17β雌二醇对氧化应激诱导的H9C2细胞凋亡的影响及其调控机制的研究
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摘要
缺血性心脏病及其引起的心肌梗死和充血性心力衰竭是疾病死亡的首要原因。绝经前女性缺血性心脏病的发病率明显低于男性,绝经后女性发病率迅速上升,甚至与男性相同,提示女性性激素,如雌激素对心血管具有有益的作用,流行病学证据、动物以及体外实验也支持雌激素具有心血管保护作用。基础研究显示雌激素在生物的发育、生长以及分化中具有重要作用,能够通过调控多种信号通路影响细胞的增殖及凋亡,其中包括转化生长因子β(Transforming growth factor-β,TGF-β)信号转导通路。关于二者对肿瘤细胞的相互影响及调控机制研究较多,但对于二者对心肌细胞影响研究较少。氧化应激是多种心脏疾病引起心肌肥厚、凋亡以及重构共同的病理生理机制,目前已有研究发现雌激素具有一定的抗氧化作用。本研究以来源于大鼠胚胎心肌细胞的H9c2细胞为模型,观察雌激素对过氧化氢诱导的心肌细胞氧化应激和凋亡的影响,及其对TGF-β信号通路的作用,探讨雌激素调控心肌细胞凋亡的机制及其生物学意义。
目的:观察雌二醇(Estradiol,E_2)对过氧化氢诱导的心肌细胞氧化应激和凋亡的影响,及对TGF-β1信号通路的作用,探讨雌激素调控心肌细胞凋亡的机制及其生物学意义。
方法:以大鼠胚胎心肌细胞系H9c2细胞作为细胞模型,1)采用MTT法以及流式细胞仪检测法,以过氧化氢作(H_2O_2)为外源性氧化应激刺激,观察其对H9c2细胞活性和凋亡、细胞内活性氧(ROS)水平的影响,以及E2对心肌细胞的保护作用;RT-PCR法检测H9c2细胞中雌激素受体α(ERα)和雌激素受体β(ERβ)mRNA的表达情况;2)Realtime RT-PCR以及Western blot法检测E_2对H9c2细胞TGF-β1,转化生长因子βⅠ型受体(TβRⅠ)以及转化生长因子βⅡ型受体(TβRⅡ)mRNA以及蛋白表达的影响,Western blot检测E_2对TβR受体后磷酸化smad2/3水平的影响。
结果:1)MTT分析显示外源性H_2O_2呈浓度依赖性诱导H9c2细胞活性下降(P＜0.05),选择100μM H_2O_2处理4小时作为后续试验的诱导条件;单纯应用不同浓度E2(50-200nM)处理H9c2细胞对其活性均无明显影响(P＞0.05);应用200nM E2预处理H9c2细胞24小时可明显减轻H_2O_2对H9c2细胞活性的损伤(P＜0.05),时相效应分析显示保护作用16小时达高峰可持续至24小时;应用不同浓度E_2(50-200nM)预处理24小时,E_2呈浓度依赖性抑制H_2O_2对H9c2细胞活性的损伤;依此结果选择200nM E2预处理24小时作为后续实验的干预条件。
2)应用流式细胞仪检测H_2O_2诱导的H9c2细胞凋亡、ROS生成及E2的干预作用,结果显示,外源性H_2O_2可引起H9c2细胞凋亡及ROS生成明显增加,与对照组比较差异明显(P＜0.05);E_2预处理能够明显减轻H_2O_2诱导的H9c2细胞凋亡,差异具有显著性(P＜0.05)。
3)RT-PCR鉴定H9c2细胞ERα和ERβ的表达情况,发现H9c2细胞仅表达ERβ,几乎不表达ERα,推测E2对H9c2细胞的抗凋亡保护作用可能是通过ERβ介导。
4)Realtime RT-PCR以及Western blot检测结果显示应用200nM E2处理H9c2细胞8小时后可下调TβRⅠ以及TβRⅡmRNA的表达,处理16小时后下调TβRⅠ以及TβRⅡ蛋白表达,但是对于TGF-β1mRNA及蛋白表达均无明显影响,给予ER阻断剂ICI 182,780阻断E_2与ER的结合,能够消除E2对TβRI及TβRⅡmRNA的下调作用。
5)Western blot检测p-smad2/3蛋白水平显示,E2处理16小时后p-smad2/3蛋白水平降低。
结论:
1)外源性H_2O_2引起氧化应激能够引起细胞内ROS增加,损伤H9c2细胞活性,增加H9c2细胞的凋亡。外源性E2本身并不能影响H9c2细胞凋亡,但能够降低H9c2细胞对H_2O_2的敏感性,保护细胞抵抗氧化应激引起的凋亡。
2)外源性E2对H9c2细胞的TβRⅠ以及TβRⅡ具有下调作用,阻断ER可消除E2对TβRⅠ和TβRⅡ的下调作用;E2可进一步通过TGF-β通路降低受体后磷酸化smad2/3的水平;H9c2细胞仅表达ERβ,提示E2可能是通过ERβ发挥其对TGF-β通路的调控作用。
Backgound:Ischemic heart disease with subsequent myocardial infarction and congestive heart failure is the leading cause of mortality.The incidence of ischemic heart disease is significantly lower in women than in men until menopause,after which the cardiovascular risk of women accelerates to equal that of men.This observation suggests a possibility that female sex hormones,such as estrogen,may have a favorable cardiovascular role.There is evidence from epidemiological,animal,and in vitro studies that estrogen may be cardioprotective.However,the mechanisms involved in this process,are poorly understood.It has been show that estradiol has important role on development, growth and differentiation of organism.It can affect proliferation and apoptosis of cells by modulating several signaling pathway,including transforming growth factor-β(TGF-β)signaling.There are several studies on the interaction between E_2 and TGF-βsignaling in tumor cells,but few in myocardial cells.Oxidative stress is the common pathophysiologic mechanism of myocardial hypertrophy, apoptosis and remodeling caused by miscellaneous heart diseases.It is indicated that estradiol has somewhat anti-oxidative effect.So we observed the effect of estradiol on oxidative stress and apoptosis of myocardial cell and on modulating TGF-βsignaling pathway,in order to explore the mechanism of myocardial cell apoptosis modulated by estradiol and its biological significance.
Objective:To explore the mechanism of apoptosis modulated by estradiol in myocardial cells and its biological significant by observing the effect of estradiol on oxidative stress and apoptosis of myocardial cells induced by H_2O_2 and its modulate effect on TGF-βsignaling pathway.
Methods:Use H9c2 cells,a clonal line derived from embryonic rat heart,as the cell model.1)To observe the effect on cell viability,apoptosis and generation of reactive oxygen speciesk(ROS)in H9c2 cells stimulated by H_2O_2 and the protective effect of estradiol on myocardial cells by means of MTT assay and flow cytometry.Expression of ERαmRNA and ERβmRNA「in H9C2 cells was detected by RT-PCR.2)Real-time RT-PCR and Western blot were performed to examine The expression of TGF-β1,TβRⅠand TβRⅡin H9c2 cells treated with 200riM E2 for different hours.The level of phosphorylation of Smad2/3 was detected by western blot.
Results:
1)MTT assay shows that exogenous H_2O_2 induces decreasing of cell viability of H9C2 cells in a dose-dependent manner(P＜0.05),and 100μM H_2O_2 treating for 4 hours was chosen as inducing condition for the following experiments.Various concentrations of E_2 alone has no effect on the cell viability of H9c2 cells(P＞0.05).Exogenous H_2O_2 increasing the apoptosis rate and ROS generation.While H9c2 cells pretreated with 200nM E_2 for 24 hours significantly attenuate the cell viability injury caused by H_2O_2.Time-phase analysis shows that the maximal protective effect achieved at 16 hours,and last for 24 hours.E_2 inhibited the cell viability injury caused by H_2O_2 in a dose dependent manner.So we choose 200nM E_2 treating for 24 hours as the intervention treatment in the follow experiment.
2)Exogenous H_2O_2 may induce increased apoptosis and ROS generation of H9c2 cells,there is a significant difference compared with control group.E_2 pretreatment can significantly extenuate apoptosis and ROS generation of H9C2 cells induced by H_2O_2(P＜0.05).
3)Determination of estradiol receptor expression of H9C2 cells by RT-PCR finds that H9C2 cells only express ERβ,almost express no ERα,and suggests that anti-apoptosis effect of estradiol on H9C2 cells may be mediated by ERβ.
4)Detection of Realtime RT-PCR and western blot finds that H9C2 cells pretreated by estradiol for 8 hours may downregulate the expression of TβRⅠand TβRⅡmRNA,and downregulate of protein expression occurred at 16 hours.But it has no effect on the expression of TGF-β1.ER blocker ICI182,780 can block the binding of E_2 and ER,and can delete the downregulation effect of E_2 on TβRⅠand TβRⅡ.
5)Western blot exanmination shows that the p-smad2/3 level in H9c2 cells decreased pretreated by estradiol for 16 hours H9c2 cells.
Conclusion:
1)Oxidative stress caused by exogenous H_2O_2 can increase the ROS generation,damage the cell viability and induce apoptosis of H9c2 cells. Exogenous E2 alone has no effect on cell viability and apoptosis of H9C2 cells, but it can decrease the sensitivity of H9C2 cells to H_2O_2,hence,protect cells against apoptosis caused by oxidative stress.
2)Exogenous E_2 has downregulation effect on TβRⅠand TβRⅡof H9c2 cells,blocking ER may delete this effect of estradiol。H9c2 cells express ERβonly,suggests that E_2 may exert modulating effect on TGF-βpathway through ERβ。
目的:观察雌二醇(Estradiol,E_2)对过氧化氢诱导的心肌细胞氧化应激和凋亡的影响,及对TGF-β1信号通路的作用,探讨雌激素调控心肌细胞凋亡的机制及其生物学意义。
方法:以大鼠胚胎心肌细胞系H9c2细胞作为细胞模型,1)采用MTT法以及流式细胞仪检测法,以过氧化氢作(H_2O_2)为外源性氧化应激刺激,观察其对H9c2细胞活性和凋亡、细胞内活性氧(ROS)水平的影响,以及E2对心肌细胞的保护作用;RT-PCR法检测H9c2细胞中雌激素受体α(ERα)和雌激素受体β(ERβ)mRNA的表达情况;2)Realtime RT-PCR以及Western blot法检测E_2对H9c2细胞TGF-β1,转化生长因子βⅠ型受体(TβRⅠ)以及转化生长因子βⅡ型受体(TβRⅡ)mRNA以及蛋白表达的影响,Western blot检测E_2对TβR受体后磷酸化smad2/3水平的影响。
结果:1)MTT分析显示外源性H_2O_2呈浓度依赖性诱导H9c2细胞活性下降(P＜0.05),选择100μM H_2O_2处理4小时作为后续试验的诱导条件;单纯应用不同浓度E2(50-200nM)处理H9c2细胞对其活性均无明显影响(P＞0.05);应用200nM E2预处理H9c2细胞24小时可明显减轻H_2O_2对H9c2细胞活性的损伤(P＜0.05),时相效应分析显示保护作用16小时达高峰可持续至24小时;应用不同浓度E_2(50-200nM)预处理24小时,E_2呈浓度依赖性抑制H_2O_2对H9c2细胞活性的损伤;依此结果选择200nM E2预处理24小时作为后续实验的干预条件。
2)应用流式细胞仪检测H_2O_2诱导的H9c2细胞凋亡、ROS生成及E2的干预作用,结果显示,外源性H_2O_2可引起H9c2细胞凋亡及ROS生成明显增加,与对照组比较差异明显(P＜0.05);E_2预处理能够明显减轻H_2O_2诱导的H9c2细胞凋亡,差异具有显著性(P＜0.05)。
3)RT-PCR鉴定H9c2细胞ERα和ERβ的表达情况,发现H9c2细胞仅表达ERβ,几乎不表达ERα,推测E2对H9c2细胞的抗凋亡保护作用可能是通过ERβ介导。
4)Realtime RT-PCR以及Western blot检测结果显示应用200nM E2处理H9c2细胞8小时后可下调TβRⅠ以及TβRⅡmRNA的表达,处理16小时后下调TβRⅠ以及TβRⅡ蛋白表达,但是对于TGF-β1mRNA及蛋白表达均无明显影响,给予ER阻断剂ICI 182,780阻断E_2与ER的结合,能够消除E2对TβRI及TβRⅡmRNA的下调作用。
5)Western blot检测p-smad2/3蛋白水平显示,E2处理16小时后p-smad2/3蛋白水平降低。
结论:
1)外源性H_2O_2引起氧化应激能够引起细胞内ROS增加,损伤H9c2细胞活性,增加H9c2细胞的凋亡。外源性E2本身并不能影响H9c2细胞凋亡,但能够降低H9c2细胞对H_2O_2的敏感性,保护细胞抵抗氧化应激引起的凋亡。
2)外源性E2对H9c2细胞的TβRⅠ以及TβRⅡ具有下调作用,阻断ER可消除E2对TβRⅠ和TβRⅡ的下调作用;E2可进一步通过TGF-β通路降低受体后磷酸化smad2/3的水平;H9c2细胞仅表达ERβ,提示E2可能是通过ERβ发挥其对TGF-β通路的调控作用。
Backgound:Ischemic heart disease with subsequent myocardial infarction and congestive heart failure is the leading cause of mortality.The incidence of ischemic heart disease is significantly lower in women than in men until menopause,after which the cardiovascular risk of women accelerates to equal that of men.This observation suggests a possibility that female sex hormones,such as estrogen,may have a favorable cardiovascular role.There is evidence from epidemiological,animal,and in vitro studies that estrogen may be cardioprotective.However,the mechanisms involved in this process,are poorly understood.It has been show that estradiol has important role on development, growth and differentiation of organism.It can affect proliferation and apoptosis of cells by modulating several signaling pathway,including transforming growth factor-β(TGF-β)signaling.There are several studies on the interaction between E_2 and TGF-βsignaling in tumor cells,but few in myocardial cells.Oxidative stress is the common pathophysiologic mechanism of myocardial hypertrophy, apoptosis and remodeling caused by miscellaneous heart diseases.It is indicated that estradiol has somewhat anti-oxidative effect.So we observed the effect of estradiol on oxidative stress and apoptosis of myocardial cell and on modulating TGF-βsignaling pathway,in order to explore the mechanism of myocardial cell apoptosis modulated by estradiol and its biological significance.
Objective:To explore the mechanism of apoptosis modulated by estradiol in myocardial cells and its biological significant by observing the effect of estradiol on oxidative stress and apoptosis of myocardial cells induced by H_2O_2 and its modulate effect on TGF-βsignaling pathway.
Methods:Use H9c2 cells,a clonal line derived from embryonic rat heart,as the cell model.1)To observe the effect on cell viability,apoptosis and generation of reactive oxygen speciesk(ROS)in H9c2 cells stimulated by H_2O_2 and the protective effect of estradiol on myocardial cells by means of MTT assay and flow cytometry.Expression of ERαmRNA and ERβmRNA「in H9C2 cells was detected by RT-PCR.2)Real-time RT-PCR and Western blot were performed to examine The expression of TGF-β1,TβRⅠand TβRⅡin H9c2 cells treated with 200riM E2 for different hours.The level of phosphorylation of Smad2/3 was detected by western blot.
Results:
1)MTT assay shows that exogenous H_2O_2 induces decreasing of cell viability of H9C2 cells in a dose-dependent manner(P＜0.05),and 100μM H_2O_2 treating for 4 hours was chosen as inducing condition for the following experiments.Various concentrations of E_2 alone has no effect on the cell viability of H9c2 cells(P＞0.05).Exogenous H_2O_2 increasing the apoptosis rate and ROS generation.While H9c2 cells pretreated with 200nM E_2 for 24 hours significantly attenuate the cell viability injury caused by H_2O_2.Time-phase analysis shows that the maximal protective effect achieved at 16 hours,and last for 24 hours.E_2 inhibited the cell viability injury caused by H_2O_2 in a dose dependent manner.So we choose 200nM E_2 treating for 24 hours as the intervention treatment in the follow experiment.
2)Exogenous H_2O_2 may induce increased apoptosis and ROS generation of H9c2 cells,there is a significant difference compared with control group.E_2 pretreatment can significantly extenuate apoptosis and ROS generation of H9C2 cells induced by H_2O_2(P＜0.05).
3)Determination of estradiol receptor expression of H9C2 cells by RT-PCR finds that H9C2 cells only express ERβ,almost express no ERα,and suggests that anti-apoptosis effect of estradiol on H9C2 cells may be mediated by ERβ.
4)Detection of Realtime RT-PCR and western blot finds that H9C2 cells pretreated by estradiol for 8 hours may downregulate the expression of TβRⅠand TβRⅡmRNA,and downregulate of protein expression occurred at 16 hours.But it has no effect on the expression of TGF-β1.ER blocker ICI182,780 can block the binding of E_2 and ER,and can delete the downregulation effect of E_2 on TβRⅠand TβRⅡ.
5)Western blot exanmination shows that the p-smad2/3 level in H9c2 cells decreased pretreated by estradiol for 16 hours H9c2 cells.
Conclusion:
1)Oxidative stress caused by exogenous H_2O_2 can increase the ROS generation,damage the cell viability and induce apoptosis of H9c2 cells. Exogenous E2 alone has no effect on cell viability and apoptosis of H9C2 cells, but it can decrease the sensitivity of H9C2 cells to H_2O_2,hence,protect cells against apoptosis caused by oxidative stress.
2)Exogenous E_2 has downregulation effect on TβRⅠand TβRⅡof H9c2 cells,blocking ER may delete this effect of estradiol。H9c2 cells express ERβonly,suggests that E_2 may exert modulating effect on TGF-βpathway through ERβ。
引文
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37.Yamamoto T,Saatcioglu F and Matsuda T.Cross-talk between bone morphogenic proteins and estrogen receptor signaling.Endocrinology.2002;143(7):2635-2642.
38.Javelaud D and Mauviel A.Crosstalk mechanisms between the mitogen-activated protein kinase pathways and Smad signaling downstream of TGF-beta:implications for carcinogenesis.Oncogene.2005;24(37):5742-5750.
39.Gilad L A,Bresler T,Gnainsky J,et al.Regulation of vitamin D receptor expression via estrogen-induced activation of the ERK 1/2 signaling pathway in colon and breast cancer cells.J Endocrinol.2005;185(3):577-592.
40.Ju H,Zhao S,Tappia P S,et al.Expression of Gq alpha and PLC-beta in scar and border tissue in heart failure due to myocardial infarction.Circulation.1998;97(9):892-899.
41.Pfeffer J M,Fischer T A and Pfeffer M A.Angiotensin-converting enzyme inhibition and ventricular remodeling after myocardial infarction.Annu Rev Physiol.1995;57:805-826.
42.Sutton M G and Sharpe N.Left ventricular remodeling after myocardial infarction:pathophysiology and therapy.Circulation.2000;101(25):2981-2988.
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44.Weber K T.Extracellular matrix remodeling in heart failure:a role for de novo angiotensin Ⅱ generation.Circulation.1997;96(11):4065-4082.
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46.Roberts A B.Molecular and cell biology of TGF-beta.Miner Electrolyte Metab.1998;24(2-3):111-119.
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48.Massague J.The transforming growth factor-beta family.Annu Rev Cell Biol.1990;6:597-641.
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51.Breitkopf K,Lahme B,Tag C G,et al.Expression and matrix deposition of latent transforming growth factor beta binding proteins in normal and fibrotic rat liver and transdifferentiating hepatic stellate cells in culture.Hepatology.2001;33(2):387-396.
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53.Moren A,Olofsson A,Stenman G,et al.Identification and characterization of LTBP-2,a novel latent transforming growth factor-beta-binding protein.J Biol Chem.1994;269(51):32469-32478.
54.Yin W,Smiley E,Germiller J,et al.Isolation of a novel latent transforming growth factor-beta binding protein gene(LTBP-3).J Biol Chem.1995;270(17):10147-10160.
55.Giltay R,Kostka G and Timpl R.Sequence and expression of a novel member(LTBP-4)of the family of latent transforming growth factor-beta binding proteins.FEBS Lett.1997;411(2-3):164-168.
56.Mangasser-Stephan K and Gressner A M.Molecular and functional aspects of latent transforming growth factor-beta binding protein:just a masking protein?Cell Tissue Res.1999;297(3):363-370.
57.Bissell D M,Wang S S,Jarnagin W R,et al.Cell-specific expression of transforming growth factor-beta in rat liver.Evidence for autocrine regulation of hepatocyte proliferation.J Clin Invest.1995;96(1):447-455.
58.O'Grady P,Huang S S and Huang J S.Expression of a new type high molecular weight receptor(type V receptor)of transforming growth factor beta in normal and transformed cells.Biochem Biophys Res Commun.1991;179(1):378-385.
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61.Massague J.TGF-beta signal transduction.Annu Rev Biochem.1998;67:753-791.
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63.Schmierer B and Hill C S.TGFbeta-SMAD signal transduction:molecular specificity and functional flexibility.Nat Rev Mol Cell Biol.2007;8(12):970-982.
64.Feng X H and Derynck R.Specificity and versatility in tgf-beta signaling through Smads.Annu Rev Cell Dev Biol.2005;21:659-693.
65.Massague J,Seoane J and Wotton D.Smad transcription factors.Genes Dev.2005;19(23):2783-2810.
66.Rahimi R A and Leof E B.TGF-beta signaling:a tale of two responses.J Cell Biochem.2007;102(3):593-608.
67.Luo K and Lodish H F.Positive and negative regulation of type Ⅱ TGF-beta receptor signal transduction by autophosphorylation on multiple serine residues.Embo J.1997;16(8):1970-1981.
68.Wang T,Li B Y,Danielson P D,et al.The immunophilin FKBP12 functions as a common inhibitor of the TGF beta family type I receptors.Cell.1996;86(3):435-444.
69.Chen Y G,Liu F and Massague J.Mechanism of TGFbeta receptor inhibition by FKBP12.Embo J.1997;16(13):3866-3876.
70.Lawler S,Feng X H,Chen R H,et al.The type Ⅱ transforming growth factor-beta receptor autophosphorylates not only on serine and threonine but also on tyrosine residues.J Biol Chem.1997;272(23):14850-14859.
71.Galliher A J and Schiemann W P.Beta3 integrin and Src facilitate transforming growth factor-beta mediated induction of epithelial-mesenchymal transition in mammary epithelial cells.Breast Cancer Res.2006;8(4):R42.
72.Galliher A J and Schiemann W P.Src phosphorylates Tyr284 in TGF-beta type Ⅱ receptor and regulates TGF-beta stimulation of p38 MAPK during breast cancer cell proliferation and invasion.Cancer Res.2007;67(8):3752-3758.
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76.Chen R H and Derynck R.Homomeric interactions between type Ⅱ transforming growth factor-beta receptors.J Biol Chem.1994;269(36):22868-22874.
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80.Feng X H and Derynck R.Ligand-independent activation of transforming growth factor(TGF)beta signaling pathways by heteromeric cytoplasmic domains of TGF-beta receptors.J Biol Chem.1996;271(22):13123-13129.
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84.Derynck R,Zhang Y and Feng X H.Smads:transcriptional activators of TGF-beta responses.Cell.1998;95(6):737-740.
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88.Hayashida T,Decaestecker M and Schnaper H W.Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta-dependent responses inhuman mesangial cells.Faseb J.2003;17(11):1576-1578.
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36.Wu L,Wu Y,Gathings B,et al.Smad4 as a transcription corepressor for estrogen receptor alpha.J Biol Chem.2003;278(17):15192-15200.
37.Yamamoto T,Saatcioglu F and Matsuda T.Cross-talk between bone morphogenic proteins and estrogen receptor signaling.Endocrinology.2002;143(7):2635-2642.
38.Javelaud D and Mauviel A.Crosstalk mechanisms between the mitogen-activated protein kinase pathways and Smad signaling downstream of TGF-beta:implications for carcinogenesis.Oncogene.2005;24(37):5742-5750.
39.Gilad L A,Bresler T,Gnainsky J,et al.Regulation of vitamin D receptor expression via estrogen-induced activation of the ERK 1/2 signaling pathway in colon and breast cancer cells.J Endocrinol.2005;185(3):577-592.
40.Ju H,Zhao S,Tappia P S,et al.Expression of Gq alpha and PLC-beta in scar and border tissue in heart failure due to myocardial infarction.Circulation.1998;97(9):892-899.
41.Pfeffer J M,Fischer T A and Pfeffer M A.Angiotensin-converting enzyme inhibition and ventricular remodeling after myocardial infarction.Annu Rev Physiol.1995;57:805-826.
42.Sutton M G and Sharpe N.Left ventricular remodeling after myocardial infarction:pathophysiology and therapy.Circulation.2000;101(25):2981-2988.
43.Ju H,Zhao S,Jassal D S,et al.Effect of ATI receptor blockade on cardiac collagen remodeling after myocardial infarction.Cardiovasc Res.1997;35(2):223-232.
44.Weber K T.Extracellular matrix remodeling in heart failure:a role for de novo angiotensin Ⅱ generation.Circulation.1997;96(11):4065-4082.
45.Hao J,Wang B,Jones S C,et al.Interaction between angiotensin Ⅱ and Smad proteins in fibroblasts in failing heart and in vitro.Am J Physiol Heart Circ Physiol.2000;279(6):H3020-3030.
46.Roberts A B.Molecular and cell biology of TGF-beta.Miner Electrolyte Metab.1998;24(2-3):111-119.
47.Roberts A B and Sporn M B.Physiological actions and clinical applications of transforming growth factor-beta(TGF-beta).Growth Factors.1993;8(1):1-9.
48.Massague J.The transforming growth factor-beta family.Annu Rev Cell Biol.1990;6:597-641.
49.Oklu R and Hesketh R.The latent transforming growth factor beta binding protein(LTBP)family.Biochem J.2000;352 Pt 3:601-610.
50.Olofsson A,Ichijo H,Moren A,et al.Efficient association of an amino-terminally extended form of human latent transforming growth factor-beta binding protein with the extracellular matrix.J Biol Chem.1995;270(52):31294-31297.
51.Breitkopf K,Lahme B,Tag C G,et al.Expression and matrix deposition of latent transforming growth factor beta binding proteins in normal and fibrotic rat liver and transdifferentiating hepatic stellate cells in culture.Hepatology.2001;33(2):387-396.
52.Kanzaki T,Olofsson A,Moren A,et al.TGF-beta 1 binding protein:a component of the large latent complex of TGF-beta 1 with multiple repeat sequences.Cell.1990;61(6):1051-1061.
53.Moren A,Olofsson A,Stenman G,et al.Identification and characterization of LTBP-2,a novel latent transforming growth factor-beta-binding protein.J Biol Chem.1994;269(51):32469-32478.
54.Yin W,Smiley E,Germiller J,et al.Isolation of a novel latent transforming growth factor-beta binding protein gene(LTBP-3).J Biol Chem.1995;270(17):10147-10160.
55.Giltay R,Kostka G and Timpl R.Sequence and expression of a novel member(LTBP-4)of the family of latent transforming growth factor-beta binding proteins.FEBS Lett.1997;411(2-3):164-168.
56.Mangasser-Stephan K and Gressner A M.Molecular and functional aspects of latent transforming growth factor-beta binding protein:just a masking protein?Cell Tissue Res.1999;297(3):363-370.
57.Bissell D M,Wang S S,Jarnagin W R,et al.Cell-specific expression of transforming growth factor-beta in rat liver.Evidence for autocrine regulation of hepatocyte proliferation.J Clin Invest.1995;96(1):447-455.
58.O'Grady P,Huang S S and Huang J S.Expression of a new type high molecular weight receptor(type V receptor)of transforming growth factor beta in normal and transformed cells.Biochem Biophys Res Commun.1991;179(1):378-385.
59.Mitchell E J,Lee K and O'Connor-McCourt M D.Characterization of transforming growth factor-beta(TGF-beta)receptors on BeWo choriocarcinoma cells including the identification of a novel 38-kDa TGF-beta binding glycoprotein.Mol Biol Cell.1992;3(11):1295-1307.
60.Segarini P R.TGF-beta receptors:a complicated system of multiple binding proteins.Biochim Biophys Acta.1993;1155(3):269-275.
61.Massague J.TGF-beta signal transduction.Annu Rev Biochem.1998;67:753-791.
62.Eickelberg O,Centrella M,Reiss M,et al.Betaglycan inhibits TGF-beta signaling by preventing type I-type Ⅱ receptor complex formation.Glycosaminoglycan modifications alter betaglycan function.J Biol Chem. 2002;277(1):823-829.
63.Schmierer B and Hill C S.TGFbeta-SMAD signal transduction:molecular specificity and functional flexibility.Nat Rev Mol Cell Biol.2007;8(12):970-982.
64.Feng X H and Derynck R.Specificity and versatility in tgf-beta signaling through Smads.Annu Rev Cell Dev Biol.2005;21:659-693.
65.Massague J,Seoane J and Wotton D.Smad transcription factors.Genes Dev.2005;19(23):2783-2810.
66.Rahimi R A and Leof E B.TGF-beta signaling:a tale of two responses.J Cell Biochem.2007;102(3):593-608.
67.Luo K and Lodish H F.Positive and negative regulation of type Ⅱ TGF-beta receptor signal transduction by autophosphorylation on multiple serine residues.Embo J.1997;16(8):1970-1981.
68.Wang T,Li B Y,Danielson P D,et al.The immunophilin FKBP12 functions as a common inhibitor of the TGF beta family type I receptors.Cell.1996;86(3):435-444.
69.Chen Y G,Liu F and Massague J.Mechanism of TGFbeta receptor inhibition by FKBP12.Embo J.1997;16(13):3866-3876.
70.Lawler S,Feng X H,Chen R H,et al.The type Ⅱ transforming growth factor-beta receptor autophosphorylates not only on serine and threonine but also on tyrosine residues.J Biol Chem.1997;272(23):14850-14859.
71.Galliher A J and Schiemann W P.Beta3 integrin and Src facilitate transforming growth factor-beta mediated induction of epithelial-mesenchymal transition in mammary epithelial cells.Breast Cancer Res.2006;8(4):R42.
72.Galliher A J and Schiemann W P.Src phosphorylates Tyr284 in TGF-beta type Ⅱ receptor and regulates TGF-beta stimulation of p38 MAPK during breast cancer cell proliferation and invasion.Cancer Res.2007;67(8):3752-3758.
73.Wrana J L,Attisano L,Wieser R,et al.Mechanism of activation of the TGF-beta receptor.Nature.1994;370(6488):341-347.
74.Lee M K,Pardoux C,Hall M C,et al.TGF-beta activates Erk MAP kinase signalling through direct phosphorylation of ShcA.Embo J.2007;26(17):3957-3967.
75.Henis Y I,Moustakas A,Lin H Y,et al.The types Ⅱ and Ⅲ transforming growth factor-beta receptors form homo-oligomers.J Cell Biol.1994;126(1):139-154.
76.Chen R H and Derynck R.Homomeric interactions between type Ⅱ transforming growth factor-beta receptors.J Biol Chem.1994;269(36):22868-22874.
77.Luo K and Lodish H F.Signaling by chimeric erythropoietin-TGF-beta receptors:homodimerization of the cytoplasmic domain of the type Ⅰ TGF-beta receptor and heterodimerization with the type Ⅱ receptor are both required for intracellular signal transduction.Embo J.1996;15(17):4485-4496.
78.Attisano L,Wrana J L,Montalvo E,et al.Activation of signalling by the activin receptor complex.Mol Cell Biol.1996;16(3):1066-1073.
79.Wieser R,Wrana J L and Massague J.GS domain mutations that constitutively activate T beta R-I,the downstream signaling component in the TGF-beta receptor complex.Embo J.1995;14(10):2199-2208.
80.Feng X H and Derynck R.Ligand-independent activation of transforming growth factor(TGF)beta signaling pathways by heteromeric cytoplasmic domains of TGF-beta receptors.J Biol Chem.1996;271(22):13123-13129.
81.Matsuzaki K,Date M,Furukawa F,et al.Regulatory mechanisms for transforming growth factor beta as an autocrine inhibitor in human hepatocellular carcinoma:implications for roles of smads in its growth.Hepatology.2000;32(2):218-227.
82.Muro-Cacho C A,Rosario-Ortiz K,Livingston S,et al.Defective transforming growth factor beta signaling pathway in head and neck squamous cell carcinoma as evidenced by the lack of expression of activated Smad2.Clin Cancer Res.2001;7(6):1618-1626.
83.Itoh S,Landstrom M,Hermansson A,et al.Transforming growth factor betal induces nuclear export of inhibitory Smad7.J Biol Chem.1998;273(44):29195-29201.
84.Derynck R,Zhang Y and Feng X H.Smads:transcriptional activators of TGF-beta responses.Cell.1998;95(6):737-740.
85.Carrillo C,Cafferata E G,Genovese J,et al.TGF-betal up-regulates the mRNA for the Na+/Ca2+ exchanger in neonatal rat cardiac myocytes.Cell Mol Biol(Noisy-le-grand).1998;44(3):543-551.
86.Mulder K M.Role of Ras and Mapks in TGFbeta signaling.Cytokine Growth Factor Rev.2000;11(1-2):23-35.
87.Derynck R and Zhang Y E.Smad-dependent and Smad-independent pathways in TGF-beta family signalling.Nature.2003;425(6958):577-584.
88.Hayashida T,Decaestecker M and Schnaper H W.Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta-dependent responses inhuman mesangial cells.Faseb J.2003;17(11):1576-1578.
89.Border W A and Noble N A.Transforming growth factor beta in tissue fibrosis.NEngl J Med.1994;331(19):1286-1292.
90.Moses H L,Yang E Y and Pietenpol J A.TGF-beta stimulation and inhibition of cell proliferation:new mechanistic insights.Cell.1990;63(2):245-247.
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