title: A dominant negative PPARgamma mutant shows altered cofactor recruitment and inhibits adipogenesis in 3T3-L1 cells.
abstract: OBJECTIVE
  PPARgamma, a member of the nuclear hormone receptor family of transcription factors, plays a key role in adipocyte differentiation and insulin sensitivity. The aim of this study was to identify a potential dominant negative murine PPARgamma mutant and to characterize the in vitro functional properties of this mutant.
  
  
  METHODS
  In vitro transient transfections and mammalian two-hybrid assays in TSA201 cells were used to characterize the transcriptional activity of the L466A mutant and to study the molecular interaction of transcriptional cofactors with the L466A mutant in an attempt to elucidate the mechanism of its dominant negative activity. Adenoviral constructs expressing PPARgamma wild-type (AdWT) or the L466A mutant (AdL466A) were infected into the murine 3T3-L1 cell line to study the mutant's effect on adipogenesis.
  
  
  RESULTS
  The L466A mutant alone is transcriptionally defective. However, it retains DNA binding and inhibits the ligand-dependent and -independent activity of the wild-type receptor, consistent with dominant negative properties. In mammalian two-hybrid studies, the L466A mutant does not bind nuclear receptor coactivators. However, it more avidly recruits corepressors due to enhanced binding to the corepressor ID1 domain, leading to pronounced transcriptional repression. The AdL466A mutant inhibits adipogenesis induced by either a differentiation cocktail or by thiazolidinedione ligand. AdL466A infection also blocked the upregulation of the adipocyte marker genes aP2 and adipsin.
  
  
  CONCLUSIONS
  We conclude that the L466A PPARgamma mutant possesses potent dominant negative activity based on preferential corepressor recruitment and it inhibits adipogenesis and the expression of adipocyte-specific genes.
authors.affiliation: Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Medical School, Chicago, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Park
authors.forename: B D
authors.initials: BD
authors.lastname: Freedman
authors.forename: E J
authors.initials: EJ
authors.lastname: Lee
authors.forename: S
authors.initials: S
authors.lastname: Park
authors.forename: J L
authors.initials: JL
authors.lastname: Jameson
mesh_terms: D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics; D000273:Adipose Tissue / Q000187:drug effects / Q000254:growth & development / Q000502:physiology*; D000818:Animals; D002454:Cell Differentiation; D011416:Complement Factor D; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005799:Genes, Dominant / Q000235:genetics*; D006728:Hormones / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D009154:Mutation / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection; D014280:Triglycerides / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D006728:Hormones; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D014280:Triglycerides; D012697:Serine Endopeptidases; D011416:Complement Factor D; C493491:complement factor D, mouse