Re-expression of GATA2 cooperates with peroxisome proliferator-activated receptor-gamma depletion to revert the adipocyte phenotype

Abstract

Nuclear peroxisome proliferator-activated receptor-gamma (PPARgamma) is required for adipocyte differentiation, but its role in mature adipocytes is less clear. Here, we report that knockdown of PPARgamma expression in 3T3-L1 adipocytes returned the expression of most adipocyte genes to preadipocyte levels. Consistently, down-regulated but not up-regulated genes showed strong enrichment of PPARgamma binding. Surprisingly, not all adipocyte genes were reversed, and the adipocyte morphology was maintained for an extended period after PPARgamma depletion. To explain this, we focused on transcriptional regulators whose adipogenic regulation was not reversed upon PPARgamma depletion. We identified GATA2, a transcription factor whose down-regulation early in adipogenesis is required for preadipocyte differentiation and whose levels remain low after PPARgamma knockdown. Forced expression of GATA2 in mature adipocytes complemented PPARgamma depletion and impaired adipocyte functionality with a more preadipocyte-like gene expression profile. Ectopic expression of GATA2 in adipose tissue in vivo had a similar effect on adipogenic gene expression. These results suggest that PPARgamma-independent down-regulation of GATA2 prevents reversion of mature adipocytes after PPARgamma depletion.

FIGURE 1.

PPARγ is required for adipocyte differentiation but not for the maintenance of mature adipocyte phenotypes. A, 3T3-L1 cells at 4, 6, or 14 days after initiation of differentiation were electroporated with control or PPARγ siRNA oligonucleotides and reseeded. Phase-contrast microscopy was performed 48 h later. B, the corresponding protein expression of PPARγ and RAN at indicated times was analyzed by immunoblotting (IB) experiments.

FIGURE 2.

PPARγ depletion in differentiated adipocytes reverses most adipogenic gene expression. A, heat map of genes with differential expression in preadipocytes (Preads) and adipocytes electroporated with control or PPARγ siRNA oligonucleotides (genes with a -fold change ≥7 between all conditions) determined by microarray. B, representative expression of adipocyte- and preadipocyte-specific genes measured by qPCR. C, GO analysis of these genes divided into two groups according to indicated patterns of regulation. D, localization of PPARγ binding to up- and down-regulated genes after PPARγ depletion in relation to distance to their transcriptional start sites (TSS; Pos. 0). Diff, differentiation; Kd, knockdown.

FIGURE 3.

PPARγ depletion in differentiated adipocytes does not entirely reverse the adipogenic gene expression. A, heat map of genes with differential expression in preadipocytes (Preads) and adipocytes electroporated with control or PPARγ siRNA oligonucleotides (with a -fold change ≥2 with the same orientation between differentiation and knockdown or with a -fold change ≥2 between at least two conditions) determined by microarray. B, GO analysis of these genes divided into two groups according to indicated patterns of regulation. C, heat map of transcription factors expressed in preadipocytes and known to control adipocyte differentiation. D and E, mRNA and protein expression of Gata2 in preadipocytes (Pre), control, or PPARγ siRNA-treated adipocytes measured by qPCR and immunoblotting (IB).

FIGURE 4.

Forced GATA2 protein expression in differentiated adipocytes complements PPARγ knockdown. A, ectopic expression of GFP or GATA2 in adipocytes is shown. B and C, differentiated adipocytes were electroporated with control or PPARγ siRNA oligonucleotides and infected with GFP-expressing (Adeno-GFP) or GATA2-expressing (Adeno-GATA2) adenoviruses. 5 days later, mRNA expression of the indicated genes was determined by qPCR, and cell morphology was assessed by phase-contrast microscopy. Data are mean ± S.D. (^*, p < 0.05; #, p < 0.05 versus control knockdown). IB, immunoblot.

FIGURE 5.

Combined PPARγ depletion and GATA2 overexpression reduce lipolytic capacity of adipocytes. Glycerol release after β-adrenergic stimulation was measured and is expressed relative to total cell protein. Data are mean ± S.D. (^*, p < 0.05). Adeno-GFP, GFP-expressing adenoviruses; Adeno-GATA2, GATA2-expressing adenoviruses.

FIGURE 6.

Ectopic expression of GATA2 in adipose tissue in vivo decreases adipogenic gene expression. A and B, epididymal fat pads were injected with adenoviruses expressing GFP (Adeno-GFP) or GATA2 (Adeno-GATA2). After 14 days, GFP expression was visualized by UV light, and GATA2 overexpression was confirmed by immunoblotting (IB). Relative mRNA expression of PPARγ2, aP2, and Ces3 was determined by qPCR (B). Data are mean ± S.E. (^*, p < 0.05).