Block of Nuclear Receptor Ubiquitination
2007; Elsevier BV; Volume: 282; Issue: 16 Linguagem: Inglês
10.1074/jbc.m609149200
ISSN1083-351X
AutoresDavide Genini, Carlo V. Catapano,
Tópico(s)NF-κB Signaling Pathways
ResumoPeroxisome proliferator-activated receptor δ (PPARδ) is a ligand-activated transcription factor involved in many physiological and pathological processes. PPARδ is a promising therapeutic target for metabolic, chronic inflammatory, and neurodegenerative disorders. However, limited information is available about the mechanisms that control the activity of this nuclear receptor. Here, we examined the role of the ubiquitinproteasome system in PPARδ turnover. The receptor was ubiquitinated and subject to rapid degradation by the 26 S proteasome. Unlike most nuclear receptors that are degraded upon ligand binding, PPARδ ligands inhibited the ubiquitination of the receptor, thereby preventing its degradation. Ligand binding was required for inhibition of the ubiquitination since disruption of the ligand binding domain abolished the effect. Site-directed mutagenesis showed that the DNA binding domain was also required, indicating that ligands preferentially stabilized the DNA-bound receptor. In contrast, the activation function-2 domain and co-repressor binding site were not involved in ligand-induced stabilization. Block of ubiquitination by ligands may be an essential step to avoid rapid degradation of a receptor, like PPARδ, with a very short half-life and sustain its transcriptional activity once it is engaged in transcriptional activation complexes. Peroxisome proliferator-activated receptor δ (PPARδ) is a ligand-activated transcription factor involved in many physiological and pathological processes. PPARδ is a promising therapeutic target for metabolic, chronic inflammatory, and neurodegenerative disorders. However, limited information is available about the mechanisms that control the activity of this nuclear receptor. Here, we examined the role of the ubiquitinproteasome system in PPARδ turnover. The receptor was ubiquitinated and subject to rapid degradation by the 26 S proteasome. Unlike most nuclear receptors that are degraded upon ligand binding, PPARδ ligands inhibited the ubiquitination of the receptor, thereby preventing its degradation. Ligand binding was required for inhibition of the ubiquitination since disruption of the ligand binding domain abolished the effect. Site-directed mutagenesis showed that the DNA binding domain was also required, indicating that ligands preferentially stabilized the DNA-bound receptor. In contrast, the activation function-2 domain and co-repressor binding site were not involved in ligand-induced stabilization. Block of ubiquitination by ligands may be an essential step to avoid rapid degradation of a receptor, like PPARδ, with a very short half-life and sustain its transcriptional activity once it is engaged in transcriptional activation complexes. Peroxisome proliferator-activated receptors (PPARs) 2The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-activated receptor response elements; ADRP, adipose differentiation-related protein; AF, activation function; CMV, cytomegalovirus; Ub, ubiquitin; UPS, ubiquitin-proteasome system; HA, hemagglutinin; RT, reverse transcription. belong to the superfamily of nuclear hormone receptors and act as ligand-activated transcription factors (1Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2744) Google Scholar). PPARs are activated by a diverse group of lipophilic compounds, including long-chain fatty acids, prostaglandins, and leukotrienes (2Chawla A. Repa J.J. Evans R.M. Mangelsdorf D.J. Science. 2001; 294: 1866-1870Crossref PubMed Scopus (1696) Google Scholar). PPARs form heterodimers with the retinoic X receptor and bind DNA in correspondence to specific PPAR response elements (PPRE) located in the promoter of target genes (1Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2744) Google Scholar). Unliganded receptors maintain the promoter in a repressive or inactive state (3Glass C.K. Ogawa S. Nat. Rev. Immunol. 2006; 6: 44-55Crossref PubMed Scopus (364) Google Scholar). Ligand binding induces a conformational remodeling of the receptor, resulting in the release of co-repressor molecules and recruitment of co-activators necessary for transcriptional activation (3Glass C.K. Ogawa S. Nat. Rev. Immunol. 2006; 6: 44-55Crossref PubMed Scopus (364) Google Scholar). The PPAR subfamily includes three isotypes, named α, δ (or β), and γ, that share extensive structural homology (1Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2744) Google Scholar). Although all three isotypes act as lipid sensors and are involved in various aspects of lipid metabolism, they have distinct tissue distribution, ligand specificity, and functions (2Chawla A. Repa J.J. Evans R.M. Mangelsdorf D.J. Science. 2001; 294: 1866-1870Crossref PubMed Scopus (1696) Google Scholar, 4Kersten S. Desvergne B. Wahli W. Nature. 2000; 405: 421-424Crossref PubMed Scopus (1678) Google Scholar). PPARα is involved in fatty acid metabolism, and high affinity ligands of this receptor, like fenofibrate and bezafibrate, are effective hypolipidemic drugs (5Lefebvre P. Chinetti G. Fruchart J.C. Staels B. J. Clin. Investig. 2006; 116: 571-580Crossref PubMed Scopus (754) Google Scholar). PPARγ prevalently controls lipid and glucose metabolism, and PPARγ agonists, like rosiglitazone and pioglitazone, are widely used anti-diabetic drugs (6Semple R.K. Chatterjee V.K. O'Rahilly S. J. Clin. Investig. 2006; 116: 581-589Crossref PubMed Scopus (681) Google Scholar). PPARδ, which is the less studied of the three isotypes, has been implicated in wound healing, inflammatory responses, and embryo implantation in addition to lipid metabolism (4Kersten S. Desvergne B. Wahli W. Nature. 2000; 405: 421-424Crossref PubMed Scopus (1678) Google Scholar, 7Barish G.D. Narkar V.A. Evans R.M. J. Clin. Investig. 2006; 116: 590-597Crossref PubMed Scopus (569) Google Scholar). PPARδ is a potential therapeutic target for diseases such as atherosclerosis and other inflammatory, metabolic, and neurodegenerative disorders (7Barish G.D. Narkar V.A. Evans R.M. J. Clin. Investig. 2006; 116: 590-597Crossref PubMed Scopus (569) Google Scholar). PPARδ has also been associated with cancer (8Michalik L. Desvergne B. Wahli W. Nat. Rev. Cancer. 2004; 4: 61-70Crossref PubMed Scopus (513) Google Scholar). PPARδ is overexpressed in colon, endometrial, and head and neck cancers (9He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1036) Google Scholar, 10Gupta R.A. Tan J. Krause W.F. Geraci M.W. Willson T.M. Dey S.K. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13275-13280Crossref PubMed Scopus (359) Google Scholar, 11Tong B.J. Tan J. Tajeda L. Das S.K. Chapman J.A. DuBois R.N. Dey S.K. Neoplasia. 2000; 2: 483-490Crossref PubMed Google Scholar, 12Jaeckel E.C. Raja S. Tan J. Das S.K. Dey S.K. Girod D.A. Tsue T.T. Sanford T.R. Arch. Otolaryngol Head Neck Surg. 2001; 127: 1253-1259Crossref PubMed Scopus (81) Google Scholar). PPARδ agonists stimulate proliferation and survival of cancer cells in vitro (13Cutler N.S. Graves-Deal R. LaFleur B.J. Gao Z. Boman B.M. Whitehead R.H. Terry E. Morrow J.D. Coffey R.J. Cancer Res. 2003; 63: 1748-1751PubMed Google Scholar, 14Shureiqi I. Jiang W. Zuo X. Wu Y. Stimmel J.B. Leesnitzer L.M. Morris J.S. Fan H.Z. Fischer S.M. Lippman S.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9968-9973Crossref PubMed Scopus (204) Google Scholar, 15Glinghammar B. Skogsberg J. Hamsten A. Ehrenborg E. Biochem. Biophys. Res. Commun. 2003; 308: 361-368Crossref PubMed Scopus (78) Google Scholar, 16Stephen R.L. Gustafsson M.C. Jarvis M. Tatoud R. Marshall B.R. Knight D. Ehrenborg E. Harris A.L. Wolf C.R. Palmer C.N. Cancer Res. 2004; 64: 3162-3170Crossref PubMed Scopus (153) Google Scholar) and promote tumor growth in mice (17Gupta R.A. Wang D. Katkuri S. Wang H. Dey S.K. DuBois R.N. Nat. Med. 2004; 10: 245-247Crossref PubMed Scopus (255) Google Scholar, 18Yin Y. Russell R.G. Dettin L.E. Bai R. Wei Z.L. Kozikowski A.P. Kopleovich L. Glazer R.I. Cancer Res. 2005; 65: 3950-3957Crossref PubMed Scopus (88) Google Scholar, 19Wang D. Wang H. Shi Q. Katkuri S. Walhi W. Desvergne B. Das S.K. Dey S.K. DuBois R.N. Cancer Cell. 2004; 6: 285-295Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Consistent with a tumor promoting role, PPARδ has been shown to increase the expression of anti-apoptotic genes, like ILK (integrin-linked kinase) and PDK1 (3-phosphoinositide-dependent kinase-1), and to activate the pro-survival Akt signaling pathway in keratinocytes (20Di-Poi N. Tan N.S. Michalik L. Wahli W. Desvergne B. Mol. Cell. 2002; 10: 721-733Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). However, the involvement of this nuclear receptor in carcinogenesis is still controversial. Somatic knockdown of PPARδ decreased growth of human colon cancer xenografts in mice (21Park B.H. Vogelstein B. Kinzler K.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2598-2603Crossref PubMed Scopus (225) Google Scholar), whereas in other studies the frequency of colon polyps was not affected or even increased in PPARδ knock-out mice (22Harman F.S. Nicol C.J. Marin H.E. Ward J.M. Gonzalez F.J. Peters J.M. Nat. Med. 2004; 10: 481-483Crossref PubMed Scopus (189) Google Scholar, 23Reed K.R. Sansom O.J. Hayes A.J. Gescher A.J. Winton D.J. Peters J.M. Clarke A.R. Oncogene. 2004; 23: 8992-8996Crossref PubMed Scopus (104) Google Scholar) and upon treatment with PPARδ ligands (24Marin H.E. Peraza M.A. Billin A.N. Willson T.M. Ward J.M. Kennett M.J. Gonzalez F.J. Peters J.M. Cancer Res. 2006; 66: 4394-4401Crossref PubMed Scopus (119) Google Scholar). Thus, this nuclear receptor is involved in many critical cell functions, including proliferation, survival, and differentiation. However, the mechanisms mediating its effects and the outcome in different cells and tissues remain unclear. Although much is known about the mechanisms that regulate the activity of most nuclear receptors, very limited information is available regarding PPARδ. At the transcriptional level, PPARδ was shown to be a downstream target of the oncogenic Wnt/APC/β-catenin pathway, showing for the first time a link with colon carcinogenesis (9He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1036) Google Scholar). Activated oncogenic Ras stimulated PPARδ expression in intestinal cells (25Shao J. Sheng H. DuBois R.N. Cancer Res. 2002; 62: 3282-3288PubMed Google Scholar), whereas activator protein-1 (26Tan N.S. Michalik L. Noy N. Yasmin R. Pacot C. Heim M. Fluhmann B. Desvergne B. Wahli W. Genes Dev. 2001; 15: 3263-3277Crossref PubMed Scopus (373) Google Scholar) and C/EBPα (CCAAT/enhancer-binding protein) (27Di-Poi N. Desvergne B. Michalik L. Wahli W. J. Biol. Chem. 2005; 280: 38700-38710Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) controlled its expression in keratinocytes. Activity and expression of PPARδ were inhibited by nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors in colon cancer cells, relating this nuclear receptor to the chemopreventive effects of this class of anti-inflammatory compounds (9He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1036) Google Scholar, 14Shureiqi I. Jiang W. Zuo X. Wu Y. Stimmel J.B. Leesnitzer L.M. Morris J.S. Fan H.Z. Fischer S.M. Lippman S.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9968-9973Crossref PubMed Scopus (204) Google Scholar). Less is known about post-transcriptional mechanisms that control PPARδ protein level and activity. A better understanding of the multiple mechanisms that modulate ligand-dependent and -independent activity might provide hints into the involvement of PPARδ in human diseases and indicate ways to exploit its therapeutic potential. In this study we examined the role of the ubiquitin-proteasome system (UPS) in basal and ligand-dependent turnover of PPARδ. The UPS is the major cellular machinery responsible for degradation of proteins and determines the level of critical regulatory factors, including nuclear receptors and transcription factors (28Ciechanover A. Orian A. Schwartz A.L. BioEssays. 2000; 22: 442-451Crossref PubMed Scopus (706) Google Scholar, 29Conaway R.C. Brower C.S. Conaway J.W. Science. 2002; 296: 1254-1258Crossref PubMed Scopus (348) Google Scholar, 30Muratani M. Tansey W.P. Nat. Rev. Mol. Cell Biol. 2003; 4: 192-201Crossref PubMed Scopus (681) Google Scholar, 31Pickart C.M. Cell. 2004; 116: 181-190Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). We found that PPARδ was ubiquitinated and subject to rapid degradation by the 26 S proteasome. However, unlike most nuclear receptors that are degraded upon ligand binding, PPARδ was stabilized by its ligands. PPARδ agonists inhibited ubiquitination of the receptor with consequent block of its degradation. Thus, PPARδ ligands have a dual effect on the receptor. Ligands induce conformational changes, allowing co-activator binding and promoter transactivation, and at the same time prevent ubiquitination of the receptor engaged in transcriptional activation complexes. Block of ubiquitination may be an essential step to avoid rapid degradation of a receptor with very short half-life, like PPARδ, and sustain its transcriptional activity. Cell Lines, Plasmids, and Chemicals—Human osteosarcoma U2OS cells and non-small cell lung cancer H358, H441, and A549 cells were purchased from American Type Culture Collection (LGC Promochem, Molsheim Cedex, F) and maintained in RPMI supplemented with 10% fetal bovine serum. In all experiments involving incubation with PPARδ ligands, cells were grown in phenol red-free RPMI supplemented with 5% charcoal-stripped serum (HyClone, Logan, UT). Full-length human wild type PPARδ (a gift of Bert Vogelstein, John Hopkins University, Baltimore, MD) was subcloned into pCMV (Stratagene, La Jolla, CA) and pcDNA3.1/His (Invitrogen) expression vectors. The C91A/C94A, F270A, and L432A/E435A PPARδ mutants and the truncated form of PPARδ-(1–299) were generated by site-directed mutagenesis of pcDNA3.1/His-PPARδ using the QuikChange site-directed mutagenesis kit (Stratagene). The HA-Ub expression vector was kindly provided by Ron R. Kopito (Stanford University, Stanford, CA), and the PPREx3-tk-Luc reporter vector was a gift of Ronald M. Evans (Salk Institute, La Jolla, CA). Cells were transfected with expression and reporter vectors using Lipofectamine 2000 (Invitrogen). Cells stably expressing PPARδ were obtained upon transfection with pCMV-PPARδ vector and selection in the presence of 600 μg/ml G418 (Calbiochem). GW501516 (Alexis, Lausanne, CH), L165041 (Sigma), cPGI2 (carboprostacyclin; Biomol, Plymouth Meeting, PA), and prostaglandin E2 (Cayman Chemical, Ann Arbor, MI) were dissolved in Me2SO. Proteasome inhibitors PS341 and MG132 were dissolved in Me2SO. Puromycin dihydrochloride (Sigma) was prepared in water. Reporter Assay—Cells (2 × 104 cells/well) were plated in 48-well plates and transfected with the PPREx3-tk-Luc, pRL-SV40 vectors and, when indicated, with PPARδ expression vectors. Dual-luciferase reporter assay was performed according to manufacturer's instructions (Promega, Catalys AG, Wallisellen, CH) using a Turner luminometer (Turner Design, Sunnyvale, CA). Data were normalized for Renilla luciferase activity used as control for transfection efficiency. PPARδ Half-life—U2OS cells were grown in 100-mm dishes to 90% confluence. Cells were transfected with 3 μg of His-PPARδ expression vector. The next day cells were plated into 6-well plates at a concentration of 3.5 × 105 cells/well. After overnight incubation with or without a PPARδ ligand, cells were treated with 50 μm puromycin and harvested at the indicated times for Western blotting. PPARδ Ubiquitination—U2OS cells were transfected with His-PPARδ and HA-Ub and incubated with proteasome inhibitors and/or PPARδ ligands. After 24 h cells were lysed in a denaturing buffer consisting of 8 m urea, 0.1 m Na2HPO4/NaH2PO4, and 10 mm imidazole, pH 8.0. His-PPARδ was pulled down using His-select nickel affinity gel (Sigma) starting with ∼100 μg of protein from cell lysates. After multiple washes with urea buffer, proteins were eluted from the beads with the same buffer supplemented with 250 mm imidazole, pH 6.0. Aliquots of whole lysate and flow-through samples corresponding to 15 μg of proteins and an equivalent fraction of the eluates were loaded on gels and analyzed by Western blotting. Western Blotting—Cells were lysed in a buffer containing 25 mm Tris-HCl, pH 7.4, 150 mm KCl, 5 mm EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors mixture (Roche Applied Science). To isolate nuclear and cytoplasmic fractions, cells were lysed in 10 mm Tris-HCl, pH 8.0, 7.5 mm ammonium sulfate, 1 mm EDTA, 0.025% Nonidet P-40, and 1 mm dithiothreitol as previously described (32Rodriguez-Vilarino S. Arribas J. Arizti P. Castano J.G. J. Biol. Chem. 2000; 275: 6592-6599Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). After incubation on ice for 5 min, sucrose (0.3 m final concentration) was added to the cell homogenate, and the cellular fractions were separated by centrifugation at 4000 × g for 10 min at 4 °C. Protein concentration was determined using a BCA assay (Pierce). Proteins were loaded on 10% polyacrylamide gels and analyzed by immunoblotting with antibodies against PPARδ (H-74, Santa Cruz Biotechnology, Santa Cruz, CA), tubulin (DM1B, Calbiochem), His tag (H1029, Sigma), FLAG tag (M2, Sigma) and HA tag (Roche Applied Science). Horseradish peroxidase-conjugated secondary antibodies and the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences) were used for detection. Band intensity was assessed with the AlphaImager 3400 and AlphaEase Software (Alpha Innotech, San Leandro, CA). RNA Isolation and Analysis—Cells (1 × 106 cells/flask) were incubated with PPARδ ligands overnight and proteasome inhibitors for 4 h. For RNA interference studies, cells were transfected with 100 nm small interfering RNA (Ambion Ltd., Huntingdon UK) specific for PPARδ (siPPARδ) or the firefly luciferase gene (siGL3) using Lipofectamine 2000, incubated for 48 h, and then treated with ligands and/or proteasome inhibitors. Total RNA was isolated using Trizol (Invitrogen) and further purified with RNeasy MiniKit (Qiagen). RNA concentration was determined using a NanoDrop spectrophotometer. RT-PCR was performed using the SuperScript One-Step RT-PCR system (Invitrogen) and gene-specific primers for the adipose differentiation-related protein (ADRP), glyceraldehyde-3-phosphate dehydrogenase, and PPARδ. PCR products were run on 2% agarose gels, stained with ethidium bromide, and visualized using the AlphaImager 3400. Band intensity was determined using the AlphaEase software. For quantitative real time RT-PCR, 1 μg of total RNA was reverse-transcribed using the SuperScript First-Strand Synthesis system (Invitrogen). Real time PCR was performed on a 7900HT Fast Real Time PCR System (Applied Biosystems, Foster City, CA) using primer sets for ADRP and β-actin and Absolute SYBR Green ROX Mix (ABgene, Epsom, UK). Standard curves were generated for each primer set, and β-actin RNA was used as control to quantify ADRP RNA. Sequences of primers and small interfering RNAs are available as supplemental Table S1. PPARδ Ligands Stabilize the Receptor—Ligand-dependent transactivation of nuclear receptors is often associated with proteasome-mediated degradation of the receptor (33Rochette-Egly C. J. Biol. Chem. 2005; 280: 32565-32568Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 34Dennis A.P. Lonard D.M. Nawaz Z. O'Malley B.W. J. Steroid Biochem. Mol. Biol. 2005; 94: 337-346Crossref PubMed Scopus (52) Google Scholar). Ligand-induced proteolysis serves as a common mechanism to terminate transcriptional activity of the ligand-activated receptors (33Rochette-Egly C. J. Biol. Chem. 2005; 280: 32565-32568Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 34Dennis A.P. Lonard D.M. Nawaz Z. O'Malley B.W. J. Steroid Biochem. Mol. Biol. 2005; 94: 337-346Crossref PubMed Scopus (52) Google Scholar). To determine whether PPARδ was subject to a similar ligand-dependent control, human lung cancer cell lines expressing different levels of the receptor were incubated overnight with a PPARδ-selective ligand. PPARδ protein level increased after incubation with L165041 in cells expressing high (H441) and low (H358) levels of the receptor, whereas there was no effect in A549 cells in which PPARδ was undetectable in the immunoblots both before and after ligand treatment (Fig. 1A). The effect of the ligand on the receptor levels correlated well with the ligand ability to transactivate the PPAR-responsive reporter in these cells (Fig. 1B). Ligand treatment increased reporter activity ∼13- and 5-fold in H441 and H358 cells, respectively. The effect on A549 and U2OS cells was minimal (∼2-fold), in agreement with the very low levels of PPARδ in these cells. To better differentiate between transcriptional and post-translational effects, in subsequent experiments we used U2OS cells expressing recombinant PPARδ from a heterologous promoter. Increased levels of PPARδ were seen when PPARδ-expressing U2OS cells were treated with synthetic agonists (i.e. GW501516 and L165041) and a stable prostaglandin analogue (cPGI2 (carboprostacyclin)) that binds to PPARδ (Fig. 1, C and D). On the other hand treatment of cells with prostaglandin E2, a prostaglandin that activates PPARδ indirectly and does not bind to the receptor (19Wang D. Wang H. Shi Q. Katkuri S. Walhi W. Desvergne B. Das S.K. Dey S.K. DuBois R.N. Cancer Cell. 2004; 6: 285-295Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar), did not have any effect on the receptor protein level, suggesting that direct binding to the receptor was required to increase its stability. A time course experiment in PPARδ-expressing U2OS cells showed that the ligand acted rapidly (Fig. 1D). A substantial increase of PPARδ (∼2-fold) was seen already after 4 h, and the levels continued to increase (up to ∼4-fold) after 24 h. The effect on the protein level was reversible, and PPARδ returned to base-line levels within 4 h after the removal of the ligand (Fig. 1E). The effect seen in cells expressing PPARδ from a heterologous promoter made it unlikely that the increased protein level was a consequence of enhanced transcription. In fact, PPARδ transcript levels measured by RT-PCR were identical in PPARδ-expressing U2OS cells incubated with or without ligand (supplemental Fig. S1). The data described above suggested that ligands could affect PPARδ levels by influencing protein turnover. To estimate PPARδ half-life, we transiently transfected U2OS cells with a His-PPARδ expression vector and monitored protein level by Western blotting after the addition of the protein synthesis inhibitor puromycin. His-tagged PPARδ level was greatly reduced within 1 h of puromycin treatment (Fig. 2A). The estimated PPARδ half-life under these conditions was ∼30 min (Fig. 2C). Next, His-PPARδ expressing U2OS cells were incubated with puromycin in the presence of L165041 to determine the effects of the ligand on the receptor half-life. Incubation with L165041 increased PPARδ protein level and completely prevented the decline induced by puromycin (Fig. 2, B and C). Thus, the ligand increased the receptor protein level by extending its half-life considerably. PPARδ Turnover Is Controlled by the Ubiquitin-Proteasome Pathway—The 26 S proteasome is the major cellular complex responsible for the degradation of proteins, including nuclear receptors and transcription factors (28Ciechanover A. Orian A. Schwartz A.L. BioEssays. 2000; 22: 442-451Crossref PubMed Scopus (706) Google Scholar, 29Conaway R.C. Brower C.S. Conaway J.W. Science. 2002; 296: 1254-1258Crossref PubMed Scopus (348) Google Scholar, 30Muratani M. Tansey W.P. Nat. Rev. Mol. Cell Biol. 2003; 4: 192-201Crossref PubMed Scopus (681) Google Scholar, 31Pickart C.M. Cell. 2004; 116: 181-190Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). To determine whether proteasome-mediated degradation played a role in controlling the turnover of PPARδ, H441 and H358 cells expressing, respectively, high and low levels of the receptor were treated with the proteasome inhibitor PS341. In both cell lines PPARδ protein level increased, consistent with reduced degradation of the receptor in the presence of the proteasome inhibitor (Fig. 3A). Similar results were obtained by treating cells with another proteasome inhibitor, MG132 (data not shown). To further study the role of the 26 S proteasome in PPARδ turnover, U2OS cells expressing recombinant His-PPARδ were treated with PS341, and protein level was determined at different times by Western blotting. As shown in Fig. 3B, treatment with PS341 led to a significant accumulation of PPARδ (∼3-fold) already after 4 h of incubation. Next, U2OS cells were treated either with puromycin, PS341, or both compounds together (Fig. 3C). PPARδ level decreased rapidly in the presence of puromycin, whereas treatment with puromycin and PS341 resulted in a higher level of the receptor, indicating that PPARδ turnover was under the control of the proteasome. A necessary step for targeting proteins to the proteasome is the covalent attachment of ubiquitin (Ub) chains catalyzed by Ub ligases (28Ciechanover A. Orian A. Schwartz A.L. BioEssays. 2000; 22: 442-451Crossref PubMed Scopus (706) Google Scholar). To determine whether PPARδ was ubiquitinated, U2OS cells were transfected with His-PPARδ and HA-Ub expression vectors and then incubated with a proteasome inhibitor for 4 h. His-tagged PPARδ was pulled down using nickel affinity gel under denaturing conditions. This strategy avoided the use anti-PPARδ antibodies that had revealed limited specificity in preliminary immunoprecipitation experiments. In addition, it used highly stringent conditions to minimize non-covalent interactions of the receptor with potential ubiquitinated protein partners. Whole cell lysate and flow-through samples were examined along with the eluates to control for quantitative recovery of His-PPARδ throughout the procedure (Fig. 3D, lanes 1–4). The specificity of the pulldown was demonstrated using cells transfected with Histag empty vector (lanes 1, 3, and 5). In His-PPARδ-expressing cells, high molecular weight protein species were detected with the anti-HA antibody indicating the presence of PPARδ with covalently linked poly-Ub chains (lane 6). No such bands were detected in the eluate from empty vector-transfected cells (lane 5). Proteasome activity is required for receptor turnover, and its inhibition generally leads to reduced transcriptional activity of nuclear receptors (34Dennis A.P. Lonard D.M. Nawaz Z. O'Malley B.W. J. Steroid Biochem. Mol. Biol. 2005; 94: 337-346Crossref PubMed Scopus (52) Google Scholar, 35Perissi V. Aggarwal A. Glass C.K. Rose D.W. Rosenfeld M.G. Cell. 2004; 116: 511-526Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). To determine whether a functional proteasome was required for PPARδ transcriptional activity, U2OS cells stably expressing recombinant PPARδ and H441 cells were transfected with a PPAR-responsive reporter and incubated with PS341 in the presence or absence of a PPARδ ligand (Fig. 4A). GW501516 increased reporter activity ∼ 6 and 12-fold in U2OS and H441 cells, respectively. PS341 induced a ∼2–3-fold increase in the absence of the ligand in both cell lines. The combination of PS341 and ligand increased reporter activity by ∼13 and 26-fold in U2OS and H441, respectively. Thus, inhibition of proteasome activity did not negatively affect ligand-dependent activation of PPARδ. Ligands and proteasome inhibitors had a positive and apparently synergistic effect on the activity of both recombinant and endogenous PPARδ in the reporter assays. To determine whether ligands and proteasome inhibitors had similar effects on transcription of endogenous PPARδ target genes, H358 and H441 cells were treated as described above, and the level of the ADRP RNA was monitored by real time RT-PCR or conventional RT-PCR. The ADRP gene contains a PPRE, and its transcription is activated by PPARδ agonists (36Lee C.H. Chawla A. Urbiztondo N. Liao D. Boisvert W.A. Evans R.M. Curtiss L.K. Science. 2003; 302: 453-457Crossref PubMed Scopus (522) Google Scholar). In both cell lines PPARδ ligand increased ADRP RNA levels (by ∼10 and 25-fold in H358 and H441 cells, respectively). This effect was enhanced in the presence of the proteasome inhibitor (up to ∼25 and 100-fold in H358 and H441, respectively) (Fig. 4B). Neither the ligand nor proteasome inhibitor affected endogenous PPARδ RNA levels in H358 and H441 cells, confirming that they acted post-transcriptionally, increasing the protein level of endogenous PPARδ (Fig. 4C and data not shown). The specificity of these effects was further assessed by RNA interference. The increase induced by the PPARδ ligand both alone and in combination with PS341 was attenuated in cells in which PPARδ had been silenced (up to 75%) by a small interfering RNA against PPARδ before the incubation with GW501516 and/or PS341 (Fig. 4C). Despite the evident reduction of PPARδ RNA, ligand-dependent activation of ADRP transcription was reduced only partially by RNA interference because of the stabilizing effect of the ligand and proteasome inhibitor at the protein level. Taken together, these data indicate that inhibition of the proteasome did not reduce PPARδ activity but, along with PPARδ ligands, led to the accumulation of transcriptionally competent receptor. To determine whether the accumulated protein had also the proper
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