Cytoplasmic localization of pregnane X receptor and ligand-dependent nuclear translocation in mouse liver.
The pregnane X receptor (PXR) plays an important role in the response to xenobiotics and endogenous toxins. We have used a specific anti-PXR antibody in the Western blotting of mouse liver nuclear extracts to show that PXR is accumulated in the nucleus after treatment with 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN), followed by an increase in Cyp3a11 mRNA. Expression of wild type PXR and various mutants as green fluorescent fusion proteins in mouse livers showed that PXR was retained in the cytoplasm from where PCN treatment translocated PXR into the nucleus. Furthermore, the xenochemical response signal, the nuclear translocation signal, and the activation function 2 domain were all required for the nuclear translocation to occur. Immunoprecipitation experiments using the hsp90 antibody demonstrated the presence of PXR in a complex with the endogenous cytoplasmic constitutive active/androstane receptor retention protein (CCRP) in HepG2 cells. Fluorescence resonance energy transfer analysis of mouse liver sections after co-expression of cyan fluorescent protein-CCRP and yellow fluorescent protein-PXR also indicated that CCRP and PXR were closely associated in vivo. Overexpression of exogenous CCRP increased the cytoplasmic level of the PXR.CCRP.hsp90 complex, whereas a decrease in endogenous CCRP by treatment with small interfering RNA for CCRP repressed the PXR-mediated reporter activity in HepG2 cells. We conclude that the CCRP mediates the retention of PXR in the cytosol and modulates the activation of PXR in response to PCN treatment.
Squires
E James
EJ
Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Sueyoshi
Tatsuya
T
Negishi
Masahiko
M
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003593:Cytoplasm / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins; D006801:Humans; D047468:Immunoprecipitation; D008024:Ligands; D008099:Liver / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D008954:Models, Biological; D018832:Molecular Chaperones; D009154:Mutation; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis; D010957:Plasmids / Q000378:metabolism; D000077297:Pregnane X Receptor; D011285:Pregnenolone Carbonitrile / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis* / Q000378:metabolism*; D011987:Receptors, Steroid / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors; D014162:Transfection
D018076:DNA, Complementary; C478938:Dnajc7 protein, mouse; D006360:Heat-Shock Proteins; D008024:Ligands; D008565:Membrane Proteins; D018832:Molecular Chaperones; D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D014157:Transcription Factors; D011285:Pregnenolone Carbonitrile; D049452:Green Fluorescent Proteins; D001189:Aryl Hydrocarbon Hydroxylases; C496508:Cyp3a11 protein, mouse; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D005982:Glutathione Transferase