Cytoplasmic Localization of Pregnane X Receptor and Ligand-dependent Nuclear Translocation in Mouse Liver

Abstract

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-3β-ol-20-one-16α-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. 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-3β-ol-20-one-16α-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. The pregnane X receptor (PXR, 1The abbreviations used are: PXR, pregnane X receptor; hPXR, human PXR; mPXR, mouse PXR; AF2, activation function 2; CAR, constitutive active/androstane receptor; mCAR, mouse CAR; CCRP, cytoplasmic CAR retention protein; FRET, fluorescence resonance energy transfer; GR, glucocorticoid receptor; hsp90, heat shock protein 90; NLS, nuclear localization sequence; PB, phenobarbital; PCN, 5-pregnen-3β-ol-20-one-16α-carbonitrile; TPR, tetratricopeptide repeat; VDR, vitamin D receptor; XRS, xenochemical response sequence; Me2SO, dimethyl sulfoxide; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MOPS, 4-morpholinepropanesulfonic acid; GST, glutathione S-transferase; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; siRNA, small interfering RNA; ROI, region of interest. NR1I2) and constitutive active/androstane receptor (CAR, NR1I3) are involved in the primary response to xenobiotics and endogenous toxins (1Goodwin B. Redinbo M.R. Kliewer S.A. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 1-23Crossref PubMed Scopus (318) Google Scholar, 2Sueyoshi T. Negishi M. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 123-143Crossref PubMed Scopus (335) Google Scholar). These receptors respond to ligands by activating the expression of genes encoding enzymes involved in phase I (oxidation) and phase II (conjugation) metabolism as well as proteins involved in the efflux of toxins from the cell (3Honkakoski P. Sueyoshi T. Negishi M. Ann. Med. 2003; 35: 172-182Crossref PubMed Scopus (158) Google Scholar, 4Sonoda J. Rosenfeld J.M. Xu L. Evans R.M. Xie W. Curr. Drug Metab. 2003; 4: 59-72Crossref PubMed Scopus (84) Google Scholar, 5Swales K. Negishi M. Mol. Endcrinol. 2004; 18: 1589-1598Crossref PubMed Scopus (135) Google Scholar). CAR is normally sequestered in the cytoplasm of untreated liver cells and translocates to the nucleus after exposure to PB and PB-like chemicals (6Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (487) Google Scholar). The cytoplasmic CAR retention protein (CCRP and designated Dnajc7 in the official gene symbol in the NCBI data base) has been shown to maintain the cytoplasmic localization of CAR by forming a complex with CAR and hsp90 (7Kobayashi K. Sueyoshi T. Inoue K. Moore R. Negishi M. Mol. Pharmacol. 2003; 64: 1-7Crossref PubMed Scopus (154) Google Scholar, 8Yoshinari K. Kobayashi K. Moore R. Kawamoto T. Negishi M. FEBS Lett. 2003; 548: 17-20Crossref PubMed Scopus (142) Google Scholar). Other nuclear receptors, such as the glucocorticoid receptor (GR), vitamin D receptor (VDR), and aryl hydrocarbon receptor also exist as complexes with receptor-specific co-chaperones and hsp90 that play an important role in locating these receptors in the cytoplasm of unstimulated cells (9Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1537) Google Scholar). These co-chaperones, including immunophilin-FK506-binding proteins and hepatitis B virus protein X-associated protein 2, contain multiple tetratricopeptide repeat (TPR) motifs, which are 34 amino acid sequences that form a pair of anti-parallel α helices that mediate protein-protein interactions and the assembly of multiprotein complexes (10D'Andrea L.D. Regan L. Trends Biochem. Sci. 2003; 28: 655-662Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar). The movement of proteins between the nucleus and the cytoplasm is an energy-dependent process determined by both nuclear localization sequence (NLS) and nuclear export sequence on the protein (11Hood J.K. Silver P.A. Curr. Opin. Cell Biol. 1999; 11: 241-247Crossref PubMed Scopus (114) Google Scholar). The typical NLS consists of a single or repeat cluster of basic amino acids that associate with factors such as importins, which carry the proteins into the nucleus through a nuclear pore complex. The typical nuclear export sequence is a leucine-rich region and has been identified on a number of proteins, including the aryl hydrocarbon receptor (12Ikuta T. Eguchi H. Tachibana T. Yoneda Y. Kawajiri K. J. Biol. Chem. 1998; 273: 2895-2904Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 13Berg P. Pongratz I. J. Biol. Chem. 2001; 276: 43231-43238Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). After binding to ligand, the nuclear receptor GR moves along cytoskeletal tracks toward the nucleus by connecting with dynein motors (reviewed in Ref. 13Berg P. Pongratz I. J. Biol. Chem. 2001; 276: 43231-43238Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The C-terminal AF2 domain is required for ligand-dependent transcriptional activation by steroid hormone and many other nuclear receptors (14Pratt W.B. Galigniana M.D. Harrell J.M. DeFranco D.B. Cell. Signal. 2004; 16: 857-872Crossref PubMed Scopus (239) Google Scholar), and it is displaced upon the binding of ligand, allowing it to interact with co-activators. The removal of the AF2 domain prevents the nuclear translocation of the VDR and GR (15Glass C.K. Rose D.W. Rosenfeld M.G. Curr. Opin. Cell Biol. 1997; 9: 222-232Crossref PubMed Scopus (601) Google Scholar, 16Racz A. Barsony J. J. Biol. Chem. 1999; 274: 19352-19360Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). However, the AF2 domain is not required for the nuclear translocation of CAR following PB exposure (17Zelko I. Sueyoshi T. Kawamoto T. Moore R. Negishi M. Mol. Cell. Biol. 2001; 21: 2838-2846Crossref PubMed Scopus (142) Google Scholar). The nuclear translocation of CAR following drug exposure is dependent on the xenochemical response sequence (XRS), a leucine-rich sequence near the C terminus conserved as (L/M)XXLXXL (17Zelko I. Sueyoshi T. Kawamoto T. Moore R. Negishi M. Mol. Cell. Biol. 2001; 21: 2838-2846Crossref PubMed Scopus (142) Google Scholar) in mouse and human CAR and PXR. However, mutations in the key leucine residues in the XRS did not affect the formation of heterodimers between CAR and RXR or the co-activation of CAR by SRC-1. This suggests that the XRS regulates the translocation but not the activation of CAR. In contrast to CAR, PXR is generally thought to be primarily retained in the nucleus where it activates the expression of genes such as Cyp3a11 after binding ligands such as PCN. Thus, the possibility that co-chaperones could regulate the cellular localization of PXR has not been investigated. However, recent results of the immunostaining of mouse liver sections using a commercially available antibody suggest that mPXR may be located in the cytosol of untreated liver cells (18Kawana K. Ikuta T. Kobayashi Y. Gotoh O. Takeda K. Kawajiri K. Mol. Pharmacol. 2003; 63: 524-531Crossref PubMed Scopus (101) Google Scholar); however, additional supporting experiments, such as Western blot analysis, were not presented. Moreover, in transformed cells, human hPXR (SXR) is located primarily in the nucleus, and mutation of specific amino acids in the so-called NLS region of the DNA binding domain of hPXR resulted in cytoplasmic localization of the receptor in transformed cells (18Kawana K. Ikuta T. Kobayashi Y. Gotoh O. Takeda K. Kawajiri K. Mol. Pharmacol. 2003; 63: 524-531Crossref PubMed Scopus (101) Google Scholar). However, the role of the NLS as well as other specific motifs (AF2 and XRS) in the nuclear translocation of mPXR in the liver following drug treatment remains unexplored. Our goal was to use more definitive methods to determine whether mPXR was located in the cytoplasm of untreated liver and translocated to the nucleus only after exposure to xenochemicals. To achieve this, we produced specific antibodies against the hinge region of mPXR and used these antibodies in the Western blotting of liver nuclear extracts prepared from control mice and those treated with PCN. We also investigated the interaction of mPXR with CCRP in the cytosol to determine whether CCRP played a role in maintaining mPXR in the cytosol. We then looked at the NLS, XRS, and the AF2 which may be involved in the nuclear translocation of mPXR in response to drug to determine whether these also the of mPXR with we investigated the role of endogenous CCRP in the activation of mPXR in response to drug treatment. antibody was in using the hinge region to as the The antibody was from by using the to antibody and antibody were produced as (7Kobayashi K. Sueyoshi T. Inoue K. Moore R. Negishi M. Mol. Pharmacol. 2003; 64: 1-7Crossref PubMed Scopus (154) Google Scholar, P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar). antibody was from number control from and antibody was from B was from encoding mPXR, hPXR, mCAR, and CCRP were using the and into to and mPXR and CCRP were also using an and an and into or expression to an fusion with green fluorescent The reporter was by B. Mol. Pharmacol. 1999; PubMed Scopus Google Scholar). The following mutants were using the and the or the mutations or in the XRS region and or in the NLS were by and prepared using a Expression of PXR and CCRP in in of various and in mouse liver in and by was as (7Kobayashi K. Sueyoshi T. Inoue K. Moore R. Negishi M. Mol. Pharmacol. 2003; 64: 1-7Crossref PubMed Scopus (154) Google Scholar, T. Moore R. Negishi M. 2002; PubMed Scopus Google Scholar). were the of mice using the in gene to the the mice were with PCN, PB, or and sections were by using a analysis, were and after using of a between and were to of the and using by of was for multiple liver cells, and was with the where is fluorescence the and is that of after the The between the and was using an at which energy transfer is in of J. D.W. Scholar). and cells were in with and of and the cells were with using to the cells were and a the cells were with and with a in and as in K. Sueyoshi T. Inoue K. Moore R. Negishi M. Mol. Pharmacol. 2003; 64: 1-7Crossref PubMed Scopus (154) Google and R.M. 11: PubMed Scopus Google Scholar). The was at for to a nuclear and the was at for to a for use in The was in in and then in and The was at for in the presence of and for at to a nuclear of extracts were prepared on methods K. M. Cell. Full Text PDF PubMed Scopus Google Scholar, T. Kobayashi R. K. K. Moore R. T. H. Negishi M. Mol. Cell. Biol. PubMed Scopus Google Scholar). was in of 2 and using a on The was then a of the and at for at The was in of at for in the presence of and at for The was against and or as a was to 2 of protein from HepG2 cells and at of a of protein protein in was and the was for at The was by and with of and The proteins were from the with and to Western Western were on a in or a in and to using the transfer The was then for in a of and followed with primary antibody in the and with antibody the of proteins, the were with in a of and with The protein were on the using In the were with Western blot and with fusion protein was and using (7Kobayashi K. Sueyoshi T. Inoue K. Moore R. Negishi M. Mol. Pharmacol. 2003; 64: 1-7Crossref PubMed Scopus (154) Google Scholar). mPXR and were produced from and using the along with or to the was with the mPXR or in and for at The was then by and in the were from the by for at in and on a in The was then with and and the proteins by and cells were in with and of and to The cells were with or the and as a control using to the RNA interfering experiments, HepG2 cells were with against CCRP from or an using to the After of the were treated with PCN or and after a the activity of the reporter and the control was using the reporter The of reporter activity to control was for liver were in and RNA was prepared to the of RNA was used as a for using the with as of the was used for with an sequence using and for mouse or as a mPXR in the of after Drug determine the cellular localization of endogenous mPXR in mouse liver in Western blotting of liver nuclear extracts was using an antibody against the hinge region of mPXR of mPXR were in nuclear extracts from untreated mice or mice treated with and increased following PCN treatment but not after PB treatment. the blot was and then using antibody against mouse CAR, of CAR were in nuclear extracts from with of PCN treatment. The results from the Western blot analysis have that mPXR in the nucleus of liver cells following PCN treatment. This nuclear of mPXR in a were from untreated mice at various to after treatment with PCN and nuclear were prepared and used for Western blotting using The results show that of mPXR in the nucleus increased after treatment with PCN and were at the of after treatment. of mPXR in the nuclear extracts at after PCN treatment and by the To the for gene activation with the of mPXR in the nucleus after treatment with PCN, of were in the livers by of increased to a at the after PCN treatment and untreated control at after treatment. This that activation of the gene after mPXR in the nucleus in response to PCN treatment. Thus, mPXR moves into the nucleus in response to PCN activates of and then is from the nucleus. of mPXR investigated the cellular localization of mPXR by in of mPXR in mouse In untreated was the cytoplasm of the liver cell However, in liver sections from mice that been treated with PCN, was in the nucleus. This cytoplasmic retention and nuclear translocation of are of with the endogenous To the of mPXR that affect nuclear translocation in we mPXR and various mutants as fusion proteins in mouse These mPXR mutants in the XRS region or the NLS region in the DNA binding domain or the C-terminal AF2 region We then the of cells with mPXR in the the with nuclear and cytoplasmic and the with primarily nuclear localization of mPXR type was located primarily in the cytoplasm in untreated mouse liver and translocated to the nucleus after treatment with PCN. However, mutations in key amino acid residues in the XRS or NLS region or of the AF2 domain nuclear translocation in in response to treatment with PCN. The of the NLS and XRS mutants whereas the AF2 more nuclear which was not by PCN treatment. These results suggest that the XRS, NLS, and AF2 are all required for the nuclear translocation of mPXR after PCN treatment in vivo. of CCRP in of is involved in maintaining the cytoplasmic localization of CAR (7Kobayashi K. Sueyoshi T. Inoue K. Moore R. Negishi M. Mol. Pharmacol. 2003; 64: 1-7Crossref PubMed Scopus (154) Google Scholar). We that CCRP could also to mPXR and was involved in the retention of mPXR in the a we used a to that to mPXR as well as binding to CAR we and in HepG2 cells and that a complex in the cytosol with hsp90 that be by antibody Furthermore, the of CCRP to mPXR used in the HepG2 cells is the retention of mPXR in the cytosol is also We then did a analysis of mouse liver sections after co-expression of and The results indicated that CCRP and mPXR are closely associated in to to occur. of YFP, at produced a from to increase the from After of YFP, did not and the The and was and after in of the cell After the with a increase in the of of in cells from to which that and were an of of these results demonstrated that mPXR a complex with CCRP that may the retention of mPXR in the cytoplasm of liver of mPXR and CCRP and were the using the in gene and liver sections were prepared for and protein and after of the of YFP, from the from and the from After the of YFP, not that from is The of the in the of of and from of of a cell 2, and and the cell The are and fluorescent by the in after the and that mPXR and CCRP could form a complex with hsp90, we and various mutants in HepG2 cells along with to whether the motifs XRS, and were involved in the formation of the complex. Immunoprecipitation of with hsp90 antibody demonstrated that the various mPXR mutants all form a complex with CCRP and hsp90 in the cytosol with Thus, the of binding of mPXR mutants to CCRP in the complex is not with the of the mPXR that translocates to the nucleus after PCN as shown in Thus, the retention of mPXR in the cytosol of HepG2 cells not the of nuclear translocation of mPXR in liver in vivo. of CCRP in the of HepG2 cells with and and the complex with the Western blot was with the endogenous CCRP that was along with the could be that the endogenous CCRP also a complex with endogenous hsp90 and the mPXR in HepG2 cells Immunoprecipitation experiments using antibody also that hsp90 was along with and not We then to determine whether CCRP the activation of PXR in response to drug treatment. for CCRP was in HepG2 cells, a decrease in endogenous CCRP protein was by Western blotting whereas was on of mPXR in the nucleus. The decrease in CCRP expression by treatment resulted in an response in a reporter for mouse PXR with a more response with PCN with PCN. In the response of human PXR to activation by was also by treatment with for This demonstrated that CCRP plays a role in the activation of both mouse and human following treatment with We produced an antibody against the hinge region of mPXR and used antibody in Western to show that the of mPXR increase in the nucleus of mouse liver cells after PCN followed by activation of a and then of mPXR decrease in the nucleus. Expression of in mouse liver in that mPXR is in the cytoplasm of untreated liver cells and is translocated in the nucleus after PCN treatment. These data that nuclear translocation of mPXR as with other nuclear receptors such as CAR, GR, and VDR, as a primary in the activation of PXR into transformed cells normally in the nucleus drug treatment. and (18Kawana K. Ikuta T. Kobayashi Y. Gotoh O. Takeda K. Kawajiri K. Mol. Pharmacol. 2003; 63: 524-531Crossref PubMed Scopus (101) Google Scholar) identified a NLS region in hPXR (SXR) resulted in of the receptor in the cytosol of transformed cells. that the NLS is in the translocation of human PXR in cells. also that hPXR with the AF2 domain was accumulated in the nucleus, that the AF2 domain was not for nuclear The results of experiments, in which various mutants of mPXR were in mouse liver in that the nuclear translocation not only the NLS but also the AF2 In to the NLS and the AF2 mPXR the XRS that is to regulate the nuclear translocation of both mouse and human CAR in mouse liver in (17Zelko I. Sueyoshi T. Kawamoto T. Moore R. Negishi M. Mol. Cell. Biol. 2001; 21: 2838-2846Crossref PubMed Scopus (142) Google Scholar). Our experiments that the XRS is also required for the nuclear translocation of mPXR to in response to PCN treatment. The between the of and (18Kawana K. Ikuta T. Kobayashi Y. Gotoh O. Takeda K. Kawajiri K. Mol. Pharmacol. 2003; 63: 524-531Crossref PubMed Scopus (101) Google Scholar) and the may be to between hPXR and mPXR, cell between the of of PXR in the nucleus and nuclear translocation of PXR in response to drug treatment. all of the NLS, XRS, and AF2 are required for nuclear translocation of mPXR in may all function in the of nuclear translocation of In contrast to mPXR, not the NLS or the AF2 domain for nuclear only the binding domain the XRS CAR AF2 domain or the C-terminal of the domain translocated to the nucleus in mouse liver in following treatment with CAR (17Zelko I. Sueyoshi T. Kawamoto T. Moore R. Negishi M. Mol. Cell. Biol. 2001; 21: 2838-2846Crossref PubMed Scopus (142) Google Scholar, T. Moore R. Negishi M. 2002; PubMed Scopus Google Scholar). The NLS region of CAR may not be for nuclear translocation of the of a conserved by in and mouse CAR with PXR and VDR (18Kawana K. Ikuta T. Kobayashi Y. Gotoh O. Takeda K. Kawajiri K. Mol. Pharmacol. 2003; 63: 524-531Crossref PubMed Scopus (101) Google Scholar). In a hPXR the NLS of nuclear translocation as with Sueyoshi and M. The AF2 domain regulates receptor function and is by the binding of The binding of GR and VDR to into the nucleus A. Barsony J. J. Biol. Chem. 1999; 274: 19352-19360Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In the of nuclear translocation of CAR may be the CAR PB not to CAR, and the AF2 domain is not required for translocation of CAR. PCN to mPXR and the translocation the AF2 the for nuclear translocation may be to that of GR and CCRP mouse of human A. J. DNA Cell Biol. PubMed Scopus Google a of the which proteins that are involved in the and protein was identified as a CAR binding protein by of a mouse liver using CAR as CCRP, as a mediates the formation of the complex in the cytosol of HepG2 cells (7Kobayashi K. Sueyoshi T. Inoue K. Moore R. Negishi M. Mol. Pharmacol. 2003; 64: 1-7Crossref PubMed Scopus (154) Google Scholar). We have shown that mPXR also to the CCRP to form a complex the cytosol along with hsp90, the retention of mPXR in the cytosol of HepG2 cells. In analysis that CCRP and mPXR are closely associated in mouse liver in vivo. CCRP has also been to to the GR and is required at a expression for activation of the GR A. T. J. 2003; PubMed Scopus Google Scholar). to the GR we have that the of genes is by CCRP in HepG2 cells. These suggest that CCRP may play a role not only in the cytoplasmic retention of the receptors, but also in activation in the nucleus. The role of hsp90 and proteins in the formation of complexes with proteins, such as steroid receptors, and movement to the nucleus has been (14Pratt W.B. Galigniana M.D. Harrell J.M. DeFranco D.B. Cell. Signal. 2004; 16: 857-872Crossref PubMed Scopus (239) Google Scholar). The GR a complex with hsp90 and which are proteins that the complex to dynein proteins for movement along to the nucleus. However, the by which CCRP regulates the retention and activation of these receptors remains an for In we have shown that mPXR is located in the cytoplasm of untreated liver cells and is in the nucleus following drug treatment. mPXR a complex with CCRP and hsp90 that the receptor in the cytosol. The formation of the complex is not dependent on the XRS, NLS, or AF2 in However, these are all required for the nuclear translocation of Thus, CCRP is involved in maintaining mPXR in the the binding of mPXR to CCRP not regulate the nuclear translocation of mPXR in response to PCN treatment. However, CCRP the activation of mPXR in response to drug treatment. This of the cytoplasmic localization of mPXR in untreated liver and the role of CCRP on the of nuclear localization and activation of PXR following drug treatment.