ATRA诱导小鼠间充质干细胞棕色成脂分化的分子机制
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摘要
目的研究全反式维甲酸(ATRA)或骨形态发生蛋白7(BMP7)诱导小鼠间充质干细胞定向分化为成熟棕色脂肪细胞的作用及机制。方法采用ATRA或BMP7对小鼠间充质干细胞C3H10T1/2进行体外诱导,使其向棕色脂肪细胞定向分化,比较二者诱导分化棕色成脂效果;采用油红O染色鉴定棕色脂肪细胞成脂能力,线粒体染色检测棕色脂肪细胞富集的线粒体,免疫印迹法检测脂肪分化相关基因、棕色脂肪标志基因表达,采用实时荧光定量聚合酶链反应(PCR)检测BMP7、线粒体相关基因及棕色脂肪细胞分化抑制因子Necdin、Wnt10a及Pref1的表达情况。结果 ATRA诱导C3H10T1/2细胞向棕色脂肪细胞分化的最适浓度为1～2μmol/L。采用1μmol/L ATRA预处理3 d(ATAR组)后,与对照组[采用二甲基亚砜(DMSO)处理]比较,ATRA组线粒体相关基因CyctoC、Tfam及Nrf1的mRNA相对表达量明显升高(P<0.05),棕色脂肪细胞分化抑制因子Necdin和其下游靶基因Pref1、Wnt10a的mRNA相对表达量明显降低(P<0.05),BMP7 mRNA相对表达量明显升高(P<0.05)。不同浓度的ATRA均可使BMP7蛋白表达量升高,且呈剂量效应。采用1μmol/L ATRA预处理0、24和48 h,BMP7蛋白表达量逐渐升高,Necdin蛋白表达量逐渐降低。ATRA与BMP7的诱导效果一致。结论 ATRA具有诱导小鼠间充质干细胞C3H10T1/2分化为棕色脂肪细胞的能力,其机制可能是通过调控BMP7,诱导棕色前体脂肪细胞定向分化。
Objective To investigate the effect and mechanism of all-trans retinoic acid(ATRA) or bone morphogenetic protein 7(BMP7) on the development of brown adipocytes in C3H10T1/2 mesenchymal stem cells. Methods C3H10T1/2 mesenchymal stem cells were pretreated with ATRA or BMP7,followed by an adipogenic cocktail. To evaluate the adipogenesis,oil red O staining was used to identify brown adipogenic capacity. Mitochondrial track staining was used to analyze mitochondrial biogenesis in mature brown adipocytes.The expressions of BMP7,brown adipogenic inhibitors(Necdin,Wnt 10 a and Pref 1) and brown adipocyterelated gene were determined by real-time ?uorescence quantitation polymerase chain reaction(PCR)and western blotting. Results The optimal concentration of ATRA in promoting C3H10T1/2 brown adipogenesis was 1-2 μmol/L.After pretreatment for 3 d with 1 μmol/L ATRA(ATRA group),the mitochondrial biogenesis gene mRNA expressions of CyctoC,Tfam and Nrf1 were increased(P<0.05),the mRNA expressions of brown adipogenic inhibitor Necdin and its downstream target genes(Pref1 and Wnt10 a) were decreased(P<0.05),and the mRNA expression of BMP7 gene was increased(P<0.05),compared with control group treated by dimethyl sulfoxide(DMSO). Different concentrations of ATRA can induce BMP7 protein expression in a dose-response manner. After the pretreatment with 1 μmol/L ATRA for 0,24 and 48 h,BMP7 protein expression was increased,and Necdin protein expression was decreased. The differentiation effect of ATRA and BMP7 was comparable. Conclusions ATRA promotes brown adipogenesis of C3H10T1/2 mesenchymal stem cells via the activation of BMP7 and reduces negative modulators of brown preadipocyte differentiation.
Objective To investigate the effect and mechanism of all-trans retinoic acid(ATRA) or bone morphogenetic protein 7(BMP7) on the development of brown adipocytes in C3H10T1/2 mesenchymal stem cells. Methods C3H10T1/2 mesenchymal stem cells were pretreated with ATRA or BMP7,followed by an adipogenic cocktail. To evaluate the adipogenesis,oil red O staining was used to identify brown adipogenic capacity. Mitochondrial track staining was used to analyze mitochondrial biogenesis in mature brown adipocytes.The expressions of BMP7,brown adipogenic inhibitors(Necdin,Wnt 10 a and Pref 1) and brown adipocyterelated gene were determined by real-time ?uorescence quantitation polymerase chain reaction(PCR)and western blotting. Results The optimal concentration of ATRA in promoting C3H10T1/2 brown adipogenesis was 1-2 μmol/L.After pretreatment for 3 d with 1 μmol/L ATRA(ATRA group),the mitochondrial biogenesis gene mRNA expressions of CyctoC,Tfam and Nrf1 were increased(P<0.05),the mRNA expressions of brown adipogenic inhibitor Necdin and its downstream target genes(Pref1 and Wnt10 a) were decreased(P<0.05),and the mRNA expression of BMP7 gene was increased(P<0.05),compared with control group treated by dimethyl sulfoxide(DMSO). Different concentrations of ATRA can induce BMP7 protein expression in a dose-response manner. After the pretreatment with 1 μmol/L ATRA for 0,24 and 48 h,BMP7 protein expression was increased,and Necdin protein expression was decreased. The differentiation effect of ATRA and BMP7 was comparable. Conclusions ATRA promotes brown adipogenesis of C3H10T1/2 mesenchymal stem cells via the activation of BMP7 and reduces negative modulators of brown preadipocyte differentiation.
引文
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[16]ZHANG H,GUAN M,TOWNSEND K L,et al.MicroRNA-455 regulates brown adipogenesis via a novel HIF1an-AMPK-PGC1αsignaling network[J].EMBO Rep,2015,16(10):1378-1393.
[2]TALLQUIST M D,WEISMANN K E,HELLSTR?MM,et al.Early myotome specification regulates PDGFA expression and axial skeleton development[J].Development,2000,127(23):5059-5070.
[3]SHAPIRA S N,SEALE P.Transcriptional control of brown and beige fat development and function[J].Obesity(Silver Spring),2019,27(1):13-21.
[4]TSENG Y H,KOKKOTOU E,SCHULZ T J,et al.New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure[J].Nature,2008,454(7207):1000-1004.
[5]PARALKAR V M,GRASSER W A,MANSOLF AL,et al.Regulation of BMP-7 expression by retinoic acid and prostaglandin E(2)[J].J Cell Physiol,2002,190(2):207-217.
[6]GRCEVIC′D,MARUSIC′A,GRAHOVAC B,et al.Expression of bone morphogenetic proteins in acute promyelocytic leukemia before and after combined all transretinoic acid and cytotoxic treatment[J].Leuk Res,2003,27(8):731-738.
[7]NIXON B T,GREEN H.Growth hormone promotes the differentiation of myoblasts and preadipocytes generated by azacytidine treatment of 10T1/2 cells[J].Proc Nat Acad Sci U S A,1984,81(11):3429-3432.
[8]ONG F J,AHMED B A,ORESKOVICH S M,et al.Recent advances in the detection of brown adipose tissue in adult humans:a review[J].Clin Sci(Lond),2018,132(10):1039-1054.
[9]TOURNIAIRE F,MUSINOVIC H,GOURANTON E,et al.All-trans retinoic acid induces oxidative phosphorylation and mitochondria biogenesis in adipocytes[J].J Lipid Res,2015,56(6):1100-1109.
[10]TSENG Y H,BUTTE A J,KOKKOTOU E,et al.Prediction of preadipocyte differentiation by gene expression reveals role of insulin receptor substrates and necdin[J].Nat Cell Biol,2005,7(6):601-611.
[11]MARLATT K L,RAVUSSIN E.Brown adipose tissue:an update on recent findings[J].Curr Obes Rep,2017,6(4):389-396.
[12]MOREB J S,UCAR-BILYEU D A,KHAN A.Use of retinoic acid/aldehyde dehydrogenase pathway as potential targeted therapy against cancer stem cells[J].Cancer Chemother Pharmacol,2017,79(2):295-301.
[13]PUIGSERVER P,VáZQUEZ F,BONET M L,et al.In vitro and in vivo induction of brown adipocyte uncoupling protein(thermogenin)by retinoic acid[J].Biochem J,1996,317(Pt 3):827-833.
[14]STONE R L,BERNLOHR D A.The molecular basis for inhibition of adipose conversion of murine 3T3-L1 cells by retinoic acid[J].Differentiation,1990,45(2):119-127.
[15]ABD ELDAIM M A,MATSUOKA S,OKAMATSU-OGURAY,et al.Retinoicaci dmodulateslipid accumulation glucose concentration dependently through inverse regulation of SREBP-1 expression in 3T3L1adipocytes[J].Genes Cells,2017,22(6):568-582.
[16]ZHANG H,GUAN M,TOWNSEND K L,et al.MicroRNA-455 regulates brown adipogenesis via a novel HIF1an-AMPK-PGC1αsignaling network[J].EMBO Rep,2015,16(10):1378-1393.