Proteasomal degradation of retinoid X receptor α reprograms transcriptional activity of PPARγ in obese mice and humans
Bruno Lefebvre, … , Bart Staels, Philippe Lefebvre
Bruno Lefebvre, … , Bart Staels, Philippe Lefebvre
Published April 1, 2010
Citation Information: J Clin Invest. 2010;120(5):1454-1468. https://doi.org/10.1172/JCI38606.
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Research Article Endocrinology

Proteasomal degradation of retinoid X receptor α reprograms transcriptional activity of PPARγ in obese mice and humans

Abstract

Obese patients have chronic, low-grade inflammation that predisposes to type 2 diabetes and results, in part, from dysregulated visceral white adipose tissue (WAT) functions. The specific signaling pathways underlying WAT dysregulation, however, remain unclear. Here we report that the PPARγ signaling pathway operates differently in the visceral WAT of lean and obese mice. PPARγ in visceral, but not subcutaneous, WAT from obese mice displayed increased sensitivity to activation by its agonist rosiglitazone. This increased sensitivity correlated with increased expression of the gene encoding the ubiquitin hydrolase/ligase ubiquitin carboxyterminal esterase L1 (UCH-L1) and with increased degradation of the PPARγ heterodimerization partner retinoid X receptor α (RXRα), but not RXRβ, in visceral WAT from obese humans and mice. Interestingly, increased UCH-L1 expression and RXRα proteasomal degradation was induced in vitro by conditions mimicking hypoxia, a condition that occurs in obese visceral WAT. Finally, PPARγ-RXRβ heterodimers, but not PPARγ-RXRα complexes, were able to efficiently dismiss the transcriptional corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) upon agonist binding. Increasing the RXRα/RXRβ ratio resulted in increased PPARγ responsiveness following agonist stimulation. Thus, the selective proteasomal degradation of RXRα initiated by UCH-L1 upregulation modulates the relative affinity of PPARγ heterodimers for SMRT and their responsiveness to PPARγ agonists, ultimately activating the PPARγ-controlled gene network in visceral WAT of obese animals and humans.

Authors

Bruno Lefebvre, Yacir Benomar, Aurore Guédin, Audrey Langlois, Nathalie Hennuyer, Julie Dumont, Emmanuel Bouchaert, Catherine Dacquet, Luc Pénicaud, Louis Casteilla, Francois Pattou, Alain Ktorza, Bart Staels, Philippe Lefebvre

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Figure 5

RXRα exerts a repressive effect on PPARγ-mediated transcription.

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RXRα exerts a repressive effect on PPARγ-mediated transcription. (A) RXR...
(A) RXR and PPAR isotype expression in differentiated 3T3-L1 adipocytes. Whole cell extracts (50 g) from control or RSG-treated (2 hours) cells were analyzed by Western blotting. (B) Promoter occupancy by RXR and PPAR isotypes. Differentiated 3T3-L1 cells were treated as in A. RXR and PPAR association to the aP2 and Adpn PPREs was detected by ChIP assay. (C) Rxrα knockdown enhanced Pparγ responsiveness to RSG in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with control, scrambled, or anti-Rxra siRNAs and treated with 1 μM RSG 24 hours later. mRNAs were assayed by QPCR for aP2, Adpn, and GyK transcripts. Results are expressed relative to untreated cells. Data represent mean ± SEM (n = 3). **P < 0.01, ***P < 0.005. (D) Overexpression of RXRα blunted induction of PPARγ target genes in 3T3-L1 CAR adipocytes. 3T3-L1 CAR cells were differentiated and transduced at day 5 with adenovirus encoding GFP, Rxrα, or Rxrβ. mRNA quantification at day 7 was as in C. Data represent mean ± SEM (n = 3). **P < 0.01, ***P < 0.005. (E) Expression levels of RXR isotypes in Min6, 3T3-L1, HepG2, and C2C12 cells. Total RNA from each cell type was analyzed by QPCR for their absolute content in RXR mRNAs. (F) Min6, undifferentiated 3T3-L1, C2C12, and HepG2 cells were transfected with the PPRE-driven J6 tk-Luc reporter gene and stimulated with increasing concentrations of RSG. Luciferase activities are expressed relative to basal expression of the unstimulated reporter system (DMSO), arbitrarily set to 1. (G) Rxrα overexpression blunted Pparγ responsiveness to RSG in Min6 cells. Min6 cells were transfected with the J6 tk-Luc reporter gene and with 30 ng of either RXRα or RXRβ expression vector or 15 ng of each. Luciferase activities were assayed and graphed as in F. (H) Rxrα overexpression blunted Pparγ responsiveness to RSG in COS cells. Experimental conditions were as in F. (I) RXRβ confers RSG responsiveness to a positionally restricted PPARγ-RXR heterodimer. PPARγ and mutated RXRα or RXRβ (both binding to a glucocorticoid response element half site; ref. 61) were overexpressed in HeLa cells, and the transcriptional activity of the heterodimer was monitored using a reporter gene driven by a composite GRE-PPRE or PPRE-GRE tk-Luc reporter gene. Results are expressed as in F. Data represent mean ± SEM (n = 3). ***P < 0.005.