c-Jun氨基末端激酶信号通路对缺氧上调血管内皮细胞钙/钙调蛋白依赖性丝氨酸蛋白激酶表达的影响

Involvement of JNK signal pathway in hypoxia related upregulation of calcium/calmodulin-dependent serine protein kinase in endothelial cells

目的了解缺氧对钙/钙调蛋白依赖性丝氨酸蛋白激酶(CASK)表达的影响及c-Jun氨基末端激酶(JNK)信号通路在其中的作用。方法将人血管内皮细胞株EA.hy926进行缺氧处理3 h后继续常规培养0、12、24、48、72 h,同时设常氧培养对照。采用蛋白质印迹法检测CASK的表达。构建CASK启动子区荧光素酶报告基因质粒。用其转染细胞后,于常氧及缺氧培养1、3、6、12 h裂解细胞提取总蛋白,检测报告基因萤火虫荧光素酶及内参照海肾素荧光素酶活性,并用蛋白质印迹法检测JNK磷酸化情况。在培养的细胞中分别加入不同剂量(0、10、100 nmol/L,1、10μmol/L)的JNK抑制剂SP600125预处理1 h后再缺氧培养3 h,观察其对CASK表达的影响。结果缺氧处理后常规培养0～72 h,细胞CASK持续保持高表达,并明显高于常氧组。随着缺氧时间延长荧光素酶相对活性普遍增加,均高于常氧组(0.010±0.003,P<0.01),且缺氧12 h达峰值(0.192±0.023)。JNK的磷酸化随缺氧时间延长逐渐增强。加入SP600125后,CASK的表达显著降低,并呈剂量-效应依赖性,其浓度为10μmol/L时,抑制效率达高峰。结论缺氧上调血管内皮细胞CASK的表达部分依赖于JNK信号通路活化。

To investigate the expression of calcium/calmodulin-dependent serine protein kinase （CASK） induced by short-term hypoxia, and to explore the role of JNK pathway in this signal event. Methods EA. hy926 cells were cultured in normoxic condition for 0 ,12 ,24 ,48 ,72 h after being exposed to hypoxic condition for 3 h, then the cellular lysates were extracted. CASK promoter luciferase reporter recombinant was constructed and transfected into EA. hy926 cells for 48h. Cellular lysates were extracted 1,3,6, 12 h after hypoxia treatment and were used to detect firefly luciferase activity and rinella luciferase activity with luminometer. EA. hy926 cells were cultured under hypoxic condition for 1, 3,6,12 h or under normoxic condition, then the cell lysates were extracted and used to detect phospho-JNK with Western blot. EA. hy926 cells were pretreated with different concentrations of JNK specific inhibitor SP 600125 （ 0,10,100nmol/L and 1,10 μmol/L） 1 h before hypoxic treatment of various duration, and the cell lysates were extracted to detect CASK expression with Western blot. Results CASK expression was obviously elevated by hypoxia, and the high expression sustained for 72 h when the hypoxic cells were cultured in normal conditions, and it was significantly higher than that of normal controls. Dual luciferase reporter assay showed that CASK promoter activity was significantly increased after hypoxia（0. 010 ± 0. 003, P 〈 0.01 ）, and it reached the peak 12 hrs after hypoxia （0. 192 ± 0. 023, P 〈 0.01 ）. The phosphorylation of JNK was enhanced with the prolongation of hypoxic time. CASK protein expression was suppressed by JNK specific inhibitor SP600125 in a dose dependent manner, and it decreased to the lowest level with 10 μmol/L SP600125 pretreatment. Conclusion JNK signal pathway is involved in short-term hypoxia related CASK upregulation.