Localization of VEGFR-2 and PLD2 in endothelial caveolae is involved in VEGF-induced phosphorylation of MEK and ERK
Localization of VEGFR-2 and PLD2 in endothelial caveolae is involved in VEGF-induced phosphorylation of MEK and ERK
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DOI：
10.1152/ajpheart.00786.2003
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发表时间：
2004-05-01
影响因子：
4.8
通讯作者：
Koh, GY
中科院分区：
文献类型：
--
作者：
Cho, CH;Lee, CS;Koh, GY
为了阐明小凹在 VEGF/VEGF 受体 2 (VEGFR-2) 介导的信号级联中的作用，对原代培养的人脐静脉内皮细胞 (HUVEC) 进行分级分离，以分离出富含小凹的细胞膜。有趣的是，VEGFR-2、磷脂酶 D-2 (PLD2) 和 Ras 在富含小凹的级分中富集。此外，VEGF 以时间和剂量依赖性方式增加 HUVEC 中的 PLD 活性，而 VEGFR-1 胎盘生长因子特异性配体不会改变 PLD 活性。 PLD 抑制剂 1-丁醇几乎完全抑制 VEGF 诱导的 ERK 磷酸化和细胞增殖，而 1-丁醇的阴性对照 3-丁醇则没有产生显着变化。磷脂酸的添加消除了 1-丁醇诱导的抑制。使用几种抑制剂的药理学分析表明 PKC-delta 调节 VEGF 诱导的 PLD/ERK 激活。因此，PLD2可能参与内皮细胞中由VEGF/VEGFR-2/PKC-delta途径诱导的MEK/ERK信号级联。用胆固醇消耗剂甲基-β-环糊精 (MbetaCD) 进行预处理几乎完全分解了小凹结构，而向 MbetaCD 处理的细胞中添加胆固醇则恢复了小凹结构。 MbetaCD 预处理很大程度上消除了 VEGF 对 MEK/ERK 的磷酸化，而添加胆固醇则恢复了 VEGF 诱导的 MEK/ERK 磷酸化。这些结果表明完整的小凹是 VEGF/VEGFR-2 介导的 MEK/ERK 信号级联反应所必需的。
To clarify the role of caveolae in VEGF/VEGF receptor-2 (VEGFR-2)-mediated signaling cascades, primary cultured human umbilical vein endothelial cells (HUVECs) were fractionated to isolate caveolae-enriched cell membranes. Interestingly, VEGFR-2, phospholipase D-2 (PLD2), and Ras were enriched in caveolae-enriched fractions. Moreover, VEGF increased PLD activity in a time- and dose-dependent manner in HUVECs, whereas a ligand specific for VEGFR-1 placental growth factor did not change PLD activity. A PLD inhibitor, 1-butanol, almost completely suppressed VEGF-induced ERK phosphorylation and cellular proliferation, whereas the negative control for 1-butanol, 3-butanol, did not produce significant changes. Addition of phosphatidic acid negated the 1-butanol-induced suppression. Pharmacological analyses using several inhibitors indicated that PKC-delta regulates the VEGF-induced activation of PLD/ERK. Thus PLD2 could be involved in MEK/ERK signaling cascades that are induced by the VEGF/VEGFR-2/PKC-delta pathway in endothelial cells. Pretreatment with the cholesterol depletion agent methyl-beta-cyclodextrin (MbetaCD) almost completely disassembled caveolar structures, whereas the addition of cholesterol to MbetaCD-treated cells restored caveolar structures. Pretreatment with MbetaCD largely abolished phosphorylation of MEK/ERK by VEGF, whereas the addition of cholesterol restored VEGF-induced MEK/ERK phosphorylations. These results indicate that intact caveolae are required for the VEGF/VEGFR-2-mediated MEK/ERK signaling cascade.