Regulation and binding of pregnane X receptor by nuclear receptor corepressor silencing mediator of retinoid and thyroid hormone receptors (SMRT).
The pregnane X receptor (PXR) is an orphan nuclear receptor predominantly expressed in liver and intestine. PXR coordinates hepatic responses to prevent liver injury induced by environmental toxins. PXR activates cytochrome P450 3A4 gene expression upon binding to rifampicin (Rif) and clotrimazole (CTZ) by recruiting transcriptional coactivators. It remains unclear whether and how PXR regulates gene expression in the absence of ligand. In this study, we analyzed interactions between PXR and the silencing mediator of retinoid and thyroid hormone receptors (SMRT) and determined the role of SMRT in regulating PXR activity. We show that SMRT interacts with PXR in glutathione S-transferase pull-down, yeast two-hybrid, and mammalian two-hybrid assays. The interaction is mediated through the ligand-binding domain of PXR and the SMRTs' nuclear receptor-interacting domain 2. The PXR-SMRT interaction is sensitive to species-specific ligands, and Rif causes an exchange of the corepressor SMRT with the p160 coactivator known as receptor-associated coactivator 3 (RAC3). Deletion of the PXR's activation function 2 helix enhances SMRT binding and abolishes ligand-dependent dissociation of SMRT. Coexpression of PXR with SMRT results in colocalization at discrete nuclear foci. Finally, transient transfection assays show that overexpression of SMRT inhibits PXR's transactivation of the Cyp3A4 promoter, whereas silencing of SMRT enhances the reporter expression. Taken together, our results suggest that the corepressor SMRT may bind to and regulate the transcriptional activity of PXR.
Johnson
David R
DR
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.
Li
Chia-Wei
CW
Chen
Liuh-Yow
LY
Ghosh
Jagadish C
JC
Chen
J Don
JD
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D056985:Nuclear Receptor Co-Repressor 2; D000077297:Pregnane X Receptor; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D004268:DNA-Binding Proteins; C539130:NCOR2 protein, human; D056985:Nuclear Receptor Co-Repressor 2; D000077297:Pregnane X Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D012097:Repressor Proteins