p120-catenin in vascular development and endothelial adhesion strengthening Público

Abstract

Adhesion between endothelial cells is subject to dynamic regulation to enable both the protection of vascular barrier function and the controlled passage of solutes and white blood cells between the bloodstream and surrounding tissues. Endothelial adherens junctions contribute to vascular morphogenesis and the maintenance of tissue integrity through the homophilic, calcium-dependent binding of vascular endothelial (VE)-cadherin molecules on adjacent cells. p120-catenin (p120) binds to the cadherin cytoplasmic tail and has been shown to regulate the stability of cadherins at the cell surface by preventing their endocytosis and subsequent degradation. The interaction between p120 and cadherins has also been shown to support strong adhesion in cell-based assays. However, the specific role of p120 in vascular development and the nature of its contribution to adhesion strengthening in an endothelial context are not fully understood.

The purpose of this research was to explore the role of p120 in vascular morphogenesis by using a mouse developmental model, and to further explore the contribution of the p120-VE-cadherin interaction in strengthening of cell adhesion. These experiments reveal that p120 is essential to vascular development in the mouse, and that its conditional endothelial knockout results in embryonic lethality in midgestation due to hemorrhaging and defects in the remodeling and organization of vascular networks in the yolk sac, placenta and embryo proper. The loss of endothelial p120 results in a specific reduction in VE-cadherin and N-cadherin levels in vivo and is accompanied by a decrease in pericyte recruitment to microvessels in mutant tissues. Furthermore, we identify a proliferation defect in endothelial cells lacking p120, and we show that this effect occurs independently of RhoA regulation but is dependent on the expression of VE-cadherin.

Using biophysical approaches, we also present evidence that p120 binding to the VE-cadherin tail supports increased adhesion strength by promoting cell spreading, and we propose that this occurs by localized Rac1 activation and actin reorganization at the cell membrane. These studies reveal that endothelial p120 plays an indispensible role in mammalian development by promoting the angiogenic remodeling, expansion, and stabilization of blood vessels in a cadherin-dependent manner.

Table of Contents

Table of Contents
Chapter 1: Adherens junctions in the vascular endothelium...1

Part 1: Cadherins and intercellular adherens junctions...2

1.1 Adherens junctions and the cadherin family...2
1.2 Catenins...5

1.2.1 β-catenin...6
1.2.2 p120-catenin...7

1.3 Rho family GTPases...9
1.4 Regulation of cadherin function by catenins...11

Part 2: Adherens junctions in the vascular endothelium: development, maturation, and pathology...16

1.5 The vascular endothelium...16
1.6 Vasculogenesis and early vascular development...17
1.7 Angiogenesis and blood vessel maturation...21
1.8 Endothelial junctions in vascular disorders and diseases...26
1.9 Cadherins, catenins, and cancer...28

Chapter 2: p120-catenin is required for mouse vascular development...38

2.1 Introduction...39
2.2 Materials and Methods...42
2.3 Results...48

2.3.1 Loss of endothelial p120-catenin is embryonic lethal...48
2.3.2 Deletion of endothelial p120 causes defects in microvascular patterning and hemorrhages...50
2.3.3 Cadherin expression and pericyte recruitment are decreased in p120-null endothelial tissues...52
2.3.4 Primary endothelial cells lacking p120 exhibit proliferation defects...53

2.4 Discussion...56

Chapter 3: p120-catenin and β-catenin differently regulate endothelial cell spreading and adhesion strengthening...85

3.1 Introduction...86
3.2 Materials and Methods...89
3.3 Results...95

3.3.1 Chimeric adhesion receptors are expressed at comparable levels at the cell surface...95
3.3.2 Chimeric receptors directly interact with catenins and colocalize with them at the adhesive interface...95
3.3.3 The catenin-binding domain of VE-cadherin is necessary for strong adhesion...97
3.3.4 p120 binding to the VE-cadherin tail is necessary to promote cell spreading...99

3.4 Discussion...101

Chapter 4: Summary and future directions...123

4.1 Overview of this dissertation...124
4.2 Future directions...127

Chapter 5: References...132

Table of Figures
Figure 1.1 The adherens junction complex...32
Figure 1.2 Model of cadherin recycling and stabilization at the membrane by p120...34
Figure 1.3 Model illustrating the processes of vasculogenesis, angiogenesis, and vascular wall stabilization...36
Figure 2.1 Endothelial p120 conditional knockout is embryonic lethal...61
Figure 2.2 p120 is successfully ablated in conditional mutant mice but deletion is mosaic in some animals...63
Figure 2.3 Major vessels form normally in p120 endothelial conditional mutants...65
Figure 2.4 p120-null mutants exhibit decreased microvascular density and disorganized vascular networks...67
Figure 2.5 Yolk sac vessels of p120 conditional mutant mice reveal angiogenic remodeling defects...69
Figure 2.6 Conditional mutant mice exhibit hemorrhages...71
Figure 2.7 VE-cadherin levels are decreased in embryonic tissues lacking endothelial p120...73
Figure 2.8 Claudin 5 expression is not altered by endothelial p120 deletion...75
Figure 2.9 N-cadherin is absent in endothelial cells lacking p120 in vivo...77
Figure 2.10 Reduced pericyte recruitment in brains of mutant mice...79
Figure 2.11 Ablation of p120 in cultured mouse endothelial cells leads to decreased VE-cadherin levels and reduced proliferation...81
Figure 2.12 Re-expression of p120 rescues VE-cadherin expression and restores proliferation rates in p120-null endothelial cells....83
Figure 3.1 Diagram of chimeric receptors combining the interleukin-2 receptor extracellular domain and the VE-cadherin cytoplasmic tail...107
Figure 3.2 Chimeric receptors are expressed at comparable levels both in total protein and at the plasma membrane...109
Figure 3.3 Cytoplasmic domains of chimeric receptors interact directly with p120 and β-catenin at the site of adhesion...111
Figure 3.4 Diagram of the hydrodynamic spinning disk apparatus for measuring cell adhesion strength...113
Figure 3.5 Linkage between cadherins and the actin cytoskeleton is necessary to strengthen steady-state adhesion...115
Figure 3.6 The interaction between p120 and the cadherin juxtamembrane domain is required to promote cell spreading...117
Figure 3.7 Cell spreading is impaired in p120-null endothelial cells...119
Figure 3.8 Interaction between p120 and cadherins strengthens cell adhesion by promoting cell spreading...121

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Cell
Palavra-chave	catenin vascular development cadherin endothelial cells
Committee Chair / Thesis Advisor	Kowalczyk, Andrew, Emory University
Committee Members	Caspary, Tamara, Emory University Nickerson, John, Emory University Faundez, Victor, Emory University Fritz, Andreas, Emory University

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