title: Altering PPARgamma ligand selectivity impairs adipogenesis by thiazolidinediones but not hormonal inducers.
abstract: Peroxisome proliferator-activated receptor gamma (PPARgamma) acts as a ligand-dependent transcription factor with a key role in mediating adipocyte differentiation and insulin sensitivity. Recently, we and others have shown that PPARgamma recruits the nuclear corepressors NCoR and silencing mediator for retinoid and thyroid hormone receptors (SMRT) to modulate adipogenesis. While the synthetic ligands for PPARgamma, the thiazolidinediones (TZD), are widely used in the treatment of type 2 diabetes mellitus, the biologically relevant endogenous PPARgamma ligand involved in adipogenesis remains unidentified. To further understand the role of ligand binding and corepressor interaction in PPARgamma-mediated adipogenesis, a mutation was introduced in the ligand-binding domain (LBD) of murine PPARgamma. PPARgammamut was created via two amino acid substitutions known to be major determinants of ligand selectivity among PPAR isotypes, H323Y and R288M. These mutations alter PPARgamma to the corresponding residues of the PPARalpha. Characterizing the in vitro functional properties of this mutant, we show that PPARgammamut preferentially responds to the PPARalpha agonist, WY-14643, over the TZD, pioglitazone. When expressed in 3T3-L1 preadipocytes using recombinant adenovirus, wild-type PPARgamma leads to adipocyte formation with both hormonal and TZD treatment. PPARgammamut blocks the upregulation of adipocyte-specific proteins by TZD, but surprisingly, not by standard hormonal inducers. Our data suggest that TZDs and the purported endogenous ligand do not interact in the same way with the PPARgamma LBD. We propose that the endogenous ligand has distinct properties that allow for promiscuity within the hydrophobic PPAR ligand-binding pocket, yet fosters appropriate cofactor recruitment and release to allow adipogenesis to proceed.
authors.affiliation: Section of Endocrinology, Department of Medicine, The University of Chicago, Chicago, Illinois, USA.
authors.forename: Shanika P
authors.initials: SP
authors.lastname: Samarasinghe
authors.forename: Maria M
authors.initials: MM
authors.lastname: Sutanto
authors.forename: Arpad M
authors.initials: AM
authors.lastname: Danos
authors.forename: Daniel N
authors.initials: DN
authors.lastname: Johnson
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Brady
authors.forename: Ronald N
authors.initials: RN
authors.lastname: Cohen
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D019943:Amino Acid Substitution; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy; D005786:Gene Expression Regulation / Q000187:drug effects; D020868:Gene Silencing; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D056985:Nuclear Receptor Co-Repressor 2; D047495:PPAR gamma / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000502:physiology; D012097:Repressor Proteins / Q000235:genetics; D045162:Thiazolidinediones / Q000627:therapeutic use; D014162:Transfection
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D007328:Insulin; C539131:Ncor2 protein, mouse; D056985:Nuclear Receptor Co-Repressor 2; D047495:PPAR gamma; D011988:Receptors, Thyroid Hormone; D012097:Repressor Proteins; D045162:Thiazolidinediones; D003907:Dexamethasone; D005982:Glutathione Transferase