Effect Of Akt/mTOR/c-Myc Signaling On CD137 Signaling Mediated Aerobic Glycolysis Of Mouse PAEC

ObjectiveTo observe whether the CD137 signaling promoting cell proliferation by modulating aerobic glycolysis through Akt/mTOR/c-Myc pathway.Methods 1.Mouse pulmonary artery endothelial cell line was used to establish cell model.2.Flow Cytometry and western blot were used separately to measure the level of membrane CD137 and total CD137 of PAEC.3.The protein levels of glycolytic enzymes such as hexokinase(HK2),fructose-2,6-diphosphatase 3(PFKFB3)were measured by Western blot.The enzyme activity of HK2 and PFKFB3 was detected by enzymatic analysis.4.Glucose oxidase method was used for measuring glucose uptake rate and lactate colorimetric assay for lactic acid production.5.The Phospo-level and total protein level of Akt/mTOR and protein level of c-Myc were detected by western blot.6.Cell Counting Kit 8 test and EdU incorporation were applied to detect proliferation of PAEC.7.Flow Cytometry was used to measure cell cycle of PAEC.Results1.Compared with control group,TNF-?(10ng/ml),ET-1(10 mmol/L)and 5-HT((1?mol/L)significantly increased the expression of CD137 membrane protein and total protein in the PAECs(P<0.05).2.Activation of CD137 signal promotes PAEC aerobic glycolysis: western blot showed that the protein level of key glycolytic enzymes HK2,PFKFB3 of the CD137 agonist group was significantly increased compared with the control group;The results of enzymatic analysis showed that the activity of key enzymes of HK2 and PFKFB3 in the CD137 agonist group was obviously increased compared with the control group.Compared with the control group,the glucose uptake and lactic acid production in the CD137 agonist group increased significantly.3.Activation of CD137 signal promotes PAEC proliferation in a time-dependent manner,and pre-incubation of glycolytic inhibitor 2-DG blocks the promoting effects on proliferation of CD137 signaling: activation of CD137 signaling promotes the expression of cyclinD1 protein,decreased the expression of P21 and P27,G1 phase negative regulatory proteins.CD137 signaling mediated p21 and p27 downregulation and cyclinD1 upregulation were reversed by 2-DG,a glycolysis inhibitor.Consistent with cell cycle protein,flow cytometry analysis showed that more PAECs entered S phase from G0/G1 phase upon activation of CD137 signaling,while pre-incubation of 2-DG block the cell cycle in the G1 phase.4.CD137 signaling activates the expression of Akt/ mTOR and c-Myc: compared with the control group,the expression of phosphorylated Akt(p-Akt)and mTOR(p-mTOR)protein were significantly increased after activation of CD137 signaling,and protein expression o f c-Myc increased(P < 0.05).5.Akt inhibitor can attenuate the expression of mTOR and c-Myc induced by CD137 signaling: pre-incubation of Akt inhibitor MK-2206 followed by activating CD137 signaling,not only inhibited the expression of p-Akt,but also decreased the activation of p-mTOR and the expression of c-Myc.6.mTOR inhibitor reduced the expression of c-Myc and increased glycolysis induced by CD137 signaling in vitro: pre-incubation of mTOR inhibitor rapamycin,then activating CD137 signaling,the expression of c-Myc decreased,and the expression of glycolysis key enzymes HK2,PFKFB3 declined.7.c-Myc inhibitor can reduce the enhanced glycolysis induced by CD137 signaling in vitro: pre-incubation of c-Myc inhibitor,followed by activation of CD137 signaling,led to the decreased glucose enzyme activity of glycolytic key enzyme HK2,PFKFB3,glucose consumption and lactate production,significantly(P < 0.05).ConclusionsCD137 signaling regulates aerobic glycolysis of mouse PAEC,promoting cell proliferation by Akt/mTOR/c-Myc signaling.