Fu Panfeng, Usatyuk Peter V, Jacobson Jeffrey, Cress Anne E, Garcia Joe G N, Salgia Ravi, Natarajan Viswanathan
Department of Pharmacology, University of Illinois, Chicago, Illinois, USA.
Department of Medicine, University of Illinois, Chicago, Illinois, USA.
Pulm Circ. 2015 Dec;5(4):619-30. doi: 10.1086/683693.
Paxillin is a multifunctional and multidomain focal adhesion adaptor protein. It serves as an important scaffolding protein at focal adhesions by recruiting and binding to structural and signaling molecules. Paxillin tyrosine phosphorylation at Y31 and Y118 is important for paxillin redistribution to focal adhesions and angiogenesis. Hepatocyte growth factor (HGF) and sphingosine-1-phosphate (S1P) are potent stimulators of lamellipodia formation, a prerequisite for endothelial cell migration. The role played by paxillin and its tyrosine phosphorylated forms in HGF- or S1P-induced lamellipodia formation and barrier function is unclear. HGF or S1P stimulated lamellipodia formation, tyrosine phosphorylation of paxillin at Y31 and Y118, and c-Abl in human lung microvascular endothelial cells (HLMVECs). Knockdown of paxillin with small interfering RNA (siRNA) or transfection with paxillin mutants (Y31F or Y118F) mitigated HGF- or S1P-induced lamellipodia formation, translocation of p47 (phox) to lamellipodia, and reactive oxygen species (ROS) generation in HLMVECs. Furthermore, exposure of HLMVECs to HGF or S1P stimulated c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 in a time-dependent fashion, and down-regulation of c-Abl with siRNA attenuated HGF- or S1P-mediated lamellipodia formation, translocation of p47 (phox) to lamellipodia, and endothelial barrier enhancement. In vivo, knockdown of paxillin with siRNA in mouse lungs attenuated ventilator-induced lung injury. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates HGF- or S1P-mediated lamellipodia formation, ROS generation in lamellipodia, and endothelial permeability.
桩蛋白是一种多功能、多结构域的粘着斑衔接蛋白。它通过招募并结合结构和信号分子，在粘着斑处作为重要的支架蛋白发挥作用。桩蛋白在Y31和Y118位点的酪氨酸磷酸化对于其重新分布到粘着斑和血管生成很重要。肝细胞生长因子（HGF）和1-磷酸鞘氨醇（S1P）是片状伪足形成的有效刺激物，而片状伪足形成是内皮细胞迁移的前提条件。桩蛋白及其酪氨酸磷酸化形式在HGF或S1P诱导的片状伪足形成和屏障功能中所起的作用尚不清楚。HGF或S1P刺激人肺微血管内皮细胞（HLMVECs）形成片状伪足、桩蛋白在Y31和Y118位点的酪氨酸磷酸化以及c-Abl的激活。用小干扰RNA（siRNA）敲低桩蛋白或转染桩蛋白突变体（Y31F或Y118F）可减轻HGF或S1P诱导的HLMVECs片状伪足形成、p47（phox）向片状伪足的转位以及活性氧（ROS）的产生。此外，将HLMVECs暴露于HGF或S1P会以时间依赖性方式刺激c-Abl介导的桩蛋白在Y31和Y118位点的酪氨酸磷酸化，用siRNA下调c-Abl会减弱HGF或S1P介导的片状伪足形成、p47（phox）向片状伪足的转位以及内皮屏障增强。在体内，用siRNA敲低小鼠肺中的桩蛋白可减轻呼吸机诱导的肺损伤。总之，这些结果表明，c-Abl介导的桩蛋白在Y31和Y118位点的酪氨酸磷酸化调节HGF或S1P介导的片状伪足形成、片状伪足中的ROS产生以及内皮通透性。
