ABSTRACT
This study reports that Cyp3a41 gene contains 13 exons and is localized on the chromosome 5. CYP3A41 is a female-specific isoform that is predominantly expressed in the liver. Estrogen signaling is not responsible for its female specificity. CYP3A41 expression in kidney and brain is observed only in 50% of mice examined. PXR mediates dexamethasone-dependent suppression of CYP3A41. In contrast to CYP3A11, CYP3A41 expression is not induced by pregnenolone-16alpha-carbonitrile (PCN) in wild-type mice, but is significantly suppressed by PCN in PXR(-/-) mice. Phenobarbital and TCPOBOP induce CYP3A11 expression only in the presence of CAR, but have no effect on CYP3A41 expression. Immunoblot and erythromycin demethylase activity analysis reveal robust CYP3A induction after PCN treatment, which is poorly correlated to CYP3A41. These findings suggest a differential role for CAR/PXR in regulating individual CYP3A isoforms by previously characterized CYP3A inducers.
Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Cytochrome P-450 Enzyme System/genetics , Oxidoreductases, N-Demethylating/genetics , Oxidoreductases/genetics , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Steroid/physiology , Transcription Factors/physiology , Animals , Aryl Hydrocarbon Hydroxylases/biosynthesis , Constitutive Androstane Receptor , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/biosynthesis , Dexamethasone/pharmacology , Dose-Response Relationship, Drug , Female , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/physiology , Isoenzymes/biosynthesis , Isoenzymes/genetics , Isoenzymes/physiology , Male , Membrane Proteins , Mice , Mice, Inbred C57BL , Mice, Knockout , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Oxidoreductases/biosynthesis , Oxidoreductases, N-Demethylating/biosynthesis , Pregnane X Receptor , Receptors, Cytoplasmic and Nuclear/biosynthesis , Receptors, Cytoplasmic and Nuclear/deficiency , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Steroid/biosynthesis , Receptors, Steroid/deficiency , Receptors, Steroid/genetics , Transcription Factors/deficiency , Transcription Factors/geneticsABSTRACT
The pregnane X receptor (PXR) is the molecular target for catatoxic steroids such as pregnenolone 16alpha-carbonitrile (PCN), which induce cytochrome P450 3A (CYP3A) expression and protect the body from harmful chemicals. In this study, we demonstrate that PXR is activated by the toxic bile acid lithocholic acid (LCA) and its 3-keto metabolite. Furthermore, we show that PXR regulates the expression of genes involved in the biosynthesis, transport, and metabolism of bile acids including cholesterol 7alpha-hydroxylase (Cyp7a1) and the Na(+)-independent organic anion transporter 2 (Oatp2). Finally, we demonstrate that activation of PXR protects against severe liver damage induced by LCA. Based on these data, we propose that PXR serves as a physiological sensor of LCA, and coordinately regulates gene expression to reduce the concentrations of this toxic bile acid. These findings suggest that PXR agonists may prove useful in the treatment of human cholestatic liver disease.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cholestasis, Intrahepatic/metabolism , Lithocholic Acid/metabolism , Liver/injuries , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Steroid/physiology , Animals , Cholestasis, Intrahepatic/prevention & control , Cholesterol 7-alpha-Hydroxylase/genetics , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Female , Gene Expression Regulation, Enzymologic , Lithocholic Acid/pharmacology , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Oxidoreductases, N-Demethylating/genetics , Pregnane X Receptor , Pregnenolone Carbonitrile/pharmacology , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/genetics , Receptors, Steroid/metabolismABSTRACT
Recently, we reported the cloning of the nuclear orphan receptor TAK1. In this study, we characterized the sequence requirements for optimal TAK1 binding and analyzed the repression of the peroxisome proliferator-activated receptor alpha (PPARalpha) signaling pathway by TAK1. Site selection analysis showed that TAK1 has the greatest affinity for direct repeat-1 response elements (RE) containing AGGTCAAAGGTCA (TAK1-RE) to which it binds as a homodimer. TAK1 is a very weak inducer of TAK1-RE-dependent transcriptional activation. We observed that TAK1, as PPARalpha, is expressed within rat hepatocytes and is able to bind the peroxisome proliferator response elements (PPREs) present in the promoter of the PPARalpha target genes rat enoyl-CoA hydratase (HD) and peroxisomal fatty acyl-CoA oxidase (ACOX). TAK1 is unable to induce PPRE-dependent transcriptional activation and represses PPARalpha-mediated transactivation through these elements in a dose-dependent manner. Two-hybrid analysis showed that TAK1 does not form heterodimers with either PPARalpha or retinoid X receptor (RXRalpha), indicating that this repression does not involve a mechanism by which TAK1 titrates out PPARalpha or RXRalpha from PPAR.RXR complexes. Further studies demonstrated that the PPARalpha ligand 8(S)-hydroxyeicosatetraenoic acid strongly promotes the interaction of PPARalpha with the co-activator RIP-140 but decreases the interaction of PPARalpha with the co-repressor SMRT. In contrast, TAK1 interacts with RIP-140 but not with SMRT and competes with PPARalpha for RIP-140 binding. These observations indicated that the antagonistic effects of TAK1 on PPARalpha.RXRalpha transactivation act at least at two levels in the PPARalpha signaling pathway: competition of TAK1 with PPARalpha.RXR for binding to PPREs as well as to common co-activators, such as RIP-140. Our results suggest an important role for TAK1 in modulating PPARalpha-controlled gene expression in hepatocytes.
Subject(s)
Nerve Tissue Proteins/physiology , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Steroid/physiology , Receptors, Thyroid Hormone/physiology , Transcription Factors/genetics , Transcriptional Activation/physiology , Animals , Base Sequence , Binding, Competitive , Cell Line , DNA-Binding Proteins/metabolism , Dimerization , Gene Expression Regulation/physiology , Liver/metabolism , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Rats , Receptors, Steroid/metabolism , Receptors, Thyroid Hormone/metabolismABSTRACT
Steroid hormones exert profound effects on differentiation, development, and homeostasis in higher eukaryotes through interactions with nuclear receptors. We describe a novel orphan nuclear receptor, termed the pregnane X receptor (PXR), that is activated by naturally occurring steroids such as pregnenolone and progesterone, and synthetic glucocorticoids and antiglucocorticoids. PXR exists as two isoforms, PXR.1 and PXR.2, that are differentially activated by steroids. Notably, PXR.1 is efficaciously activated by pregnenolone 16alpha-carbonitrile, a glucocorticoid receptor antagonist that induces the expression of the CYP3A family of steroid hydroxylases and modulates sterol and bile acid biosynthesis in vivo. Our results provide evidence for the existence of a novel steroid hormone signaling pathway with potential implications in the regulation of steroid hormone and sterol homeostasis.
