The analysis of atypical PKC (aPKC) in endothelial cells during angiogenesis

血管生成过程中内皮细胞非典型PKC（aPKC）分析

• 批准号: 259157124
• 负责人: Dr. Masanori Nakayama
• 金额: --
• 依托单位: Max-Planck-Institut für Herz- und Lungenforschung
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/259157124?language=en
• 关键词: analysis; atypical; PKC; aPKC; endothelial

During tissue morphogenesis and in tissue homeostasis, cell behaviors like migration and polarization are regulated simultaneously and in a tightly coordinated fashion. Endothelial cells (ECs) form tubular structures thereby contribute to closed circulation system. While ECs in established blood vessels show apical-basal polarity, endothelia acquire front-rear polarity for migratory behavior during angiogenesis. Cell polarity switching is regulated in this process. The formation of motile and filopodia-carrying endothelial sprouts is controlled by vascular endothelial growth factor (VEGF) together with ephrin-B2, a transmembrane ligand for Eph family receptor tyrosine kinases. Endothelial cell-to-cell interactions among sprouts trigger the phenotypic change of EC from motile to quiescent behavior. VE-cadherin-mediated contact formation is crucial for this process. Subsequently, ECs acquire apical-basal polarity again to facilitate vascular lumen. I have identified PAR-3 and the cargo adaptor protein Dab2 as ephrin-B2 interacting proteins. PAR-3/Dab2/ephrin-B2 interactions regulate VEGFR2/3 endocytosis. It is well known that PAR-3 forms a polarity protein complex, called the PAR complex together with PAR-6 and atypical protein kinase C (aPKC), and regulates epithelial junction formation by controlling actin cytoskeleton. Active aPKC, which I found to be much more abundant in the maturating vascular plexus, antagonizes VEGFR2/3 internalization by phosphorylating Dab2. This process contributes to the observed regional differences in VEGFR2/3 internalization. Thus, regionally distinct behaviors of ECs in the growing vasculature are not only modulated by the VEGF-A gradient, but also intrinsic properties of the endothelium. However the role of PAR-3 and aPKC in endothelial cell-to-cell junctions in vivo remains elusive. Furthermore, molecular machinery controlling spatially activated aPKC during angiogenesis is largely unknown.Here we found that adherence junction visualized by VE-cadherin and tight junction visualized by JAM-A in the growing vasculature in PAR-3 and aPKC endothelial specific-inducible mutant mice did not show prominent defects. On the other hand, the Claudin-5 transcription, the more matured EC tight junction marker, was compromised in aPKC but not in PAR-3 mutants. To gain further insight into mode of action of aPKC in ECs, we would like to propose to two projects by using endothelial specific-inducible mutant mice as a model system. 1) What is the role of junction mediated aPKC activation in the growing vasculature? 2) How does aPKC control junction maturation by regulating Claudin-5 transcription in vivo? The process of blood vessel formation is key in development and in the pathogenesis of diseases. Thus, by identifying the molecular mechanisms underlying aPKC signal transduction, this project will elucidate important insight into the function of in both physiological and pathological angiogenesis.

在组织形态发生和组织稳态中，细胞行为如迁移和极化同时受到调节，并且以紧密协调的方式进行。内皮细胞（EC）形成管状结构，从而有助于封闭的循环系统。在血管生成过程中，内皮细胞的迁移行为表现为前-后极性，而血管内皮细胞表现为顶-底极性。细胞极性转换在这个过程中受到调节。血管内皮生长因子（VEGF）和ephrin-B2（Eph家族受体酪氨酸激酶的跨膜配体）共同控制着运动和携带丝状伪足的内皮芽的形成。芽之间的内皮细胞与细胞的相互作用触发EC从运动到静止行为的表型变化。VE-钙粘蛋白介导的接触形成对于这一过程至关重要。随后，EC再次获得顶-底极性以促进血管腔。我已经确定PAR-3和货物衔接蛋白Dab 2作为肝配蛋白-B2相互作用蛋白。PAR-3/Dab 2/ephrin-B2相互作用调节VEGFR 2/3内吞作用。众所周知，PAR-3与PAR-6和非典型蛋白激酶C（aPKC）形成极性蛋白复合物，称为PAR复合物，并通过控制肌动蛋白细胞骨架来调节上皮连接的形成。我发现在成熟的血管丛中更丰富的活性aPKC通过磷酸化Dab 2拮抗VEGFR 2/3内化。这一过程有助于观察到的VEGFR 2/3内化的区域差异。因此，在生长的血管系统中EC的区域性不同行为不仅受到VEGF-A梯度的调节，而且还受到内皮细胞的内在特性的调节。然而，PAR-3和aPKC在体内内皮细胞-细胞连接中的作用仍然是难以捉摸的。此外，在血管生成过程中控制空间激活的aPKC的分子机制在很大程度上是未知的。在这里，我们发现，在PAR-3和aPKC内皮特异性诱导突变小鼠的血管生长中，通过VE-钙粘蛋白和JAM-A观察到的粘附连接和紧密连接没有显示出明显的缺陷。另一方面，Claudin-5转录，更成熟的EC紧密连接标记，在aPKC中受损，但在PAR-3突变体中没有。为了进一步了解aPKC在内皮细胞中的作用方式，我们以内皮细胞特异性诱导突变小鼠为模型系统，提出了两个方案。1)连接介导的aPKC激活在血管生长中的作用是什么？2)在体内aPKC如何通过调节Claudin-5转录来控制连接成熟？血管形成的过程是疾病发展和发病机制的关键。因此，通过确定潜在的aPKC信号转导的分子机制，该项目将阐明重要的洞察在生理和病理血管生成的功能。