羊膜上皮细胞水通道蛋白3表达的cAMP-PKA信号通路调控机制

摘要

目的 探讨羊膜上皮细胞水通道蛋白3(AQP3)表达过程中环磷酸腺苷-蛋白激酶A(cAMP-PKA)信号传导通路的调控机制.方法 选择2012年l至11月温州医科大学附属第二医院无并发症、羊水量正常的足月孕妇30例,剖宫产时取羊膜进行上皮细胞培养,并进行如下分组实验:(1)毛喉素组:不同浓度的毛喉素(forskolin,0、2.5、5、50、100 μmol/L)作用羊膜上皮细胞2h,再以最适宜浓度的forskolin作用不同时间(0、1、2、10、20h)；(2)SP-cAMP组:不同浓度的PKA激动剂SP-cAMP(0、2.5、5、50、100 μmol/L)作用羊膜上皮细胞2h,再以最适宜浓度的SP-cAMP作用不同时间(0、1、2、10、20 h)；(3)H-89组:不同浓度的PKA抑制剂H-89(0、5、10、50、100 μmol/L)作用人羊膜上皮细胞2h,再以最适宜浓度H-89作用不同时间(0、1、2、10、20 h).采用ELISA方法检测各组细胞内CAMP水平及PKA活性,免疫细胞化学染色法检测AQP3表达定位,蛋白印迹法检测总cAMP反应元件结合蛋白(CREB)、磷酸化CREB(p-CREB)和AQP3表达水平,采用细胞计数试剂盒(CCK-8)检测细胞增殖率.结果 (1)各组羊膜上皮细胞的胞质及胞膜均有AQP3蛋白表达.(2)各组不同浓度forskolin、SP-cAMP和H-89作用羊膜上皮细胞后,细胞增殖率无明显变化,分别比较,差异均无统计学意义(P＞0.05).(3)不同浓度forskolin作用羊膜上皮细胞2h,总CREB表达水平比较,差异无统计学意义(P＞0.05).cAMP水平、PKA活性、p-CREB和AQP3表达水平分别比较,差异均有统计学意义(P＜0.05),2.5、5、50μmol/L forskolin作用后,上述4种因子均高于0μmol/L(P＜0.05)；而5 μmol/L明显高于2.5、50 μmol/L(P＜0.05).forskolin作用的最适宜浓度为5μmol/L.(4)不同浓度SP-cAMP作用羊膜上皮细胞2h,细胞中cAMP和总CREB表达水平分别比较,差异均无统计学意义(P＞0.05).PKA活性、p-CREB和AQP3表达水平比较,差异均有统计学意义(P＜0.05)；SP-cAMP浓度为5、50 μmol/L时,上述3种因子高于0μmol/L(P＜0.05)；50 μmol/L时明显高于5 μmol/L(P ＜0.05).SP-cAMP作用的最适宜浓度为50 μmol/L.(5)不同浓度H-89作用羊膜上皮细胞2h,细胞中cAMP水平和总CREB表达水平比较,差异均无统计学意义(P＞0.05).PKA活性、p-CREB和AQP3表达水平比较,差异均有统计学意义(P ＜0.05)；H-89浓度为10、50和100μmol/L时,上述3种因子均低于0 μmol/L(P＜0.05),10 μmol/L时明显低于50和100 μmol/L(P＜0.05).H-89作用的最适宜浓度为10 μmol/L.(6)5 μmol/L forskolin联合10 μmol/L H-89作用2h后,羊膜上皮细胞中总CREB表达水平比较,差异无统计学意义(P＞0.05).p-CREB与AQP3表达水平比较,低于5 μmol/L forskolin作用2 h(P ＜0.05),但高于10 μmol/L H-89作用2 h(P ＜0.05).结论 cAMP-PKA信号传导通路对羊膜上皮细胞中AQP3蛋白的表达起调控作用.%Objective To investigate the expression of aquaporins-3 (AQP3) in amniotic epithelial cells regulated by cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signal pathway and to explore the mechanisms of its expression.Methods The amniotic epithelial cells were collected from 30 patients who underwent elective caesarean sections at term with normal amniotic fluid volume and primarily cultured.The cultured cells were treated with (1) forskolin groups: different concentration (0,2.5,5,50 or 100 μmol/L) of forskolin treated cells for 2 hours,and the optimal concentration of forskolin treated cells with different time (0,1,2,10 or 20 hours) ; (2)SP-cAMP groups: different concentration (0,2.5,5,50 or 100 μmol/L) of SP-cAMP treated cells for 2 hours,and the optimal concentration of SP-cAMP treated cells with different time (0,1,2,10 or 20 hours); (3)H-89 groups: different concentration (0,5,10,50 or 100 μmol/L) of H-89 treated cells for 2 hours,and the optimal concentration of H-89 treated cells with different time (0,1,2,10 or 20 hours).The level of intracellular cAMP and activity of PKA were detected by using ELISA,and immunohistochemistry was used to detect the localization of AQP3,the protein expression of total cAMP-response element binding protein (CREB) and phospho-CREB (p-CREB) and AQP3 were assessed by western blot analysis.Cell proliferation was assessed by cell counting kit-8 (CCK-8)assay.Results (1) The brown staining of AQP3 was detected in both cell membrane and cytoplasm in each group.(2) There was no significant change of the cell proliferation rate among groups with different concentration of forskolin,SP-cAMP and H-89 treatment (P ＞ 0.05).(3) After different concentration of forskolin treated 2 hours,the expression of total CREB had no significant difference among them(P ＞ 0.05).While the expression of cAMP level,PKA activity,p-CREB and AQP3 protein were significantly changed,which were higher in 2.5 μmol/L,5 μmol/L,50 μmol/L forskolin group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 5 μmol/L forskolin group were higher than that in 2.5 μmol/L and 50 μmol/L (P ＜ 0.05).The optimal forskolin concentration was 5 μmol/L.(4) After different concentration of SP-cAMP treated 2 hours,the expression of total CREB and cAMP level had no significant difference among them (P ＞ 0.05),while the expression of PKA activity,p-CREB and AQP3 protein were significantly changed,which were higher in 5 μμmol/L,50 μmol/L SP-cAMP group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 50 μmol/L SP-cAMP group were higher than that in 5 μmol/L (P ＜0.05).The optimal SP-cAMP concentration was 50 μmol/L (5) After different concentration of H-89 treated 2 hours,the expression of total CREB and cAMP level had no significant difference among them (P ＞ 0.05),while the expression of PKA activity,p-CREB and AQP3 protein were significantly changed,which were lower in 10 μmol/L,50 μmol/L and 100 μmol/L H-89 group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 10 μmol/L H-89 group were lower than that in 50 μmol/L,100 μmol/L (P ＜ 0.05).The optimal H-89 concentration was 10 μmol/L.(6) p-CREB and AQP3 protein expression were significantly lower in 5 μmol/L forskolin combined 10 μmol/L H-89 incubating 2 hours group when compared with 5 μmol/L forskolin,but higher than that in 10 μmol/L H-89 treated group (P ＜ 0.05).Total CREB was no significant difference among the three groups (P ＞ 0.05).Conclusion cAMP-PKA signal transduction pathway may regulate AQP3 protein expression in human amniotic epithelial cells.