VEGF regulation of endothelial nitric oxide synthase in glomerular endothelial cells
VEGF regulation of endothelial nitric oxide synthase in glomerular endothelial cells
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DOI：
10.1111/j.1523-1755.2005.00575.x
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发表时间：
2005-10-01
影响因子：
19.6
通讯作者：
Kasinath, BS
中科院分区：
文献类型：
--
作者：
Feliers, D;Chen, XY;Kasinath, BS
血管内皮生长因子对肾小球内皮细胞内皮型一氧化氮合酶的调节。血管内皮生长因子(VEGF)对内皮型一氧化氮合酶(ENOS)的调控及其信号转导途径在肾小球内皮细胞(GENC)中的作用尚未见报道。使用从tsA58 Immortomice(R)培养的GENC。免疫印迹和细胞内Western印迹分析检测血管内皮细胞生长因子受体信号通路和内皮型一氧化氮合酶磷酸化的变化。采用免疫激活法和磷酸化特异性抗体免疫印迹法检测蛋白激酶活性。血管内皮生长因子可迅速诱导1型和2型血管内皮生长因子受体酪氨酸磷酸化。血管内皮生长因子诱导血管内皮生长因子受体2(VEGF-R2)与胰岛素受体底物(IRS-1)和磷脂酰肌醇3‘-激酶(PI3K)之间的物理结合，增强了VEGF-R2免疫沉淀物中PI3K的活性。血管内皮生长因子以PI3K依赖的方式刺激Akt的磷酸化。血管内皮细胞生长因子增加Ser1177上eNOS的磷酸化。ENOS的激活与一氧化氮的产生有关，通过中等亚硝酸盐含量来衡量。探讨了血管内皮细胞生长因子刺激eNOS的信号转导机制。VEGF-R2抑制剂SU1498、PI3K抑制剂LY294002以及携带显性Akt负性突变体的腺病毒感染细胞，均可阻断VEGF诱导的eNOS磷酸化，表明eNOS的激活需要VEGF-R2/IRS-1/PI3K/Akt轴的参与。血管内皮生长因子还以时间依赖的方式激活细胞外信号调节蛋白激酶(ERK)，ERK上游抑制剂PD098059可阻断血管内皮生长因子刺激的eNOS对Ser1177的磷酸化，表明ERK参与了血管内皮生长因子对eNOS的调节。LY294002可抑制ERK的磷酸化，提示在经VEGF处理的GENC中，ERK位于PI3K下游。我们的研究结果表明，在Genc中，血管内皮细胞生长因子刺激血管内皮生长因子-R2/IRS-1/PI3K/Akt轴，通过ERK信号通路调节Ser1177上eNOS的磷酸化。
VEGF regulation of endothelial nitric oxide synthase in glomerular endothelial cells.Background. Vascular endothelial growth factor (VEGF) regulation of endothelial nitric oxide synthase (eNOS) and signaling pathways involved have not been well studied in glomerular endothelial cells (GENCs).Methods. GENCs grown from tsA58 Immortomice (R) were used. Immunoblotting and in-cell Western blot analysis were employed to assess changes in VEGF receptor signaling pathway and eNOS phosphorylation of ser1177. Immunokinase assay and immunoblotting with phosphospecific antibodies were performed to assess activity of kinases.Results. VEGF rapidly induced tyrosine phosphorylation of type 1 and type 2 VEGF receptors. Physical association between VEGF-receptor 2 (VEGF-R2) and insulin receptor substrate (IRS-1) and phosphatidylinositol 3'-kinase (PI3K) was induced by VEGF, which augmented PI3K activity in VEGF-R2 immunoprecipitates. VEGF stimulated Akt phosphorylation in a PI3K-dependent manner. VEGF increased eNOS phosphorylation on Ser1177. Activation of eNOS was associated with nitric oxide generation as measured by medium nitrite content. Signaling mechanisms involved in VEGF stimulation of eNOS were explored. VEGF-induced eNOS phosphorylation was abolished by SU1498, a VEGF-R2 inhibitor, LY294002, a PI3K inhibitor, and infection of cells with an adenovirus carrying a dominant negative-mutant of Akt, demonstrating the requirement of the VEGF-R2/IRS-1/PI3K/Akt axis for activation of eNOS. VEGF also activated extracellular signal-regulated protein kinase (ERK) in a time-dependent manner; and VEGF-stimulated eNOS phosphorylation on Ser1177 was prevented by PD098059, an upstream inhibitor of ERK, demonstrating that ERK was involved in VEGF regulation of eNOS. ERK phosphorylation was abolished by LY294002, suggesting ERK was downstream of PI3K in VEGF-treated GENC.Conclusions. Our data demonstrate that in GENC, VEGF stimulates VEGF-R2/IRS-1/PI3K/Akt axis to regulate eNOS phosphorylation on Ser1177 in conjunction with the ERK signaling pathway.