棕榈酸激活人脐静脉内皮细胞PP2C降低eNOS Ser1177的磷酸化

Palmitic acid decreases phosphorylation of eNOS Ser1177 by activating protein phosphatase 2C (PP2C) of human umbilical vein endothelial cells

目的探讨棕榈酸(PA)激活人脐静脉内皮细胞(HUVEC)蛋白磷酸酶2C(PP2C)对内皮一氧化氮合酶第1177位丝氨酸(eNOS Ser1177)磷酸化的调控作用。方法HUVEC随机分为对照组、PA组、特异性PP2C抑制剂血根碱(San)联合PA组以及转染PP2Cα特异性小干扰RNA(siRNA)组。采用Western blot法检测eNOS总蛋白、eNOS Ser1177磷酸化水平和PP2Cα蛋白水平,二氨基荧光素-FM二乙酸酯(DAF-FM DA)负载法检测细胞内一氧化氮(NO)含量,免疫共沉淀法观察eNOS与PP2C之间的共定位关系。结果与对照组相比,PA处理组eNOS Ser1177磷酸化水平及NO含量均降低,San预处理组可逆转以上变化。敲低PP2Cα蛋白表达水平后,eNOS Ser1177磷酸化水平增加。eNOS与PP2C在细胞内共定位。结论PA通过激活HUVEC的PP2C引起eNOS Ser1177磷酸化水平降低。

Objective To investigate the roles of protein phosphatase 2C(PP2C)activated by palmitic acid(PA)in the phosphorylation modulation of endothelial nitric oxide(eNOS)at the site of serine 1177(eNOS Ser1177)in human umbilical vein endothelial cells(HUVECs).Methods HUVECs were randomly divided into control group,PA group,specific PP2C inhibitor sanguinarine(San)combined with PA group and PP2Cαspecific small interference RNA(siRNA)transfection group.The protein expression of total eNOS,phosphorylated eNOS Ser1177 and PP2Cαwere detected by Western blotting.The intracellular NO content was measured by DAF-FM DA.The co-localization of eNOS protein and PP2C protein was observed by co-immunoprecipitation.Results Compared with the control group,the phosphorylation levels of eNOS Ser1177 and NO content decreased significantly in the PA group.The PP2C inhibitor sanguinarine reversed PA-induced decrease of eNOS Ser1177 phosphorylation level and NO content.The phosphorylation levels of eNOS Ser1177 were enhanced dramatically after PP2Cαprotein was knocked down by specific siRNA.The eNOS protein and PP2C protein co-localized in endothelial cells.Conclusion PA reduces the phosphorylation level of endothelial eNOS Ser1177 in HUVECs by activating PP2C.