scholarly journals Vascular Expression of Permeability-Resistant Occludin Mutant Preserves Visual Function in Diabetes

2021 ◽  
Author(s):  
Andreia Goncalves ◽  
Alyssa Dreffs ◽  
Cheng-mao Lin ◽  
Sarah Sheskey ◽  
Natalie Hudson ◽  
...  
Keyword(s):  
Tight Junction ◽  
Visual Function ◽  
Vision Loss ◽  
Neurovascular Unit ◽  
Barrier Properties ◽  
Dependent Manner ◽  
Specific Expression ◽  
Edema Formation ◽  
Junction Protein ◽  
Vascular Expression

Diabetic retinopathy is one of the leading causes of vision loss and blindness. Extensive pre-clinical and clinical evidence exists for both vascular and neuronal pathology. However, the relationship of these changes in the neurovascular unit and impact on vision remains to be determined. Here, we investigate the role of tight junction protein occludin phosphorylation at S490 in modulating barrier properties and its impact on visual function. Conditional vascular expression of the phosphorylation resistant Ser490 to Ala (S490A) form of occludin preserved tight junction organization and reduced VEGF-induced permeability and edema formation after intra-ocular injection. In the retinas of streptozotocin-induced diabetic mice, endothelial specific expression of the S490A form of occludin completely prevented diabetes-induced permeability to labeled dextran and inhibited leukostasis. Importantly, vascular-specific expression of the occludin mutant completely blocked the diabetes-induced decrease in visual acuity and contrast sensitivity. Together, these results reveal that occludin acts to regulate barrier properties downstream of VEGF in a phosphorylation dependent manner and that loss of inner blood-retinal barrier (iBRB) integrity induced by diabetes contributes to vision loss.

scholarly journals Vascular Expression of Permeability-Resistant Occludin Mutant Preserves Visual Function in Diabetes

2021 ◽  
Author(s):  
Andreia Goncalves ◽  
Alyssa Dreffs ◽  
Cheng-mao Lin ◽  
Sarah Sheskey ◽  
Natalie Hudson ◽  
...  
Keyword(s):  
Tight Junction ◽  
Visual Function ◽  
Vision Loss ◽  
Neurovascular Unit ◽  
Barrier Properties ◽  
Dependent Manner ◽  
Specific Expression ◽  
Edema Formation ◽  
Junction Protein ◽  
Vascular Expression

Diabetic retinopathy is one of the leading causes of vision loss and blindness. Extensive pre-clinical and clinical evidence exists for both vascular and neuronal pathology. However, the relationship of these changes in the neurovascular unit and impact on vision remains to be determined. Here, we investigate the role of tight junction protein occludin phosphorylation at S490 in modulating barrier properties and its impact on visual function. Conditional vascular expression of the phosphorylation resistant Ser490 to Ala (S490A) form of occludin preserved tight junction organization and reduced VEGF-induced permeability and edema formation after intra-ocular injection. In the retinas of streptozotocin-induced diabetic mice, endothelial specific expression of the S490A form of occludin completely prevented diabetes-induced permeability to labeled dextran and inhibited leukostasis. Importantly, vascular-specific expression of the occludin mutant completely blocked the diabetes-induced decrease in visual acuity and contrast sensitivity. Together, these results reveal that occludin acts to regulate barrier properties downstream of VEGF in a phosphorylation dependent manner and that loss of inner blood-retinal barrier (iBRB) integrity induced by diabetes contributes to vision loss.

scholarly journals Interendothelial Claudin-5 Expression Depends on Cerebral Endothelial Cell–Matrix Adhesion by β1-Integrins

2011 ◽  
Vol 31 (10) ◽  
pp. 1972-1985 ◽  
Author(s):  
Takashi Osada ◽  
Yu-Huan Gu ◽  
Masato Kanazawa ◽  
Yoshiaki Tsubota ◽  
Brian T Hawkins ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tight Junction ◽  
Barrier Properties ◽  
Permeability Barrier ◽  
Dependent Manner ◽  
Matrix Adhesion ◽  
Cell Matrix ◽  
Microvessel Permeability ◽  
Cell Matrix Interactions

The hypothesis tested by these studies states that in addition to interendothelial cell tight junction proteins, matrix adhesion by β1-integrin receptors expressed by endothelial cells have an important role in maintaining the cerebral microvessel permeability barrier. Primary brain endothelial cells from C57 BL/6 mice were incubated with β1-rintegrin function-blocking antibody (Ha2/5) or isotype control and the impacts on claudin-5 expression and microvessel permeability were quantified. Both flow cytometry and immunofluorescence studies demonstrated that the interendothelial claudin-5 expression by confluent endothelial cells was significantly decreased in a time-dependent manner by Ha2/5 exposure relative to isotype. Furthermore, to assess the barrier properties, transendothelial electrical resistance and permeability measurements of the monolayer, and stereotaxic injection into the striatum of mice were performed. Ha2/5 incubation reduced the resistance of endothelial cell monolayers significantly, and significantly increased permeability to 40 and 150 k Da dextrans. Ha2/5 injection into mouse striatum produced significantly greater IgG extravasation than the isotype or the control injections. This study demonstrates that blockade of β1-integrin function changes interendothelial claudin-5 expression and increases microvessel permeability. Hence, endothelial cell-matrix interactions via β1-integrin directly affect interendothelial cell tight junction claudin-5 expression and brain microvascular permeability.

scholarly journals Role of Tricellular Tight Junction Protein Lipolysis-Stimulated Lipoprotein Receptor (LSR) in Cancer Cells

2019 ◽  
Vol 20 (14) ◽  
pp. 3555 ◽  
Author(s):  
Takayuki Kohno ◽  
Takumi Konno ◽  
Takashi Kojima
Keyword(s):  
Tight Junction ◽  
Cancer Progression ◽  
Epithelial Barrier ◽  
Dependent Manner ◽  
Tight Junction Proteins ◽  
Barrier Dysfunction ◽  
Endometrial Cells ◽  
Functional Changes ◽  
Junction Protein ◽  
Junction Proteins

Maintaining a robust epithelial barrier requires the accumulation of tight junction proteins, LSR/angulin-1 and tricellulin, at the tricellular contacts. Alterations in the localization of these proteins temporarily cause epithelial barrier dysfunction, which is closely associated with not only physiological differentiation but also cancer progression and metastasis. In normal human endometrial tissues, the endometrial cells undergo repeated proliferation and differentiation under physiological conditions. Recent observations have revealed that the localization and expression of LSR/angulin-1 and tricellulin are altered in a menstrual cycle-dependent manner. Moreover, it has been shown that endometrial cancer progression affects these alterations. This review highlights the differences in the localization and expression of tight junction proteins in normal endometrial cells and endometrial cancers and how they cause functional changes in cells.

Faecal Proteases from Pouchitis Patients Activate Protease Activating Receptor-2 to Disrupt the Epithelial Barrier

2019 ◽  
Vol 13 (12) ◽  
pp. 1558-1568 ◽  
Author(s):  
Sarit Hoffman ◽  
Nathaniel Aviv Cohen ◽  
Ian M Carroll ◽  
Hagit Tulchinsky ◽  
Ilya Borovok ◽  
...  
Keyword(s):  
Tight Junction ◽  
Proteolytic Activity ◽  
Epithelial Barrier ◽  
Dependent Manner ◽  
Epithelial Permeability ◽  
Tight Junction Proteins ◽  
Microbial Composition ◽  
Junction Protein ◽  
Activating Receptor ◽  
Junction Proteins

Abstract Background and Aims The pathogenesis of pouch inflammation may involve epithelial barrier disruption. We investigated whether faecal proteolytic activity is increased during pouchitis and results in epithelial barrier dysfunction through protease activating receptor [PAR] activation, and assessed whether the intestinal microbiome may be the source of the proteases. Methods Faecal samples were measured for protease activity using a fluorescein isothiocyanate [FITC]-casein florescence assay. Caco-2 cell monolayers were exposed to faecal supernatants to assess permeability to FITC-dextran. Tight junction protein integrity and PAR activation were assessed by immunoblot and immunofluorescence. A truncated PAR2 protein in Caco-2 cells was achieved by stable transfection using CRISPR/Cas9 plasmid. PAR2 activation in pouch biopsies was examined using antibodies directed to the N-terminus of the protein. Microbial composition was analysed based on 16S rRNA gene sequence analysis. Results Ten pouchitis patients, six normal pouch [NP] patients and nine healthy controls [HC] were recruited. The pouchitis patients exhibited a 5.19- and 5.35-fold higher faecal protease [FP] activity [p ≤ 0.05] compared to the NP and HC participants, respectively. The genus Haemophilus was positively associated with FP activity [R = 0.718, false discovery rate < 0.1]. Faecal supernatants from pouchitis patients activated PAR2 on Caco-2 monolayers, disrupted tight junction proteins and increased epithelial permeability. PAR2 truncation in Caco-2 abrogated faecal protease-mediated permeability. Pouch biopsies obtained from pouchitis patients, but not from NP patients, displayed PAR2 activation. Conclusions Protease-producing bacteria may increase faecal proteolytic activity that results in pouch inflammation through disruption of tight junction proteins and increased epithelial permeability in a PAR2-dependent manner. This mechanism may initiate or propagate pouch inflammation.

Effects of selective hypothermia on blood-brain barrier integrity and tight junction protein expression levels after intracerebral hemorrhage in rats

2013 ◽  
Vol 394 (10) ◽  
pp. 1317-1324 ◽  
Author(s):  
Hongwei Sun ◽  
Ying Tang ◽  
Xiqin Guan ◽  
Lanfeng Li ◽  
Desheng Wang
Keyword(s):  
Intracerebral Hemorrhage ◽  
Tight Junction ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Edema Formation ◽  
Expression Levels ◽  
Blood Brain ◽  
Tnf Α ◽  
Junction Protein ◽  
Local Hypothermia

Abstract Hypothermia has neuroprotective effects on global cerebral ischemic injuries. However, its efficacy after intracerebral hemorrhage (ICH) is inconclusive. In this study, bacterial collagenase was used to induce ICH stroke in male Wistar rats. We assessed the effects of normothermia and 4 h of local hypothermia (∼33.2°C) initiated 1, 6, or 12 h after collagenase infusion on hemorrhage volume and neurological outcomes. Following early cooling initiated after 1 h, blood-brain barrier (BBB) disruption and brain water content were tested. Furthermore, the expression levels of tight junction (TJ) proteins (claudin 5 and occludin) and the proinflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were determined using Western blotting, real-time quantitative PCR, and immunohistochemical staining at 1 and 3 d after ICH. Early local hypothermia tends to reduce hemorrhagic volume and neurological deficits, but the difference is not statistically significant compared with other groups. However, early hypothermia significantly reduces BBB disruption, edema formation, the expression levels of IL-1β and TNF-α, and the loss of TJ proteins. Together, these data suggest that local hypothermia is an effective treatment for edema formation and BBB disruption via the upregulation of TJ proteins and the suppression of TNF-α and IL-1β.

scholarly journals The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state

2008 ◽  
Vol 181 (4) ◽  
pp. 683-695 ◽  
Author(s):  
Le Shen ◽  
Christopher R. Weber ◽  
Jerrold R. Turner
Keyword(s):  
Tight Junction ◽  
Diffusion Constant ◽  
Tight Junction Protein ◽  
Dependent Manner ◽  
Zonula Occludens ◽  
Determine Function ◽  
Junction Protein ◽  
Associated Proteins ◽  
Energy Dependent ◽  
Darby Canine Kidney

The tight junction defines epithelial organization. Structurally, the tight junction is comprised of transmembrane and membrane-associated proteins that are thought to assemble into stable complexes to determine function. In this study, we measure tight junction protein dynamics in live confluent Madin–Darby canine kidney monolayers using fluorescence recovery after photobleaching and related methods. Mathematical modeling shows that the majority of claudin-1 (76 ± 5%) is stably localized at the tight junction. In contrast, the majority of occludin (71 ± 3%) diffuses rapidly within the tight junction with a diffusion constant of 0.011 μm2s−1. Zonula occludens-1 molecules are also highly dynamic in this region, but, rather than diffusing within the plane of the membrane, 69 ± 5% exchange between membrane and intracellular pools in an energy-dependent manner. These data demonstrate that the tight junction undergoes constant remodeling and suggest that this dynamic behavior may contribute to tight junction assembly and regulation.

scholarly journals Tension-dependent stretching and folding of ZO-1 controls the localization of its interactors

2017 ◽  
Author(s):  
Domenica Spadaro ◽  
Shimin Le ◽  
Thierry Laroche ◽  
Isabelle Mean ◽  
Lionel Jond ◽  
...  
Keyword(s):  
Tight Junction ◽  
Molecular Mechanisms ◽  
Magnetic Tweezers ◽  
Dependent Manner ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Epithelial Homeostasis ◽  
Junction Protein ◽  
Tensile Forces

Tensile forces regulate epithelial homeostasis, but the molecular mechanisms behind this regulation are poorly understood. Using structured illumination microscopy and proximity ligation assays we show that the tight junction protein ZO-1 undergoes actomyosin tension-dependent stretching and folding in vivo. Magnetic tweezers experiments using purified ZO-1 indicate that pN-scale tensions (~2-4 pN) are sufficient to maintain the stretched conformation of ZO-1, while keeping its structured domains intact. Actomyosin tension and substrate stiffness regulate the localization and expression of the transcription factor DbpA and the tight junction membrane protein occludin in a ZO-1/ZO-2-dependent manner, resulting in modulation of gene expression, cell proliferation, barrier function and cyst morphogenesis. Interactions between the N-terminal (ZPSG) and C-terminal domains of ZO-1 prevent binding of DbpA to the ZPSG, and folding is antagonized by heterodimerization with ZO-2. We propose that tensile forces regulate epithelial homeostasis by activating ZO proteins through stretching, to modulate their protein interactions and downstream signaling.

scholarly journals Optimal liver metabolism and proliferation require the tight junction protein claudin-3

2021 ◽  
Vol 108 (Supplement_4) ◽  
Author(s):  
F A Baier ◽  
D Sanchez-Taltavull ◽  
C Gómez Castellà ◽  
F Jebbawi ◽  
A Keogh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tight Junction ◽  
Energy Homeostasis ◽  
Transmembrane Protein ◽  
Parenchymal Cell ◽  
Cell Cycle Gene ◽  
Tight Junction Proteins ◽  
Specific Expression ◽  
Junction Protein ◽  
Junction Proteins

Abstract Objective The expression of hepatic tight junction proteins and their contribution to homeostasis and regeneration remained largely unexplored. Here, we determine the cell type specific expression of tight junction genes in murine livers. We further explore the regulation and functional importance of the transmembrane protein CLDN3 in normal and regenerating livers. Methods Murine livers were used for tissue- and single cell RNA-seq. CLDN3 localization was determined by immunofluorescence. CLDN3+/+ or CLDN3-/- livers were analysed by electron microscopy, fluorescence-activated cell sorting and liquid chromatography mass spectrometry. Lipid content was quantified with oil-red. Mice were subjected to 2/3 partial hepatectomy. Proliferation was quantified with Ki67 and pHH3 stainings. Cell cycle gene expression was determined by RT-qPCR. Barrier impairments were assessed with total bile acid measurements. Differential gene expression was analysed by tissue RNAseq with DESeq2. Results We determined the profile of tight junction gene expression the main liver cell types, showing that tight junction transcripts can be found in hepatocytes and cholangiocytes but also on non-parenchymal cell populations. CLDN3 was among the highly expressed- and regulated genes in native and regenerating livers. CLDN3 had a zonated expression pattern. CLDN3-/- mice had microscopically intact tight junctions, but showed significantly downregulated hepatic energy metabolism and suboptimal cell proliferation in the regeneration model. Conclusion Our data suggests a functional role of CLDN3 for maintenance of energy homeostasis and optimal regeneration, proving that the function of hepatic tight junction proteins extends beyond basic membrane sealing.

scholarly journals Paracingulin Regulates the Activity of Rac1 and RhoA GTPases by Recruiting Tiam1 and GEF-H1 to Epithelial Junctions

2008 ◽  
Vol 19 (10) ◽  
pp. 4442-4453 ◽  
Author(s):  
Laurent Guillemot ◽  
Serge Paschoud ◽  
Lionel Jond ◽  
Andrea Foglia ◽  
Sandra Citi
Keyword(s):  
Tight Junction ◽  
Small Gtpases ◽  
Cell Junctions ◽  
Dependent Manner ◽  
Junction Protein ◽  
Junction Formation ◽  
Epithelial Junctions ◽  
Junctional Protein

Small GTPases control key cellular events, including formation of cell–cell junctions and gene expression, and are regulated by activating and inhibiting factors. Here, we characterize the junctional protein paracingulin as a novel regulator of the activity of two small GTPases, Rac1 and RhoA, through the functional interaction with their respective activators, Tiam1 and GEF-H1. In confluent epithelial monolayers, paracingulin depletion leads to increased RhoA activity and increased expression of mRNA for the tight junction protein claudin-2. During tight junction assembly by the calcium-switch, Rac1 shows two transient peaks of activity, at earlier (10–20 min) and later (3–8 h) time points. Paracingulin depletion reduces such peaks of Rac1 activation in a Tiam1-dependent manner, resulting in a delay in junction formation. Paracingulin physically interacts with GEF-H1 and Tiam1 in vivo and in vitro, and it is required for their efficient recruitment to junctions, based on immunofluorescence and biochemical experiments. Our results provide the first description of a junctional protein that interacts with GEFs for both Rac1 and RhoA, and identify a novel molecular mechanism whereby Rac1 is activated during junction formation.

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