotides
-
PPARGC1A protein, human
-
Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha
-
Phosphoproteins
-
Pregnane X Receptor
-
RNA, Messenger
-
RNA, Small Interfering
-
Receptors, Cytoplasmic and Nuclear
-
Receptors, Steroid
-
Recombinant Proteins
-
Transcription Factors
-
nuclear receptor subfamily 0, group B, member 2
-
Green Fluorescent Proteins
-
Sepharose
-
Cytochrome P-450 Enzyme System
-
Cholesterol
-
CYP3A protein, human
-
Cytochrome P-450 CYP3A
-
Cholesterol 7-alpha-Hydroxylase
-
Steroid 12-alpha-Hydroxylase
-
Glutathione Transferase
-
Phosphoenolpyruvate Carboxykinase (GTP)
-
Glutathione
-
Glucose
-
Rifampin