Inhibition of MEK/ERK1/2 signalling alters endothelial nitric oxide synthase activity in an agonist-dependent manner
Inhibition of MEK/ERK1/2 signalling alters endothelial nitric oxide synthase activity in an agonist-dependent manner
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DOI：
10.1042/bj20060371
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发表时间：
2006-09-01
影响因子：
4.1
通讯作者：
Bird, An M.
中科院分区：
文献类型：
--
作者：
Cale, Jacqueline M.;Bird, An M.
eNOS（内皮型一氧化氮合酶）活性以复杂的方式通过酰化、蛋白质-蛋白质相互作用、细胞内运输和磷酸化等进行后调节。调节eNOS活性的信号通路包括磷酸肌醇3-激酶/Akt、环核苷酸依赖性激酶[PKA（蛋白激酶A）和PKG]、PKC以及ERK（细胞外信号调节激酶）。ERK在eNOS激活中的作用仍然存在争议。在本研究中，我们研究了ERK 1/2在HUVEC-CS [转化的HUVEC（人脐静脉内皮细胞）]以及广泛用于eNOS研究的模型瞬时转染的COS-7细胞中eNOS活化中的作用。U 0126预处理HUVEC-CS可增强ATP刺激的eNOS活性，与细胞内Ca ~（2+）浓度（[Ca ~（2+）]（i））变化无关。在瞬时表达绵羊eNOS的COS-7细胞中，U 0126增强A23187刺激的eNOS活性，但抑制ATP刺激的活性。五个关键eNOS残基磷酸化的补偿性变化不能解释A23187刺激活性的变化。然而，在ATP的情况下，改变的磷酸化和[Ca 2 +]（i）的变化可能部分有助于U 0126活性的抑制。最后，7 eNOS丙氨酸突变体的推定ERK 1/2的目标，并与A23187和ATP处理U 0126预处理eNOS活性的影响进行了测量。T97 A-eNOS是在U 0126预处理和eNOS激活的ATP刺激后与野生型显著不同的唯一构建体。在本研究中，在COS-7细胞中eNOS活性被增强或抑制，表明MEK/ERK 1/2信号传导[其中MEK是MAPK（促分裂原活化蛋白激酶）/ERK激酶]对eNOS的激动剂依赖性和复杂的机制，包括[Ca 2 +]（i）、磷酸化和可能的细胞内运输。
eNOS (endothelial nitric oxide synthase) activity is post-translationally regulated in a complex fashion by acylation, protein-protein interactions, intracellular trafficking and phosphorylation, among others. Signalling pathways that regulate eNOS activity include phosphomositide 3-kinase/Akt, cyclic nucleotide-dependent kinases [PKA (protein kinase A) and PKG], PKC, as well as ERKs (extracellular-signal-regulated kinases). The role of ERKs in eNOS activation remains controversial. In the present study, we have examined the role of ERK1/2 in eNOS activation in HUVEC-CS [transformed HUVEC (human umbilical-vein endothelial cells)] as well as a widely used model for eNOS study, transiently transfected COS-7 cells. U0126 pretreatment of HUVEC-CS potentiated ATP-stimulated eNOS activity, independent of changes in intracellular Ca2+ concentration ([Ca2+](i)). In COS-7 cells transiently expressing ovine eNOS, U0126 potentiated A23187-stimulated eNOS activity, but inhibited ATP-stimulated activity. Compensatory changes in phosphorylation of five key eNOS residues did not account for changes in A23187-stimulated activity. However, in the case of ATP, altered phosphorylation and changes in [Ca2+](i) may partially contribute to U0126 inhibition of activity. Finally, seven eNOS alanine mutants of putative ERK1/2 targets were generated and the effects of U0126 pretreatment on eNOS activity were gauged with A23187 and ATP treatment. T97A-eNOS was the only construct significantly different from wild-type after U0126 pretreatment and ATP stimulation of eNOS activation. In the present study, eNOS activity was either potentiated or inhibited in COS-7 cells, suggesting agonist dependence for MEK/ERK1/2 signalling [where MEK is MAPK (mitogen-activated protein kinase)/ERK kinase] to eNOS and a complex mechanism including [Ca2+](i), phosphorylation and, possibly, intracellular trafficking.