Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
PLoS One. 2011;6(8):e22334. doi: 10.1371/journal.pone.0022334. Epub 2011 Aug 1.
Tumor Endothelial Marker 8/Anthrax Toxin Receptor 1 (TEM8/ANTXR1) expression is induced in the vascular compartment of multiple tumors and therefore, is a candidate molecule to target tumor therapies. This cell surface molecule mediates anthrax toxin internalization, however, its physiological function in blood vessels remains largely unknown. We identified the chicken chorioallantoic membrane (CAM) as a model system to study the endogenous function of TEM8 in blood vessels as we found that TEM8 expression was induced transiently between day 10 and 12 of embryonic development, when the vascular tree is undergoing final development and growth. We used the cell-binding component of anthrax toxin, Protective Antigen (PA), to engage endogenous TEM8 receptors and evaluate the effects of PA-TEM8 complexes on vascular development. PA applied at the time of highest TEM8 expression reduced vascular density and disrupted hierarchical branching as revealed by quantitative morphometric analysis of the vascular tree after 48h. PA-dependent reduced branching phenotype was partially mimicked by Wnt3a application and ameliorated by the Wnt antagonist, Dikkopf-1. These results implicate TEM8 expression in endothelial cells in regulating the canonical Wnt signaling pathway at this day of CAM development. Consistent with this model, PA increased beta catenin levels acutely in CAM blood vessels in vivo and in TEM8 transfected primary human endothelial cells in vitro. TEM8 expression in Hek293 cells, which neither express endogenous PA-binding receptors nor Wnt ligands, stabilized beta catenin levels and amplified beta catenin-dependent transcriptional activity induced by Wnt3a. This agonistic function is supported by findings in the CAM, where the increase in TEM8 expression from day 10 to day 12 and PA application correlated with Axin 2 induction, a universal reporter gene for canonical Wnt signaling. We postulate that the developmentally controlled expression of TEM8 modulates endothelial cell response to canonical Wnt signaling to regulate vessel patterning and density.
肿瘤内皮标志物 8/炭疽毒素受体 1（TEM8/ANTXR1）在多种肿瘤的血管部位表达，因此是一种靶向肿瘤治疗的候选分子。这种细胞表面分子介导炭疽毒素的内化，然而，其在血管中的生理功能在很大程度上尚不清楚。我们发现鸡胚绒毛尿囊膜（CAM）是研究 TEM8 在血管中内源性功能的模型系统，因为我们发现 TEM8 的表达在胚胎发育的第 10 天到第 12 天之间短暂诱导，此时血管树正在进行最后的发育和生长。我们使用炭疽毒素的细胞结合成分，保护性抗原（PA），来结合内源性 TEM8 受体，并评估 PA-TEM8 复合物对血管发育的影响。在 TEM8 表达最高时施加 PA 会降低血管密度并破坏层次分支，这可以通过对血管树进行 48 小时后的定量形态计量分析来揭示。PA 依赖性分支减少表型部分模拟 Wnt3a 的应用，并通过 Wnt 拮抗剂 Dikkopf-1 得到改善。这些结果表明，在 CAM 发育的这一天，内皮细胞中的 TEM8 表达参与调节经典 Wnt 信号通路。与该模型一致，PA 在体内 CAM 血管中和体外 TEM8 转染的原代人内皮细胞中急性增加β-catenin 水平。在 Hek293 细胞中，既不表达内源性 PA 结合受体也不表达 Wnt 配体的 TEM8 表达稳定了β-catenin 水平，并放大了 Wnt3a 诱导的β-catenin 依赖性转录活性。这一激动作用得到了 CAM 中的发现的支持，其中从第 10 天到第 12 天 TEM8 表达的增加和 PA 的施加与 Axin 2 的诱导相关，Axin 2 是经典 Wnt 信号的通用报告基因。我们假设 TEM8 的发育调控表达调节内皮细胞对经典 Wnt 信号的反应，从而调节血管模式和密度。
