Brd2 inhibits adipogenesis via the ERK1/2 signaling pathway in 3T3-L1 adipocytes

Abstract

Bromodomain-containing protein 2 (Brd2) is a nuclear serine/threonine kinase involved in transcriptional regulation. In 3T3-L1 adipocytes, Brd2 normally co-represses PPARγ (peroxisome proliferator-activated receptor gamma) and inhibits adipogenesis. Here, we show that Brd2 over-expression in preadipocytes inhibits their differentiation into adipocytes, while Brd2 knockdown promotes adipogenic differentiation in vitro and forces cells to undergo adipogenesis independent of the MDI (methyisobutylxanthane, dexamethasone and insulin) induction. In this study, the two key transcription factors for adipogenesis, PPARγ and C/EBPα (CCAAT/enhancer binding protein-α) were persistently expressed during the differentiation of preadipocytes to mature adipocytes in Brd2 knockdown 3T3-L1 cells, but their expression was inhibited in cells in which Brd2 was overexpressed. To investigate the role of Brd2 in signal transduction we examined the expression of several signaling molecules involved in the regulation of gene expression and cell differentiation by immunoblotting assay. Down-regulation of Brd2 expression in 3T3-L1 cells led to a decrease in extracellular signal-regulated kinase1/2 (ERK1/2) activity and, conversely, the up-regulation of Brd2 leads to increase in ERK1/2 phosphorylation. Nevertheless, changes in Brd2 expression do not affect the activities of JNK and p38 MAPK. In addition, the phosphorylation of Rafs is not affected by changes in Brd2 expression in 3T3-L1 cells. MEK inhibitor UO126 partly restores differentiation of 3T3-L1 cells that overexpress Brd2. In conclusion, these results indicate that Brd2 regulates ERK1/2 activity independently of Raf signaling in 3T3-L1 adipocytes.

Figure 1. Characteristics of 3T3-L1 cells with Brd2 knockdown.

3T3-L1 cells were transfected with Brd2 shRNA (SiBrd2) or scramble shRNA (SiC) plasmids respectively. (A) Adipogenic differentiation of 3T3-L1 adipocytes from pre-adipocytes and Oil Red O staining on day 4 of treatment reveals lipids in cells that have undergone differentiation. (B) Quantification of lipid content in transfected 3T3-L1 cells after adipogenic differentiation (n = 3; **P < 0.01). (C) 3T3-L1 cells transfected with Brd2 shRNA (SiBrd2) or scramble shRNA (SiC) plasmids, respectively, cultured without MDI induction for 8 days, and then stained with Oil Red O. (D) Quantification of lipid content in transfected 3T3-L1 cells after adipogenic differentiation (n = 3; **P < 0.01).

Figure 2. Characteristics of 3T3-L1 cells with overexpressed Brd2.

3T3-L1 cells transfected with pmBrd2 and control (pcDNA3.1), respectively. (A) Adipogenic differentiation of 3T3-L1 adipocytes from pre-adipocytes and Oil Red O staining on day 4 of treatment reveals lipids in cells that have undergone differentiation. (B) Quantification of lipid content in transfected 3T3-L1 cells after adipogenic differentiation (n = 3; **P < 0.01). (C) 3T3-L1 cells transfected with pmBrd2 and pcDNA3.1 respectively induced with MDI, and on day 8, stained with Oil Red O. (D) Quantification of lipid content in transfected 3T3-L1 cells after adipogenic differentiation (n = 3; **P < 0.01).

Figure 3. Effective inhibition and increased Brd2 expression in 3T3-L1 adipocytes.

(A) WT 3T3-L1 adipocytes were transfected with the Brd2 shRNA (SiBrd2), scramble shRNA (SiC) plasmids, pmBrd2 and control (pcDNA3.1), respectively. After transfection (48 h), cells were passaged, and treated with selection of antibiotics G418 for stable transfection. Single colonies were expanded and cells were harvested to examine Brd2 expression by immunoblotting with anti-Brd2 antibody. (B) Brd2 expression levels were quantified by densitometry in arbitrary units (AU) using ImageJ software and normalized to the amount of β-actin. Data are expressed as mean ± SEM of at least three experiments. **P < 0.01 by Student t-test.

Figure 4. Analysis of Brd2 influence on PPARγ and C/EBPα expression.

(A) PPARγ and C/EBPα mRNA expression in Brd2 knockdown 3T3-L1 cells, and (B) in Brd2 overexpression 3T3-L1 cells, as detected by reverse transcription PCR at different time points during adipogenic differentiation. mRNA expression was analyzed using reverse transcription PCR. (C and D) Quantitative real-time PCR expression analysis of PPARγ and C/EBPα in 3T3-L1 cells with Brd2 shRNA, and (E&F) with pmBrd2, relative mRNA levels were determined by △△Ct =△Ct, _sample —△Ct, _reference and β-actin was used as the reference gene. (n = 3, **P < 0.01). (G) The protein levels of PPARγ and C/EBPα in 3T3-L1 cells, which were transfected with pSiBrd2 or pSiC respectively, and (H) in 3T3-L1 cells which were transfected with pmBrd2 and control (pcDNA3.1) by western blotting at different time points during adipogenic differentiation. Each blot is representative of those obtained from triplicate experiments.

Figure 5. Activation of MAPKs in transfected 3T3-L1 adipocytes.

Western blotting was performed with phosphorylated ERK1/2 (Thr202/Tyr204) and total ERK1/2 antibodies, anti-phosphop-JNK at Thr183/Tyr185 and total JNK, anti-phosphop-p38 MAPK at Thr180/Tyr182 and total p38 MAPK antibodies. (A) 3T3-L1 cells were transfected with Brd2 shRNA (SiBrd2) or control (SiC) plasmids, and (B) ERK1/2 phosphorylation was quantified by densitometry in arbitrary units (AU) using ImageJ software, normalized to total amount of ERK1/2. (C) 3T3-L1 cells were transfected with pmBrd2 or control, and (D) ERK1/2 phosphorylation was quantified using the same method as in (B). Each blot is representative of those obtained from triplicate experiments; data are expressed as mean ± SEM. **P < 0.01, Student t-test.

Figure 6. Brd2 activates ERK1/2 in a Raf-independent manner.

Expression of phosphorylated A-Raf (Ser299), phosphorylated B-Raf (Ser445), and phosphorylated C-Raf (Ser338) was examined in protein lysates isolated from transfected cells. (A) Indicative blot representing the expression of target protein after transfection with pSiBrd2 or pSiC. (B) Raf phosphorylation was quantified by densitometry in arbitrary units (AU) using ImageJ software, normalized to β-actin. (C) Indicative blot representing the expression of target proteins after transfection with pmBrd2 or pcDNA3.1. Western blotting was performed with a Raf Family Antibody Sampler Kit. (D) Rafs phosphorylation was quantified using the same method as (B). Each blot is representative of those obtained from triplicate experiments, data are expressed as mean ± SEM. ns, not significant as determined by Student t test compared to controls.

Figure 7. Inhibition of MEK signaling leads to the resaturation of differentiation in cells with Brd2 overexpression.

3T3-L1 cells transfected with pmBrd2 were induced to differentiate in the presence of UO126 or dimethylsulfoxide (DMSO) as a vehicle, and WT 3T3-L1 as a control. (A) Adipogenic differentiation of 3T3-L1 adipocytes from pre-adipocytes was performed and on day +8, cells were fixed, and then stained with Oil Red O. (B) Quantification of lipid content in transfected 3T3-L1 cells after adipogenic differentiation. (C) Adipogenic differentiation of 3T3-L1 adipocytes from pre-adipocytes was performed and on day +8, Western blotting was performed with p-PPARγ, PPARγ, 422/aP2 and β-actin antibodies. (D) 422/aP2 expression levels were quantified by densitometry in arbitrary units (AU) using ImageJ software, normalized to the amount of β-actin. All data are expressed as mean ± SEM and representative results of at least three experiments are shown. **P < 0.01, Student t-test.