Coactivation of the N-terminal Transactivation of Mineralocorticoid Receptor by Ubc9
2006; Elsevier BV; Volume: 282; Issue: 3 Linguagem: Inglês
10.1074/jbc.m607741200
ISSN1083-351X
AutoresKenichi Yokota, Hirotaka Shibata, Isao Kurihara, Sakiko Kobayashi, Noriko Suda, Ayano Murai‐Takeda, Ikuo Saito, Hirochika Kitagawa, Shigeaki Kato, Takao Saruta, Hiroshi Itoh,
Tópico(s)Heat shock proteins research
ResumoMolecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood. Various transcription factors are post-translationally modified by small ubiquitin-related modifier-1 (SUMO-1). We investigated the role of the SUMO-1-conjugating enzyme Ubc9 in MR transactivation. Yeast two-hybrid, GST-pulldown, and coimmunoprecipitation assays showed that Ubc9 interacted with N-terminal MR-(1-670). Endogenous Ubc9 is associated with stably expressing MR in 293-MR cells. Transient transfection assays in COS-1 cells showed that Ubc9 increased MR transactivation of reporter constructs containing MRE, ENaC, or MMTV promoter in a hormone-sensitive manner. Moreover, reduction of Ubc9 protein levels by small interfering RNA attenuated hormonal activation of a reporter construct as well as an endogenous target gene by MR. A sumoylation-inactive mutant Ubc9(C93S) similarly interacted with MR and potentiated aldosterone-dependent MR transactivation. An MR mutant in which four lysine residues within sumoylation motifs were mutated into arginine (K89R/K399R/K494R/K953R) failed to be sumoylated, but Ubc9 similarly enhanced transactivation by the mutant MR, indicating that sumoylation activity is dispensable for coactivation capacity of Ubc9. Coexpression of Ubc9 and steroid receptor coactivator-1 (SRC-1) synergistically enhanced MR-mediated transactivation in transient transfection assays. Indeed, chromatin immunoprecipitation assays demonstrated that endogenous MR, Ubc9, and SRC-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner. Coimmunoprecipitation assays showed a complex of MR, Ubc9, and SRC-1 in mammalian cells, and the endogenous proteins were colocalized in the nuclei of the mouse collecting duct cells. These findings support a physiological role of Ubc9 as a transcriptional MR coactivator, beyond the known SUMO E2-conjugating enzyme. Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood. Various transcription factors are post-translationally modified by small ubiquitin-related modifier-1 (SUMO-1). We investigated the role of the SUMO-1-conjugating enzyme Ubc9 in MR transactivation. Yeast two-hybrid, GST-pulldown, and coimmunoprecipitation assays showed that Ubc9 interacted with N-terminal MR-(1-670). Endogenous Ubc9 is associated with stably expressing MR in 293-MR cells. Transient transfection assays in COS-1 cells showed that Ubc9 increased MR transactivation of reporter constructs containing MRE, ENaC, or MMTV promoter in a hormone-sensitive manner. Moreover, reduction of Ubc9 protein levels by small interfering RNA attenuated hormonal activation of a reporter construct as well as an endogenous target gene by MR. A sumoylation-inactive mutant Ubc9(C93S) similarly interacted with MR and potentiated aldosterone-dependent MR transactivation. An MR mutant in which four lysine residues within sumoylation motifs were mutated into arginine (K89R/K399R/K494R/K953R) failed to be sumoylated, but Ubc9 similarly enhanced transactivation by the mutant MR, indicating that sumoylation activity is dispensable for coactivation capacity of Ubc9. Coexpression of Ubc9 and steroid receptor coactivator-1 (SRC-1) synergistically enhanced MR-mediated transactivation in transient transfection assays. Indeed, chromatin immunoprecipitation assays demonstrated that endogenous MR, Ubc9, and SRC-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner. Coimmunoprecipitation assays showed a complex of MR, Ubc9, and SRC-1 in mammalian cells, and the endogenous proteins were colocalized in the nuclei of the mouse collecting duct cells. These findings support a physiological role of Ubc9 as a transcriptional MR coactivator, beyond the known SUMO E2-conjugating enzyme. The human mineralocorticoid receptor (MR, 2The abbreviations used are: MR, mineralocorticoid receptor; GST, glutathione S-transferase; SF-1, steroidogenic factor-1; SUMO, small ubiquitin-related modifier; ENaC, epithelial sodium channel; MMTV, mouse mammary tumor virus; SRC-1, steroid receptor coactivator-1; E1, SUMO-activating enzyme; E2, SUMO carrier protein; E3, SUMO-protein isopeptide ligase; IP, immunoprecipitation; Sgk, serum- and glucocorticoid-regulated kinase; p/CIP, p300/CBP cointegrator protein; CBP, cAMP-response element-binding protein-binding protein; EGFP, enhanced green fluorescent protein; DsRed, Discosoma sp. Red; YFP, yellow fluorescent protein; ChIP, chromatin immunoprecipitation; WB, Western blot; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA; HA, hemagglutinin. NR3C2), a ligand-dependent transcription factor that belongs to the nuclear receptor superfamily, mediates most of the known effects of aldosterone (1Arriza J.L. Weinberger C. Cerelli G. Glaser T.M. Handelin B.L. Housman D.E. Evans R.M. Science. 1987; 237: 268-275Crossref PubMed Scopus (1651) Google Scholar, 2Le Menuet D. Viengchareun S. Muffat-Joly M. Zennaro M.C. Lombes M. Mol. Cell. Endocrinol. 2004; 217: 127-136Crossref PubMed Scopus (36) Google Scholar). Besides its involvement in the regulation of electrolyte balance in epithelial cells, most notably in the distal collecting duct of the kidney and the colon, MR is also present in a variety of nonepithelial cells, such as cardiomyocytes and neurons (2Le Menuet D. Viengchareun S. Muffat-Joly M. Zennaro M.C. Lombes M. Mol. Cell. Endocrinol. 2004; 217: 127-136Crossref PubMed Scopus (36) Google Scholar, 3Funder J.W. Exp. Opin. Investig. Drugs. 2003; 12: 1963-1969Crossref PubMed Scopus (14) Google Scholar, 4Fiebeler A. Haller H. 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Antibodies—Goat anti-human MR (N-17) antibody, goat anti-human SRC-1 (M-341) antibody, and normal goat IgG were obtained from Santa Cruz Biotechnology. Mouse monoclonal anti-Xpress IgG was obtained from Invitrogen. Mouse anti-Ubc9 antibody was obtained from Pharmingen. Rabbit anti-HA antibody was obtained from Clontech. Mouse anti α-tubulin antibody was obtained from Oncogene. Cell Culture, Transfections, and Luciferase Assays—COS-1 cells, COS-7 cells, and HEK293 cells were routinely maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Twenty four hours before transfection, 5 × 104 cells per well of a 24-well dish were plated in the medium. All transfections were carried out by using Lipofectamine 2000 (Invitrogen) with 0.3 μg/well of the luciferase reporter, 0.01 μg/well of pRL-null internal control plasmids, and the indicated amounts of expression plasmids according to the manufacturer's instructions. After 18-24 h, the medium was changed to DMEM with 10% fetal bovine serum and 10-8 m aldosterone or vehicle. After an additional 24 h, cell extracts were assayed for both Firefly and Renilla luciferase activities with a dual-luciferase reporter assay system (Promega). Relative luciferase activity was determined as ratio of Firefly/Renilla luciferase activities, and data were expressed as the mean (±S.D.) of triplicate values obtained from a representative experiment that was independently repeated at least three times. Generation of Human MR Expressing Cell Line, 293-MR—Human 293F embryonic kidney cells and transformants were routinely maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone, UT). To establish stable transformants (293-MR cells), parent 293F cells were transfected with pNTAP-hMR (Stratagene) with Lipofectamine Plus reagents (Invitrogen) and cultured for 2 weeks in the presence of 700 μg/ml G418 for transformant selection as described previously (46Kitagawa H. Fujiki R. Yoshimura K. Mezaki Y. Uematsu Y. Matsui D. Ogawa S. Unno K. Okubo M. Tokita A. Nakagawa T. Ito T. Ishimi Y. Nagasawa H. Matsumoto T. Yanagisawa J. Kato S. Cell. 2003; 113: 905-917Abstract Full Text PDF PubMed Scopus (248) Google Scholar, 47Watanabe M. Yanagisawa J. Kitagawa H. Takeyama K. Ogawa S. Arao Y. Suzawa M. Kobayashi Y. Yano T. Yoshikawa H. Masuhiro Y. Kato S. EMBO J. 2001; 20: 1341-1352Crossref PubMed Scopus (246) Google Scholar, 48Yanagisawa J. Kitagawa H. Yanagida M. Wada O. Ogawa S. Nakagomi M. Oishi H. Yamamoto Y. Nagasawa H. McMahon S.B. Cole M.D. Tora L. Takahashi N. Kato S. Mol. Cell. 2002; 9: 553-562Abstract Full Text PDF PubMed Scopus (146) Google Scholar). Individual colonies were selected and expanded for further analysis. Plasmid Constructs—Several Ubc9 constructs, such as pcDNA3.1/His-Ubc9, pcDNA3.1/His-Ubc9(C93S), pGADT7-Ubc9, pGEX4T-1-Ubc9, pGEX4T-1-Ubc9(C93S), and pEGFP-Ubc9 were described previously (49Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar, 50Kurihara I. Shibata H. Kobayashi S. Suda N. Ikeda Y. Yokota K. Murai A. Saito I. Rainey W.E. Saruta T. J. Biol. Chem. 2005; 280: 6721-6730Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). pCMV-YFP-SRC-1 was a generous gift from Dr. Toshihiko Yanase (Kyushu University). 3xMRE-E1b-Luc and pCR3.1-SRC-1 were generous gifts from Dr. Bert W. O'Malley (Baylor College of Medicine, Houston). pGL3-MMTV (-1146/+88)-Luc was a generous gift from Dr. Jorma J. Palvimo (University of Helsinki, Finland). pRShMR was a generous gift from Dr. Ronald M. Evans (The Salk Institute for Biological Studies). pcDNA-HA-SUMO-1 was a generous gift from Dr. Ronald T. Hay (University of St. Andrews). pGL3-ENaC(-1388/+55)-Luc (51Mick V.E. Itani O.A. Loftus R.W. Husted R.F. Schmidt T.J. Thomas C.P. Mol. Endocrinol. 2001; 15: 575-588Crossref PubMed Scopus (107) Google Scholar) was a generous gift from Dr. Christie P. Thomas (University of Iowa College of Medicine). Several MR fragments, such as MR-(1-984), - (1-670), and -(671-984), were subcloned into pGBKT7, pcDNA3.1/His, and pDsRed vectors using a PCR amplification with primers containing oligonucleotide linkers of restriction enzyme sites. In detail, each MR fragment was first obtained by PCR amplification with primers containing oligonucleotide linkers of restriction enzyme sites (SmaI-SalI for pGBKT7, KpnI-XhoI for pcDNA3.1/His, and XhoI-XmaI for pDsRed), followed by TA cloning into pCRII-TOPO vector (Invitrogen). These pCR-TOPO-MR constructs were then digested with SmaI-SalI, KpnI-XhoI, or XhoI-XmaI and subcloned into the pGBKT7 yeast expression vector (Clontech), pcDNA3.1/His, or pDsRed2-C1 (Clontech) mammalian expression vector (Invitrogen). pRShMR-KRmut, in which the lysine residues positioned at 89, 399, 494, and 953 amino acids were substituted for arginine residues, was generated by the QuickChange site-directed mutagenesis kit (Stratagene). DNA sequencing of all the constructs was confirmed by ABI PRISM dye terminator cycle sequencing analysis (Amersham Biosciences). Yeast Two-hybrid Assay—Yeast two-hybrid assays were used to determine interaction of MR with Ubc9. Yeast Y187 cells were transformed with yeast expression plasmids encoding hMR and Ubc9. β-Galactosidase activity in liquid culture was determined with chlorophenol red β-d-galactopyranoside as described previously (49Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. 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Nawaz Z. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Endocrinol. 1997; 11: 714-724Crossref PubMed Scopus (149) Google Scholar). GST pulldown assay was performed as described (49Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar, 52Shibata H. Nawaz Z. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Endocrinol. 1997; 11: 714-724Crossref PubMed Scopus (149) Google Scholar), with modifications: 50 μl of glutathione-Sepharose beads 4B (Amersham Biosciences) stored in beads incubation buffer (50 mm potassium phosphate buffer (pH 7.4), 100 mm NaCl, 1 mm MgCl2, 10% glycerol, and 0.1% Tween 20) were incubated with bacterial extracts containing GST fusion proteins together with beads incubation buffer for 30-60 min at room temperature. Preparation of bacteria extracts containing GST fusion protein was as described previously (49Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar, 52Shibata H. Nawaz Z. Tsai S.Y. O'Malley B.W. Tsai M.J. Mol. Endocrinol. 1997; 11: 714-724Crossref PubMed Scopus (149) Google Scholar). The supernatant was then removed, and the beads were washed three times with beads incubation buffer. In vitro-translated 35S-labeled proteins were obtained by using TnT Coupled Reticulocyte Lysate Systems (Promega). Crude lysates were incubated with the beads in 200 μl of beads incubation buffer for 60 min at 4 °C with a circle rotator. Finally the beads were washed five times with 1 ml of beads incubation buffer, and the proteins were solubilized in SDS loading buffer and analyzed on SDS-PAGE (12.5% polyacrylamide gel). The input lanes contained 20% of the labeled protein used for binding. Western Blot Analysis and Coimmunoprecipitation—The cells were lysed with lysis buffer (10 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, 2 mm phenylmethylsulfonyl fluoride), and Western blots were performed before the immunoprecipitation (IP) steps to confirm protein expression by corresponding antibodies as described previously (49Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar, 50Kurihara I. Shibata H. Kobayashi S. Suda N. Ikeda Y. Yokota K. Murai A. Saito I. Rainey W.E. Saruta T. J. Biol. Chem. 2005; 280: 6721-6730Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The same samples for the Western blots were diluted to 1 ml in IP buffer (20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 10 mm dithiothreitol, 5 ng/μl aprotinin, 0.5 mm phenylmethylsulfonyl fluoride, 0.1% Tween 20) and precleared with protein G plus-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA), and antibodies were added for 1 h. Immune complexes were adsorbed to protein G plus-agarose beads and washed four times in IP buffer. Proteins were then separated on 12.5% polyacrylamide gels and transferred onto Hybond ECL nitrocellulose membranes (Amersham Biosciences). The primary antibodies used for immunoprecipitation were anti-MR (Santa Cruz Biotechnology) or anti-Xpress (Invitrogen) antibodies, and antibodies used for the Western blots were anti-MR, anti-Xpress, anti-HA (Oncogene), and anti-SRC-1 (Santa Cruz Biotechnology) antibodies. RNA Interference—COS-7 cells were transfected with siRNAs, and luciferase assays were performed as described previously (50Kurihara I. Shibata H. Kobayashi S. Suda N. Ikeda Y. Yokota K. Murai A. Saito I. Rainey W.E. Saruta T. J. Biol. Chem. 2005; 280: 6721-6730Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). COS-7 cells were plated into 24-well plates, grown until reaching 70-80% confluence, and transfected with 30 pmol of negative control sequence, Ubc9-specific siRNA duplex using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Whole cell extracts were prepared as described previously as follows: siRNA Ubc9#1 sense, 5′-GGC CAG CCA UCA CAA UCA ATT-3′; siRNA Ubc9#1 antisense, 5′-UUG AUU GUG AUG GCU GGC CTC-3′; siRNA Ubc9#2 sense, 5′-GGA ACU UCU AAA UGA ACC ATT-3′; siRNA Ubc9#2 antisense, 5′-UGG UUC AUU UAG AAG UUC CTG-3′; and Silencer Negative Control #1 siRNA (Ambion) were used. Chromatin Immunoprecipitation (ChIP) Assay—ChIP assay was performed as described previously (50Kurihara I. Shibata H. Kobayashi S. Suda N. Ikeda Y. Yokota K. Murai A. Saito I. Rainey W.E. Saruta T. J. Biol. Chem. 2005; 280: 6721-6730Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The cross-linked sheared chromatin solution was used for immunoprecipitation with 3 μg of anti-MR (N-17) antibody (Santa Cruz Biotechnology), anti-Ubc9 antibody (Pharmingen), or normal IgG. The immunoprecipitated DNAs were purified by phenol/chloroform extraction, precipitated by ethanol, and amplified by PCR using primers flanking the MRE region or control region as follows: ENaC MRE sense primer, 5′-TTC CTT TCC AGC GCT GGC CAC-3′ (-1567/-1547); ENaC MRE antisense primer, 5′-CCT CCA ACC TTG TCC AGA CCC-3′ (-1317-1297); ENaC control sense primer, 5′-ATG GGC ATG GCC AGG-3′ (+1/+15); ENaC control antisense primer, 5′-CCT GCT CCT CAC GCT-3′ (+251/+265). DNA samples with serial dilution were amplified by PCR to determine the linear range for the amplification (data not shown). Immunohistochemical Staining—Tissues were isolated from 2-month-old wild type male mice, fixed in 4% paraformaldehyde overnight at 4 °C, dehydrated in graded ethanol, and then processed for paraffin embedding. Sections (7 μm) were incubated for 2 h in blocking buffer, which is constituted by 1% bovine serum albumin, 5% normal donkey serum, 20 μg/ml donkey anti-mouse IgG Fab fragment (Jackson Immuno-Research, West Grove, PA), and 0.02% Triton X-100 in phosphate-buffered saline, and followed by incubation with the primary antibodies overnight at 4 °C. Primary antibodies used in this experiment were as follows: mouse anti-MR, rabbit anti-Ubc9 (Santa Cruz Biotechnology), and rabbit anti-SRC-1 (Santa Cruz Biotechnology), and the optimized dilutions were 1:50-1:100. After incubation with the combination of primary antibodies (MR and Ubc9, MR and SRC-1), slides were incubated with Alexa Fluor 488-conjugated donkey anti-mouse IgG and Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Molecular Probes, Eugene, OR) for 30 min at 4 °C. 4′,6-Diamino-2-phenylindole (DAPI) was used for nuclear staining. Fluorescence Imaging—The images of EGFP-tagged Ubc9 and YFP-tagged SRC-1 were described previously (49Kobayashi S. Shibata H. Kurihara I. Yokota K. Suda N. Saito I. Saruta T. J. Mol. Endocrinol. 2004; 32: 69-86Crossref PubMed Scopus (32) Google Scholar, 54Saitoh M. Takayanagi R. Goto K. Fukamizu A. Tomura A. Yanase T. Nawata H. Mol. Endocrinol. 2002; 16: 694-706Crossref PubMed Scopus (68) Google Scholar). HEK293 cells were transiently transfected with expression vectors of pDsRed-MR with pEGFP-Ubc9 or pYFP-SRC-1. Live cell microscopy of DsRed fusion, EGFP fusion, or YFP fusion proteins was performed on a confocal microscope (Axiovert 100 M, Carl Zeiss Co., Ltd.). Imaging for DsRed and EGFP or YFP was performed by excitation with 543 and 488 nm, respectively, from an argon laser, and the emissions were viewed through band passes ranging from 550 to 600 and 500-550 nm, respectively, by band pass regulation with LSM510 (Carl Zeiss Co., Ltd.). All images were processed as tagged image file format (TIFF) files on Photoshop 7.0 using standard imageprocessing techniques. Semiquantitative RT-PCR—The effect of endogenous Ubc9 on the endogenous Sgk, Ubc9, and GAPDH levels in the presence of 10-8 m aldosterone was investigated by semiquantitative RT-PCR. For semiquantitative RT-PCR, total RNA was extracted from 293-MR cells and reverse-transcribed as described previously. Procedures of RT-PCR were performed as described previously elsewhere. Preliminary experiments were conducted to ensure linearity for the semiquantitative procedures. Hot start PCR was performed by heat-activating AmpliTaq Gold DNA polymerase (PerkinElmer Life Sciences) at 94 °C for 4 min. Optimized cycling condition was 30 cycles (for Sgk) or 20 cycles (for Ubc9 and glyceraldehyde-3-phosphate dehydrogenase) for 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C. Oligonucleotide prime
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