Rab5a-mediated localization of claudin-1 is regulated by proteasomes in endothelial cells

2011 ◽  
Vol 300 (1) ◽  
pp. C87-C96 ◽  
Author(s):  
Machiko Asaka ◽  
Tetsuaki Hirase ◽  
Aiko Hashimoto-Komatsu ◽  
Koichi Node
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Tight Junction ◽  
Endothelial Permeability ◽  
Ubiquitin Proteasome System ◽  
Cell Contact ◽  
Major Barrier ◽  
Tight Junction Permeability ◽  
Interfering Rna

Tight junctions composed of transmembrane proteins, including claudin, occludin, and tricellulin, and peripheral membrane proteins are a major barrier to endothelial permeability, whereas the role of claudin in the regulation of tight junction permeability in nonneural endothelial cells is unclear. This study demonstrates that claudin-1 is dominantly expressed and depletion of claudin-1 using small interfering RNA (siRNA) increased tight junction permeability in EA hy.926 cells, indicating that claudin-1 is a crucial regulator of endothelial tight junction permeability. The ubiquitin-proteasome system has been implicated in the regulation of endocytotic trafficking of plasma membrane proteins. Therefore, the involvement of proteasomes in the localization of claudin-1 was investigated by pharmacological and genetic inhibition of proteasomes using a proteasome inhibitor, N-acetyl-Leu-Leu-Nle-CHO, and siRNA against the β5-subunit of the 20S proteasome, respectively. Claudin-1 was localized at cell-cell contact sites in control cells. Claudin-1 was localized in the cytoplasm in association with Rab5a and EEA-1, a marker of early endosome, following inhibition of proteasomes. Depletion of Rab5a using siRNA reversed the localization of claudin-1 induced by inhibition of proteasomes. These data suggest that proteasomes regulate claudin-1 localization at the plasma membrane, which changes upon proteasomal inhibition to a Rab5a-mediated endosomal localization.

Evidence that tyrosine phosphorylation may increase tight junction permeability

1995 ◽  
Vol 108 (2) ◽  
pp. 609-619 ◽  
Author(s):  
J.M. Staddon ◽  
K. Herrenknecht ◽  
C. Smales ◽  
L.L. Rubin
Keyword(s):  
Endothelial Cells ◽  
Tight Junction ◽  
Tyrosine Phosphorylation ◽  
Mdck Cells ◽  
Tyrosine Phosphatase ◽  
Adherens Junction ◽  
Brain Endothelial Cells ◽  
Phenylarsine Oxide ◽  
Phosphatase Inhibitor ◽  
Tight Junction Permeability

Tight junction permeability control is important in a variety of physiological and pathological processes. We have investigated the role of tyrosine phosphorylation in the regulation of tight junction permeability. MDCK epithelial cells and brain endothelial cells were grown on filters and tight junction permeability was determined by transcellular electrical resistance (TER). The tyrosine phosphatase inhibitor pervanadate caused a concentration- and time-dependent decrease in TER in both MDCK and brain endothelial cells. However, as expected, pervanadate resulted in the tyrosine phosphorylation of many proteins; hence interpretation of its effects are extremely difficult. Phenylarsine oxide, a more selective tyrosine phosphatase inhibitor, caused the tyrosine phosphorylation of relatively few proteins as analyzed by immunoblotting of whole cell lysates. This inhibitor, like pervanadate, also elicited a decrease in TER in the two cell types. In the MDCK cells, the action of phenylarsine oxide could be reversed by the subsequent addition of the reducing agent 2,3-dimercaptopropanol. Immunocytochemistry revealed that phenylarsine oxide rapidly stimulated the tyrosine phosphorylation of proteins associated with intercellular junctions. Because of the known influence of the adherens junction on tight junctions, we analyzed immunoprecipitates of the E-cadherin/catenin complex from MDCK cells treated with phenylarsine oxide. This revealed an increase in the tyrosine phosphorylation of beta-catenin, but not of alpha-catenin. However, the tight junction associated protein ZO-1 was also tyrosine phosphorylated after PAO treatment. These data indicate that tight junction permeability may be regulated via mechanisms involving tyrosine phosphorylation of adherens junction and tight junction proteins.

scholarly journals Intracellular ascorbate tightens the endothelial permeability barrier through Epac1 and the tubulin cytoskeleton

2016 ◽  
Vol 311 (4) ◽  
pp. C652-C662 ◽  
Author(s):  
William H. Parker ◽  
Elizabeth Meredith Rhea ◽  
Zhi-Chao Qu ◽  
Morgan R. Hecker ◽  
James M. May
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Cyclic Amp ◽  
Umbilical Vein ◽  
Endothelial Permeability ◽  
Cell Contact ◽  
Permeability Barrier ◽  
Basal Cells ◽  
Microtubule Stabilization ◽  
Umbilical Vein Endothelial Cells

Vitamin C, or ascorbic acid, both tightens the endothelial permeability barrier in basal cells and also prevents barrier leak induced by inflammatory agents. Barrier tightening by ascorbate in basal endothelial cells requires nitric oxide derived from activation of nitric oxide synthase. Although ascorbate did not affect cyclic AMP levels in our previous study, there remains a question of whether it might activate downstream cyclic AMP-dependent pathways. In this work, we found in both primary and immortalized cultured endothelial cells that ascorbate tightened the endothelial permeability barrier by ∼30%. In human umbilical vein endothelial cells, this occurred at what are likely physiologic intracellular ascorbate concentrations. In so doing, ascorbate decreased measures of oxidative stress and also flattened the cells to increase cell-to-cell contact. Inhibition of downstream cyclic AMP-dependent proteins via protein kinase A did not prevent ascorbate from tightening the endothelial permeability barrier, whereas inhibition of Epac1 did block the ascorbate effect. Although Epac1 was required, its mediator Rap1 was not activated. Furthermore, ascorbate acutely stabilized microtubules during depolymerization induced by colchicine and nocodazole. Over several days in culture, ascorbate also increased the amount of stable acetylated α-tubulin. Microtubule stabilization was further suggested by the finding that ascorbate increased the amount of Epac1 bound to α-tubulin. These results suggest that physiologic ascorbate concentrations tighten the endothelial permeability barrier in unstimulated cells by stabilizing microtubules in a manner downstream of cyclic AMP that might be due both to increasing nitric oxide availability and to scavenging of reactive oxygen or nitrogen species.

scholarly journals Dephosphorylation of the catenins p120 and p100 in endothelial cells in response to inflammatory stimuli

1999 ◽  
Vol 338 (2) ◽  
pp. 471-478 ◽  
Author(s):  
Marianne J. RATCLIFFE ◽  
Caroline SMALES ◽  
James M. STADDON
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Tight Junction ◽  
Adherens Junction ◽  
Receptor Activation ◽  
Protein Kinase C Inhibitors ◽  
Kinase C ◽  
Inflammatory Stimuli ◽  
Tight Junction Permeability

Inflammatory mediators such as histamine and thrombin increase the tight-junction permeability of endothelial cells. Tight-junction permeability may be independently controlled, but is dependent on the adherens junction, where adhesion is achieved through homotypic interaction of cadherins, which in turn are associated with cytoplasmic proteins, the catenins. p120, also termed p120cas/p120ctn, and its splice variant, p100, are catenins. p120, originally discovered as a substrate of the tyrosine kinase Src, is also a target for a protein kinase C-stimulated pathway in epithelial cells, causing its serine/threonine dephosphorylation. The present study shows that pharmacological activation of protein kinase C stimulated a similar pathway in endothelial cells. Activation of receptors for agents such as histamine (H1), thrombin and lysophosphatidic acid in the endothelial cells also caused serine/threonine dephosphorylation of p120 and p100, suggesting physiological relevance. However, protein kinase C inhibitors, although blocking the effect of pharmacological activation of protein kinase C, did not block the effects due to receptor activation. Calcium mobilization and the myosin-light-chain-kinase pathway do not participate in p120/p100 signalling. In conclusion, endothelial cells possess protein kinase C-dependent and -independent pathways regulating p120/p100 serine/threonine phosphorylation. These data describe a new connection between inflammatory agents, receptor-stimulated signalling and pathways potentially influencing intercellular adhesion in endothelial cells.

scholarly journals Lysophosphatidic Acid Increases Tight Junction Permeability in Cultured Brain Endothelial Cells

2002 ◽  
Vol 68 (3) ◽  
pp. 991-1000 ◽  
Author(s):  
Charlotte Schulze ◽  
Caroline Smales ◽  
Lee L. Rubin ◽  
James M. Staddon
Keyword(s):  
Endothelial Cells ◽  
Tight Junction ◽  
Lysophosphatidic Acid ◽  
Brain Endothelial Cells ◽  
Tight Junction Permeability

scholarly journals Rab22a Regulates the Recycling of Membrane Proteins Internalized Independently of Clathrin

2004 ◽  
Vol 15 (8) ◽  
pp. 3758-3770 ◽  
Author(s):  
Roberto Weigert ◽  
Albert Chi Yeung ◽  
Jean Li ◽  
Julie G. Donaldson
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Tubular Structures ◽  
Tubule Formation ◽  
Early Endosomes ◽  
Interfering Rna ◽  
Negative Mutant

Plasma membrane proteins that are internalized independently of clathrin, such as major histocompatibility complex class I (MHCI), are internalized in vesicles that fuse with the early endosomes containing clathrin-derived cargo. From there, MHCI is either transported to the late endosome for degradation or is recycled back to the plasma membrane via tubular structures that lack clathrin-dependent recycling cargo, e.g., transferrin. Here, we show that the small GTPase Rab22a is associated with these tubular recycling intermediates containing MHCI. Expression of a dominant negative mutant of Rab22a or small interfering RNA-mediated depletion of Rab22a inhibited both formation of the recycling tubules and MHCI recycling. By contrast, cells expressing the constitutively active mutant of Rab22a exhibited prominent recycling tubules and accumulated vesicles at the periphery, but MHCI recycling was still blocked. These results suggest that Rab22a activation is required for tubule formation and Rab22a inactivation for final fusion of recycling membranes with the surface. The trafficking of transferrin was only modestly affected by these treatments. Dominant negative mutant of Rab11a also inhibited recycling of MHCI but not the formation of recycling tubules, suggesting that Rab22a and Rab11a might coordinate different steps of MHCI recycling.

scholarly journals Clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins and preimplantation development

Development ◽  
2021 ◽  
Vol 148 (14) ◽  
Author(s):  
Akihito Morita ◽  
Yuhkoh Satouh ◽  
Hidetaka Kosako ◽  
Hisae Kobayashi ◽  
Akira Iwase ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Ubiquitin Proteasome System ◽  
Maternal Plasma ◽  
Early Embryogenesis ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Selective Degradation ◽  
Kinase C ◽  
Ubiquitin Proteasome

ABSTRACT Fertilization triggers significant cellular remodeling through the oocyte-to-embryo transition. In this transition, the ubiquitin-proteasome system and autophagy are essential for the degradation of maternal components; however, the significance of degradation of cell surface components remains unknown. In this study, we show that multiple maternal plasma membrane proteins, such as the glycine transporter GlyT1a, are selectively internalized from the plasma membrane to endosomes in mouse embryos by the late two-cell stage and then transported to lysosomes for degradation at the later stages. During this process, large amounts of ubiquitylated proteins accumulated on endosomes. Furthermore, the degradation of GlyT1a with mutations in potential ubiquitylation sites was delayed, suggesting that ubiquitylation may be involved in GlyT1a degradation. The clathrin inhibitor blocked GlyT1a internalization. Strikingly, the protein kinase C (PKC) activator triggered the heterochronic internalization of GlyT1a; the PKC inhibitor markedly blocked GlyT1a endocytosis. Lastly, clathrin inhibition completely blocked embryogenesis at the two-cell stage and inhibited cell division after the four-cell stage. These findings demonstrate that PKC-dependent clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins during oocyte-to-embryo transition and early embryogenesis.

Abstract WMP3: Exosomes Derived From Stroke Activated Platelets Impair Cerebral Endothelial Permeability

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Chao Li ◽  
Chunyang Wang ◽  
Li Zhang ◽  
Chopp Michael ◽  
Zheng Gang Zhang
Keyword(s):  
Endothelial Cells ◽  
Tight Junction ◽  
Western Blot ◽  
Blot Analysis ◽  
Endothelial Permeability ◽  
Aged Rats ◽  
Tight Junction Proteins ◽  
Adult Rats ◽  
Activated Platelets ◽  
Junction Proteins

Introduction: Stroke activated platelets damage the blood brain barrier (BBB). Mechanisms underlying this process remain to be investigated. The present study tested a hypothesis that exosomes, nano size extracellular vesicles, derived from stroke-activated platelets promote BBB leakage. Methods: Platelets were harvested from blood samples collected from non-stroke adult and aged male rats and from respective rats subjected to 4h of middle cerebral artery occlusion (MCAO) (n=6/group). Exosomes were then isolated from the platelets by means of differential ultracentrifugation. Primary cerebral endothelial cells harvested from healthy adult rats were treated with platelet exosomes. Endothelial permeability and trans-endothelial electrical resistance (TEER) were assayed. Western blot analysis was performed to measure pro-coagulation and tight junction proteins. Results: Platelet exosomes (P-Exos) had an average size from 70 to 82 nm among adult and aged rats and these exosomes contained platelet exosomes markers: CD41, CD63, CD9 and Alix. P-Exos derived from non-ischemic adult and aged rats exhibited TEER at 154±18 and 159 ±16 Ω.cm 2 , respectively, and did not increase FITC-dextran leakage. However, P-Exos from ischemic adult rats significantly (p<0.05) decreased TEER (118 ±11 vs 154±18Ω.cm 2 in non-ischemia) and increased FITC-dextran leakage by 178%. P-Exos from aged ischemic rats further significantly decreased TEER (98 ±9 Ω.cm 2 ) and augmented dextran leakage to 216%. Western blot analysis of endothelial cells showed that P-Exos from ischemic adult rats significantly increased pro-inflammation proteins, ICAM-1, TNF-a and NFkB p65 and decreased tight junction proteins, ZO-1 and occludin, whereas P-Exos from ischemic aged rats further significantly increased and decreased pro-inflammation proteins and junction proteins. Conclusion: These data provide new insights into how stroke activated platelets mediate BBB disruption and promote endothelial proinflammatory protein expression via their released exosomes and how aging further negatively impacts cerebral endothelial cells.

When Capillary Permeability Increases

Physiology ◽  
1987 ◽  
Vol 2 (1) ◽  
pp. 16-18
Author(s):  
C Crone
Keyword(s):  
Endothelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Cell Shape ◽  
Capillary Permeability ◽  
Signal Molecule ◽  
Small Pore ◽  
Myosin Filaments ◽  
Tight Junction Permeability

Capillary permeability is usually ascribed to a number of "small" pores. In this article the small-pore pathway is identified as interruptions in the tight junctions between endothelial cells. Modulation of capillary permeability involves control of tight junction permeability with cytosolic Ca2+ as the important signal molecule. Actin and myosin filaments are probably involved in the process, which may implicate subtle changes in cell shape.

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