Degradation of the Peroxisome Proliferator-activated Receptor γ Is Linked to Ligand-dependent Activation

Abstract

The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) γ is a ligand-activated transcription factor that regulates several crucial biological processes such as adipogenesis, glucose homeostasis, and cell growth. It is also the functional receptor for a new class of insulin-sensitizing drugs, the thiazolidinediones, now widely used in the treatment of type 2 diabetes mellitus. Here we report that PPARγ protein levels are significantly reduced in adipose cells and fibroblasts in response to specific ligands such as thiazolidinediones. Studies with several doses of different ligands illustrate that degradation of PPARγ correlates well with the ability of ligands to activate this receptor. However, analyses of PPARγ mutants show that, although degradation does not strictly depend on the transcriptional activity of the receptor, it is dependent upon the ligand-gated activation function 2 (AF2) domain. Proteasome inhibitors inhibited the down-regulation of PPARγ and ligand activation enhanced the ubiquitination of this receptor. These data indicate that, although ligand binding and activation of the AF2 domain increase the transcriptional function of PPARγ, these same processes also induce ubiquitination and subsequent degradation of this receptor by the proteasome. The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) γ is a ligand-activated transcription factor that regulates several crucial biological processes such as adipogenesis, glucose homeostasis, and cell growth. It is also the functional receptor for a new class of insulin-sensitizing drugs, the thiazolidinediones, now widely used in the treatment of type 2 diabetes mellitus. Here we report that PPARγ protein levels are significantly reduced in adipose cells and fibroblasts in response to specific ligands such as thiazolidinediones. Studies with several doses of different ligands illustrate that degradation of PPARγ correlates well with the ability of ligands to activate this receptor. However, analyses of PPARγ mutants show that, although degradation does not strictly depend on the transcriptional activity of the receptor, it is dependent upon the ligand-gated activation function 2 (AF2) domain. Proteasome inhibitors inhibited the down-regulation of PPARγ and ligand activation enhanced the ubiquitination of this receptor. These data indicate that, although ligand binding and activation of the AF2 domain increase the transcriptional function of PPARγ, these same processes also induce ubiquitination and subsequent degradation of this receptor by the proteasome. peroxisome proliferator-activated receptor γ thiazolidinedione retinoic X receptor 15-deoxy-Δ12,14-prostaglandin J2 estrogen receptor retinoic acid receptor progesterone receptor Dulbecco's modified Eagle's medium mitogen-activated protein kinase phosphate-buffered saline N-acetyl leucine leucine norleucinal CAAT/enhancer-binding protein cAMP response element binding protein pioglitazone rosiglitazone The peroxisome proliferator-activated receptor (PPAR) γ,1 a member of the nuclear hormone receptor family of transcription factors, has recently been implicated in the regulation of a variety of biological processes. Two forms of PPARγ, PPARγ1 and PPARγ2, are generated by alternative splicing. PPARγ2 bears an additional 30 amino acids at the amino terminus and is predominantly expressed in adipose tissue. PPARγ plays a central regulatory role in adipogenesis, where it acts in concert with members of the CAAT/enhancer-binding protein (C/EBP) family of transcription factors (reviewed in Ref. 1.Wu Z. Puigserver P. Spiegelman B.M. Curr. Opin. Cell Biol. 1999; 11: 689-694Crossref PubMed Scopus (108) Google Scholar). The expression of PPARγ mRNA and protein are induced early during adipogenesis. Many of the adipocyte-specific genes harbor PPARγ binding sites in their promoters or upstream enhancer regions. Gain-of-function experiments have shown that PPARγ is sufficient to induce adipogenesis, specifically, ectopic expression and activation of PPARγ in fibroblasts efficiently induces an adipocytic phenotype (2.Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3098) Google Scholar). Recent genetic studies conclusively demonstrate that PPARγ is also absolutely required for fat cell formation. Cells lacking both alleles for PPARγ do not differentiate into adipocytes in vitro orin vivo; a PPARγ−/− mouse lived only briefly after birth and lacked visible fat pads (3.Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1216) Google Scholar, 4.Barak Y. Nelson M.C. Ong E.S. Jones Y.Z. Ruiz-Lozano P. Chien K.R. Koder A. Evans R.M. Mol. Cell. 1999; 4: 585-595Abstract Full Text Full Text PDF PubMed Scopus (1636) Google Scholar, 5.Rosen E.D. Sarraf P. Troy A.E. Bradwin G. Moore K. Milstone D.S. Spiegelman B.M. Mortensen R.M. Mol. Cell. 1999; 4: 611-617Abstract Full Text Full Text PDF PubMed Scopus (1637) Google Scholar). The gene dosage of PPARγ is apparently important, because differentiation of cells that contain only a single PPARγ allele shows a phenotype intermediate to wild-type and null cells (3.Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1216) Google Scholar, 5.Rosen E.D. Sarraf P. Troy A.E. Bradwin G. Moore K. Milstone D.S. Spiegelman B.M. Mortensen R.M. Mol. Cell. 1999; 4: 611-617Abstract Full Text Full Text PDF PubMed Scopus (1637) Google Scholar). PPARγ has also been implicated in the regulation of systemic insulin sensitivity. This was first suggested when PPARγ was found to be the functional receptor for a group of synthetic insulin-sensitizing agents, the thiazolidinediones (TZDs), which are currently used for the treatment of type 2 diabetes mellitus (6.Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3446) Google Scholar, 7.Henry R.R. Endocrinol. Metab. Clin. North Am. 1997; 26: 553-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). This role is now supported by the finding that certain mutations within PPARγ are associated with severe insulin resistance and diabetes mellitus, even though these patients are not obese (8.Barroso I. Gurnell M. Crowley V.E. Agostini M. Schwabe J.W. Soos M.A. Maslen G.L. Williams T.D. Lewis H. Schafer A.J. Chatterjee V.K. O'Rahilly S. Nature. 1999; 402: 880-883Crossref PubMed Scopus (1151) Google Scholar). Although expressed at lower levels than in adipose cells, PPARγ has also been implicated in the growth and/or differentiation of several cell types such as monocytes, breast, and colonic epithelium. The ability of PPARγ to arrest growth in many of these cell types has suggested a possible connection to tumor biology (reviewed in Refs. 9.Ricote M. Huang J.T. Welch J.S. Glass C.K. J. Leukoc. Biol. 1999; 66: 733-739Crossref PubMed Scopus (281) Google Scholar,10.Rocchi S. Auwerx J. Ann. Med. 1999; 31: 342-351Crossref PubMed Scopus (81) Google Scholar). Indeed, loss-of-function mutations of PPARγ have been found in human colon cancers, suggesting a possible role as a tumor suppressor in this cell lineage (11.Sarraf P. Mueller E. Smith W.M. Wright H.M. Kum J.B. Aaltonen L.A. de la Chapelle A. Spiegelman B.M. Eng C. Mol. Cell. 1999; 3: 799-804Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). Recently, clinical data show that treatment of liposarcoma patients with TZDs induced tumor differentiation and a reduction in tumor cell growth (12.Demetri G.D. Fletcher C.D. Mueller E. Sarraf P. Naujoks R. Campbell N. Spiegelman B.M. Singer S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3951-3956Crossref PubMed Scopus (464) Google Scholar). PPARγ is a ligand-activated transcription factor that binds to DR-1 sites as a heterodimeric complex with the retinoic X receptor (RXR). Synthetic ligands for PPARγ are the aforementioned TZDs (e.g. troglitazone, pioglitazone, and rosiglitazone) and certain nonsteroidal anti-inflammatory drugs (6.Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3446) Google Scholar, 13.Lehmann J.M. Lenhard J.M. Oliver B.B. Ringold G.M. Kliewer S.A. J. Biol. Chem. 1997; 272: 3406-3410Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar). Natural ligands include 15-deoxy-Δ12,14-prostaglandin J2(15-dPGJ2), certain polyunsaturated fatty acids such as linoleic acid, and endogenous constituents of oxidized low density lipoprotein particles such as 9- and 13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatetraenoic acid (14.Nagy L. Tontonoz P. Alvarez J.G. Chen H. Evans R.M. Cell. 1998; 93: 229-240Abstract Full Text Full Text PDF PubMed Scopus (1579) Google Scholar, 15.Kliewer S.A. Lenhard J.M. Willson T.M. Patel I. Morris D.C. Lehmann J.M. Cell. 1995; 83: 813-819Abstract Full Text PDF PubMed Scopus (1861) Google Scholar, 16.Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2718) Google Scholar, 17.Huang J.T. Welch J.S. Ricote M. Binder C.J. Willson T.M. Kelly C. Witztum J.L. Funk C.D. Conrad D. Glass C.K. Nature. 1999; 400: 378-382Crossref PubMed Scopus (768) Google Scholar). All of these natural compounds are low affinity ligands. So far, there is no consensus on the existence or the nature of a high affinity endogenous ligand. To date, the major regulatory events identified in PPARγ function are ligand binding, coactivator docking, and phosphorylation at an inhibitory serine (serine 112 of murine PPARγ) by mitogen-activated protein (MAP) kinase (18.Camp H.S. Tafuri S.R. J. Biol. Chem. 1997; 272: 10811-10816Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 19.Hu E. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; PubMed Scopus Google Scholar, J. G. A. S. S. D. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). for nuclear binding of ligands induces a in a in the terminus of the ligand binding domain S. R. Willson T.M. Glass C.K. Nature. 1998; PubMed Scopus Google Scholar). This also as the AF2 is a for a of that transcription of or with the transcription (reviewed in Ref. J. Z. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). PPARγ also is to regulation of serine 112 in murine PPARγ2 by and protein in a of PPARγ activity (18.Camp H.S. Tafuri S.R. J. Biol. Chem. 1997; 272: 10811-10816Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 19.Hu E. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; PubMed Scopus Google Scholar, J. G. A. S. S. D. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google at in by ligand binding D. M.A. Nature. 1998; PubMed Scopus Google Scholar). a regulatory for PPARγ ligand the of degradation of this receptor by the proteasome. ligands induce both the transcriptional activation and the of the receptor, these illustrate a for the transcriptional activity of was and and rosiglitazone A. was a R. The inhibitors and N-acetyl leucine leucine norleucinal for was and and insulin and cells for as H. O. Cell. Full Text PDF Scopus Google Scholar). and inhibitors at the and for the in the both in the modified Eagle's medium or the of growth of PPARγ alleles is the of an The for wild-type PPARγ (2.Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3098) Google E. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; PubMed Scopus Google (2.Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3098) Google have been was by to To PPARγ degradation in cells, and or at by with for to the after and cell after 2 and ligands and inhibitors after for the in the AF2 of PPARγ is for the of PPARγ of PPARγ with of the sites of the and binding ligand binding, and AF2 cells with expression for PPARγ wild-type or PPARγ mutants in the of a with PPARγ response and an expression for after the cells for with or rosiglitazone Cell for and cells with expression for PPARγ type or mutants and for with or cell in with an cells with expression for and or Cells for with or cell in with an ubiquitination of cells with expression for or the of was used as a Cells for with or and was with an after of the cell with a PPARγ of the of PPARγ cells with an expression for the cells for with the compounds or and cell in for the of adipocytes for 30 with the of and for 30 with or cell in with To for transcriptional activity of PPARγ, cells, in cell in with PPARγ, and DR-1 and of after cells to ligands for and for and activity a and in Cells in phosphate-buffered saline and in with 2 for and of the was by and for the same of protein was by after acid E. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; PubMed Scopus Google Scholar). as E. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; PubMed Scopus Google Scholar). The PPARγ is in a E. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; PubMed Scopus Google and cAMP response element binding protein of was a S. with an in of of a of protein was for 2 with by and as was the to the of was for PPARγ mRNA with an of the PPARγ as (2.Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3098) Google Scholar). To the of PPARγ ligands on the of this receptor, adipocytes with or pioglitazone, a ligand of the cell by with an PPARγ2 as a in as shown the is an of PPARγ, by on serine 112 E. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; PubMed Scopus Google Scholar). The lower the of this protein and treatment with pioglitazone in a of PPARγ with the of this receptor 2 and The levels of the of PPARγ, and not when the cells to This that the in protein is or specific for To the of PPARγ protein is to specific ligand binding, adipocytes with several different TZDs troglitazone, and rosiglitazone) and the natural ligand we also used a of that no binds to PPARγ, the ligand and the ligand with the different TZDs and with in a of the of PPARγ with treatment with the or and not PPARγ and the of receptor, with and the of the PPARγ protein to be specific for PPARγ ligands. The experiments with of ligand and that are the of of these ligands for PPARγ for rosiglitazone to for a response was on adipocytes to the this reduced of protein and the affinity for a ligand. shown in an of pioglitazone on PPARγ levels be at be at and which is to of T.M. S.R. Moore L.B. Kliewer S.A. Lehmann J.M. J. Med. Chem. 1996; PubMed Scopus Google Scholar). Recently, D. M.A. Nature. 1998; PubMed Scopus Google that the of PPARγ binds to ligand with lower affinity than the ligand to the of the the of the receptor correlates well with ligand and Mol. Endocrinol. 1998; PubMed Google and H.S. Tafuri S.R. 1999; PubMed Scopus Google recently that PPARγ mRNA levels are upon of cells to specific the of protein shown be a of mRNA To we first studies of the of PPARγ protein and PPARγ mRNA in response to a PPARγ ligand. protein and of adipocytes after or of treatment with pioglitazone or shows that, in with the a treatment of adipocytes with a PPARγ mRNA and both of PPARγ However, of pioglitazone treatment PPARγ mRNA of the of the PPARγ protein has by this the was These data that PPARγ ligands have an on PPARγ protein that is at the mRNA To the of PPARγ ligands at transcriptional and levels we expressed PPARγ with the which is not to regulation by PPARγ ligands. with the or with a PPARγ expression for with pioglitazone and by and be in 2 PPARγ mRNA and protein only be in cells the expression for that the PPARγ mRNA is expressed at levels in the or of ligand 2 and However, the treatment with the of PPARγ protein was the of PPARγ was 2 and to is for endogenous PPARγ in adipocytes the protein levels of transcription factors, and not by the treatment 2 These data demonstrate an of PPARγ ligands on PPARγ protein that is at the mRNA in these show that the of PPARγ protein is not to To the for PPARγ we wild-type PPARγ and alleles into cells Although the experiments indicate that the of PPARγ is and forms upon ligand binding these To and this we used an allele of PPARγ with the serine 112 phosphorylation to E. 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The also binds to PPARγ ligands with affinity as the wild-type receptor not of these mutants no transcriptional activation ligand was to the levels of not These data indicate that transcriptional activation of the receptor is not a for However, an AF2 domain is suggesting that a of the AF2 and/or of to the AF2 is for the to with the AF2 of nuclear hormone are and of a coactivator or the degradation of Although to nuclear hormone in a is in that it is a the is by ligand shown in down-regulation in the of degradation of These illustrate that protein down-regulation is not to ligand binding is associated with a of the AF2 domain of the receptor. Two different of with the activity of the AF2 not ligand binding, in the or binding of protein the by which PPARγ is the regulates the of many transcription factors A. J. 1998; PubMed Scopus Google we and the cells for in the or of ligand with or is to also we also the of which does not degradation C. L. K. R. L. D. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). shown in down-regulation was inhibited by inhibitors was not by to the of the levels of PPARγ are by the of the inhibitors for the This that the of the receptor is also by the proteasome. To the of the in degradation of PPARγ in cells with pioglitazone or treatment with or the To cell by the inhibitors in fat cells, we used a treatment with inhibitors and PPARγ ligands at shown in PPARγ is these with the inhibited the down-regulation These experiments that PPARγ is by the upon activation in both fibroblasts and fat are to their degradation and be by the of high Ref. M. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). 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Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). is required to the role of the AF2 in the of a of that there in The complex biology of PPARγ is only to be However, it is that ligands that activate this receptor are Many patients with type 2 diabetes mellitus glucose the of synthetic PPARγ and clinical data in patients with insulin resistance R.R. Endocrinol. Metab. Clin. North Am. 1997; 26: 553-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). differentiation of human in patients with liposarcoma was (12.Demetri G.D. Fletcher C.D. Mueller E. Sarraf P. Naujoks R. Campbell N. Spiegelman B.M. Singer S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3951-3956Crossref PubMed Scopus (464) Google the that PPARγ ligands also have in the PPARγ ligands also several in monocytes, differentiation to and M. Huang J.T. Welch J.S. Glass C.K. J. Leukoc. Biol. 1999; 66: 733-739Crossref PubMed Scopus (281) Google Scholar,10.Rocchi S. Auwerx J. Ann. Med. 1999; 31: 342-351Crossref PubMed Scopus (81) Google Scholar). However, even in many clinical where have been such as in type 2 the of PPARγ ligands are many patients do not a response to the of insulin or drugs R.R. Endocrinol. Metab. Clin. North Am. 1997; 26: 553-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). an of to a response the PPARγ response and of the receptor induced by PPARγ ligands the of of receptor and this the members of the Spiegelman for we and for the