mTORC1/2 play central roles as signaling hubs of cell growth and metabolism and are therapeutic targets for several diseases.However,the human genetic evidence linking mutations of mTORC1/2 to obesity remains elusive....mTORC1/2 play central roles as signaling hubs of cell growth and metabolism and are therapeutic targets for several diseases.However,the human genetic evidence linking mutations of mTORC1/2 to obesity remains elusive.Using whole-exome sequencing of 1944 cases with severe obesity and 2161 healthy lean controls,we identify a rare RICTOR p.I116V variant enriched in 9 unrelated cases.In Rictor null mouse embryonic fibroblasts,overexpression of the RICTOR p.I116V mutant increases phosphorylation of AKT,a canonical mTORC2 substrate,compared with wild-type RICTOR,indicating a gain-of-function change.Consistent with the human obesity phenotype,the knock-in mice carrying homogenous Rictor p.I116V variants gain more body weight under a high-fat diet.Additionally,the stromal vascular fraction cells derived from inguinal white adipose tissue of knock-in mice display an enhanced capacity for adipocyte differentiation via AKT activity.These findings demonstrate that the rare gain-of-function RICTOR p.I116V mutation activates AKT signaling,promotes adipogenesis,and contributes to obesity in humans.展开更多
目的:探讨沉默雷帕霉素不敏感的哺乳动物雷帕霉素靶蛋白伴侣(rapamycin insensitive companion of mammalian target of rapamycin,RICTOR)基因表达后食管鳞癌细胞对依维莫司敏感性的变化,及其可能的分子机制。方法:蛋白质印迹法检测食...目的:探讨沉默雷帕霉素不敏感的哺乳动物雷帕霉素靶蛋白伴侣(rapamycin insensitive companion of mammalian target of rapamycin,RICTOR)基因表达后食管鳞癌细胞对依维莫司敏感性的变化,及其可能的分子机制。方法:蛋白质印迹法检测食管鳞癌ECa109、EC9706、TE1、KYSE450和KYSE790细胞中RICTOR蛋白的表达情况。采用脂质体转染法将携带有RICTOR-shRNA和Control-shRNA(对照组)的重组质粒分别转入ECa109细胞中,并筛选稳定转染的细胞株(命名为ECa109-RICTORshRNA和ECa109-control-shRNA)。采用CCK-8法检测沉默RICTOR基因表达后依维莫司对ECa109细胞增殖的影响,再用蛋白质印迹法检测磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB,又称Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)通路中相关因子RICTOR、Akt、磷酸化Akt(phospho-Akt,p-Akt)(Ser473)、核糖体蛋白S6激酶70 kDa肽(ribosome protein subunit 6 kinase of 70 kDa,p70S6K)、p-p70S6K、40 kDa富含脯氨酸的Akt底物(proline-rich Akt substrate of 40 kDa,PRAS40)和p-PRAS40(r246)蛋白的表达水平。最后,用ECa109-RICTOR-shRNA和ECa109-control-shRNA细胞建立裸鼠移植瘤模型,并用依维莫司进行治疗,观察依维莫司对裸鼠体内移植瘤生长的影响。结果:RICTOR蛋白在5株食管鳞癌细胞中均有表达,且在ECa109细胞中表达水平相对较高。与ECa109-control-shRNA细胞相比,ECa109-RICTOR-shRNA细胞的增殖速度明显变慢(P<0.05);依维莫司对ECa109-RICTOR-shRNA和ECa109-control-shRNA细胞的增殖均有明显抑制作用,依维莫司对ECa109-RICTOR-shRNA和ECa109-controlshRNA细胞的半数抑制浓度(half maximal inhibitory concentration,IC50)值分别为(17.68±1.25)μmol/L和(36.84±1.57)μmol/L。沉默RICTOR基因表达后,ECa109细胞中p-Akt(Ser473)和p-PRAS40(r246)蛋白的表达水平明显降低(P值均<0.001),而依维莫司使p-p70S6K蛋白的表达水平下调(P<0.001),但促进了p-Akt(Ser473)和p-PRAS40(r246)蛋白表达水平的上调(P值均<0.001);与ECa109-controlshRNA+依维莫司组相比,ECa109-RICTOR-shRNA+依维莫司组细胞中p-Akt(Ser473)和p-PRAS40(r246)蛋白的表达水平明显降低(P值均<0.001),p-p70S6K蛋白的表达水平无明显变化(P>0.05),提示RICTOR-shRNA能够抑制依维莫司引起的Akt和PRAS40蛋白的磷酸化激活。RICTOR-shRNA和依维莫司均能抑制裸鼠体内ECa109细胞移植瘤的生长(P值均<0.05),并以RICTOR-shRNA联合依维莫司对肿瘤生长的抑制作用最强(P<0.001)。结论:沉默RICTOR基因表达能够提高ECa109细胞对依维莫司的敏感性,其分子机制可能与下调RICTOR表达抑制了依维莫司引起的Akt和PRAS40的磷酸化有关。展开更多
基金supported by grants from National Key Research and Development Program of China(2022YFC2505201)National Natural Science Foundation of China(92157204,91957124,and 82250901)+1 种基金Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support(20161306,20171903 Round 2)Innovative Research Team of High-level Local University in Shanghai,and Program of Shanghai Academic Research Leader(20XD1403200,23XD1422400).
文摘mTORC1/2 play central roles as signaling hubs of cell growth and metabolism and are therapeutic targets for several diseases.However,the human genetic evidence linking mutations of mTORC1/2 to obesity remains elusive.Using whole-exome sequencing of 1944 cases with severe obesity and 2161 healthy lean controls,we identify a rare RICTOR p.I116V variant enriched in 9 unrelated cases.In Rictor null mouse embryonic fibroblasts,overexpression of the RICTOR p.I116V mutant increases phosphorylation of AKT,a canonical mTORC2 substrate,compared with wild-type RICTOR,indicating a gain-of-function change.Consistent with the human obesity phenotype,the knock-in mice carrying homogenous Rictor p.I116V variants gain more body weight under a high-fat diet.Additionally,the stromal vascular fraction cells derived from inguinal white adipose tissue of knock-in mice display an enhanced capacity for adipocyte differentiation via AKT activity.These findings demonstrate that the rare gain-of-function RICTOR p.I116V mutation activates AKT signaling,promotes adipogenesis,and contributes to obesity in humans.
