蛋白磷酸酶4在棕榈酸降低人脐静脉内皮细胞eNOS Ser633位点磷酸化中的作用

目的:探讨蛋白磷酸酶4(protein phosphatase 4,PP4)在棕榈酸(palmitic acid,PA)引起内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)Ser633位点磷酸化水平降低中的调控作用。方法:选用人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)为研究对象,分别用终浓度为25μmol/L、50μmol/L、100μmol/L和200μmol/L的PA处理HUVECs 36 h,另用100μmol/L PA处理HUVECs 12 h、24 h、36 h和48 h,用蛋白磷酸酶2A(protein phosphatase 2A,PP2A)家族抑制剂福司曲星(fostriecin,FST)20 nmol/L或冈田酸(okadaic acid,OA)5 nmol/L分别预处理细胞30 min,然后用蛋白磷酸酶4催化亚基(protein phosphatase 4 catalytic subunit,PP4c)小干扰RNA(small interfering RNA,siRNA)和蛋白磷酸酶2A催化亚基(protein phosphatase 2A catalytic subunit,PP2Ac)siRNA分别转染HUVECs。用Western blot法检测eNOS总蛋白、PP4c和PP2Ac蛋白表达水平及eNOS Ser633磷酸化水平,用DAF-FM DA荧光探针检测细胞内一氧化氮(nitric oxide,NO)的含量。结果:(1)与control组比较,PA(终浓度25μmol/L、50μmol/L、100μmol/L和200μmol/L)处理HUVECs 36 h后,eNOS Ser633磷酸化水平均显著降低(P<0.05),100μmol/L PA处理HUVECs 24 h、36 h和48 h后eNOS Ser633磷酸化水平均显著降低(P<0.05);各组间eNOS总蛋白表达无显著差异。(2)与control组相比,FST和OA预处理均可逆转PA处理引起的eNOS Ser633磷酸化水平降低(P<0.05)和细胞内NO产量减少(P<0.05);各组间eNOS总蛋白表达无显著差异。(3)与siControl组相比,si-PP4c转染组PP4c蛋白表达水平显著降低(P<0.05),同时eNOS Ser633磷酸化水平显著增高(P<0.05);si-PP2Ac转染组PP2Ac蛋白表达水平显著降低(P<0.05),但eNOS Ser633磷酸化水平表达无显著差异;各组间eNOS总蛋白表达无显著差异。结论:PA可显著降低HUVECs中eNOS Ser633磷酸化水平及NO产量,其机制可能是由于PA诱导PP2A家族中的PP4而不是PP2A激活所致。

Roles of protein phosphatase 4 in down-regulation of eNOS Ser633 phosphorylation induced by palmitic acid in human umbilical vein endothelial cells

AIM:To investigate the roles of protein phosphatase 4(PP4)in down-regulation of endothelial nitric oxide synthase(eNOS)Ser633 phosphorylation induced by palmitic acid(PA).METHODS:Human umbilical vein endothelial cells(HUVECs)were treated with PA at 25 μmol/L,50 μmol/L,100 μmol/L and 200μmol/L for 36 h,or treated with PA at 100 μmol/L for 12 h,24 h,36 h and 48 h.Protein phosphatase 2 A(PP2 A)family inhibitor fostriecin(FST,20 nmol/L)or okadaic acid(OA,5 nmol/L)was selected to pretreat the HUVECs for 30 min.Protein phosphatase4 catalytic subunit(PP4 c)siRNA or protein phosphatase 2 A catalytic subunit(PP2 Ac)siRNA was transfected into the HUVECs.The protein expression levels of of eNOS,PP4 c and PP2 Ac,as well as the level of eNOS Ser633 phosphorylation,were detected by Western blot.The intracellular nitric oxide(NO)content was measured by DAF-FM DA.RESULTS:(1)Compared with control group,the levels of eNOS Ser633 phosphorylation were decreased in PA groups in which the HUVECs were treated with 25 μmol/L,50 μmol/L,100 μmol/L and 200 μmol/L PA for 36 h(P<0.05)and100 μmol/L PA for 24 h,36 h and 48 h(P<0.05).No significant difference in the level of total eNOS protein expression among all the groups was observed.(2)Compared with control group,both FST and OA pretreatment reversed the reduction of eNOS Ser633 phosphorylation(P<0.05)and the decrease in intracellular NO content(P<0.05)induced by PA.No significant difference in the level of total eNOS protein expression among all the groups was observed.(3)Compared with si-Control group,the PP4 c protein expression was significantly reduced(P<0.05),while the level of eNOS Ser633 phosphorylation was significantly increased in si-PP4 c group(P<0.05).Although the levels of PP2 Ac protein expression declined significantly(P<0.05),the level of eNOS Ser633 phosphorylation remained unchanged in si-PP2 Ac group.No significant differencein the level of total eNOS protein expression among all the groups was found.CONCLUSION:PA significantly reduces the level of eNOS Ser633 phosphorylation and the content of NO in the HUVECs,which may be due to PA inducing the activation of the PP2 A family member PP4 rather than PP2 A.