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. 2015 Aug;56(8):1471-80.
doi: 10.1194/jlr.M057679. Epub 2015 Jun 25.

Foxc2 enhances proliferation and inhibits apoptosis through activating Akt/mTORC1 signaling pathway in mouse preadipocytes

Lu Gan  1 , Zhenjiang Liu  1 , Wei Jin  1 , Zhongjie Zhou  1 , Chao Sun  1
Affiliations

Foxc2 enhances proliferation and inhibits apoptosis through activating Akt/mTORC1 signaling pathway in mouse preadipocytes

Lu Gan et al. J Lipid Res. 2015 Aug.

Abstract

Forkhead box C2 (Foxc2) protein is a transcription factor in regulation of development, metabolism, and immunology. However, the regulatory mechanisms of Foxc2 on proliferation and apoptosis of preadipocytes are unclear. In this study, we found that high-fat-diet-induced obesity elevated the expression of Foxc2 and cyclin E after 6 weeks. Additionally, Foxc2 suppressed preadipocyte differentiation, increased cell counts and augmented G1-S transition of preadipocytes, along with the elevation of cyclin E expression and the reduction levels of p27 and p53. Furthermore, Foxc2 knockdown reduced early apoptotic cells with accompanying reduction of mitochondrial membrane potential and increased fragmentation of genomic DNA. We show that Foxc2 reduces the expression of Bax, caspase-9, and caspase-3 in both serum-starved and palmitic acid-induced cell apoptotic models, which confirms the anti-apoptotic role of Foxc2. Moreover, the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)C1 signaling pathway and the ERK/mTORC1 signaling pathway were activated along with preadipocyte proliferation in response to Foxc2 overexpression, whereas apoptosis marker genes were downregulated during this process. Those effects were blocked by the interference of Foxc2 or signal pathways specific inhibitors. These data collectively reveal that Foxc2 enhances proliferation of preadipocytes and inhibits apoptosis of preadipocytes by activating the Akt/mTORC1 and ERK/mTORC1 signaling pathways.

Keywords: cell death; cell signaling; fat; forkhead box C2; protein kinase B/mammalian target of rapamycin C1 signaling pathway; transcription factor.

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Figures

Fig. 1.
Fig. 1.
Foxc2 inhibited mouse preadipocyte differentiation and increased cell counts. A: Body weight of male mice fed a HFD (n = 18 each). B: Food intake of male mice fed a HFD and a chow diet (n = 18 each). C: Relative Foxc2 mRNA expression in both chow diet and HFD groups (n = 18 each). D: Relative cyclin E mRNA expression in both chow diet and HFD groups (n = 18 each). E: Transfection efficiencies of overexpression Foxc2 (pcDNA-Foxc2) and shRNA-Foxc2 in mouse preadipocytes (n = 3). F: BODIPY staining of differentiation preadipocytes after transfection with pcDNA-Foxc2 and shRNA-Foxc2 for 48 h (n = 3). G: Relative mRNA expression of PPARγ, FAS, and HSL after transfection with pcDNA-Foxc2 and shRNA-Foxc2 for 48 h (n = 3). H: Cell counting after cells were cultured for 6, 12, 24, and 48 h in groups: control group, pcDNA-Foxc2 group, and shRNA-Foxc2 group (n = 3 each). I: Cell counting after serum-starved cells were cultured for 6, 12, 24, and 48 h in groups: control group, pcDNA-Foxc2 group, and shRNA-Foxc2 group (n = 3 each).Values are mean ± SD. *P < 0.05, **P < 0.01 compared with the control.
Fig. 2.
Fig. 2.
Foxc2 enhanced preadipocyte proliferation and induced cell cycle transition. Mouse preadipocytes were pretreated with either pcDNA-Foxc2 or shRNA-Foxc2 plasmids. A: Cell cycle analyzed in flow cytometry and the percentages of cell counts at different phases (n = 3). B: The protein levels of p27, p53, cyclin E, and β-actin in normal growth medium (n = 3). C: The protein expression levels of p27, p53, cyclin E, and β-actin in serum-starved medium for 6 h (n = 3). The total β-actin was used as the loading control. Values are mean ± SD. *P < 0.05, **P < 0.01 compared with the control.
Fig. 3.
Fig. 3.
Foxc2 inhibited preadipocyte apoptosis. Mouse preadipocytes were pretreated with either pcDNA-Foxc2 or shRNA-Foxc2 plasmids. A: Images of mitochondrial membrane integrity under fluorescence microscope after dyeing with JC-1. Scale bar: 100 μm (n = 3). B: Cell counts by flow cytometry after staining with JC-1. The shift of green and red fluorescence intensities were quantitated by subtracting the mean of treated cultures from the mean of the control. The ratio of red/green fluorescence intensity was then used to quantitate the potential of mitochondrial membrane (n = 3). C: Images of preadipocyte apoptosis under fluorescence microscope after TUNEL staining. Fluorescence intensity was used to quantitate the TUNEL-positive cells. Scale bar: 100 μm (n = 3). D: Annexin V/PI staining analysis of apoptosis. The apoptotic rates were determined by flow cytometry (n = 3). E: Protein expressions of Bax, Bcl-2, caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9, and β-actin. The expression level of total β-actin was used as the loading control (n = 3). Values are mean ± SD. *P < 0.05, **P < 0.01 compared with the control.
Fig. 4.
Fig. 4.
Foxc2 attenuated serum-starved and palmitate-induced preadipocyte apoptosis. Mouse preadipocytes were pretreated with either pcDNA-Foxc2 or shRNA-Foxc2. A: Protein expression of Bax, Bcl-2, caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9, and β-actin after treatment with palmitic acid (PA) for 24 h (n = 3). B: Protein expression of Bax, Bcl-2, caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9, and β-actin after serum starvation treatment for 24 h. The expression level of total β-actin was used as the loading control (n = 3). Values are mean ± SD. *P < 0.05 compared with the control.
Fig. 5.
Fig. 5.
Foxc2 regulated proliferation and apoptosis of preadipocytes via the mTORC1 pathway. Mouse preadipocytes were pretreated with pcDNA-Foxc2, shRNA-Foxc2, MK-2206, U0126, and rapamycin, respectively. A: Representative immunoblots and densitometric quantification for p-IRS1, p-Aktser473, p-FoxO3a, and p-mTORSer2448 (n = 3) with or without MK-2206. B: Representative immunoblots and densitometric quantification for cyclin E, p53, and caspase-3 (n = 3) with or without MK-2206. C: Representative immunoblots and densitometric quantification for p-ERK1/2, raptor, and p-mTORSer2448 (n = 3) with or without U0126. D: Representative immunoblots and densitometric quantification for cyclin E and caspase-9 (n = 3) with or without U0126. E: Representative immunoblots and densitometric quantification for p-AktSer473, p-ERK1/2, raptor, and p-mTORSer2448 (n = 3) with or without MK-2206 or U0126. F: Representative immunoblots and densitometric quantification for raptor, p-mTORSer2448, and p-S6K1Thr389 (n = 3) with or without rapamycin. G: Representative immunoblots and densitometric quantification for p27 and caspase-9 (n = 3) with or without rapamycin. The level of total β-actin was used as the loading control. Values are mean ± SD. *P < 0.05, **P < 0.01 compared with the control.
Fig. 6.
Fig. 6.
Summary of the effects of Foxc2 on proliferation and apoptosis of adipocytes. Forced expression of Foxc2 promoted preadipocyte proliferation and inhibited preadipocyte apoptosis via the activation of the Akt/mTORC1 and ERK/mTORC1 signaling pathways.
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