Mucosal Biology Research Center, and Departments of Medicine and Pathology, University of Maryland, School of Medicine, Baltimore, Maryland 21201, USA.
Am J Physiol Lung Cell Mol Physiol. 2011 Jul;301(1):L79-90. doi: 10.1152/ajplung.00287.2010. Epub 2011 Apr 29.
Thrombospondin-1 (TSP1) is a multidomain protein that contains epidermal growth factor (EGF)-like repeats that indirectly activate the EGF receptor (EGFR) and selected downstream signaling pathways. In these studies, we show that TSP1 opens the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls) in a dose-, time-, and protein tyrosine kinase (PTK)-dependent manner. TSP1 increased tyrosine phosphorylation of proteins enriched to intercellular boundaries including the zonula adherens (ZA) proteins, vascular endothelial-cadherin, γ-catenin, and p120 catenin. In HMVEC-Ls, EGFR and ErbB2 are expressed at low levels, and both heterodimerize and tyrosine autophosphorylate in response to TSP1. Prior EGFR-selective PTK inhibition with AG1478 or ErbB2-selective PTK inhibition with AG825 protected against TSP1-induced tyrosine phosphorylation of ZA proteins and barrier disruption. Preincubation of HMVEC-Ls with an EGFR ectodomain-blocking antibody also prevented TSP1-induced opening of the paracellular pathway. Therefore, in HMVEC-Ls, TSP1 increases tyrosine phosphorylation of ZA proteins and opens the paracellular pathway, in part, through EGFR/ErbB2 activation. Surprisingly, recombinant TSP1 EGF-like repeats 1-3 and the high-affinity EGFR ligands, EGF, TGF-α, and amphiregulin, each failed to increase paracellular permeability. However, HMVEC-Ls in which EGFR was overexpressed became responsive to the EGF-like repeats of TSP1 as well as to EGF. These studies indicate that TSP1 disrupts the endothelial barrier through EGFR/ErbB2 activation although additional signals are necessary in cells with low receptor expression.
血小板反应蛋白-1（TSP1）是一种具有表皮生长因子（EGF）样重复序列的多功能蛋白，可间接激活表皮生长因子受体（EGFR）和选定的下游信号通路。在这些研究中，我们表明 TSP1 以剂量、时间和蛋白酪氨酸激酶（PTK）依赖的方式打开人肺微血管内皮细胞（HMVEC-L）的细胞旁途径。TSP1 增加了富含细胞间边界的蛋白质的酪氨酸磷酸化，包括粘着连接（ZA）蛋白、血管内皮钙粘蛋白、γ-连环蛋白和 p120 连环蛋白。在 HMVEC-L 中，EGFR 和 ErbB2 的表达水平较低，两者均可异二聚化并对 TSP1 发生酪氨酸自磷酸化。用 AG1478 进行 EGFR 选择性 PTK 抑制或用 AG825 进行 ErbB2 选择性 PTK 抑制可防止 TSP1 诱导的 ZA 蛋白酪氨酸磷酸化和屏障破坏。HMVEC-L 预先用 EGFR 细胞外结构域阻断抗体孵育也可防止 TSP1 诱导的细胞旁途径开放。因此，在 HMVEC-L 中，TSP1 通过 EGFR/ErbB2 激活增加 ZA 蛋白的酪氨酸磷酸化并打开细胞旁途径。令人惊讶的是，重组 TSP1 EGF 样重复 1-3 和高亲和力 EGFR 配体 EGF、TGF-α 和 Amphiregulin 均未能增加细胞旁通透性。然而，EGFR 过表达的 HMVEC-L 对 TSP1 的 EGF 样重复序列以及 EGF 变得有反应。这些研究表明，尽管在受体表达水平较低的细胞中需要其他信号，但 TSP1 通过 EGFR/ErbB2 激活破坏内皮屏障。
