信号转导和转录活化因子3通过趋化因子CX3C配体1促进血管内皮细胞增殖迁移
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摘要
信号转导和转录活化因子3 (STAT3)与趋化因子CX3C配体1 (Fractalkine/CX3CL1)在血管炎症和损伤中起重要作用,为了探讨STAT3是否通过CX3CL1促进血管内皮细胞增殖和迁移,在血管内皮细胞(HUVEC)中过表达或敲降STAT3,通过quantitative real-time PCR、Western blotting实验确定STAT3对CX3CL1表达的影响。构建含有STAT3结合位点及突变STAT3结合位点的CX3CL1启动子荧光素酶报告基因质粒,利用荧光素酶活性分析实验研究STAT3对CX3CL1启动子转录活性的作用。利用MTT实验检测过表达或敲降STAT3或CX3CL1对血管内皮细胞增殖率的影响。利用划痕实验检测过表达或敲降STAT3或CX3CL1对血管内皮细胞迁移率的影响。结果显示,过表达STAT3可以促进CX3CL1表达,敲降STAT3可以使CX3CL1表达下调。STAT3可以直接结合到CX3CL1的启动子促进其转录激活,其促进作用依赖于CX3CL1启动子上的GAS位点。敲降STAT3可以抑制血管内皮细胞的迁移,过表达CX3CL1拮抗该抑制作用。总结得出,STAT3通过结合到CXCL1启动子促进CX3CL1转录与表达进而促进血管内皮的增殖与迁移。
Signal transducer and activator of transcription 3(STAT3) and Chemokine CX3C ligand 1(Fractalkine/CX3CL1)play important roles in vascular inflammation and injury. To study if STAT3 promotes vascular endothelial cell proliferation and migration through fractalkine, we overexpressed or knocked down STAT3 in vascular endothelial cells, and used quantitative real-time PCR and Western blotting to determine the effect of STAT3 on fractalkine expression. The wild type and STAT3 binding site mutant fractalkine promoter luciferase reporter plasmids were constructed, and luciferase activity assays were used to explore the effect of STAT3 on the transcriptional activity of the fractalkine promoter. MTT assays were used to detect the effect of overexpression or knockdown of STAT3 or fractalkine on the proliferation rate of vascular endothelial cells. Scratch assays were used to detect the effect of overexpression or knockdown of STAT3 or fractalkine on vascular endothelial cell migration. There results showed that overexpression of STAT3 could promote fractalkine expression,and knockdown of STAT3 could down-regulate fractalkine expression. STAT3 could directly bind to the promoter of fractalkine to promote its transcriptional activity via binding the GAS site of the fractalkine promoter. Knockdown of STAT3 could inhibit the migration of vascular endothelial cell, and overexpression of fractalkine antagonized this inhibition. Our data concluded that STAT3 promotes the proliferation and migration of vascular endothelial cell by binding the GAS site of the fractalkine promoter to promote fractalkine transcriptional activity and expression.
Signal transducer and activator of transcription 3(STAT3) and Chemokine CX3C ligand 1(Fractalkine/CX3CL1)play important roles in vascular inflammation and injury. To study if STAT3 promotes vascular endothelial cell proliferation and migration through fractalkine, we overexpressed or knocked down STAT3 in vascular endothelial cells, and used quantitative real-time PCR and Western blotting to determine the effect of STAT3 on fractalkine expression. The wild type and STAT3 binding site mutant fractalkine promoter luciferase reporter plasmids were constructed, and luciferase activity assays were used to explore the effect of STAT3 on the transcriptional activity of the fractalkine promoter. MTT assays were used to detect the effect of overexpression or knockdown of STAT3 or fractalkine on the proliferation rate of vascular endothelial cells. Scratch assays were used to detect the effect of overexpression or knockdown of STAT3 or fractalkine on vascular endothelial cell migration. There results showed that overexpression of STAT3 could promote fractalkine expression,and knockdown of STAT3 could down-regulate fractalkine expression. STAT3 could directly bind to the promoter of fractalkine to promote its transcriptional activity via binding the GAS site of the fractalkine promoter. Knockdown of STAT3 could inhibit the migration of vascular endothelial cell, and overexpression of fractalkine antagonized this inhibition. Our data concluded that STAT3 promotes the proliferation and migration of vascular endothelial cell by binding the GAS site of the fractalkine promoter to promote fractalkine transcriptional activity and expression.
引文
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[24]Matsuura T,Ichinose S,Akiyama M,et al.Involvement of CX3CL1 in the migration of osteoclast precursors across osteoblast layer stimulated by interleukin‐1?.JCell Physiol,2017,232(7):1739-1745.
[25]Garcia GE,Xia YY,Chen SZ,et al.NF-κB-dependent fractalkine induction in rat aortic endothelial cells stimulated by IL-1β,TNF-α,and LPS.J Leukocyte Biol,2000,67(4):577-584.
[26]Jang J,Yoon Y,Oh DJ.A calpain inhibitor protects against fractalkine production in lipopolysaccharidetreated endothelial cells.Kidney Res Clin Pract,2017,36(3):224-231.
[2]Kiu H,Nicholson SE.Biology and significance of the JAK/STAT signalling pathways.Growth Factors,2012,30(2):88-106.
[3]O’Shea JJ,Schwartz DM,Villarino AV,et al.The JAK-STAT pathway:impact on human disease and therapeutic intervention.Annu Rev Med,2015,66:311-328.
[4]Zhao GB,Zhu GW,Huang YJ,et al.IL-6 mediates the signal pathway of JAK-STAT3-VEGF-C promoting growth,invasion and lymphangiogenesis in gastric cancer.Oncol Rep,2016,35(3):1787-1795.
[5]postolakis S,Spandidos D.Chemokines and atherosclerosis:focus on the CX3CL1/CX3CR1 pathway.Acta Pharmacol Sin,2013,34(10):1251-1256.
[6]Flierl U,Bauersachs J,Sch?fer A.Modulation of platelet and monocyte function by the chemokine fractalkine(CX3CL1)in cardiovascular disease.Eur J Clin Invest,2015,45(6):624-633.
[7]Wong BW,Wong D,Mcmanus BM.Characterization of fractalkine(CX3CL1)and CX3CR1 in human coronary arteries with native atherosclerosis,diabetes mellitus,and transplant vascular disease.Cardiovasc Pathol,2002,11(6):332-338.
[8]Furio E,García-Fuster MJ,Redon J,et al.CX3CR1/CX3CL1 Axis mediates platelet-leukocyte adhesion to arterial endothelium in younger patients with a history of idiopathic deep vein thrombosis.Thromb Hhemostasis,2018,118(3):562-571.
[9]Ou D,He RZ.Research progress of cytokines in the pathogenesis of pulmonary hypertension.Guangzhou Med J,2016,47(2):100-102(in Chinese).欧东,贺仁忠.细胞因子在肺动脉高压发病中作用的研究进展.广州医药,2016,47(2):100-102.
[10]Rowinska Z,Koeppel TA,Sanati M,et al.Role of the CX3C chemokine receptor CX3CR1 in the pathogenesis of atherosclerosis after aortic transplantation.PLoSONE,2017,12(2):e0170644.
[11]McDermott DH,Fong AM,Yang Q,et al.Chemokine receptor mutant CX3CR1-M280 has impaired adhesive function and correlates with protection from cardiovascular disease in humans.J Clin Invest,2003,111(8):1241-1250.
[12]Gan AM,Butoi E,Manea A,et al.Functional analysis of the fractalkine gene promoter in human aortic smooth muscle cells exposed to proinflammatory conditions.FEBS J,2015,281(17):3869-3881.
[13]Park HJ,Zhang YL,Georgescu SP,et al.Human umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new insights into the relationship between lipid metabolism and angiogenesis.Stem Cell Rev,2006,2(2):93-101.
[14]O'Shea JJ,Plenge R.JAK and STAT signaling molecules in immunoregulation and immune-mediated disease.Immunity,2012,36(4):542-550.
[15]Matsui F,Meldrum KK.The role of the Janus kinase family/signal transducer and activator of transcription signaling pathway in fibrotic renal disease.J Surg Res,2012,178(1):339-345.
[16]Li F,Xia K,Ali Sheikh MS,et al.Retinol binding protein 4 promotes hyperinsulinism-induced proliferation of rat aortic smooth muscle cells.Mol Med Rep,2014,9(5):1634-1640.
[17]Lim S,Park S.Role of vascular smooth muscle cell in the inflammation of atherosclerosis.BMB Rep,2014,47(1):1-7.
[18]Huang LY,Ma BW,Ma JW,et al.Fractalkine/CX3CR1axis modulated the development of pancreatic ductal adenocarcinoma via JAK/STAT signaling pathway.Biochem Biophys Res Commun,2017,493(4):1510-1517.
[19]Clark AK,Malcangio M.Fractalkine/CX3CR1 signaling during neuropathic pain.Front Cell Neurosci,2014,8:121.
[20]Yin XY,Zhang SF,Yu D,et al.Circulating fractalkine levels predict the development of the metabolic syndrome.Int J Endocrinol,2014,2014:715148.
[21]Zhang HL,Guo CF,Wu DJ,et al.Hydrogen sulfide inhibits the development of atherosclerosis with suppressing CX3CR1 and CX3CL1 expression.PLoSONE,2012,7(7):e41147.
[22]Imai T,Yasuda N.Therapeutic intervention of inflammatory/immune diseases by inhibition of the fractalkine(CX3CL1)-CX3CR1 pathway.Inflammat Regenerat,2016,36:9.
[23]Ebaid H,Salem A,Sayed A,et al.Whey protein enhances normal inflammatory responses during cutaneous wound healing in diabetic rats.Lipids Health Dis,2011,10(1):235-235.
[24]Matsuura T,Ichinose S,Akiyama M,et al.Involvement of CX3CL1 in the migration of osteoclast precursors across osteoblast layer stimulated by interleukin‐1?.JCell Physiol,2017,232(7):1739-1745.
[25]Garcia GE,Xia YY,Chen SZ,et al.NF-κB-dependent fractalkine induction in rat aortic endothelial cells stimulated by IL-1β,TNF-α,and LPS.J Leukocyte Biol,2000,67(4):577-584.
[26]Jang J,Yoon Y,Oh DJ.A calpain inhibitor protects against fractalkine production in lipopolysaccharidetreated endothelial cells.Kidney Res Clin Pract,2017,36(3):224-231.