p120-Catenin prevents neutrophil transmigration independently of RhoA inhibition by impairing Src dependent VE-cadherin phosphorylation

Leukocyte transendothelial migration (TEM) is regulated by several signaling pathways including Src family kinases (SFK) and the small RhoGTPases. Previous studies have shown that vascular endothelial-cadherin (VE-cad) forms a complex with β-,γ-, and p120-catenins and this complex disassociates to form a transient gap during leukocyte TEM. Additionally, p120-catenin (p120-1A) overexpression in human umbilical vein endothelial cells (HUVEC) stabilizes VE-cad surface expression, prevents tyrosine phosphorylation of VE-cad, and inhibits leukocyte TEM. Based on reports showing that p120 overexpression in fibroblasts or epithelial cells inhibits RhoA and activates Rac and Cdc42 GTPases, and on other reports showing that RhoA activation in endothelial cells is necessary for leukocyte TEM, we reasoned that p120 overexpression inhibited TEM through inhibition of RhoA. To test this idea, we overexpressed a mutant p120 isoform, p120-4A, which does not interact with RhoA. p120-4A colocalized with VE-cad in HUVEC junctions and enhanced VE-cad surface expression, similar to overexpression of p120-1A. Interestingly, overexpression of either p120-4A or p120-1A dramatically blocked TEM, and overexpression of p120-1A in HUVEC did not affect RhoA basal activity or activation of RhoA and Rac induced by thrombin or ICAM-1 crosslinking. In contrast, biochemical studies revealed that overexpression of p120-1A reduced activated pY416-Src association with VE-cad. In summary, p120 overexpression inhibits neutrophil TEM independently of an effect on RhoA or Rac and instead blocks TEM by preventing VE-cad tyrosine phosphorylation and association of active Src with the VE-cad complex.

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Effect of melatonin on EGF- and VEGF-induced monolayer permeability of HUVECs

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00542.2018 ◽

2019 ◽

Vol 316 (5) ◽

pp. H1178-H1191 ◽

Cited By ~ 2

Author(s):

Ling Yang ◽

Yujie Zhang ◽

Yadong Ma ◽

Jun Du ◽

Luo Gu ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Vascular Permeability ◽

Tyrosine Phosphorylation ◽

Umbilical Vein ◽

Vascular Endothelial ◽

Phosphatidylinositol 3 Kinase ◽

Human Umbilical Vein ◽

Monolayer Permeability ◽

Umbilical Vein Endothelial Cells

Melatonin is a natural hormone involved in the regulation of circadian rhythm, immunity, and cardiovascular function. In the present study, we focused on the mechanism of melatonin in the regulation of vascular permeability. We found that melatonin could inhibit both VEGF- and EGF-induced monolayer permeability of human umbilical vein endothelial cells (HUVECs) and change the tyrosine phosphorylation of vascular-endothelial (VE-)cadherin, which was related to endothelial barrier function. In addition, phospho-AKT (Ser473) and phospho-ERK(1/2) played significant roles in the regulation of VE-cadherin phosphorylation. Both the phosphatidylinositol 3-kinase/AKT inhibitor LY49002 and MEK/ERK inhibitor U0126 could inhibit the permeability of HUVECs, but with different effects on tyrosine phosphorylation of VE-cadherin. Melatonin can influence the two growth factor-induced phosphorylation of AKT (Ser473) but not ERK(1/2). Our results show that melatonin can inhibit growth factor-induced monolayer permeability of HUVECs by influencing the phosphorylation of AKT and VE-cadherin. Melatonin can be a potential treatment for diseases associated with abnormal vascular permeability. NEW & NOTEWORTHY We found that melatonin could inhibit both EGF- and VEGF-induced monolayer permeability of human umbilical vein endothelial cells, which is related to phosphorylation of vascular-endothelial cadherin. Blockade of phosphatidylinositol 3-kinase/AKT and MEK/ERK pathways could inhibit the permeability of human umbilical vein endothelial cells, and phosphorylation of AKT (Ser473) might be a critical event in the changing of monolayer permeability and likely has cross-talk with the MEK/ERK pathway.

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In Vitro Degradation of Endothelial Catenins by a Neutrophil Protease

The Journal of Cell Biology ◽

10.1083/jcb.140.2.403 ◽

1998 ◽

Vol 140 (2) ◽

pp. 403-407 ◽

Cited By ~ 71

Author(s):

Thomas Moll ◽

Elisabetta Dejana ◽

Dietmar Vestweber

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Total Cell ◽

Polymorphonuclear Cells ◽

Vascular Endothelial ◽

In Vitro Degradation ◽

Endothelial Cell Surface ◽

Vascular Endothelial Cadherin ◽

Umbilical Vein Endothelial Cells

It has been recently proposed that adhesion of polymorphonuclear cells (PMNs) to human umbilical vein endothelial cells leads to the disorganization of the vascular endothelial cadherin–dependent endothelial adherens junctions. Combined immunofluorescence and biochemical data suggested that after adhesion of PMNs to the endothelial cell surface, β-catenin, as well as plakoglobin was lost from the cadherin/catenin complex and from total cell lysates. In this study we present data that strongly suggest that the adhesion-dependent disappearance of endothelial catenins is not mediated by a leukocyte to endothelium signaling event, but is due to the activity of a neutrophil protease that is released upon detergent lysis of the cells.

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p120-Catenin regulates leukocyte transmigration through an effect on VE-cadherin phosphorylation

Blood ◽

10.1182/blood-2008-03-147181 ◽

2008 ◽

Vol 112 (7) ◽

pp. 2770-2779 ◽

Cited By ~ 88

Author(s):

Pilar Alcaide ◽

Gail Newton ◽

Scott Auerbach ◽

Seema Sehrawat ◽

Tanya N. Mayadas ◽

...

Keyword(s):

Surface Expression ◽

Vascular Endothelial ◽

Gap Formation ◽

P120 Catenin ◽

Leukocyte Transmigration ◽

Vascular Endothelial Cadherin ◽

Leukocyte Transendothelial Migration ◽

Endothelial Junctions ◽

Functional Consequences ◽

Time Required

Abstract Vascular endothelial–cadherin (VE-cad) is localized to adherens junctions at endothelial cell borders and forms a complex with α-, β-, γ-, and p120-catenins (p120). We previously showed that the VE-cad complex disassociates to form short-lived “gaps” during leukocyte transendothelial migration (TEM); however, whether these gaps are required for leukocyte TEM is not clear. Recently p120 has been shown to control VE-cad surface expression through endocytosis. We hypothesized that p120 regulates VE-cad surface expression, which would in turn have functional consequences for leukocyte transmigration. Here we show that endothelial cells transduced with an adenovirus expressing p120GFP fusion protein significantly increase VE-cad expression. Moreover, endothelial junctions with high p120GFP expression largely prevent VE-cad gap formation and neutrophil leukocyte TEM; if TEM occurs, the length of time required is prolonged. We find no evidence that VE-cad endocytosis plays a role in VE-cad gap formation and instead show that this process is regulated by changes in VE-cad phosphorylation. In fact, a nonphosphorylatable VE-cad mutant prevented TEM. In summary, our studies provide compelling evidence that VE-cad gap formation is required for leukocyte transmigration and identify p120 as a critical intracellular mediator of this process through its regulation of VE-cad expression at junctions.

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Synthesis and Surface Expression of CD14 by Human Endothelial Cells

Infection and Immunity ◽

10.1128/iai.69.1.479-485.2001 ◽

2001 ◽

Vol 69 (1) ◽

pp. 479-485 ◽

Cited By ~ 62

Author(s):

Hubertus P. A. Jersmann ◽

Charles S. T. Hii ◽

Greg L. Hodge ◽

Antonio Ferrante

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Surface Expression ◽

Significant Loss ◽

Vascular Endothelial ◽

Serum Factors ◽

Membrane Bound ◽

Umbilical Vein Endothelial Cells ◽

Assay Conditions

ABSTRACT Previous studies have reported that human vascular endothelial cells lack the membrane-bound lipopolysaccharide (LPS) receptor, CD14 (mCD14). By optimizing assay conditions, including the selection of anti-CD14 monoclonal antibody, we now demonstrate that human umbilical vein endothelial cells (HUVEC) express CD14 on the cell surface. Single-passage HUVEC showed approximately 20 times less expression of CD14 than monocytes. Interestingly, there was significant loss of surface CD14 expression with increasing numbers of culture passages. Evidence for synthesis of CD14 by HUVEC was provided by the finding that l-[35S]methionine was incorporated into CD14. In addition, the expression of CD14 on HUVEC was upregulated by LPS, lysophosphatidic acid, and tissue culture supplements, and this upregulation was dependent on protein synthesis. Furthermore, the results imply that mCD14 is required for LPS-induced activation of endothelial cells in the absence of serum and that it acts in concert with serum factors (soluble CD14). Our results provide evidence that CD14 is expressed by endothelial cells and suggest that the previous inability to observe expression of this molecule has been due to culture and staining conditions. This finding has important implications for the understanding of the mechanisms by which LPS stimulates endothelial cells and the management of sepsis caused by gram-negative bacteria.

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