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• W1981062818 abstract "Covalent modification of many transcription factors with SUMO-1 is emerging as a key role of trans-activational regulation. Here, we demonstrate that peroxisome proliferator-activated receptor (PPAR) γ, which is a ligand-activated nuclear receptor, is modified by SUMO-1. Sumoylation of PPARγ mainly occurs at a lysine residue within the activation function 1 domain. Furthermore, we show that the PIAS family proteins, PIAS1 and PIASxβ, function as E3 ligases (ubiquitin-protein isopeptide ligase) for PPARγ. PPARγ interacts directly with PIASxβ in a ligand-independent manner. Analysis using a PPARγ mutant with a disrupted sumoylation site shows that modification of PPARγ by SUMO-1 represses its transcriptional activity. Interestingly, PIASxβ and Ubc9 enhance the transcriptional activity of PPARγ independent of PPARγ sumoylation. Furthermore, PPARγ ligand-induced apoptosis in a human hepatoblastoma cell line, HepG2, is significantly enhanced by ectopic production of the sumoylation-mutant PPARγ. These results suggest that the PPARγ-dependent transactivation pathway seems to be modulated by SUMO-1 modification and may serve as a novel target for apoptosis-induction therapy in cancer cells. Covalent modification of many transcription factors with SUMO-1 is emerging as a key role of trans-activational regulation. Here, we demonstrate that peroxisome proliferator-activated receptor (PPAR) γ, which is a ligand-activated nuclear receptor, is modified by SUMO-1. Sumoylation of PPARγ mainly occurs at a lysine residue within the activation function 1 domain. Furthermore, we show that the PIAS family proteins, PIAS1 and PIASxβ, function as E3 ligases (ubiquitin-protein isopeptide ligase) for PPARγ. PPARγ interacts directly with PIASxβ in a ligand-independent manner. Analysis using a PPARγ mutant with a disrupted sumoylation site shows that modification of PPARγ by SUMO-1 represses its transcriptional activity. Interestingly, PIASxβ and Ubc9 enhance the transcriptional activity of PPARγ independent of PPARγ sumoylation. Furthermore, PPARγ ligand-induced apoptosis in a human hepatoblastoma cell line, HepG2, is significantly enhanced by ectopic production of the sumoylation-mutant PPARγ. These results suggest that the PPARγ-dependent transactivation pathway seems to be modulated by SUMO-1 modification and may serve as a novel target for apoptosis-induction therapy in cancer cells. PPARγ 1The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; GST, glutathione S-transferase; HA, hemagglutinin; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; SUMO, small ubiquitin-like modifier; STAT, signal transducers and activators of transcription; PIAS, protein inhibitors of activated STAT; AF, activation function; HEK cells, human embryonic kidney cells. is a member of the nuclear hormone receptor super-family of ligand-activated transcription factors (1Schoonjans K. Martin G. Staels B. Auwerx J. Curr. Opin. Lipidol. 1997; 8: 159-166Crossref PubMed Scopus (469) Google Scholar) which regulates diverse biological functions including cell differentiation, growth inhibition, lipid metabolism, and apoptosis (2Tsubouchi Y. Sano H. Kawahito Y. Mukai S. Yamada R. Kohno M. Inoue K. Hla T. Kondo M. Biochem. Biophys. Res. Commun. 2000; 270: 400-405Crossref PubMed Scopus (310) Google Scholar, 3Yang W.L. Frucht H. Carcinogenesis. 2001; 22: 1379-1383Crossref PubMed Scopus (191) Google Scholar, 4Nolan J.J. Ludvik B. Beerdsen P. Joyce M. Olefsky J. N. Engl. J. Med. 1994; 331: 1188-1193Crossref PubMed Scopus (922) Google Scholar, 5Tontonoz P. Singer S. Forman B.M. Sarraf P. Fletcher J.A. Fletcher C.D. Brun R.P. Mueller E. Altiok S. Oppenheim H. Evans R.M. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 237-241Crossref PubMed Scopus (619) Google Scholar). Two isoforms of PPARγ, PPARγ1 and PPARγ2, are generated by alternative promoter usage. PPARγ2, which contains an additional 30 amino acid residues at the amino terminus compared with PPARγ1, is predominantly expressed in adipose tissue, whereas PPARγ1 is widely expressed (6Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2005) Google Scholar). The role of PPARγ in adipogenesis has been extensively studied. Many adipocyte-specific genes, such as adipocytokines, contain PPARγ-responsible elements in their promoter and/or upstream enhancer regions (7Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1057) Google Scholar, 8Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11807) Google Scholar, 9Friedman J.M. Halaas J.L. Nature. 1998; 395: 763-770Crossref PubMed Scopus (4541) Google Scholar, 10Maeda K. Okubo K. Shimomura I. Funahashi T. Matsuzawa Y. Matsubara K. Biochem. Biophys. Res. Commun. 1996; 221: 286-289Crossref PubMed Scopus (1853) Google Scholar). PPARγ plays a role as a central transcription factor in cellular differentiation and lipid accumulation during adipogenesis. Recent investigations demonstrate that treatment of a variety of human cancer cell lines with PPARγ ligands leads to growth inhibition and apoptosis (2Tsubouchi Y. Sano H. Kawahito Y. Mukai S. Yamada R. Kohno M. Inoue K. Hla T. Kondo M. Biochem. Biophys. Res. Commun. 2000; 270: 400-405Crossref PubMed Scopus (310) Google Scholar, 11Eibl G. Wente M.N. Reber H.A. Hines O.J. Biochem. Biophys. Res. Commun. 2001; 287: 522-529Crossref PubMed Scopus (107) Google Scholar, 12Nagamine M. Okumura T. Tanno S. Sawamukai M. Motomura W. Takahashi N. Kohgo Y. Cancer Sci. 2003; 94: 338-343Crossref PubMed Scopus (63) Google Scholar, 13Li M.Y. Deng H. Zhao J.M. Dai D. Tan X.Y. World J. Gastroenterol. 2003; 9: 1220-1226Crossref PubMed Scopus (55) Google Scholar). The use of PPARγ ligands in the treatment of cancer is a potentially promising nontoxic and selective chemotherapeutic approach, and consequently, increased understanding of the mechanisms of PPARγ in tumor suppression is needed. Post-translational modifications regulate the function of many proteins. In the case of PPARγ, transcriptional activity is reduced by mitogen-activated protein kinase-induced phosphorylation of serine residue 112 (14Camp H.S. Tafuri S.R. J. Biol. Chem. 1997; 272: 10811-10816Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 15Adams M. Reginato M.J. Shao D. Lazar M.A. Chatterjee V.K. J. Biol. Chem. 1997; 272: 5128-5132Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 16Hu E. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; 274: 2100-2103Crossref PubMed Scopus (941) Google Scholar). Knock-in mice expressing PPARγ with a Ser → Ala mutation at this residue exhibit preserved insulin sensitivity in the setting of diet-induced obesity by changing fat cell size, generation of adiponectin, and increasing the amount of free fatty acid levels in serum (17Rangwala S.M. Rhoades B. Shapiro J.S. Rich A.S. Kim J.K. Shulman G.I. Kaestner K.H. Lazar M.A. Dev. Cell. 2003; 5: 657-663Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Recently, a number of ubiquitin-like proteins (Ubl) have been identified that are covalently linked to lysine residues in target proteins (18Muller S. Hoege C. Pyrowolakis G. Jentsch S. Nat. Rev. Mol. Cell Biol. 2001; 2: 202-210Crossref PubMed Scopus (652) Google Scholar, 19Ohsumi Y. Nat. Rev. Mol. Cell Biol. 2001; 2: 211-216Crossref PubMed Scopus (1052) Google Scholar). One Ubl, SUMO-1, also known as PIC1, UBL1, sentrin, GMP1, and SMT3, is an 11-kDa protein that is structurally homologous to ubiquitin (20Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar, 21Yeh E.T. Gong L. Kamitani T. Gene (Amst.). 2000; 248: 1-14Crossref PubMed Scopus (420) Google Scholar, 22Hay R.T. Trends Biochem. Sci. 2001; 26: 332-333Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). SUMO-1 modification plays an important role in altering the function of modified proteins, including transcriptional activation, nuclear localization, and increased turnover (23Pinsky B.A. Biggins S. Dev. Cell. 2002; 3: 4-6Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 24Seeler J.S. Dejean A. Nat. Rev. Mol. Cell Biol. 2003; 4: 690-699Crossref PubMed Scopus (581) Google Scholar, 25Muller S. Ledl A. Schmidt D. Oncogene. 2004; 23: 1998-2008Crossref PubMed Scopus (244) Google Scholar). SUMO-1 is conjugated to proteins through a series of enzymatic steps (26Melchior F. Schergaut M. Pichler A. Trends Biochem. Sci. 2003; 28: 612-618Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Initially, the ATP-dependent formation of a thioester bond between SUMO-1 and the E1 enzyme complex (SAE1·Uba2) is formed, and SUMO-1 is then transferred to the E2-conjugating enzyme Ubc9. Finally, SUMO-1 is conjugated from Ubc9 directly to a lysine residue of target proteins in vitro. The E3 ligase that conjugates SUMO-1 to target molecules in vitro and in vivo has only recently been identified (27Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar, 28Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8: 713-718Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 29Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar, 30Kagey M.H. Melhuish T.A. Wotton D. Cell. 2003; 113: 127-137Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). One group of such E3 ligases, protein inhibitor of activated STAT (PIAS) family proteins, homologous to the yeast Siz family protein, has a conserved RING finger domain that regulates transactivation of many transcription factors including STAT1 (31Rogers R.S. Horvath C.M. Matunis M.J. J. Biol. Chem. 2003; 278: 30091-30097Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 32Ungureanu D. Vanhatupa S. Kotaja N. Yang J. Aittomaki S. Janne O.A. Palvimo J.J. Silvennoinen O. Blood. 2003; 102: 3311-3313Crossref PubMed Scopus (123) Google Scholar), lymphoid enhancer factor-1 (33Sachdev S. Bruhn L. Sieber H. Pichler A. Melchior F. Grosschedl R. Genes Dev. 2001; 15: 3088-3103Crossref PubMed Scopus (466) Google Scholar), and nuclear receptors (34Kotaja N. Karvonen U. Janne O.A. Palvimo J.J. Mol. Cell. Biol. 2002; 22: 5222-5234Crossref PubMed Scopus (355) Google Scholar, 35Nishida T. Yasuda H. J. Biol. Chem. 2002; 277: 41311-41317Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) by conjugating SUMO-1. To understand the molecular mechanisms of PPARγ transcriptional function through post-translational modifications, we explored the possible modification of PPARγ by SUMO-1. In this paper we demonstrate that PPARγ is a target for SUMO-1 modification, and PIAS proteins function as E3 ligases for SUMO-1 modification. The main sumoylation site of PPARγ was mapped to a lysine residue at position 107, located in close proximity to the regulatory Ser-112. Sumoylation at this lysine residue reduced PPARγ-dependent transcriptional activation significantly. However, reporter gene assays suggested that PIAS proteins enhanced the transcriptional activity of PPARγ by a mechanism independent of PPARγ sumoylation. We also observed that a PPARγ sumoylation mutant displayed enhanced ligand-induced apoptosis in a human hepatoblastoma cell line, HepG2, which suggests a possible new target for cancer therapy. Cell Culture, Transfection, and Luciferase Reporter Assay—Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Transfection was performed using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Luciferase activity was normalized to Renilla luciferase activity derived from co-transfected pRL-CMV-Luc (Promega). All reporter assays were performed in triplicate, and S.E. are denoted by bars in the figures. Antibodies and Reagents—Rat anti-HA (3F10; Roche Applied Science), mouse and rabbit anti-FLAG (Sigma), and mouse anti-GFP (Clontech) antibodies were purchased commercially. Horseradish peroxidase-linked goat antibodies to rat IgG were from Jackson ImmunoResearch Laboratories. Horseradish peroxidase-linked goat antibodies to mouse or rabbit IgG were from Amersham Biosciences. Rosiglitazone was purchased from Alexis Biochemicals. Plasmid Construction—Plasmids producing GST fusion proteins and pcDNA3 (Invitrogen)-based plasmids expressing epitope-tagged human SUMO-1, human UBC9, and mouse PIAS families have been described previously (36Ohshima T. Shimotohno K. J. Biol. Chem. 2003; 278: 50833-50842Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). A luciferase reporter plasmid, p4xPPRE-Luc, was constructed by inserting four copies of the PPAR response element (PPRE) (5′-TTGACCTTTGACCTTTGACCTTTGACCTTAGATC-3′) into the luciferase reporter plasmid pGL2 (Promega). The mouse PPARγ2 gene containing the entire coding region was isolated by reverse transcription-PCR from 3T3-L1 cells and subcloned into epitope-tagged pcDNA3 to generate pcDNA3-FLAG-PPARγ2, -HA-PPARγ2. The cDNAs for mutant PPARγ2 with substitution of Lys-107 to Arg, PPARγ2(K/R1), and Lys-159 to Arg, PPARγ2(K/R2), were created using site-directed mutagenesis and subcloned into expression vectors to obtain pcDNA3-FLAG-PPARγ2(K/R1) and -PPARγ2(K/R2). The expression vector for PPARγ1 was obtained using PCR from PPARγ2 as a template. Expression plasmids of PPARγ-GAL4 DNA-binding fusion proteins were generated by inserting these genes into pM (Clontech). pSG5-mRXRα, encoding mouse RAR, was kindly provided by P. Chambon of the Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP/College de France, and S. Kato at the Institute of Molecular and Cellular Biosciences, The University of Tokyo. GST Pull-down Analysis—GST and GST fusion proteins were expressed in the Escherichia coli strain BL21 (DE3) and affinity-purified with glutathione-Sepharose beads according to the manufacturer's instructions (Amersham Biosciences). PPARγ2 protein was metabolically labeled in the TNT-coupled reticulocyte lysate system (Promega) with T7 RNA polymerase and [35S]methionine. GST pull-down analysis was carried out as described previously (37Ohshima T. Nakajima T. Oishi T. Imamoto N. Yoneda Y. Fukamizu A. Yagami K. Biochem. Biophys. Res. Commun. 1999; 264: 144-150Crossref PubMed Scopus (20) Google Scholar). Immunoprecipitations—HEK-293T cells (1 × 105 per 6-cm-diameter dish) were transfected using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. After incubation, cells were lysed in 1 ml of lysis buffer (25 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mm dithiothreitol, 5 mm EDTA, 10 mm N-ethylmaleimide, 200 μm indole-3-acetic acid, and a complete protease inhibitor mixture tablet (Roche Applied Science) for sumoylation analysis or radioimmune precipitation assay buffer (25 mm Tris-HCl, pH 8.0, 125 mm NaCl, 0.1% Nonidet P-40, 1 mm dithiothreitol, 1 mm EDTA, and a complete protease inhibitor mixture tablet) for co-immunoprecipitation analysis. Cell debris was removed by centrifugation for 15 min. Lysates were first cleared with protein G beads for 30 min followed by incubation with antibodies for 1 h at 4 °C. Finally, the antibody complexes were captured with protein G beads for 1 h. Beads were washed four times with the same buffer, and immunoprecipitates were eluted and analyzed by Western blot. Detection and Measurement of Apoptosis and Indirect Immunofluoresence Observation—HepG2 cells were grown to subconfluency on 8-well Lab-Tec Chamber (NUNC) in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Cells were transfected with 500 ng of pcDNA3-FLAG-PPARγ1 or -PPARγ1(K/R1) expression vectors. Forty-eight hours after transfection, rosiglitazone was added to culture medium to a final concentration at 1 μm for 24 h. Cells were fixed at room temperature with 3.7% formaldehyde for 3 min and then permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 5 min. After blocking with 3% bovine serum albumin and 0.1% Triton X-100 in phosphate-buffered saline, the cells were incubated with anti-HA antibody for 1 h at 37 °C and stained with Alexa Fluor 568 anti-rat secondary antibody (Molecular Probes) for 1 h at room temperature. Apoptotic cells were detected by using the in situ cell death detection kit, Fluorescein, following the manufacturer's instructions (Roche Applied Science). The ratio of apoptotic cells was quantitated by analysis of DNA fragmentation using the cell death detection enzyme-linked immunosorbent assay according to the manufacturer's instructions (Roche Applied Science). PPARγ Is a Substrate for SUMO-1 Modification—SUMO-1 modification of certain transcription factors including nuclear hormone receptors is known to affect transcriptional activity, and consequently, we wished to address whether PPARγ was a substrate for SUMO-1 modification. We first examined whether PPARγ is modified by SUMO-1 in cells transiently expressing FLAG-PPARγ2 and HA-SUMO-1. Western blot analysis using anti-FLAG antibody revealed the presence of FLAG-tagged PPARγ2 in all cells transfected with the plasmid expressing FLAG-PPARγ2. When HA-SUMO-1 was co-expressed, additional slower migrating bands were detected by the FLAG antibody (Fig. 1A, left panel, lane 3). Moreover, to determine whether these slower migrating bands represent PPARγ2 conjugated to SUMO-1, the membrane was re-probed with anti-HA antibody, which detects proteins conjugated to HA-SUMO-1. The result showed that the slower migrating forms of PPARγ2, about 90 and 130 kDa, were indeed sumoylated (Fig. 1A, right panel, lane 6). These data suggest that PPARγ is modified by SUMO-1 at least two sites. We next examined whether a specific PPARγ ligand, rosiglitazone, affected PPARγ sumoylation. As shown in Fig. 1B, SUMO-1-conjugated PPARγ2 was detected in cells co-producing SUMO-1. In lysates prepared from cells treated with rosiglitazone, the amount of SUMO-1-conjugated PPARγ2 was lower than in mock-treated cells (lane 2 and 4), suggesting that PPARγ ligand negatively regulates SUMO-1 conjugation to PPARγ. Two lysine residues, Lys-107 and Lys-347, in the AF1 and AF2 domains, respectively, of PPARγ2 conform to the proposed consensus motif ψKX(D/E) (where ψ is a hydrophobic amino acid residue, X represents any residue, and D or E is an acidic residue) for SUMO-1-conjugating sites (18Muller S. Hoege C. Pyrowolakis G. Jentsch S. Nat. Rev. Mol. Cell Biol. 2001; 2: 202-210Crossref PubMed Scopus (652) Google Scholar, 20Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar) (Fig. 1C). To determine whether these lysine residues are targets for sumoylation, mutants with lysine to arginine substitutions, K107R (K/R1) and K347R (K/R2) as shown in Fig. 1C, were generated and analyzed for sumoylation. Two bands migrating slower than the original band were detected with almost the same intensity as cells producing both wild type PPARγ1, PPARγ2, and the mutant PPARγ2(K/R2) (Fig. 1D). In contrast, the slowest band disappeared in cells producing the mutant PPARγ2(K/R1), and moreover, the amount of the slower migrating band was also reduced in the cells expressing the mutant PPARγ2(K/R1) (Fig. 1D, lane 2). These results imply that Lys-107 is the major site for sumoylation, and this site may function as the master switch of sumoylation because mutation of this lysine residue greatly impaired sumoylation for PPARγ. The fact that mutation of lysine residue at 347 did not affect the efficiency of sumoylation suggests the presence of lysine residues other than those not in the consensus motif for SUMO-1 modification in PPARγ. PIAS Family Proteins Act as E3 Ligases for PPARγ Sumoylation—Recent studies indicated that members of the PIAS family enhanced sumoylation of many proteins including nuclear receptors (25Muller S. Ledl A. Schmidt D. Oncogene. 2004; 23: 1998-2008Crossref PubMed Scopus (244) Google Scholar, 34Kotaja N. Karvonen U. Janne O.A. Palvimo J.J. Mol. Cell. Biol. 2002; 22: 5222-5234Crossref PubMed Scopus (355) Google Scholar, 35Nishida T. Yasuda H. J. Biol. Chem. 2002; 277: 41311-41317Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Therefore, we investigated whether PIAS family proteins function as E3 ligases for PPARγ. We generated a mutant of PIASxβ, PIASxβ(C/S), in which the conserved cysteine residue at position 353 within the RING finger domain was changed to serine. This mutant was not able to interact with Ubc9 (data not shown) and completely lacked E3 ligase function (36Ohshima T. Shimotohno K. J. Biol. Chem. 2003; 278: 50833-50842Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). SUMO-1 conjugation to PPARγ2 was analyzed in cells producing either wild-type PIASxβ, PIASxβ(C/S), or PIAS1. Small amounts of SUMO-1-conjugated PPARγ2 were detected in cells expressing only HA-SUMO-1 ectopically (Fig. 2, lane 2). PPARγ2 sumoylation was enhanced by exogenous expression of PIAS1 and PIASxβ but not PIASxβ(C/S) (lanes 3-5). These findings indicate that PIAS family proteins function as E3 ligases for PPARγ2. Next, to investigate the association of PPARγ2 with PIASxβ, we employed a GST pull-down analysis. Full-length PIASxβ expressed in bacteria as a GST fusion protein was coupled to glutathione S-Sepharose beads, and this complex was incubated with in vitro translated 35S-labeled PPARγ2 in buffer with or without rosiglitazone, a specific ligand for PPARγ. As shown Fig. 3A, PPARγ2 interacted both in the presence and absence of rosiglitazone with GST-PIASxβ but not with GST alone. To analyze the physical interaction of PPARγ2 with PIASxβ in cells, a co-immunoprecipitation experiment was conducted using extracts from HEK-293T cells co-expressing HAPPARγ2 and FLAG-PIASxβ treated with or without rosiglitazone. Proteins precipitated with anti-HA antibody were resolved by SDS-PAGE and Western blot using anti-FLAG was conducted. A FLAG-reactive species was detectable in the complex precipitated with the anti-HA antibody. The PIASxβ in the immunocomplex was increased in cells treated with rosiglitazone, indicating that the binding efficiency of PPARγ2 and PIASxβ was significantly enhanced by treatment with this ligand (Fig. 3B, lanes 2 and 3). SUMO-1 Conjugation to PPARγ Represses the Transcriptional Activity of PPARγ—To evaluate the effect of PPARγ sumoylation on its transcriptional function, we analyzed the effects of PPARγ sumoylation on expression of the p4xPPRELuc reporter gene in which the luciferase gene is driven by a PPAR-responsive promoter. NIH3T3 cells were transfected with various combinations of plasmids expressing wild-type PPARγ1, wild-type PPARγ2, PPARγ1(K/R1), PPARγ2(K/R2), and mRXRα, a component of a heterodimeric complex with PPARγ, together with p4xPPRE-Luc. Cells were then treated with or without rosiglitazone. Additional production of mRXRα in cells enhanced reporter activity by PPARγ. Reporter activity was enhanced by treatment with rosiglitazone, and this was highest in cell lysates containing PPARγ2(K/R1) (Fig. 4A). The fact that transcriptional activity of PPARγ2 was higher than that of PPARγ1 was in good agreement with a previous report (38Mueller E. Drori S. Aiyer A. Yie J. Sarraf P. Chen H. Hauser S. Rosen E.D. Ge K. Roeder R.G. Spiegelman B.M. J. Biol. Chem. 2002; 277: 41925-41930Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Next, to analyze the direct effect of the SUMO-1 conjugation-dependent transcriptional activity of PPARγ, we utilized PPARγ fused with GAL4 to analyze gene expression from pGL2-Luc containing five GAL4 binding sites in the promoter region. GAL4-PPARγ1(K/R1) and -PPARγ2(K/R1) showed about 5-fold higher luciferase activities than the activity observed by GAL4-wild-type-PPARγ1 and GAL4-wild-type-PPARγ2, respectively, in a ligand-dependent manner (Fig. 4B). Taken together, these data suggest that sumoylation of PPARγ represses the transcriptional activity of PPARγ itself. Both Ubc9 and PIASxβ Enhance PPARγ-dependent Transactivation—To further investigate the transcriptional role of sumoylated PPARγ, we examined the effects of Ubc9, an essential factor for sumoylation, on PPARγ-dependent transcription. GAL4-fused wild-type PPARγ2 and -PPARγ2(K/R1) were expressed in HEK-293T cells with increasing amounts of Ubc9 (Fig. 5A). Co-production of Ubc9 enhanced transcription by PPARγ2 and PPARγ2(K/R1) in a dose-dependent fashion. We next examined the effects of PIASxβ and PIASxβ(C/S) on the transcriptional activation of PPARγ2. Luciferase activities were significantly enhanced by the co-production of PIASxβ. However, co-production of PIASxβ(C/S) only slightly enhanced the activity (Fig. 5B). Similar results were also observed for PPARγ1 (data not shown). These data suggest that the SUMO-1 conjugation activity of Ubc9 and PIAS positively regulates PPARγ-mediated transactivation. The observation that the transcriptional activity of PPARγ2(K/R1) was not only significantly enhanced by co-production of Ubc9 but also by PIASxβ suggests that Ubc9 and PIAS proteins function as positive regulators for PPARγ-dependent transcription possibly through SUMO-1 conjugation of a factor(s) other than PPARγ involved in transcriptional regulation. Ligand-induced Apoptosis by PPARγ Is Enhanced in Cells Producing PPARγ(K/R1)—Recent studies have demonstrated that specific ligands for PPARγ inhibit cell growth and induce apoptosis in several human cancer cells (2Tsubouchi Y. Sano H. Kawahito Y. Mukai S. Yamada R. Kohno M. Inoue K. Hla T. Kondo M. Biochem. Biophys. Res. Commun. 2000; 270: 400-405Crossref PubMed Scopus (310) Google Scholar, 11Eibl G. Wente M.N. Reber H.A. Hines O.J. Biochem. Biophys. Res. Commun. 2001; 287: 522-529Crossref PubMed Scopus (107) Google Scholar, 12Nagamine M. Okumura T. Tanno S. Sawamukai M. Motomura W. Takahashi N. Kohgo Y. Cancer Sci. 2003; 94: 338-343Crossref PubMed Scopus (63) Google Scholar, 13Li M.Y. Deng H. Zhao J.M. Dai D. Tan X.Y. World J. Gastroenterol. 2003; 9: 1220-1226Crossref PubMed Scopus (55) Google Scholar). PPARγ activation seems to be important for inducing apoptosis in some cells. However, the molecular mechanisms of PPARγ-dependent apoptosis, particularly the relationship between the transcriptional activity of PPARγ and apoptosis, remain unclear. To investigate the effect of sumoylation on PPARγ-dependent apoptosis, we compared the apoptotic potential of wild-type PPARγ1 to that of PPARγ1(K/R1) in HepG2 cells. Plasmids expressing FLAG-PPARγ1 or -PPARγ1(K/R1) were transfected into HepG2 cells, and 48 h after transfection cells were treated with 1 μm rosiglitazone for 24 h. PPARγ expression in cells and apoptotic cells were detected by immunostaining and terminal dUTP nick-end labeling assay, respectively. Approximately 5% of PPARγ1-transduced cells became terminal dUTP nick-end label-positive, which stained strongly by the anti-FLAG antibody. In contrast, ∼40% of PPARγ1(K/R1)-transduced cells became terminal dUTP nick-end label-positive, and almost all cells expressed high levels of PPARγ1(K/R1) (Fig. 6A). The numbers of apoptotic cells producing PPARγ1 or PPARγ1(K/R1) were verified by measurement of the accumulation of fragmented nucleosomes. Ligand-induced apoptosis is significantly enhanced when PPARγ1(K/R1) was produced in cells (Fig. 6B). These results suggest that transcriptional activation of PPARγ is involved in enhancing ligand-mediated apoptosis. In this study we showed that sumoylation of PPARγ significantly affected its transcriptional activity. PPARγ was predominantly modified by SUMO-1 at Lys-107 within the AF1 domain. Our result suggest that there is a lysine residue(s) in addition to Lys-107 targeted for sumoylation that is likely to lie in a non-consensus SUMO-1 conjugation motif, because mutational analysis of the lysine residues lying in other consensus SUMO-1 conjugation motifs in this protein did not affect SUMO-1 conjugation (Fig. 1D). Because mutation of Lys-107 reduced SUMO-1 conjugation of PPARγ severely, Lys-107 is the primary site for modification. Similar observations of the presence of hierarchic lysine residues for SUMO-1 conjugation were reported in other proteins such as promyelocytic leukaemia protein (39Kamitani T. Kito K. Nguyen H.P. Wada H. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 26675-26682Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), androgen receptor (40Poukka H. Karvonen U. Janne O.A. Palvimo J.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14145-14150Crossref PubMed Scopus (371) Google Scholar), aryl hydrocarbon receptor (41Tojo M. Matsuzaki K. Minami T. Honda Y. Yasuda H. Chiba T. Saya H. Fujii-Kuriyama Y. Nakao M. J. Biol. Chem. 2002; 277: 46576-46585Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and DNA topoisomerase I (42Horie K. Tomida A. Sugimoto Y. Yasugi T. Yoshikawa H. Taketani Y. Tsuruo T. Oncogene. 2002; 21: 7913-7922Crossref PubMed Scopus (53) Google Scholar). PIAS1 and PIASxβ acted as E3 ligase factors for SUMO-1 conjugation to PPARγ (Fig. 2). We also showed that PIASxβ associated with PPARγ2 in vitro and in vivo in a ligand-independent manner, but the association was enhanced by the presence of the ligand in vivo (Fig. 3). Interestingly, ligand treatment led to a reduction in the amount of SUMO-1 conjugated to PPARγ2 (Fig. 1B). Because of conformational alteration of nuclear receptors, association of co-activator complexes with nuclear receptor seems to be regulated by specific ligands. Thus, it is likely that PPARγ sumoylation is suppressed by the association of the co-activator complex with the ligated PPARγ, in which the sumoylation sites of PPARγ may be masked, and/or the E3 ligase activity of PIAS proteins may be blocked. Using a reporter gene assay, we demonstrated that the transcriptional activity of PPARγ was negatively regulated by sumoylation. It has been reported that phosphorylation of Ser-112, adjacent to the sumoylation site, as revealed by this work, on PPARγ by mitogen-activated protein kinase significantly inhibited both ligand-independent and ligand-dependent transcriptional activation by PPARγ (15Adams M. Reginato M.J. Shao D. Lazar M.A. Chatterjee V.K. J. Biol. Chem. 1997; 272: 5128-5132Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). Mutation analysis of the phosphorylation site revealed that this phosphorylation-mediated transcriptional repression was not due to a reduced capacity to make PPARγ·RXRα complexes or the impairment of recognition of its DNA binding site (14Camp H.S. Tafuri S.R. J. Biol. Chem. 1997; 272: 10811-10816Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). An AF-1 domain of PPARγ may be negatively regulated by phosphorylation and sumoylation. Alternatively, SUMO-1-conjugated PPARγ may recruit the transcriptional repressor complex by providing a novel interaction site. Recently it has been shown that sumoylation of the ETS domain transcription factor, Elk-1, results in the recruitment of histone deacetylase activity to promoters (43Yang S.H. Sharrocks A.D. Mol. Cell. 2004; 13: 611-617Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Similarly, SUMO-1-conjugated PPARγ may recruit additional cellular factors that repress PPARγ-dependent transcription. Our data clearly showed that Ubc9 enhanced the transcriptional activities of both PPARγ and PPARγ(K/R1), possibly by a mechanism independent of SUMO-1 conjugation to PPARγ. PIASxβ also enhanced PPARγ activity through a RING finger domain-dependent mechanism. Thus, it seems that ectopically produced PIASxβ regulates PPARγ-mediated transactivation through not only sumoylation of PPARγ itself but also in the conjugation of SUMO-1 to another cellular factor(s) involved in transcriptional regulation. In agreement with our observations, key molecules in the SUMO-1 conjugation system, including SUMO-1, Ubc9, and PIAS, have been shown to modulate the transcriptional activities of p53 (44Schmidt D. Muller S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2872-2877Crossref PubMed Scopus (371) Google Scholar), androgen receptor (40Poukka H. Karvonen U. Janne O.A. Palvimo J.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14145-14150Crossref PubMed Scopus (371) Google Scholar), aryl hydrocarbon receptor (41Tojo M. Matsuzaki K. Minami T. Honda Y. Yasuda H. Chiba T. Saya H. Fujii-Kuriyama Y. Nakao M. J. Biol. Chem. 2002; 277: 46576-46585Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and lymphoid enhancer factor-1 (33Sachdev S. Bruhn L. Sieber H. Pichler A. Melchior F. Grosschedl R. Genes Dev. 2001; 15: 3088-3103Crossref PubMed Scopus (466) Google Scholar) even when these target molecules lacked a major sumoylation site(s) by mutation. Moreover, it has been shown that Ubc9 modulates the transcriptional activity of ETS-1- and TEL-independent of its E2 enzymatic activity (45Hahn S.L. Wasylyk B. Criqui-Filipe P. Criqui P. Oncogene. 1997; 15: 1489-1495Crossref PubMed Scopus (40) Google Scholar, 46Chakrabarti S.R. Sood R. Ganguly S. Bohlander S. Shen Z. Nucifora G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7467-7472Crossref PubMed Scopus (74) Google Scholar). In view of these reports, a mechanism(s) other than the direct SUMO-1 conjugation to PPARγ by Ubc9 and PIASxβ seem to be important for the regulation of transactivation of PPARγ. Further studies to clarify the molecular basis of the transcriptional activation of PPARγ-dependent transcription by Ubc9 and PIAS should provide significant insight. A role for PPARγ in adipogenesis is well characterized. In addition, a novel function of PPARγ in tumor pathogenesis has been reported recently, which includes PPARγ ligand-dependent growth inhibition and/or apoptosis in a variety of human cancer cells (2Tsubouchi Y. Sano H. Kawahito Y. Mukai S. Yamada R. Kohno M. Inoue K. Hla T. Kondo M. Biochem. Biophys. Res. Commun. 2000; 270: 400-405Crossref PubMed Scopus (310) Google Scholar, 11Eibl G. Wente M.N. Reber H.A. Hines O.J. Biochem. Biophys. Res. Commun. 2001; 287: 522-529Crossref PubMed Scopus (107) Google Scholar, 12Nagamine M. Okumura T. Tanno S. Sawamukai M. Motomura W. Takahashi N. Kohgo Y. Cancer Sci. 2003; 94: 338-343Crossref PubMed Scopus (63) Google Scholar, 13Li M.Y. Deng H. Zhao J.M. Dai D. Tan X.Y. World J. Gastroenterol. 2003; 9: 1220-1226Crossref PubMed Scopus (55) Google Scholar). Several studies have demonstrated that induction of apoptosis was accompanied by the up-regulation of several pro-apoptotic genes, Bax and caspase-3 and -9, and down-regulation of the anti-apoptotic gene Bcl-2 (47Michael M.S. Badr M.Z. Badawi A.F. Int. J. Mol. Med. 2003; 11: 733-736PubMed Google Scholar, 48Yoshizawa K. Cioca D.P. Kawa S. Tanaka E. Kiyosawa K. Cancer. 2002; 95: 2243-2251Crossref PubMed Scopus (69) Google Scholar), suggesting that transactivation of PPARγ is likely to regulate expression of apoptosis modulators at the transcriptional level and contribute as an important modulator of tumor suppression. In this study we demonstrated that the trans-activation function of PPARγ was up-regulated by mutation of Lys-107, the major target for sumoylation in PPARγ. BecauseHepG2 cells expressing this mutant form of PPARγ displayed enhanced PPARγ ligand-dependent apoptosis, the increased transactivation function of PPARγ seems to play an important role in inducing apoptosis. Sumoylation regulates the transacting function of PAPRγ, which could play a role in the regulation of apoptosis. We suggest here that the sumoylation of PPARγ may be a good target for a novel therapeutic agent in cancer cells." @default.
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• W1981062818 title "Transcriptional Activity of Peroxisome Proliferator-activated Receptor γ Is Modulated by SUMO-1 Modification" @default.
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