Cell−Cell Junctions of Dermal Microvascular Endothelial Cells Contain Tight and Adherens Junction Proteins in Spatial Proximity†

Biochemistry ◽  
2004 ◽  
Vol 43 (18) ◽  
pp. 5360-5369 ◽  
Author(s):  
Claas Rüffer ◽  
Anke Strey ◽  
Annette Janning ◽  
Kwang Sik Kim ◽  
Volker Gerke
Keyword(s):  
Endothelial Cells ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Spatial Proximity ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial ◽  
Adherens Junction Proteins ◽  
Junction Proteins ◽  
Cell Cell

scholarly journals Integrin regulation of cell-cell adhesion during epithelial tubule formation

2001 ◽  
Vol 114 (5) ◽  
pp. 941-952 ◽  
Author(s):  
G.K. Ojakian ◽  
D.R. Ratcliffe ◽  
R. Schwimmer
Keyword(s):  
Extracellular Matrix ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Junctional Complex ◽  
Tubule Formation ◽  
Epithelial Remodeling ◽  
Adherens Junction Proteins ◽  
Junction Proteins ◽  
E Cadherin ◽  
Cell Cell

The extracellular matrix plays an important role in regulation of epithelial development and organization. To determine more precisely the function of extracellular matrix in this process, the initial steps in collagen-mediated formation of epithelial tubules were studied using a model cell culture system. Previous studies have demonstrated that incubation of Madin-Darby canine kidney (MDCK) epithelial cells with a collagen gel overlay induces (beta)1 integrin-regulated epithelial remodeling accompanied by extensive cell rearrangements and formation of epithelial tubules. During epithelial remodeling there was extensive disruption of the epithelial junctional complex. Progressive opening of tight junctions was observed over 8 hours using transepithelial resistance measurements and immunofluorescence microscopy demonstrated that tight and adherens junction proteins were dispersed throughout the apical and basolateral membranes. Junction complex disruption allowed the formation of apical cell extensions and subsequent migration of selected cell sheets from the epithelial monolayer. Confocal microscopy demonstrated the presence of adherens junction (E-cadherin, (alpha)-catenin, (beta)-catenin, plakoglobin) and desmosomal (desmoplakin-1/2, plakoglobin) proteins on, and within, cell extensions demonstrating that cell junctions had undergone considerable disassembly. However, groups of cell extensions appeared to be associated by E-cadherin/catenin-mediated interactions. Association of E-cadherin/catenin complexes with the epithelial cytoskeleton was analyzed by differential detergent extraction. SDS-PAGE and immunoblot analysis demonstrated that adherens junction proteins were primarily cytoskeleton-associated in control cells. During integrin-regulated remodeling, there was a progressive reduction in the interaction of adherens junction proteins with the cytoskeleton suggesting that they play an important role in the maintenance of epithelial integrity. Since loss of transepithelial electrical resistance and disruption of junctional complexes were inhibited by an antifunctional integrin antibody, we propose that activation of integrin signaling pathways regulate junctional complex stability, cell-cell interactions and cell migration. These observations provide evidence that integrin-regulated MDCK epithelial tubule formation can serve as a model system for studying rearrangements of epithelial sheets which occur during development.

Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells

1997 ◽  
Vol 110 (17) ◽  
pp. 2065-2077 ◽  
Author(s):  
M.G. Lampugnani ◽  
M. Corada ◽  
P. Andriopoulou ◽  
S. Esser ◽  
W. Risau ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Molecular Organization ◽  
Beta Catenin ◽  
Cell Contacts ◽  
Cell Type Specific ◽  
Dynamic Structures ◽  
Cell Cell

In src- and ras-transformed cells, tyrosine phosphorylation of adherens junction (AJ) components is related to impairment of cell-cell adhesion. In this paper we report that in human endothelial cells (EC), tyrosine phosphorylation of AJ can be a physiological process regulated by cell density. Immunofluorescence analysis revealed that a phosphotyrosine (P-tyr) antibody could stain cell-cell junctions only in sparse or loosely confluent EC, while the staining was markedly reduced in tightly confluent cultures. This process was reversible, since on artificial wounding of EC monolayers, the cells at the migrating front reacquired P-tyr labelling at cell contacts. In EC, the major cadherin at intercellular AJ is the cell-type-specific VE-cadherin. We therefore analyzed whether this molecule was at least in part responsible for the changes in P-tyr content at cell junctions. Tyrosine phosphorylation of VE-cadherin, beta-catenin and p120, occurred in looser AJ, i.e. in recently confluent cells, and was notably reduced in tightly confluent cultures. Changes in P-tyr content paralleled changes in the molecular organization of AJ. VE-cadherin was mostly associated with beta-catenin and p120 in loose EC monolayers, while in long-confluent cells, these two catenins were largely replaced by plakoglobin. Inhibition of P-tyr phosphatases (PTPases) by PV markedly augmented the P-tyr content of VE-cadherin, which bound p120 and beta-catenin more efficiently, but not plakoglobin. Transfection experiments in CHO cells showed that p120 could bind to a VE-cadherin cytoplasmic region different from that responsible for beta-catenin binding, and PV stabilized this association. Overall these data indicate that endothelial AJ are dynamic structures that can be affected by the state of confluence of the cells. Tyrosine phosphorylation of VE-cadherin and its association to p120 and beta-catenin characterizes early cell contacts, while the formation of mature and cytoskeleton-connected junctions is accompanied by dephosphorylation and plakoglobin association.

scholarly journals Histamine stimulates phosphorylation of adherens junction proteins and alters their link to vimentin

2002 ◽  
Vol 282 (6) ◽  
pp. L1330-L1338 ◽  
Author(s):  
D. Michael Shasby ◽  
Dana R. Ries ◽  
Sandra S. Shasby ◽  
Michael C. Winter
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Adherens Junction ◽  
Vascular Endothelial ◽  
Cortical Actin ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells ◽  
Adherens Junction Proteins ◽  
Junction Proteins

Histamine increases microvascular permeability by creating small transitory (100–400 nm) gaps between adjacent endothelial cells at sites of vascular endothelial (VE)-cadherin-based adhesion. We examined the effects of histamine on the proteins within the VE-cadherin-based adherens junction in primary human umbilical vein endothelial cells. VE-cadherin is linked not only by β- and α-catenin to cortical actin but also by γ-catenin to the intermediate filament vimentin. In mature human umbilical vein cultures, the VE-cadherin immunoprecipitate contained equivalent amounts of α- and β-catenin, 130% as much β- as γ-catenin, and 50% as much actin as vimentin. Within 60 s, histamine decreased the fraction of VE-cadherin in the insoluble portion of the cell lysate by 35 ± 1.5%. At the same time, histamine decreased the amount of vimentin that immunoprecipitated with VE-cadherin by 50 ± 6%. Histamine did not affect the amount of actin or the amount of α-, β-, or γ-catenin that immunoprecipitated with VE-cadherin. Within 60 s, histamine simulated a doubling in the phosphorylation of VE-cadherin and β- and γ-catenin. The VE-cadherin immunoprecipitate contained kinase activity that phosphorylated VE-cadherin and γ-catenin in vitro.

Gα12 regulates epithelial cell junctions through Src tyrosine kinases

2003 ◽  
Vol 285 (5) ◽  
pp. C1281-C1293 ◽  
Author(s):  
Tobias N. Meyer ◽  
Jennifer Hunt ◽  
Catherine Schwesinger ◽  
Bradley M. Denker
Keyword(s):  
Epithelial Cell ◽  
Tyrosine Kinase ◽  
Mdck Cells ◽  
Adherens Junction ◽  
Paracellular Permeability ◽  
Cell Junctions ◽  
Scaffolding Protein ◽  
Junctional Complex ◽  
Adherens Junction Proteins ◽  
Junction Proteins

Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that Gα12 binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated Gα12 increases paracellular permeability in Madin-Darby canine kidney (MDCK) cells (Meyer et al. J Biol Chem 277: 24855-24858, 2002). In the present studies, we explore the effects of Gα12 expression on tight and adherens junction proteins and examine downstream signaling pathways. By confocal microscopy, we detect disrupted tight and adherens junction proteins with increased actin stress fibers in constitutively active Gα12 (QLα12)-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QLα12-expressing MDCK cells, consistent with loss of polarity. We found that the tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 reversibly abrogated the QLα12 phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QLα12-expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and [3H]mannitol flux was prevented by the inhibitors. Src activity was increased in QLα12-expressing MDCK cells as assessed by Src autophosphorylation, and β-catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that Gα12 regulates MDCK cell junctions, in part through Src tyrosine kinase pathways.

scholarly journals Caveolin-1 regulates expression of junction-associated proteins in brain microvascular endothelial cells

Blood ◽  
2006 ◽  
Vol 109 (4) ◽  
pp. 1515-1523 ◽  
Author(s):  
Li Song ◽  
Shujun Ge ◽  
Joel S. Pachter
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Adherens Junction ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Caveolin 1 ◽  
Synthetic Cell ◽  
Associated Proteins ◽  
Junctional Protein ◽  
Microvascular Endothelial

Abstract Recent evidence from this laboratory indicated that reduced expression of caveolin-1 accompanied the diminished expression of tight junction (TJ)–associated proteins occludin and zonula occludens-1 (ZO-1) following stimulation of brain microvascular endothelial cells (BMECs) with the chemokine CCL2 (formerly called MCP-1). Because attenuated caveolin-1 levels have also been correlated with heightened permeability of other endothelia, the objective of this study was to test the hypothesis that reduced caveolin-1 expression is causally linked to the action of CCL2 on BMEC junctional protein expression and barrier integrity. This was achieved using adenovirus to nondestructively deliver caveolin-1 siRNA (Ad-siCav-1) to BMEC monolayers, which model the blood-brain barrier (BBB). Treatment with siRNA reduced the caveolin-1 protein level as well as occludin and ZO-1. Additionally, occludin exhibited dissociation from the cytoskeletal framework. These changes were attended by comparable alterations in adherens junction (AJ)–associated proteins, VE-cadherin and β-catenin, increased BMEC paracellular permeability, and facilitated the ability of CCL2 to stimulate monocytic transendothelial migration. Furthermore, treating BMECs with cavtratin, a synthetic cell-permeable peptide encoding the caveolin-1 scaffolding domain, antagonized effects of both Ad-siCav-1 and CCL2. These results collectively highlight caveolin-1 loss as a critical step in CCL2-induced modulation of BMEC junctional protein expression and integrity, and possibly serve a crucial role in regulating inflammation at the BBB.

Sign in / Sign up

Export Citation Format

Share Document