The Research On Testosterone Regulating The TFPI Expession Via JNK Of Mitogen-activated Protein Kinase Signalling Pathways In Human Umbilical Vein Endothelial Cells

BackgroundMale 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.ObjectiveTo 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. MethodsHUVECs 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).ConclusionTestosterone at physiological concentrations may regulate the expression of TFPI via the JNK signalling pathways,rather than P38 signalling pathways.