http www tsbmi m utokyo ac jp Rad

－文部科学省イノベーションシステム整備事業「先端融合領域イノベーション創出拠点の形成」－ システム疾患生命科学による先端医療技術開発拠点 http: //www. tsbmi. m. u-tokyo. ac. jp/ Rad 51 is a novel key regulator of adipocyte proliferation and differentiation 松本 佐保姫 1、真鍋 一郎1 、永井 良三2 東京大学医学部循環器内科 1、自治治医科大学 2 Abstract Adipose tissue plays a central role in metabolic syndrome, by producing various adipokines and controlling systemic energy metabolism. Recent studies indicated that inflammation takes place in obese adipose tissue, which affects not only adipose function but also systemic insulin sensitivity. We developed a highthroughput, functional imaging-based screening strategy to identify new regulators involved in adipocyte differentiation and function. Using this strategy, we found that Rad 51, a key molecule of homologous-recombination and DNA-repair, was required for adipocyte differentiation. Rad 51 knockdown suppressed adipocyte differentiation in 3 T 3 -L 1 preadipocytes by inhibiting cell cycle progression at the mitotic clonal expansion, which is an essential step for adipocyte differentiation. We analyzed RAD 51 -dependent molecular steps and found that transcription factor E 2 F 4 inhibits Rad 51 expression in preadipocytes and insulin-stimulation attenuated E 2 F 4 binding to Rad 51 promoter, indicating that novel insulin/E 2 F 4/Rad 51 signaling mediates adipocyte differentiation. In obese mice, Rad 51 expression was increased in white adipose tissues (WAT). Interestingly, Rad 51+/- reduced macrophage infiltration and protected from high fat diet-induced systemic insulin resistance. These results elucidate that Rad 51 is a novel key molecule that controls diet-induced adipose tissue proliferation and inflammation, which leads to systemic insulin resistance. Method Result 1 a Well differentiated Transfection of si. RNA library b Differentiation stimulation Pre-adipocyte Fluorescence staining: Hoechst for nuclei BODYPY for lipid droplets Control d si Negative control Poorly differentiated si RAD 51 c d We used In Cell Analyzer as a device to take high speed imaging and analysis of multi-well plates. IN Cell Analyzer Result 1 Rad 51 is important for adipocyte differentiation a, si. RNA against Rad 51 (si. Rad 51) or control (si. Cntrl) was transfected into 3 T 3 -L 1 preadipocytes. After the induction of differentiation, cells were stained with BODIPY for lipid droplets (green) and DAPI for nuclei (blue). Knockdown of DNA-repair protein Rad 51 suppress adipocyte differentiation. b, During differentiation of 3 T 3 -L 1 cells, Rad 51 expression peaked at 12 to 24 h. c, Rad 51 knockdown significantly reduced expression of Cebpa, Pparg, and Fabp 4. d, Knockdown of Rad 51 inhibit cell proliferation during mitotic clonal expansion (MCE). sh. RNA library screening Schematic representation of the strategy for using a sh. RNA library to screen for genes important for adipocyte differentiation and function. We used In Cell Analyzer as a device to take high speed imaging and analysis of multi-well plates. Result 2 Result 3 b b a a c c BAT Liver WT (DIO) d E 2 F binding sites RAD 51+/(DIO) Result 2 E 2 F 4 suppresses Rad 51 expression in preadipocyte a, Relative levels of Rad 51 expression in 3 T 3 -L 1 cells 48 h after transfection with si. RNA against E 2 f 4 or control si. RNA. E 2 F 4 knock down increase Rad 51 expression. b, Relative luciferase activity of a Rad 51 promoter reporter construct (p. GL 3 -Rad 51) or promoterless p. GL 3 -basic in 3 T 3 -L 1 preadipocytes co-transfected with an E 2 f 4 -expressing vector (p. CAG-E 2 f 4) or empty vector. Forty-eight h after transfection, the cells were treated with insulin (5 mg/ml) or vehicle for an additional 24 h, and the luciferase activity was analyzed. Insulin strongly activated the Rad 51 promoter, and overexpression of E 2 F 4 inhibited that activation. c, Chromatin immunoprecipitation (Ch. IP) assays demonstrated that E 2 F 4 binds to the endogenous Rad 51 promoter in unstimulated 3 T 3 -L 1 preadipocytes and that E 2 F 4 was removed from the promoter in response to insulin. d, Schematic representation of Rad 51 promoter. WAT a, Rad 51 expression was relatively high in white adipose tissue of diet induced obese (DIO) mice and ob/ob mice. b, Weights of epidilymal white adipose tissue (epi-WAT) and subcutaneous WAT (SC-WAT) and liver from fed a normal chow diet (ND) or a highfat diet (HFD). epi-WAT mass was significantly smaller in Rad 51+/- mice. c, HE staining of sections of liver, brown adipose tissue (BAT) and WAT. Whereas number of crown-like structures was markedly reduced in Rad 51+/-, steatosis in liver and BAT was enhanced in Rad 51+/- mice. Conclusions ü Rad 51 is a novel key molecule in adipocyte differentiation by controlling cell proliferation. 循環器内科 URL: http: //www. tsbmi. m. u-tokyo. ac. jp/ TSBMI Translational Systems Biology and Medicine Initiative