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

在组织形态发生和组织稳态过程中，细胞的迁移和极化等行为是同时受到密切协调的调节的。内皮细胞（ECs）形成管状结构，有助于封闭循环系统。在血管生成过程中，内皮细胞的迁移行为表现为前后极性，而内皮细胞的迁移行为表现为顶基极性。细胞极性转换在此过程中受到调节。血管内皮生长因子（VEGF）和ephrin-B2（一种Eph家族受体酪氨酸激酶的跨膜配体）共同控制运动和携带丝状足的内皮芽的形成。芽之间的内皮细胞间相互作用触发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这一过程至关重要。随后，内皮细胞再次获得顶基极性，以促进血管腔的形成。我已经确定PAR-3和货物接头蛋白Dab2是ephrin-B2相互作用的蛋白。PAR-3/Dab2/ephrin-B2相互作用调节VEGFR2/3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。我发现活性aPKC在成熟的血管丛中更为丰富，它通过磷酸化Dab2来对抗VEGFR2/3的内化。这一过程导致了VEGFR2/3内化的区域差异。因此，内皮细胞在生长血管中的区域差异行为不仅受到VEGF-A梯度的调节，而且受到内皮细胞固有特性的调节。然而，PAR-3和aPKC在体内内皮细胞间连接中的作用尚不清楚。此外，在血管生成过程中，控制空间激活aPKC的分子机制在很大程度上是未知的。我们发现，在PAR-3和aPKC内皮特异性诱导突变小鼠的生长血管中，VE-cadherin显示的粘附连接和JAM-A显示的紧密连接没有显示出明显的缺陷。另一方面，更成熟的EC紧密连接标记Claudin-5的转录在aPKC中受到损害，而在PAR-3突变体中没有受到损害。为了进一步了解aPKC在内皮细胞中的作用方式，我们提出了两个项目，以内皮特异性诱导突变小鼠为模型系统。1)连接介导的aPKC激活在血管生长中的作用是什么？2) aPKC在体内如何通过调节Claudin-5的转录来控制连接成熟？血管的形成过程是疾病发展和发病的关键。因此，通过确定aPKC信号转导的分子机制，该项目将阐明aPKC在生理和病理血管生成中的重要功能。

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.