ADP Signaling in Vascular Endothelial Cells ADP-DEPENDENT ACTIVATION OF THE ENDOTHELIAL ISOFORM OF NITRIC-OXIDE SYNTHASE REQUIRES THE EXPRESSION BUT NOT THE KINASE ACTIVITY OF AMP-ACTIVATED PROTEIN KINASE
ADP Signaling in Vascular Endothelial Cells ADP-DEPENDENT ACTIVATION OF THE ENDOTHELIAL ISOFORM OF NITRIC-OXIDE SYNTHASE REQUIRES THE EXPRESSION BUT NOT THE KINASE ACTIVITY OF AMP-ACTIVATED PROTEIN KINASE
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DOI：
10.1074/jbc.m109.032656
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发表时间：
2009-11-20
影响因子：
4.8
通讯作者：
Michel, Thomas
中科院分区：
文献类型：
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作者：
Hess, Connie Ng;Kou, Ruqin;Michel, Thomas
ADP反应是许多心血管疾病治疗方法的基础，ADP受体拮抗剂在临床广泛使用。ADP在血小板生物学中的作用已被广泛研究，但内皮细胞中的ADP信号通路仍不完全清楚。我们发现ADP促进了一氧化氮合酶（eNOS）内皮异构体丝氨酸位点（1179）和丝氨酸位点（635）的磷酸化和丝氨酸位点（116）的去磷酸化。尽管ADP和ATP都能刺激eNOS活性，但只有ADP信号被P2Y(1)受体拮抗剂MRS 2179或用小干扰RNA （siRNA）敲低P2Y(1)显著抑制。ADP激活小GTPase Rac1，促进内皮细胞迁移。sirna介导的敲低Rac1阻断adp依赖性eNOS Ser（1179）和Ser（635）磷酸化，以及eNOS激活。我们分别使用抑制剂wortmannin、PD98059、PP2和STO-609分析了已知的调节eNOS的途径，包括磷酸肌醇3-激酶/Akt、ERK1/2、Src和钙/钙调素依赖性激酶β (CaMKK β)。这些抑制剂都没有改变adp调节的eNOS磷酸化。相比之下，sirna介导的amp活化蛋白激酶（AMPK）的敲低抑制adp依赖性eNOS Ser（635）磷酸化和eNOS活性，但不影响eNOS Ser（1179）磷酸化。重要的是，AMPK酶抑制剂化合物C对adp刺激的eNOS活性没有影响，尽管它完全阻断了AMPK活性。CaMKK β敲低抑制ADP刺激的eNOS活性，但STO-609抑制CaMKK β激酶活性不能影响ADP对eNOS的激活。这些数据表明AMPK和CaMKK β的表达，而不是激酶活性，是ADP向eNOS信号传导所必需的。
ADP responses underlie therapeutic approaches to many cardiovascular diseases, and ADP receptor antagonists are in widespread clinical use. The role of ADP in platelet biology has been extensively studied, yet ADP signaling pathways in endothelial cells remain incompletely understood. We found that ADP promoted phosphorylation of the endothelial isoform of nitric-oxide synthase (eNOS) at Ser(1179) and Ser(635) and dephosphorylation at Ser(116) in cultured endothelial cells. Although eNOS activity was stimulated by both ADP and ATP, only ADP signaling was significantly inhibited by the P2Y(1) receptor antagonist MRS 2179 or by knockdown of P2Y(1) using small interfering RNA (siRNA). ADP activated the small GTPase Rac1 and promoted endothelial cell migration. siRNA-mediated knockdown of Rac1 blocked ADP-dependent eNOS Ser(1179) and Ser(635) phosphorylation, as well as eNOS activation. We analyzed pathways known to regulate eNOS, including phosphoinositide 3-kinase/Akt, ERK1/2, Src, and calcium/calmodulin-dependent kinase kinase-beta (CaMKK beta) using the inhibitors wortmannin, PD98059, PP2, and STO-609, respectively. None of these inhibitors altered ADP-modulated eNOS phosphorylation. In contrast, siRNA-mediated knockdown of AMP-activated protein kinase (AMPK) inhibited ADP-dependent eNOS Ser(635) phosphorylation and eNOS activity but did not affect eNOS Ser(1179) phosphorylation. Importantly, the AMPK enzyme inhibitor compound C had no effect on ADP-stimulated eNOS activity, despite completely blocking AMPK activity. CaMKK beta knockdown suppressed ADP-stimulated eNOS activity, yet inhibition of CaMKK beta kinase activity using STO-609 failed to affect eNOS activation by ADP. These data suggest that the expression, but not the kinase activity, of AMPK and CaMKK beta is necessary for ADP signaling to eNOS.