A dominant negative PPARγ mutant shows altered cofactor recruitment and inhibits adipogenesis in 3T3-L1 cells

Abstract

Aims/hypothesis. PPARγ, 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 PPARγ 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 PPARγ 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. Conclusion. We conclude that the L466A PPARγ mutant possesses potent dominant negative activity based on preferential corepressor recruitment and it inhibits adipogenesis and the expression of adipocyte-specific genes. [Diabetologia (2003) 46:365-377]

A Dominant Negative Peroxisome Proliferator-activated Receptor-γ Knock-in Mouse Exhibits Features of the Metabolic Syndrome

“…1C). The PPAR␥ L466A mutant has no inherent transcriptional activity (22). The lack of homozygote viability is consistent with the embryonic death seen in homozygous PPAR␥ null animals (9).…”

“…This behavior is analogous to the properties of dominant negative thyroid receptor ␤ mutants associated with the syndrome of resistance to thyroid hormone (31). We have previously shown that the L466A mutation studied here exerts stronger dominant negative activity than the P465L mouse mutant (P467L in humans) (22). In contrast to the L466A mutant (PPARKI), a P465L dominant negative PPAR␥ knock-in mouse model does not exhibit features of the metabolic syndrome (32).…”

“…These clinical manifestations include many features of the metabolic syndrome and are thought to result from a combination of loss of function and the dominant negative effects of the mutant PPAR␥ receptors. In this study, we created a knock-in mouse model containing a PPAR␥ L466A mutation, shown previously to inhibit wildtype PPAR␥ action in vitro (22). The potent dominant negative activity of the L466A mutation, located in the transactivation domain of PPAR␥, is because of preserved DNA binding activity in combination with loss of coactivator binding but preserved corepressor recruitment (22).…”

“…In this study, we created a knock-in mouse model containing a PPAR␥ L466A mutation, shown previously to inhibit wildtype PPAR␥ action in vitro (22). The potent dominant negative activity of the L466A mutation, located in the transactivation domain of PPAR␥, is because of preserved DNA binding activity in combination with loss of coactivator binding but preserved corepressor recruitment (22). Homozygous PPAR␥ L466A knock-in mice die in utero, but heterozygous PPAR␥ L466A knock-in (PPARKI) mice are viable and manifest many of the features seen in patients with dominant negative PPAR␥ mutations.…”

Disruption of transforming growth factor β signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator–activated receptor γ

“…The expression vector pCMX-mPPAR␥ contains the fulllength PPAR␥ complementary DNA under the cytomegalovirus (CMV) promoter in the pCMX vector. Dominant-negative PPAR␥ mutant L466A was generated by polymerase chain reaction-based site-directed mutagenesis (obtained from Dr. J. Larry Jameson, Northwestern University Medical School, Chicago, IL) (33). Dominant-negative PPAR␥ (L468A/ E471A) was constructed by mutation of 2 highly conserved residues in the ligand-binding domain (obtained from Dr. Krishna K. Chatterjee, University of Cambridge, Cambridge, UK) (34).…”