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

• Applicant: Dr. Masanori Nakayama
• Subject Area: Cell Biology ; Developmental Biology
• Term: from 2014 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 259157124

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.

• DFG Programme: Research Grants