GW501516通过TGFβ-Smad3信号途径促进人脐静脉内皮细胞PAI-1的表达
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摘要
为探讨过氧化物酶体增殖物激活型受体δ(peroxisome proliferator-activated receptor-δ,PPARδ)激动剂GW501516对人脐静脉内皮细胞纤溶酶原激活物抑制剂1(PAI-1)表达的影响及机制,采用siRNA、TGFβ-Smad3信号通路阻滞剂等处理细胞,经实时定量PCR、Western blot方法分别检测细胞中PAI-1及磷酸化Smad3蛋白的表达.结果显示,与对照组比较,GW501516可诱导人脐静脉内皮细胞(HUVEC)中PAI-1表达,且此效应呈浓度和时间依赖性(P<0.05);siRNA沉默PPARδ的表达后,可阻抑GW501516对HUVEC细胞PAI-1表达的促进作用;TGFβ-Smad3信号通路抑制剂SB-431542与SIS3均可降低HUVEC细胞pSmad3蛋白的表达,而细胞PAI-1表达也随之降低.结果提示,GW501516可促进HUVEC细胞PAI-1的表达,其机制可能与TGFβ-Smad3信号通路有关.
Increased plasminogen activator inhibitor-1(PAI-1) level is the risk of thrombotic disease and atherosclerosis(As). Our research aims to study the effect and mechanism of PPARδ antagonist GW501516 on the expression of PAI-1. Firstly, human umbilical vein endothelial cells(HUVECs) were incubated with DMEM, and then treated with siRNA and TGFβ-Smad3 inhibitor, respectively. The protein and mRNA expression were examined by Western blotting assays and Real-time quantitative PCR, respectively. The result showed that GW501516 induced PAI-1 mRNA and protein expression in HUVECs compared with control groups(P < 0.05).According to PPARδ gene, the designed PPARδ siRNA primer silenced the PPARδ expression and depressed the induction of GW501516 on PAI-1 expression. Then, the HUVECs were treated by SB431542 or SIS3, which is the TGFβ-Smad3 signaling pathway blocker, and found that PAI-1 expression of cells were down-regulated.At last, we found that SB431542, SIS3 and GW501516 inhibited the expressions of pSmad3 protein. These results suggested that TGF beta-Smad3 signaling pathway involved in the regulation of GW501516-induced PAI-1 expression in HUVECs.
Increased plasminogen activator inhibitor-1(PAI-1) level is the risk of thrombotic disease and atherosclerosis(As). Our research aims to study the effect and mechanism of PPARδ antagonist GW501516 on the expression of PAI-1. Firstly, human umbilical vein endothelial cells(HUVECs) were incubated with DMEM, and then treated with siRNA and TGFβ-Smad3 inhibitor, respectively. The protein and mRNA expression were examined by Western blotting assays and Real-time quantitative PCR, respectively. The result showed that GW501516 induced PAI-1 mRNA and protein expression in HUVECs compared with control groups(P < 0.05).According to PPARδ gene, the designed PPARδ siRNA primer silenced the PPARδ expression and depressed the induction of GW501516 on PAI-1 expression. Then, the HUVECs were treated by SB431542 or SIS3, which is the TGFβ-Smad3 signaling pathway blocker, and found that PAI-1 expression of cells were down-regulated.At last, we found that SB431542, SIS3 and GW501516 inhibited the expressions of pSmad3 protein. These results suggested that TGF beta-Smad3 signaling pathway involved in the regulation of GW501516-induced PAI-1 expression in HUVECs.
引文
[1]Zhou B,Pan Y,Yu Q,et al.Fibrinogen facilitates atherosclerotic formation in Sprague-Dawley rats:a rodent model of atherosclerosis.Exp Ther Med,2013,5(3):730-734
[2]Tosetto A,Prati P,Baracchini C,et al.Association of plasma fibrinogen,C-reactive protein and G-455>A polymorphism with early atherosclerosis in the VITA Project cohort.Thromb Haemost,2011,105(2):329-335
[3]Zhou B R,Pan Y,Zhai Z M.Fibrinogen and P-selectin expression in atherosclerosis model of Sprague Dawley rat.Chin Med J(Engl),2011,124(22):3768-3772
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[6]Lee C H,Olson P,Hevener A,et al.PPARdelta regulates glucose metabolism and insulin sensitivity.Proc Natl Acad Sci USA,2006,103(9):3444-3449
[7]Hu C,Jia W,Fang Q,et al.Peroxisome proliferator-activated receptor(PPAR)delta genetic polymorphism and its association with insulin resistance index and fasting plasma glucose concentrations in Chinese subjects.Diabet Med,2006,23(12):1307-1312
[8]Lee C H,Chawla A,Urbiztondo N,et al.Transcriptional repression of atherogenic inflammation:modulation by PPARdelta.Science,2003,302(5644):453-457
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[10]Ye P,Hu X,Liu Y,et al.Activation of peroxisome proliferatoractivated receptor alpha in human endothelial cells increases plasminogen activator inhibitor type-1 expression.Chin Med J(Engl),2003,116(1):29-33
[11]Kim H J,Ham S A,Kim S U,et al.Transforming growth factor-beta1 is a molecular target for the peroxisome proliferatoractivated receptor delta.Circ Res,2008,102(2):193-200
[12]周寿红,杨旭红,宋涛,等.亚毒性剂量毒死蜱降低新西兰兔ABCA1表达加重高脂诱导的动脉粥样硬化.生物化学与生物物理进展,2011,38(1):46-54Zhou S H,Yang X H,Song T,et al.Prog Biochem Biophys,2011,38(1):46-54
[13]叶国红,韩莲花,李红霞,等.探讨血管紧张素Ⅱ对人脐静脉内皮细胞产生超氧阴离子和1型纤溶酶原激活物抑制剂的作用机制.中国循环杂志,2012,27(2):141-144Ye G H,Han L H,Li H X,et al.Chinese Circulation Journal,2012,27(2):141-144
[14]Ekmekci H,Gungor O Z,Ekmekci O B,et al.Significance of vitronectin and PAI-1 activity levels in carotid artery disease:comparison of symptomatic and asymptomatic patients.Minerva Med,2013,104(2):215-223
[15]Sakata T,Mannami T,Baba S,et al.Potential of free-form TFPI and PAI-1 to be useful markers of early atherosclerosis in a Japanese general population(the Suita Study):association with the intimal-medial thickness of carotid arteries.Atherosclerosis,2004,176(2):355-360
[16]Cesari M,Pahor M,Incalzi R A.Plasminogen activator inhibitor-1(PAI-1):a key factor linking fibrinolysis and age-related subclinical and clinical conditions.Cardiovasc Ther,2010,28(5):e72-e91
[17]Naya M,Tsukamoto T,Inubushi M,et al.Elevated plasma plasminogen activator inhibitor type-1 is an independent predictor of coronary microvascular dysfunction in hypertension.Circ J,2007,71(3):348-353
[18]欧阳琳,彭佑铭,伍国宝,等.PAI-1和ET-1在维持性血液透析患者动脉硬化中的作用.中南大学学报(医学版),2013,38(5):458-467Ouyang L,Peng Y,Wu G,et al.J Central South University (Medical Sciences),2013,38(5):458-467
[19]Ho F M,Liu S H,Liau C S,et al.High glucose-induced apoptosis in human endothelial cells is mediated by sequential activations of c-Jun NH(2)-terminal kinase and caspase-3.Circulation,2000,101(22):2618-2624
[20]Batista F A,Trivella D B,Bernardes A,et al.Structural insights into human peroxisome proliferator activated receptor delta(PPAR-delta)selective ligand binding.PLoS One,2012,7(5):e33643
[21]Sprecher D L,Massien C,Pearce G,et al.Triglyceride:high-density lipoprotein cholesterol effects in healthy subjects administered a peroxisome proliferator activated receptor delta agonist.Arterioscler Thromb Vasc Biol,2007,27(2):359-365
[22]Serrano-Marco L,Barroso E,El K I,et al.The peroxisome proliferator-activated receptor(PPAR)beta/delta agonist GW501516 inhibits IL-6-induced signal transducer and activator of transcription 3(STAT3)activation and insulin resistance in human liver cells.Diabetologia,2012,55(3):743-751
[23]Defaux A,Zurich M G,Braissant O,et al.Effects of the PPAR-beta agonist GW501516 in an in vitro model of brain inflammation and antibody-induced demyelination.J Neuroinflammation,2009,6(1):15-28
[24]Li H,Ding Y,Yi G,et al.Establishment of nasal tolerance to heat shock protein-60 alleviates atherosclerosis by inducing TGF-beta-dependent regulatory T cells.J Huazhong Univ Sci Technolog Med Sci,2012,32(1):24-30
[25]van Dijk R A,Engels C C,Schaapherder A F,et al.Visualizing TGF-beta and BMP signaling in human atherosclerosis:a histological evaluation based on Smad activation.Histol Histopathol,2012,27(3):387-396
[26]Mccaffrey T A.TGF-beta signaling in atherosclerosis and restenosis.Front Biosci(Schol Ed),2009,S1(1):236-245
[27]Edlin R S,Tsai S,Yamanouchi D,et al.Characterization of primary and restenotic atherosclerotic plaque from the superficial femoral artery:Potential role of Smad3 in regulation of SMC proliferation.J Vasc Surg,2009,49(5):1289-1295
[28]Kim H J,Kim M Y,Jin H,et al.Peroxisome proliferator-activated receptor{delta}regulates extracellular matrix and apoptosis of vascular smooth muscle cells through the activation of transforming growth factor-{beta}1/Smad3.Circ Res,2009,105(1):16-24
[2]Tosetto A,Prati P,Baracchini C,et al.Association of plasma fibrinogen,C-reactive protein and G-455>A polymorphism with early atherosclerosis in the VITA Project cohort.Thromb Haemost,2011,105(2):329-335
[3]Zhou B R,Pan Y,Zhai Z M.Fibrinogen and P-selectin expression in atherosclerosis model of Sprague Dawley rat.Chin Med J(Engl),2011,124(22):3768-3772
[4]Bicakcigil M,Tasan D,Tasdelen N,et al.Role of fibrinolytic parameters and plasminogen activator inhibitor 1(PAI-1)promoter polymorphism on premature atherosclerosis in SLE patients.Lupus,2011,20(10):1063-1071
[5]Peng Y,Liu H,Liu F,et al.Atherosclerosis is associated with plasminogen activator inhibitor type-1 in chronic haemodialysis patients.Nephrology(Carlton),2008,13(7):579-586
[6]Lee C H,Olson P,Hevener A,et al.PPARdelta regulates glucose metabolism and insulin sensitivity.Proc Natl Acad Sci USA,2006,103(9):3444-3449
[7]Hu C,Jia W,Fang Q,et al.Peroxisome proliferator-activated receptor(PPAR)delta genetic polymorphism and its association with insulin resistance index and fasting plasma glucose concentrations in Chinese subjects.Diabet Med,2006,23(12):1307-1312
[8]Lee C H,Chawla A,Urbiztondo N,et al.Transcriptional repression of atherogenic inflammation:modulation by PPARdelta.Science,2003,302(5644):453-457
[9]Banfi C,Eriksson P,Giandomenico G,et al.Transcriptional regulation of plasminogen activator inhibitor type 1 gene by insulin:insights into the signaling pathway.Diabetes,2001,50(7):1522-1530
[10]Ye P,Hu X,Liu Y,et al.Activation of peroxisome proliferatoractivated receptor alpha in human endothelial cells increases plasminogen activator inhibitor type-1 expression.Chin Med J(Engl),2003,116(1):29-33
[11]Kim H J,Ham S A,Kim S U,et al.Transforming growth factor-beta1 is a molecular target for the peroxisome proliferatoractivated receptor delta.Circ Res,2008,102(2):193-200
[12]周寿红,杨旭红,宋涛,等.亚毒性剂量毒死蜱降低新西兰兔ABCA1表达加重高脂诱导的动脉粥样硬化.生物化学与生物物理进展,2011,38(1):46-54Zhou S H,Yang X H,Song T,et al.Prog Biochem Biophys,2011,38(1):46-54
[13]叶国红,韩莲花,李红霞,等.探讨血管紧张素Ⅱ对人脐静脉内皮细胞产生超氧阴离子和1型纤溶酶原激活物抑制剂的作用机制.中国循环杂志,2012,27(2):141-144Ye G H,Han L H,Li H X,et al.Chinese Circulation Journal,2012,27(2):141-144
[14]Ekmekci H,Gungor O Z,Ekmekci O B,et al.Significance of vitronectin and PAI-1 activity levels in carotid artery disease:comparison of symptomatic and asymptomatic patients.Minerva Med,2013,104(2):215-223
[15]Sakata T,Mannami T,Baba S,et al.Potential of free-form TFPI and PAI-1 to be useful markers of early atherosclerosis in a Japanese general population(the Suita Study):association with the intimal-medial thickness of carotid arteries.Atherosclerosis,2004,176(2):355-360
[16]Cesari M,Pahor M,Incalzi R A.Plasminogen activator inhibitor-1(PAI-1):a key factor linking fibrinolysis and age-related subclinical and clinical conditions.Cardiovasc Ther,2010,28(5):e72-e91
[17]Naya M,Tsukamoto T,Inubushi M,et al.Elevated plasma plasminogen activator inhibitor type-1 is an independent predictor of coronary microvascular dysfunction in hypertension.Circ J,2007,71(3):348-353
[18]欧阳琳,彭佑铭,伍国宝,等.PAI-1和ET-1在维持性血液透析患者动脉硬化中的作用.中南大学学报(医学版),2013,38(5):458-467Ouyang L,Peng Y,Wu G,et al.J Central South University (Medical Sciences),2013,38(5):458-467
[19]Ho F M,Liu S H,Liau C S,et al.High glucose-induced apoptosis in human endothelial cells is mediated by sequential activations of c-Jun NH(2)-terminal kinase and caspase-3.Circulation,2000,101(22):2618-2624
[20]Batista F A,Trivella D B,Bernardes A,et al.Structural insights into human peroxisome proliferator activated receptor delta(PPAR-delta)selective ligand binding.PLoS One,2012,7(5):e33643
[21]Sprecher D L,Massien C,Pearce G,et al.Triglyceride:high-density lipoprotein cholesterol effects in healthy subjects administered a peroxisome proliferator activated receptor delta agonist.Arterioscler Thromb Vasc Biol,2007,27(2):359-365
[22]Serrano-Marco L,Barroso E,El K I,et al.The peroxisome proliferator-activated receptor(PPAR)beta/delta agonist GW501516 inhibits IL-6-induced signal transducer and activator of transcription 3(STAT3)activation and insulin resistance in human liver cells.Diabetologia,2012,55(3):743-751
[23]Defaux A,Zurich M G,Braissant O,et al.Effects of the PPAR-beta agonist GW501516 in an in vitro model of brain inflammation and antibody-induced demyelination.J Neuroinflammation,2009,6(1):15-28
[24]Li H,Ding Y,Yi G,et al.Establishment of nasal tolerance to heat shock protein-60 alleviates atherosclerosis by inducing TGF-beta-dependent regulatory T cells.J Huazhong Univ Sci Technolog Med Sci,2012,32(1):24-30
[25]van Dijk R A,Engels C C,Schaapherder A F,et al.Visualizing TGF-beta and BMP signaling in human atherosclerosis:a histological evaluation based on Smad activation.Histol Histopathol,2012,27(3):387-396
[26]Mccaffrey T A.TGF-beta signaling in atherosclerosis and restenosis.Front Biosci(Schol Ed),2009,S1(1):236-245
[27]Edlin R S,Tsai S,Yamanouchi D,et al.Characterization of primary and restenotic atherosclerotic plaque from the superficial femoral artery:Potential role of Smad3 in regulation of SMC proliferation.J Vasc Surg,2009,49(5):1289-1295
[28]Kim H J,Kim M Y,Jin H,et al.Peroxisome proliferator-activated receptor{delta}regulates extracellular matrix and apoptosis of vascular smooth muscle cells through the activation of transforming growth factor-{beta}1/Smad3.Circ Res,2009,105(1):16-24