Atrial natriuretic peptide-initiated cGMP pathways regulate vasodilator-stimulated phosphoprotein phosphorylation and angiogenesis in vascular endothelium
Atrial natriuretic peptide-initiated cGMP pathways regulate vasodilator-stimulated phosphoprotein phosphorylation and angiogenesis in vascular endothelium
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DOI：
10.1074/jbc.m709439200
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发表时间：
2008-02-15
影响因子：
4.8
通讯作者：
Lin, Alison J.
中科院分区：
文献类型：
--
作者：
Chen, Hongjie;Levine, Yehoshua C.;Lin, Alison J.
一氧化氮(NO)和心钠素(ANP)介导的cGMP信号转导通路在维持心血管内环境平衡中起重要作用。然而，cGMP下游的分子信号机制还不是很清楚。我们已经使用小干扰RNA(SiRNA)方法特异性地敲除了牛主动脉内皮细胞中的一系列信号蛋白，并结合生化分析和生理检测来研究cGMP介导的信号转导途径。ANP激活颗粒鸟苷环化酶(GC-A)导致细胞内cGMP显著、剂量依赖性、快速和持续增加。相反，NO对可溶性鸟苷环化酶的刺激只能产生微弱的、短暂的cGMP增加。心钠素诱导的cGMP产生通过siRNA介导的GC-A被选择性地抑制。ANP显著增强血管扩张剂刺激的磷酸蛋白(VASP)在Ser-239处的磷酸化，Vasp是cGMP依赖的蛋白激酶(PKG)的主要底物，显著影响肌动蛋白的动力学。此外，ANP诱导的Vasp在Ser-239处的磷酸化伴随着肌动蛋白应力纤维形成的增加和内皮管的形成。SiRNA介导的GC-A、Vasp或PKG基因敲除可取消ANP诱导的Vasp Ser-239磷酸化、应力纤维形成和内皮管形成。我们已经在人脐静脉内皮细胞中证明了类似的发现，ANP显著增加细胞内cGMP含量，Ser-239处Vasp的磷酸化，以及内皮管的形成。综上所述，我们的研究结果表明，ANP介导的cGMP信号转导通路调节内皮细胞中Vasp Ser-239的PKG磷酸化，导致肌动蛋白细胞骨架的重组，促进血管生成。
Nitric oxide (NO)- and atrial natriuretic peptide (ANP)-initiated cGMP signaling cascades are important in the maintenance of cardiovascular homeostasis. The molecular signaling mechanisms downstream of cGMP are not well understood, however. We have used small interfering RNA (siRNA) approaches to specifically knock down a series of signaling proteins in bovine aortic endothelial cells, and we have combined biochemical analyses with physiological assays to investigate cGMP-mediated signal transduction pathways. Activation of particulate guanylate cyclase (GC-A) by ANP leads to a substantial, dose-dependent, rapid, and sustained increase in intracellular cGMP. In contrast, stimulation of soluble guanylate cyclase by NO yields only a weak and transient increase in cGMP. ANP-induced cGMP production is selectively suppressed by siRNA-mediated knockdown of GC-A. ANP greatly enhances the phosphorylation at Ser-239 of the vasodilator-stimulated phosphoprotein (VASP), a major substrate of cGMP-dependent protein kinase (PKG) that significantly influences actin dynamics. Moreover, the ANP-induced phosphorylation of VASP at Ser-239 is accompanied by increased actin stress fiber formation and enhanced endothelial tube formation. siRNA-mediated knockdown of GC-A, VASP, or PKG abolishes ANP-induced VASP Ser-239 phosphorylation, stress fiber formation, and endothelial tube formation. We have demonstrated similar findings in human umbilical vein endothelial cells, where ANP substantially enhances intracellular cGMP content, phosphorylation of VASP at Ser-239, and endothelial tube formation. Taken together, our findings suggest that ANP-mediated cGMP signal transduction pathways regulate PKG phosphorylation of VASP Ser-239 in endothelial cells, resulting in reorganization of the actin cytoskeleton and enhancement of angiogenesis.