Protein kinase C-ε regulates sphingosine 1-phosphate-mediated migration of human lung endothelial cells through activation of phospholipase D2, protein kinase C-ζ, and Rac1
Protein kinase C-ε regulates sphingosine 1-phosphate-mediated migration of human lung endothelial cells through activation of phospholipase D2, protein kinase C-ζ, and Rac1
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DOI：
10.1074/jbc.m800250200
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发表时间：
2008-04-25
影响因子：
4.8
通讯作者：
Natarajan, Viswanathan
中科院分区：
文献类型：
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作者：
Gorshkova, Irina;He, Donghong;Natarajan, Viswanathan
1-磷酸鞘氨醇（S1 P）有效刺激内皮细胞迁移和血管生成的信号通路尚未完全确定。因此，我们研究了蛋白激酶C（PKC）亚型，磷脂酶D（PLD）和Rac在S1 P诱导的人肺动脉内皮细胞（HPAECs）迁移中的作用。S1 P诱导的迁移对S1 P（1）小干扰RNA（siRNA）和百日咳毒素敏感，表明S1 P（1）与G（i）偶联。显性负性（dn）PKC-β或-zeta的过表达，而不是PKC-α或-δ，阻断S1 P诱导的迁移。尽管S1 P激活了PLD 1和PLD 2，但通过敲低PLD 2或表达dnPLD 2而不是PLD 1来减弱S1 P诱导的迁移。阻断PKC-β而非PKC-zeta活性减弱S1 P介导的PLD刺激，表明PKC-β而非PKC-zeta位于PLD的上游。用dnRac 1或Rac 1 siRNA转染HPAEC减弱S1 P诱导的迁移。此外，转染PLD 2 siRNA，感染的HPAECs与dnPKC-zeta，或治疗与豆蔻酰化的PKC-zeta肽抑制剂废除S1 P诱导的Rac 1激活。这些结果证实S1 P通过S1 P1和Gi信号传导以激活PKC-Rac 1，随后激活PLD 2-PKC-zeta-Rac 1级联。该途径的激活对于刺激肺内皮细胞的迁移是必要的，肺内皮细胞是血管生成过程的关键组成部分。
The signaling pathways by which sphingosine 1-phosphate (S1P) potently stimulates endothelial cell migration and angiogenesis are not yet fully defined. We, therefore, investigated the role of protein kinaseC(PKC) isoforms, phospholipase D(PLD), and Rac in S1P-induced migration of human pulmonary artery endothelial cells (HPAECs). S1P-induced migration was sensitive to S1P(1) small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S1P(1) to G(i). Overexpression of dominant negative (dn) PKC-epsilon or -zeta, but not PKC-alpha or -delta, blocked S1P-induced migration. Although S1P activated both PLD1 and PLD2, S1P-induced migration was attenuated by knocking down PLD2 or expressing dnPLD2 but not PLD1. Blocking PKC-epsilon, but not PKC-zeta, activity attenuated S1P-mediated PLD stimulation, demonstrating that PKC-epsilon, but not PKC-zeta, was upstream of PLD. Transfection of HPAECs with dnRac1 or Rac1 siRNA attenuated S1P-induced migration. Furthermore, transfection with PLD2 siRNA, infection of HPAECs with dnPKC-zeta, or treatment with myristoylated PKC-zeta peptide inhibitor abrogated S1P-induced Rac1 activation. These results establish that S1P signals through S1P1 and Gi to activate PKC-epsilon and, subsequently, a PLD2-PKC-zeta-Rac1 cascade. Activation of this pathway is necessary to stimulate the migration of lung endothelial cells, a key component of the angiogenic process.