E3 ubiquitin ligase E6AP negatively regulates adipogenesis by downregulating proadipogenic factor C/EBPalpha - PubMed (original) (raw)

E3 ubiquitin ligase E6AP negatively regulates adipogenesis by downregulating proadipogenic factor C/EBPalpha

Pooja Pal et al. PLoS One. 2013.

Abstract

CCAAT/Enhancer Binding Protein Alpha (C/EBPα) is a key transcription factor involved in the adipocyte differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase inhibits adipocyte differentiation in 3T3-L1 cells as revealed by reduced lipid staining with oil red. Knock down of E6AP in mouse 3T3L1 preadipocytes is sufficient to convert them to adipocytes independent of external hormonal induction. C/EBPα protein level is drastically increased in E6AP deficient 3T3L1 preadipocytes while inverse is observed when wild type E6AP is over expressed. We show that transient transfection of wild type E6AP downregulates C/EBPα protein expression in a dose dependent manner while catalytically inactive E6AP-C843A rather stabilizes it. In addition, wild type E6AP inhibits expression of proadipogenic genes while E6AP-C843A enhances them. More importantly, overexpression of E6AP-C843A in mesenchymal progenitor cells promotes accumulation of lipid droplets while there is drastically reduced lipid droplet formation when E6AP is over expressed. Taken together, our finding suggests that E6AP may negatively control adipogenesis by inhibiting C/EBPα expression by targeting it to ubiquitin-proteasome pathway for degradation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. E6AP negatively regulates Adipogenesis.

3T3L1 cells were transfected with E6AP (2.0 µg) and E6AP-C843A (2.0 µg). Post 48 h of transfection, cells were treated with MDI for next 8 days followed by Oil red O staining.

Figure 2

Figure 2. E6AP downregulates expression of proadipogenic factor C/EBPα.

(a,b) 3T3-L1 preadipocytes were transfected with increasing amounts of E6AP (0.5 µg–2.0 µg) and E6AP-C843A (0.5 µg–2.0 µg). Post 48 h transfection, WCEs were prepared and resolved on 10% SDS-PAGE followed by immunoblotting with C/EBPα, E6AP and β actin antibodies; lysates of 293T alone and transfected with C/EBPα were used as positive and negative control, note that there is no endogenous expression of C/EBPα in 293T. E6AP promotes C/EBPα degradation through ubiquitin-proteasome pathway: (c) 3T3-L1 preadipocytes pre-treated with MDI for 48 h were transiently transfected with expression plasmids for E6AP, E6AP-C843A and His-ubiquitin. Post 48 h Transfection, WCEs were prepared and C/EBPα was co-immunoprecipitated. IgG was used as a control. Co-immunoprecipitates were resolved on 8% SDS-PAGE and blots were probed with anti-His and anti-C/EBPα antibody respectively. E6AP inhibits C/EBPα transactivation potential to activate PPRE-Luc: (d) E6AP mediated downregulation of C/EBPα curtails its transactivation potential: 3T3-L1 preadipocytess were transiently transfected with pPPRE-luc reporter and expression plasmids for C/EBPα, E6AP and E6AP-C843A. 24 h post transfection, luciferase activity was measured. MG132 and lactacytin (LCN) treatment was given 3 h prior to cell harvesting for luciferase activity measurement. Data are representative of three independent experiments. Results are given as standard error of mean (± s.e.m.); *p<0.05; **p<0.001, ***<0.0001.

Figure 3

Figure 3. E6AP knock down by siE6AP promotes adipogenesis.

(a) 3T3L1 preadipocytes pre-treated with MDI were transfected with siE6AP or scrambled siRNA, 48 h post transfection, cells were harvested, resolved on 10% SDS-PAGE and probed C/EBPα, E6AP and β-actin antibody (lysates of 293T transfected with C/EBPα were used as control) (b) 3T3L1 preadipocytes were transfected with siE6AP or scrambled siRNA. Cells were grown in the presence or absence of MDI for 10 days followed by Oil red O staining.

Figure 4

Figure 4. siE6AP mediated induction of adipogenesis is marked by increase in expression of proadipogenic factors.

(a) 3T3L1 cells were transfected with siE6AP or scrambled siRNA. Post 48 h transfection, cells were treated with MDI for until ten days; subsequently mRNA expression levels of indicated adipogenic genes was determined with qRT-PCR. Over expression of wild type E6AP down regulates while catalytically inactive E6AP-C843A up regulates proadipogneic factors: (b) 3T3L1 cells were transfected with E6AP and E6AP-C843A. After transfection cells were treated with MDI for 10 days and mRNA expression level of various adipogenic genes was determined with real time PCR. The primers used for C/EBPα, PPARγ, Leptin, SREBP1c and Adipsin listed in table 1.

Figure 5

Figure 5. E6AP over expression inhibits while E6AP-C843A promotes adipocyte differentiation of MDI treated mesenchymal stem cells isolated from murine bone marrow.

Mesenchymal progenitor cells were transfected with E6AP (2.0 µg) and E6AP-C843A (2.0 µg). Post 48 h of transfection, cells were grown in presence or absence of MDII for next 8 days followed by Oil red O staining.

Figure 6

Figure 6. Hypothetical model for E6AP mediated negative regulation of adipogenesis.

Depicts a hypothetical model suggesting higher expression of the E3 ubiquitin ligase E6AP in preadipocytes inhibits proadipogenic transcription factor C/EBPα apparently by targeting it for ubiquitin mediated degradation and thereby attenuating its transactivation potential and adipocyte differentiation.

References

    1. Gregoire FM (2001) Adipocyte differentiation: from fibroblast to endocrine cell. Exp Biol Med (Maywood) 226: 997–1002. -PubMed
    1. Attie AD, Scherer PE (2009) Adipocyte metabolism and obesity. J Lipid Res 50 Suppl: S395–399 -PMC -PubMed
    1. MacDougald OA, Lane MD (1995) Transcriptional regulation of gene expression during adipocyte differentiation. Annu Rev Biochem 64: 345–373. -PubMed
    1. MacDougald OA, Lane MD (1995) Adipocyte differentiation. When precursors are also regulators. Curr Biol 5: 618–621. -PubMed
    1. Morrison RF, Farmer SR (1999) Role of PPARgamma in regulating a cascade expression of cyclin-dependent kinase inhibitors, p18(INK4c) and p21(Waf1/Cip1), during adipogenesis. J Biol Chem 274: 17088–17097. -PubMed

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