睾酮通过MAPK信号转导通路中的JNK调节人脐静脉内皮细胞TFPI表达的机制研究
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摘要
背景男性是冠心病的一个危险因素。睾酮(Testosterone,T)是男性体内最主要的循环雄激素。睾酮在冠心病中的作用一直存在争议,是目前心血管领域研究的热点。我们前期研究发现生理浓度的睾酮(3×10-8mol/L)能够促进人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cell,HUVEC)合成、分泌组织因子途径抑制物(Tissue Factor Pathway Inhibitor,TFPI),增强内皮抗凝活性,对止凝血系统产生有益的影响。传统认为睾酮是通过与胞浆内的雄激素受体结合转入细胞核内与敏感靶基因上的雄激素反应元件(androgen response element,ARE)相结合或与其他转录因子相互作用,增强或抑制靶基因转录。近年发现睾酮还可通过激活细胞内信号通路(非传统途径)而发挥更为迅速、广阔的生理调控作用。活化丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)超家族是细胞外信号转导的重要调节因子,他包括细胞外信号调节激酶(extracellular signal-regulated kinases,ERKs)、c-Jun氨基末端激酶(the c-Jun NH2-terminal kinases,JNKs)、丝裂原活化蛋白激酶p38(p38 MAPK)。研究发现睾酮可通过激活JNK、P38发挥各种不同的功能。目前睾酮调节TFPI的机制尚未完全阐明,本实验将探究睾酮能否通过JNK、P38信号转导通路调节TFPI的表达。
目的观察生理浓度睾酮对人脐静脉内皮细胞JNK、P38磷酸化水平的影响,以及抑制JNK、P38活化对睾酮调节TFPI蛋白和mRNA表达的影响,明确睾酮是否能够通过JNK、P38信号转导通路来调节人脐静脉内皮细胞TFPI的表达。
方法将体外培养的HUVEC(3-4代)分为四组,对照组(未作处理),其他三组分别加入生理浓度睾酮、雄激素受体拮抗剂氟他胺(Flutamide,F)及生理浓度睾酮+氟他胺后,通过Western blotting方法测定JNK、P38的磷酸化水平:体外培养的HUVEC(3-4代),分为6组,分别加入培养基、生理浓度睾酮、JNK抑制剂SP、P38抑制剂SB、以及SB+T和SP+T,通过ELISSA方法观察各组TFPI的蛋白表达量。体外培养的HUVEC(3-4代),分为8组,分别加入培养基、生理浓度睾酮、氟他胺、睾酮+氟他胺、JNK抑制剂SP、P38抑制剂SB、以及SB+T和SP+T,通过real-timeRT-PCR方法观察各组TFPI的mRNA表达量。
结果生理浓度的睾酮能增加JNK、P38的磷酸化水平,有统计学意义(P＜0.05).生理浓度睾酮能够上调TFPI的蛋白和mRNA的表达量,JNK阻滞剂可以显著下调TFPI的蛋白和mRNA的表达量,并且能够逆转睾酮对TFPI的上调作用,和睾酮合用时能使TFPI的蛋白和mRNA的表达量下调(P＜0.05)。P38阻滞剂对TFPI的蛋白和mRNA的表达量无显著影响,无统计学意义(P＞0.05)。
结论生理浓度的睾酮能够促进JNK、P38活化,并且能够通过JNK信号转导通路调节人脐静脉内皮细胞TFPI的蛋白和基因表达,而P38激活后所介导的信号转导途径不影响TFPI的表达.
Background
Male gender was found to be an independent risk factor for coronary heart diseases. Testosterone is the most abundant androgen in males.But the role androgen played has been controversial,which becomes the interest of cardiovascular research.We have previously reportedthat physiological testosterone(3×10-8mol/L) had beneficial influence on hemostatic system to enhance the anticoagulant activity through stimulating the tissue factor pathway inhibitor(TFPI) levels secreted by the human umbilic vein endothial cells(HUVEC). Testosterone transfers into the nucleus through bingding intracellular receptor,then binds to specific response elements in target gene promoters,causing subsequent activation or repression of transcription.The mitogen-activated protein kinase(MAPK) superfamily consists of three main protein kinase families:the extracellular signal-regulated protein kinases(ERKs),the c-Jun N-terminal kinases(JNKs) and the p38 family of kinases.Testosterone plays various roles by the activation of JNK、P38.However,the specific mechanism of the regulation of TFPI by testosterone,till now,is still not fully understood.The purpose of the present study,therefore,is to investigate whether the testosterone regulates the TFPI expression via the JNK、P38 signalling pathways.
Objective
To investigate the effects of testosterone at physiological concentration on the phosphorylation of JNK、P38 in HUVECs,further influence on the proteins and mRNA expression of TFPI by inhibition of acration of JNK、P38,and explore whether the testosterone regulated the expression of TFPI via the JNK、P38 signalling pathways.
Methods
HUVECs within 3-4passages were cultured in 75cm~2 flasks.The cells were divided into four experimental groups:"control",the cells were incubated in the medium;"T-8",the cells were incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins;"F-5", the cells were incubated in the presence of androgen receptor antagonist(flutamide 10μmol/L) for 3h;"T-8+F-5",the cells were preincubated in the presence of androgen receptor antagonist for 3h,then incubated in the presence of physiological testosterone for 30mins.After the incubation the phosphorylation of JNK、P38 were examined by Western blotting according to the manufacture's protocol.(2) HUVECs within 3-4passages were cultured in 96-well plates.The cells were divided into six experimental groups:"control",the cells were incubated in the medium;"T-8",the cells were incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins;"SP",the cells were incubated in the presence of JNK inhibitor(SP 2.5μmol/L) for 2h;"SB,the cells were incubated in the presence of P38 inhibitor(SB 10μmol/L) for 2h;"SP+T-8",the cells were preincubated in the presence of JNK inhibitor(SP 2.5μmol/L) for 2h,the incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins; "SB+T-8",the cells were preincubated in the presence of P38 inhibitor(SB10μmol/L) for 2h,then incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins; After the incubation,the the expression of TFPI were examined by ELISA according to the manufacture's protocol.(3) HUVECs within 3-4passages were cultured in 25cm~2 flasks.The cells were divided into eight experimental groups:"control",the cells were incubated in the medium;"T-8",the cells were incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins;"F-5",the cells were incubated in the presence of androgen receptor antagonist(flutamide 10μmol/L) for 3h;"T-8+F-5",the cells were preincubated in the presence of androgen receptor antagonist for 3h,then incubated in the presence of physiological testosterone for 30mins."SP",the cells were incubated in the presence of JNK inhibitor(SP 2.5μmol/L) for 2h;"SB,the cells were incubated in the presence of P38 inhibitor(SB 10μmol/L) for 2h;"SP+T-8",the cells were preincubated in the presence of JNK inhibitor(SP 2.5μmol/L) for 2h,the incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins; "SB+T-8",the cells were preincubated in the presence of P38 inhibitor(SB10μmol/L) for 2h,the incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins; After the incubation,the the mRNA expression of TFPI was examined using RT-PCR according to the manufacture's protocol.
Results
(1)Testosterone at physiological concentrations(3×10-8 mol/L) significantly increased the phosphorylation of JNK、P38 in HUVECs;(2)the inhibitor of activation of JNK could attenuated the testosterone's effects,decreased the proteins and mRNA expression of TFPI significantly(p＜0.01).The inhibitor of activation of P38 did not influence the proteins and mRNA expression of TFPI(p＞0.05).
Conclusion
Testosterone at physiological concentrations may regulate the expression of TFPI via the JNK signalling pathways,rather than P38 signalling pathways.
目的观察生理浓度睾酮对人脐静脉内皮细胞JNK、P38磷酸化水平的影响,以及抑制JNK、P38活化对睾酮调节TFPI蛋白和mRNA表达的影响,明确睾酮是否能够通过JNK、P38信号转导通路来调节人脐静脉内皮细胞TFPI的表达。
方法将体外培养的HUVEC(3-4代)分为四组,对照组(未作处理),其他三组分别加入生理浓度睾酮、雄激素受体拮抗剂氟他胺(Flutamide,F)及生理浓度睾酮+氟他胺后,通过Western blotting方法测定JNK、P38的磷酸化水平:体外培养的HUVEC(3-4代),分为6组,分别加入培养基、生理浓度睾酮、JNK抑制剂SP、P38抑制剂SB、以及SB+T和SP+T,通过ELISSA方法观察各组TFPI的蛋白表达量。体外培养的HUVEC(3-4代),分为8组,分别加入培养基、生理浓度睾酮、氟他胺、睾酮+氟他胺、JNK抑制剂SP、P38抑制剂SB、以及SB+T和SP+T,通过real-timeRT-PCR方法观察各组TFPI的mRNA表达量。
结果生理浓度的睾酮能增加JNK、P38的磷酸化水平,有统计学意义(P＜0.05).生理浓度睾酮能够上调TFPI的蛋白和mRNA的表达量,JNK阻滞剂可以显著下调TFPI的蛋白和mRNA的表达量,并且能够逆转睾酮对TFPI的上调作用,和睾酮合用时能使TFPI的蛋白和mRNA的表达量下调(P＜0.05)。P38阻滞剂对TFPI的蛋白和mRNA的表达量无显著影响,无统计学意义(P＞0.05)。
结论生理浓度的睾酮能够促进JNK、P38活化,并且能够通过JNK信号转导通路调节人脐静脉内皮细胞TFPI的蛋白和基因表达,而P38激活后所介导的信号转导途径不影响TFPI的表达.
Background
Male gender was found to be an independent risk factor for coronary heart diseases. Testosterone is the most abundant androgen in males.But the role androgen played has been controversial,which becomes the interest of cardiovascular research.We have previously reportedthat physiological testosterone(3×10-8mol/L) had beneficial influence on hemostatic system to enhance the anticoagulant activity through stimulating the tissue factor pathway inhibitor(TFPI) levels secreted by the human umbilic vein endothial cells(HUVEC). Testosterone transfers into the nucleus through bingding intracellular receptor,then binds to specific response elements in target gene promoters,causing subsequent activation or repression of transcription.The mitogen-activated protein kinase(MAPK) superfamily consists of three main protein kinase families:the extracellular signal-regulated protein kinases(ERKs),the c-Jun N-terminal kinases(JNKs) and the p38 family of kinases.Testosterone plays various roles by the activation of JNK、P38.However,the specific mechanism of the regulation of TFPI by testosterone,till now,is still not fully understood.The purpose of the present study,therefore,is to investigate whether the testosterone regulates the TFPI expression via the JNK、P38 signalling pathways.
Objective
To investigate the effects of testosterone at physiological concentration on the phosphorylation of JNK、P38 in HUVECs,further influence on the proteins and mRNA expression of TFPI by inhibition of acration of JNK、P38,and explore whether the testosterone regulated the expression of TFPI via the JNK、P38 signalling pathways.
Methods
HUVECs within 3-4passages were cultured in 75cm~2 flasks.The cells were divided into four experimental groups:"control",the cells were incubated in the medium;"T-8",the cells were incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins;"F-5", the cells were incubated in the presence of androgen receptor antagonist(flutamide 10μmol/L) for 3h;"T-8+F-5",the cells were preincubated in the presence of androgen receptor antagonist for 3h,then incubated in the presence of physiological testosterone for 30mins.After the incubation the phosphorylation of JNK、P38 were examined by Western blotting according to the manufacture's protocol.(2) HUVECs within 3-4passages were cultured in 96-well plates.The cells were divided into six experimental groups:"control",the cells were incubated in the medium;"T-8",the cells were incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins;"SP",the cells were incubated in the presence of JNK inhibitor(SP 2.5μmol/L) for 2h;"SB,the cells were incubated in the presence of P38 inhibitor(SB 10μmol/L) for 2h;"SP+T-8",the cells were preincubated in the presence of JNK inhibitor(SP 2.5μmol/L) for 2h,the incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins; "SB+T-8",the cells were preincubated in the presence of P38 inhibitor(SB10μmol/L) for 2h,then incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins; After the incubation,the the expression of TFPI were examined by ELISA according to the manufacture's protocol.(3) HUVECs within 3-4passages were cultured in 25cm~2 flasks.The cells were divided into eight experimental groups:"control",the cells were incubated in the medium;"T-8",the cells were incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins;"F-5",the cells were incubated in the presence of androgen receptor antagonist(flutamide 10μmol/L) for 3h;"T-8+F-5",the cells were preincubated in the presence of androgen receptor antagonist for 3h,then incubated in the presence of physiological testosterone for 30mins."SP",the cells were incubated in the presence of JNK inhibitor(SP 2.5μmol/L) for 2h;"SB,the cells were incubated in the presence of P38 inhibitor(SB 10μmol/L) for 2h;"SP+T-8",the cells were preincubated in the presence of JNK inhibitor(SP 2.5μmol/L) for 2h,the incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins; "SB+T-8",the cells were preincubated in the presence of P38 inhibitor(SB10μmol/L) for 2h,the incubated in the presence of physiological testosterone(3×10-8mol/L) for 30 mins; After the incubation,the the mRNA expression of TFPI was examined using RT-PCR according to the manufacture's protocol.
Results
(1)Testosterone at physiological concentrations(3×10-8 mol/L) significantly increased the phosphorylation of JNK、P38 in HUVECs;(2)the inhibitor of activation of JNK could attenuated the testosterone's effects,decreased the proteins and mRNA expression of TFPI significantly(p＜0.01).The inhibitor of activation of P38 did not influence the proteins and mRNA expression of TFPI(p＞0.05).
Conclusion
Testosterone at physiological concentrations may regulate the expression of TFPI via the JNK signalling pathways,rather than P38 signalling pathways.
引文
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1.Davis,R.J.Signal transduction by the JNK group of MAP kinases.Cell;2000;10(3):239-252.
2.Zhang,Y.,Zhou,L.and Miller,C.A.A splicing variant of a death domain protein that is regulated by a mitogen-activated kinase is a substrate for c-Jun N-terminal kinase in the human central nervous system.Proc.Natl.1995;2(3):2586-2591.
3.Pombo,C.M.,Bonventre,J.V and Force,T.The stress-activated protein kinases are major c-Jun amino-terminal kinases activated by ischemia and reperfusion.J.Biol.Chem.1994;26(9):26546-26551.
4.Irving,E.A.and Bamford,M.Role of mitogenand stress-activated kinases in ischemic injury.J.Cereb.Blood Flow Metab.2002;2(2):631-647.
5.Dougherty,C.J.,Kubasiak,L.A and Webster,K.A.Activation of c-Jun N-terminal kinase promotes survival of cardiac myocytes after oxidative stress.Biochem.J.2002;36(2):561-571
6.Lisovsky,M.,Itoh,K.and Sokol,S.Y.Frizzled receptors activate a novel JNK-dependent pathway that may lead to apoptosis.Curr.Biol.2002;1(2):53-58.
7.Moreno,E.,Basler,K.and Morata,G.Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development.Curr.Biol.2002;20(7):1597-609.
8.New,L.and Han,J.The p38 MAP kinase pathway and its biological function.Trends Cardiovasc.Med.1998;8(2):220-229
9.Zhu,T.and Lobie,P.E.Janus kinase 2-dependent activation of p38 mitogen-activated protein kinase by growth hormone.J.Biol.Chem.2000;27(5):2103-2114.
10.Kyriakis,J.M.and Avruch,J.Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation.Physiol.Rev.2001;8(7):807-869.
11.Vera Y,Erkkila K,Wang C;Involvement of p38 mitogen-activated protein kinase and inducible nitric oxide synthase in apoptotic signaling of murine and human male germ cells after hormone deprivation.Mol Endocrinol.2006;20(7):1597-609.
12.Pinthus JH,Bryskin I,Trachtenberg J;Androgen induces adaptation to oxidative stress in prostate cancer:implications for treatment with radiation therapy.Neoplasia.2007;9(1):68-80
13.Wang M,Tsai BM,Kher A;Role of endogenous testosterone in myocardial proinflammatory and proapoptotic signaling after acute ischemia-reperfusion.Am J Physiol Heart Circ Physiol.2005;288(1):H221-6.
14. Angele MK, Nitsch S, Knoferl MW; Sex-specific p38 MAP kinase activation following trauma-hemorrhage: involvement of testosterone and estradiol. Am J Physiol Endocrinol Metab. 2003;285(1):E189-96
15. Liu L, Wang L, Zhao Y; Testosterone attenuates p38 MAPK pathway during Leishmania donovani infection of macrophages. Parasitol Res. 2006 Jul;99(2):189-93.
16. Brown CM, Xu Q, Okhubo N; Androgen-mediated immune function is altered by the apolipoprotein E gene. Endocrinology. 2007;148(7):3383-90.
17. Benten WP, Guo Z, Krucken J; Rapid effects of androgens in macrophages. Steroids. 2004;69(8-9):585-90
18. Park KM, Kim JI, Ann Y; Testosterone is responsible for enhanced susceptibility of males to ischemic renal injury. J Biol Chem. 2004;279(50):52282-92.
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1.Davis,R.J.Signal transduction by the JNK group of MAP kinases.Cell;2000;10(3):239-252.
2.Zhang,Y.,Zhou,L.and Miller,C.A.A splicing variant of a death domain protein that is regulated by a mitogen-activated kinase is a substrate for c-Jun N-terminal kinase in the human central nervous system.Proc.Natl.1995;2(3):2586-2591.
3.Pombo,C.M.,Bonventre,J.V and Force,T.The stress-activated protein kinases are major c-Jun amino-terminal kinases activated by ischemia and reperfusion.J.Biol.Chem.1994;26(9):26546-26551.
4.Irving,E.A.and Bamford,M.Role of mitogenand stress-activated kinases in ischemic injury.J.Cereb.Blood Flow Metab.2002;2(2):631-647.
5.Dougherty,C.J.,Kubasiak,L.A and Webster,K.A.Activation of c-Jun N-terminal kinase promotes survival of cardiac myocytes after oxidative stress.Biochem.J.2002;36(2):561-571
6.Lisovsky,M.,Itoh,K.and Sokol,S.Y.Frizzled receptors activate a novel JNK-dependent pathway that may lead to apoptosis.Curr.Biol.2002;1(2):53-58.
7.Moreno,E.,Basler,K.and Morata,G.Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development.Curr.Biol.2002;20(7):1597-609.
8.New,L.and Han,J.The p38 MAP kinase pathway and its biological function.Trends Cardiovasc.Med.1998;8(2):220-229
9.Zhu,T.and Lobie,P.E.Janus kinase 2-dependent activation of p38 mitogen-activated protein kinase by growth hormone.J.Biol.Chem.2000;27(5):2103-2114.
10.Kyriakis,J.M.and Avruch,J.Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation.Physiol.Rev.2001;8(7):807-869.
11.Vera Y,Erkkila K,Wang C;Involvement of p38 mitogen-activated protein kinase and inducible nitric oxide synthase in apoptotic signaling of murine and human male germ cells after hormone deprivation.Mol Endocrinol.2006;20(7):1597-609.
12.Pinthus JH,Bryskin I,Trachtenberg J;Androgen induces adaptation to oxidative stress in prostate cancer:implications for treatment with radiation therapy.Neoplasia.2007;9(1):68-80
13.Wang M,Tsai BM,Kher A;Role of endogenous testosterone in myocardial proinflammatory and proapoptotic signaling after acute ischemia-reperfusion.Am J Physiol Heart Circ Physiol.2005;288(1):H221-6.
14. Angele MK, Nitsch S, Knoferl MW; Sex-specific p38 MAP kinase activation following trauma-hemorrhage: involvement of testosterone and estradiol. Am J Physiol Endocrinol Metab. 2003;285(1):E189-96
15. Liu L, Wang L, Zhao Y; Testosterone attenuates p38 MAPK pathway during Leishmania donovani infection of macrophages. Parasitol Res. 2006 Jul;99(2):189-93.
16. Brown CM, Xu Q, Okhubo N; Androgen-mediated immune function is altered by the apolipoprotein E gene. Endocrinology. 2007;148(7):3383-90.
17. Benten WP, Guo Z, Krucken J; Rapid effects of androgens in macrophages. Steroids. 2004;69(8-9):585-90
18. Park KM, Kim JI, Ann Y; Testosterone is responsible for enhanced susceptibility of males to ischemic renal injury. J Biol Chem. 2004;279(50):52282-92.
19. Shimada K, Nakamura M, Ishida E; Requirement of c-jun for testosterone-induced sensitization to N-(4-hydroxyphenyl)retinamide-induced apoptosis. Mol Carcinog. 2003;36(3):115-22.