CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Biochemical and Biophysical Research Communications 472 (2016) 617e623 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc HMGA2 promotes adipogenesis by activating C/EBPb-mediated expression of PPARg * Yang Xi a, Wanjing Shen a, Lili Ma a, Ming Zhao a, Jiachen Zheng a, Shizhong Bu a, , ** Shinjiro Hino b, Mitsuyoshi Nakao b, c, a Diabetes Center, and Zhejiang Provincial Key Laboratory of Pathophysiology, Institute of Biochemistry and Molecular Biology, School of Medicine, Ningbo University, Ningbo 315211, China b Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, 860-0811, Japan c Core Research for Evolutional Science and Technology (CREST), Japan Agency for Medical Research and Development, Tokyo, Japan article info abstract Article history: Adipogenesis is orchestrated by a highly ordered network of transcription factors including peroxisome- Received 1 March 2016 proliferator activated receptor-gamma (PPARg) and CCAAT-enhancer binding protein (C/EBP) family Accepted 6 March 2016 proteins. High mobility group protein AT-hook 2 (HMGA2), an architectural transcription factor, has been Available online 8 March 2016 reported to play an essential role in preadipocyte proliferation, and its overexpression has been impli- cated in obesity in mice and humans. However, the direct role of HMGA2 in regulating the gene Keywords: expression program during adipogenesis is not known. Here, we demonstrate that HMGA2 is required for Adipogenesis C/EBPb-mediated expression of PPARg, and thus promotes adipogenic differentiation. We observed a HMGA2 C/EBPb transient but marked increase of Hmga2 transcript at an early phase of differentiation of mouse 3T3-L1 PPARg preadipocytes. Importantly, Hmga2 knockdown greatly impaired adipocyte formation, while its over- expression promoted the formation of mature adipocytes. We found that HMGA2 colocalized with C/ EBPb in the nucleus and was required for the recruitment of C/EBPb to its binding element at the Pparg2 promoter. Accordingly, HMGA2 and C/EBPb cooperatively enhanced the Pparg2 promoter activity. Our results indicate that HMGA2 is an essential constituent of the adipogenic transcription factor network, and thus its function may be affected during the course of obesity. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction activate the master adipogenic factor, peroxisome-proliferator activated receptor-gamma (PPARg) [3]. Because the total adipo- Cellular differentiation is a highly ordered process, in which cyte number has been intimately linked to obesity in humans, it is stepwise expression and/or activation of transcription factors (TFs) crucial to understand the precise mechanism of how preadipocytes orchestrate the timely expression of a select set of genes [1e4]. cease proliferation and initiate the adipogenic program. During adipogenesis, preadipocytes undergo cell-cycle arrest fol- High mobility group protein AT-hook 2 (HMGA2) belongs to the lowed by the reconstitution of a gene expression program, leading non-histone chromosomal high mobility group (HMG) protein to the formation of lipid-accumulating mature adipocytes [5e7]. family and functions as an architectural transcriptional factor by Upon adipogenic induction, key TFs including CCAAT-enhancer recognizing AT-rich sequences in the minor groove of DNA and binding protein (C/EBP) family factors sequentially operate to enhancing gene transcription [8e10]. HMGA2 expression is absent or very low in adult tissues, while it is highly expressed in embryos and mesenchymal stem cells, implying that HMGA2 functions at an early developmental stage [9]. A previous study demonstrated that * Corresponding author. Diabetes Center, and Zhejiang Provincial Key Laboratory transgenic expression of Hmga2 in mice caused a giant phenotype of Pathophysiology, Institute of Biochemistry and Molecular Biology, School of e Medicine, Ningbo University, Ningbo 315211, China. and hyperplasia of white adipose tissue (WAT) [11 13]. However, ** Corresponding author. Department of Medical Cell Biology, Institute of Molec- Hmga2-deficient mice exhibited a pygmy phenotype with drasti- ular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, cally reduced fat tissue and resisted against diet-induced obesity Kumamoto, 860-0811, Japan. Tel.: þ81 96 373 6800; fax: þ81 96 373 6804. [14,15]. Importantly, a recent study reported significantly elevated E-mail addresses: [email protected] (S. Bu), [email protected]. levels of HMGA2 in human WAT of individuals with obesity and/or ac.jp (M. Nakao). http://dx.doi.org/10.1016/j.bbrc.2016.03.015 0006-291X/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 618 Y. Xi et al. / Biochemical and Biophysical Research Communications 472 (2016) 617e623 type 2 diabetes mellitus [16]. This evidence indicates the direct Table 1 involvement of HMGA2 in adipose tissue development and obesity. A list of primers used in current study. A previous study demonstrated that HMGA2 promotes cell- qRT-PCR primers cycle progression by relieving retinoblastoma protein-mediated 36b4 S 50-CAACCCAGCTCTGGAGAAAC-30 0 0 repression of E2F1 activity [17]. Thus, a plausible cause of adipose 36b4 AS 5 -CCAACAGCATATCCCGAATC-3 b-actin S 50-CCAACCGTGAAAAGATGACC-30 tissue atrophy in Hmga2 knockout mice is impaired cell prolifera- 0 0 b-actin AS 5 -CCAGAGGCATACAGGGACAG-3 tion, which results in the reduction of the number of preadipocytes Adipone S 50-GGAACTTGTGCAGGTTGGAT-30 and consequent adipocytes. However, the precise function of Adipone AS 50-TCTCCAGGAGTGCCATCTCT-30 0 0 HMGA2 during the adipogenic process is still poorly understood. C/EBPb S5-ACAAGGCCAAGATGCGCAAC-3 0 0 Here, we report that a pulsatile expression of HMGA2 at the early C/EBPb AS 5 -TTCCGCAGGGTGCTGAGCT-3 Cd36 S 50-CCATTGGTGATGAAAAAGCA-30 phase of adipogenesis drives C/EBPb-mediated induction of PPARg Cd36 AS 50-ATCACCACTCCAATCCCAAG-30 and thus promotes differentiation. Our study suggests that HMGA2 Fabp4 S 50-CATCAGCGTAAATGGGGATT-30 plays an essential role in the transition of proliferative pre- Fabp4 AS 50-GTCGTCTGCGGTGATTTCAT-30 0 0 adipocytes to differentiating adipocytes, which may be a key event Hmga2 S 5 -CATCAGCCCAGGGACAAC-3 Hmga2 AS 50-GAGAGGGCTCACAGGTTGG-30 during acquisition of the obese phenotype. 0 0 Pparg2S 5-TGCAGGAGCAGAGCAAAGAG-3 Pparg2AS 50-CGGCTTCTACGGATCGAAAC-30 2. Materials and methods ChIP primers Pparg2-58kbS 50-GCATTTCAGAAGATTGGGTTTCA-30 0 0 2.1. Cell culture Pparg2-58kb AS 5 -CCAACTAAGGCATGACACTGAG-3 Pparg2-556 S 50-TTACCTGTGTGGGTAACAAAATC-30 Pparg2-468 AS 50-TGAAACTCCTAATCACACATCTG-30 Mouse 3T3-L1 cells were cultured in high glucose (4.5 g/L) Pparg2-1756 S 50-TGAACAGCAGTTGGGACTAGC-30 DMEM supplemented with 10% heat-inactivated fetal bovine serum Pparg2-1892 AS 50-TGATAAACCAGATGACCATAAAAG-30 and penicillin/streptomycin. Adipogenic differentiation of 3T3-L1 preadipocytes was performed as previously described [18]. Briefly, cells were maintained for 3 days post-confluence and the medium cDNA was cloned into the pcDNA3.1 vector (pcDNA3-C/EBPb). DNA was changed to DMEM containing 0.5 mM 3-isobutyl-1- fragments harboring C/EBPb and Pparg2 gene promoters were methylxanthine (SigmaeAldrich), 1.0 mM dexamethasone (Sigma- synthesized in vitro at Shanghai Novobio Biotechnology (Shanghai, eAldrich), and 5.0 mg/mL bovine insulin (SigmaeAldrich) for 2 China). Pparg2 promoter region containing the C/EBPb binding sites days. Cells were then cultured in a growth medium for an addi- from À363-bp to þ17-bp (from the transcription start site) was tional 3e8 days before collection. Isolation and culture of stromal cloned for analysis [21]. Newly synthesized sequences of Pparg2 vascular fractions (SVFs) from inguinal WAT of C57BL/6 mice were promoter region were cloned into the pGL3 luciferase reporter performed as previously described [18]. Lipid accumulation was vector for transient transfection studies: À363/þ17-luc and mut- evaluated by oil red O staining, and the stained lipid droplets were CEBP-363/þ17-luc (C/EBPb binding sites at À340/-336 and À327/- visualized by light microscopy and photographed. Animal experi- 323 were mutated from GCAATTTTAAAAAGCAAT to ments were conducted in accordance with the guidelines of the GCGGTTTTAAAAAGCGGT; mutated sites are underlined). The pGL3 Animal Care and Use Committee of Kumamoto University. basic vector was used as a control. Plasmid transfection experi- ments were performed using Lipofectamine 2000 according to the 2.2. Quantitative reverse transcription-polymerase chain reaction manufacturer's protocol (Invitrogen). (qRT-PCR) Total RNAs were extracted by TRIZOL (Qiagen). RNAs were 2.5. Small interfering RNA (siRNA)-mediated knockdown (KD) reverse transcribed with the Transcriptor First Strand cDNA Syn- 0 thesis Kit (Roche). For quantification, real-time PCR analysis was For the siRNA KD experiment, Hmga2 siRNAs (siHmga2-1: 5 - GACCCAAAGGCAGCAAAAATT-30; siHmga2-2: 50-GAAAGCAGA- performed using LightCycler 480 SYBR Green I Master on a Light- 0 Cycler 480 II (Roche) or ABI7500 Fast (Life Technologies). The GACCAUUGGATT-3 ) were synthesized and transfected