The ubiquitin ligase Fbxw7 controls adipocyte differentiation by targeting C/EBPalpha for degradation

Abstract

Adipose tissue controls body lipid and energy metabolism, as well as food intake, and abnormalities in adipose function play a central role in diseases such as obesity and type-2 diabetes. Adipocyte differentiation is controlled by a transcriptional cascade involving PPARgamma and members of the C/EBP family of transcription factors. Here, we demonstrate that C/EBPalpha is targeted for degradation by the ubiquitin ligase Fbxw7 in a phosphorylation-dependent manner. Importantly, inactivation of Fbxw7 is sufficient to convert mouse preadipocytes into mature adipocytes in a manner dependent on C/EBPalpha. In addition, inactivation of Fbxw7 promotes adipocyte differentiation of human adult stem cells. Taken together, our results suggest that Fbxw7 is a negative regulator of adipogenesis by targeting C/EBPalpha for degradation. This notion is supported by the observation that the expression of Fbxw7 is down-regulated during adipocyte differentiation, resulting in the accumulation of proadipogenic proteins such as C/EBPalpha. Thus, Fbxw7 could be an important regulator of energy and lipid metabolism.

Fig. 1.

Fbxw7 is a negative regulator of adipogenesis. (A) 3T3-L1 preadipocyte cells were transfected with control or Fbxw7 siRNA. Ten days following transfection, cellular lipids were visualized with Oil Red O stain, and the cells were photographed. (B) 3T3-L1 preadipocyte cells were transfected as in A and the mRNA expression of the indicated genes was determined by RT-PCR analysis (Cyclo; cyclophilin). (C) 3T3-L1 preadipocyte cells were transfected as in A and the expression of the indicated proteins was determined by Western blotting (TUB; α-tubulin). (D) NIH/3T3 cells were transfected with control or Fbxw7 siRNA and treated with a mixture of methylisobutylxanthine, dexamethasone, and insulin (MDI). Ten days following the addition of MID, the mRNA levels of the indicated genes were determined by RT-PCR analysis. (E) NIH/3T3 cells were transfected and treated as in D. Ten days following the addition of MDI, cellular lipids were visualized with Oil Red O stain, and the cells were photographed.

Fig. 2.

C/EBPα is a substrate for Fbxw7. (A) The sequence surrounding Thr222 and Thr226 in human C/EBPα is aligned to the Fbxw7 phosphodegrons of c-Myc, cyclin E and SREBP1a. (B) 3T3-L1 cells were transfected with C/EBPα or C/EBPβ in the presence of increasing amounts of Fbxw7α. The levels of C/EBPα, C/EBPβ, Fbxw7α and α-tubulin were determined by Western blotting. (C) 3T3-L1 cells were transfected with C/EBPα, either wild-type or the T222A/T226A mutant (TT/AA), in the absence or presence of Flag-Fbxw7α. The levels of C/EBPα, Fbxw7α, and α-tubulin in cell lysates were determined by Western blotting. (D) Confluent 3T3-L1 cells were treated with MG132 (25 μM) for 5 h. Total cell lysates were immunoprecipitated with anti-HA or anti-C/EBPα antibodies. Immunoprecipitated (IP) C/EBPα and Fbxw7α and the levels of C/EBPα and Fbxw7 in cell lysates (Input) were determined by Western blotting. (E) HEK293T cells were transfected with dominant-negative cullin 1 and C/EBPα, either wild-type or the T222A/T226A mutant (TT/AA), in the absence or presence of Flag-Fbxw7α. Cell lysates were immunoprecipitated with anti-Flag antibodies. Immunoprecipitated C/EBPα and Fbxw7α and the levels of C/EBPα in cell lysates were determined by Western blotting. (F) 3T3-L1 cells were tranfected with a C/EBP-responsive promoter-reporter construct in the absence or presence of C/EBPα, either wild-type or the T222A/T226A mutant (TT/AA), and increasing amounts of Fbxw7α. Thirty-six hours after transfection, the luciferase activity was measured. (G) 3T3-L1 preadipocyte cells were transfected with C/EBPα, either wild-type or the T222A/T226A mutant (TT/AA), in the absence or presence of Fbxw7α. Forty-eight hours after transfection, the mRNA expression of PPARγ and cyclophilin was determined by RT-PCR analysis.

Fig. 3.

Endogenous Fbxw7 regulates endogenous C/EBPα. (A) HepG2 cells were transfected with control or Fbxw7 siRNA. Forty-eight hours after transfection, total cell extracts were prepared from the tranfected cells and analyzed by SDS/PAGE. The expression of C/EBPα, C/EBPβ, and α-tubulin and the phosphorylation of C/EBPα (P-C/EBPα) was determined by Western blotting. (B) HepG2 cells were transfected with control or Fbxw7 siRNA. Forty-eight hours after transfection, cells were treated for the indicated times with cyclohexamide and total cell extracts were prepared from the transfected cells and analyzed by SDS/PAGE. The expression of C/EBPα and α-tubulin and the phosphorylation of C/EBPα (P-C/EBPα) were determined by Western blotting. The levels of total and phosphorylated C/EBPα are indicated. The levels of total and phosphorylated C/EBPα in the absence of cyclohexamide was set as 100.

Fig. 4.

Fbxw7 regulates adipocyte differentiation of human adult stem cells. (A) 3T3-L1 preadipocyte cells were transfected with control, Fbxw7 or Fbxw7 plus C/EBPα siRNA. Ten days following transfection, the mRNA expression of the indicated genes was determined by RT-PCR analysis. (B) 3T3-L1 preadipocyte cells were transfected with C/EBPα, either wild-type or the T222A/T226A mutant (TT/AA), in the absence or presence of Fbxw7α. Ten days after transfection, the mRNA expression of the indicated genes was determined by RT-PCR analysis. (C) 3T3-L1 preadipocytes were left untreated or allowed to differentiate to mature adipocytes. The expression of Fbxw7, C/EBPα, PPARγ and cyclophilin was determined by RT-PCR analysis (Left). 3T3-L1 preadipocytes were treated as above and total cell extracts were prepared. Fbxw7 was immunoprecipitated and the levels of Fbxw7 in the immunoprecipitates and α-tubulin in cell lysates were determined by Western blotting (Right). (D) The expression of Fbxw7, C/EBPα, PPARγ, and cyclophilin in total RNA from preadipocytes (preadip) and adipocytes (adip) obtained from human donors was analyzed by RT-PCR analysis. (E) Adipose-derived human adult stem cells were transfected with control or Fbxw7 siRNA and left untreated or treated with a mixture of methylisobutylxanthine, dexamethasone, and insulin (MDI). Ten days after the addition of MDI, the cells were photographed. (F) Adipose-derived human adult stem cells were transfected and treated as in (E). Ten days following the addition of MDI, the mRNA expression of the indicated genes was determined by RT-PCR analysis and correlated to the expression of cyclophilin in each sample. The expression of each gene in control cells in the absence of MDI was set as 1.

Fig. 5.

Fbxw7 is a negative regulator of adipocyte differentiation. High expression of the ubiquitin ligase Fbxw7 in preadipocytes promotes the phosphorylation-dependent degradation of proadipogenic transcription factor such as C/EBPα and SREBP1c, thereby attenuating adipocyte differentiation. The expression of Fbxw7 is down-regulated early during adipogenesis, leading to the stabilization of C/EBPα and SREBP1c, which in turn results in enhanced expression of proadipogenic target genes and enhanced adipogenesis.