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David A Antonetti - One of the best experts on this subject based on the ideXlab platform.
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Occludin s490 phosphorylation regulates vascular endothelial growth factor induced retinal neovascularization
American Journal of Pathology, 2016Co-Authors: Xuwen Liu, Aaron E Runkle, Thomas W. Gardner, Alyssa Dreffs, Monica Diazcoranguez, Vince A Chiodo, William W Hauswirth, David A AntonettiAbstract:Occludin is a transmembrane tight junction protein that contributes to diverse cellular functions, including control of barrier properties, cell migration, and proliferation. Vascular endothelial growth factor (VEGF) induces phosphorylation of Occludin at S490, which is required for VEGF-induced endothelial permeability. Herein, we demonstrate that Occludin S490 phosphorylation also regulates VEGF-induced retinal endothelial cell proliferation and neovascularization. Using a specific antibody, phospho-Occludin was located in centrosomes in endothelial cell cultures, animal models, and human surgical samples of retinal neovessels. Occludin S490 phosphorylation was found to increase with endothelial tube formation in vitro and in vivo during retinal neovascularization after induction of VEGF expression. More important, expression of Occludin mutated at S490 to Ala, completely inhibited angiogenesis in cell culture models and in vivo. Collectively, these data suggest a novel role for Occludin in regulation of endothelial proliferation and angiogenesis in a phosphorylation-dependent manner. These findings may lead to methods of regulating pathological neovascularization by specifically targeting endothelial cell proliferation.
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Occludin is a direct target of thyroid transcription factor 1 ttf 1 nkx2 1
Journal of Biological Chemistry, 2012Co-Authors: Aaron E Runkle, David A Antonetti, Shawn J Rice, Dustin Masser, Monte M WinslowAbstract:The thyroid transcription factor 1 gene (TTF-1 or NKX2–1) is essential to lung development; however, it is also a critical factor in lung cancer. TTF-1 is amplified in lung cancers, suggesting that it is a gain-of-function lung oncogene. Conversely, TTF-1 counters epithelial to mesenchymal transition in cell-based studies and inhibits progression of primary lung adenocarcinomas to metastases in an animal model of lung adenocarcinomas. The unifying theory regarding TTF-1 is that it exhibits both pro-oncogenic and anti-metastatic function depending on the cellular context. Occludin is the first discovered constituent of the epithelial tight junction; in recent years, a functional role of Occludin as a tumor suppressor has begun to emerge. Here, we demonstrate that TTF-1 transactivated the expression of the epithelial tight junction molecules Occludin (OCLN) and claudin-1 (CLDN1). We show that transcriptional activation occurred through a direct interaction of TTF-1 with the OCLN and CLDN1 promoters. Furthermore, in cells that lack TTF-1, exogenous TTF-1 expression dampened the inhibitory effect of TGF-β on Occludin and claudin-1 content. Using cells derived from a genetically engineered mouse model of lung adenocarcinomas, we observed that silenced TTF-1 expression down-regulated Occludin, which we supported with additional siRNA experiments. Finally, TTF-1 knockdown conferred human lung cancer cells resistance to anoikis, and expression of Occludin restored cellular sensitivity to anoikis. Overexpression of Occludin impeded migration and induced anoikis in lung carcinoma cells. Collectively, these data suggest that TTF-1 transcriptionally regulates Occludin, which represents another avenue of TTF-1-mediated metastasis suppression.
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protein kinase cβ phosphorylates Occludin regulating tight junction trafficking in vascular endothelial growth factor induced permeability in vivo
Diabetes, 2012Co-Authors: Tomoaki Murakami, Tiffany Frey, Chengmao Lin, David A AntonettiAbstract:Vascular endothelial growth factor (VEGF)–induced breakdown of the blood-retinal barrier requires protein kinase C (PKC)β activation. However, the molecular mechanisms related to this process remain poorly understood. In this study, the role of Occludin phosphorylation and ubiquitination downstream of PKCβ activation in tight junction (TJ) trafficking and endothelial permeability was investigated. Treatment of bovine retinal endothelial cells and intravitreal injection of PKCβ inhibitors as well as expression of dominant-negative kinase was used to determine the contribution of PKCβ to endothelial permeability and Occludin phosphorylation at Ser490 detected with a site-specific antibody. In vitro kinase assay was used to demonstrate direct Occludin phosphorylation by PKCβ. Ubiquitination was measured by immunoblotting after Occludin immunoprecipitation. Confocal microscopy revealed organization of TJ proteins. The results reveal that inhibition of VEGF-induced PKCβ activation blocks Occludin Ser490 phosphorylation, ubiquitination, and TJ trafficking in retinal vascular endothelial cells both in vitro and in vivo and prevents VEGF-stimulated vascular permeability. Occludin Ser490 is a direct target of PKCβ, and mutating Ser490 to Ala (S490A) blocks permeability downstream of PKCβ. Therefore, PKCβ activation phosphorylates Occludin on Ser490, leading to ubiquitination required for VEGF-induced permeability. These data demonstrate a novel mechanism for PKCβ targeted inhibitors in regulating vascular permeability.
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Occludin localizes to centrosomes and modifies mitotic entry
Journal of Biological Chemistry, 2011Co-Authors: Aaron E Runkle, Jeffrey M. Sundstrom, Kristin B Runkle, David A AntonettiAbstract:Proper control of cell cycle progression and barrier function are essential processes to the maintenance of epithelial cell homeostasis. The contribution of tight junction proteins to barrier function is well established, whereas their contribution to cell cycle control is only beginning to be understood. Centrosomes are the principal microtubule organizing centers in eukaryotic cells and centrosome duplication and separation are linked to the cell cycle and mitotic entry. Here we demonstrate that Occludin localizes with centrosomes in Madin-Darby canine kidney cells. Immunocytochemistry and biochemical fractionation studies reveal Occludin localizes with centrosomes during interphase and Occludin Ser-490 phosphorylation at centrosomes increases with mitotic entry. Stable expression of aspartic acid phosphomimetic (S490D) results in centrosomal localization of Occludin and increases cell numbers. Furthermore, we provide evidence that Occludin regulates centrosome separation and mitotic entry as the nonphosphorylatable alanine mutation (S490A) impedes centrosome separation, delays mitotic entry, and reduces proliferation. Collectively, these studies demonstrate a novel location and function for Occludin in centrosome separation and mitosis.
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Occludin phosphorylation and ubiquitination regulate tight junction trafficking and vascular endothelial growth factor induced permeability
Journal of Biological Chemistry, 2009Co-Authors: Tomoaki Murakami, Edward A Felinski, David A AntonettiAbstract:Vascular endothelial growth factor (VEGF) alters tight junctions (TJs) and promotes vascular permeability in many retinal and brain diseases. However, the molecular mechanisms of barrier regulation are poorly understood. Here we demonstrate that Occludin phosphorylation and ubiquitination regulate VEGF-induced TJ protein trafficking and concomitant vascular permeability. VEGF treatment induced TJ fragmentation and Occludin trafficking from the cell border to early and late endosomes, concomitant with increased Occludin phosphorylation on Ser-490 and ubiquitination. Furthermore, both co-immunoprecipitation and immunocytochemistry demonstrated that VEGF treatment increased the interaction between Occludin and modulators of intracellular trafficking that contain the ubiquitin interacting motif, including Epsin-1, epidermal growth factor receptor pathway substrate 15 (Eps15), and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Inhibiting Occludin phosphorylation by mutating Ser-490 to Ala suppressed VEGF-induced ubiquitination, inhibited trafficking of TJ proteins, and prevented the increase in endothelial permeability. In addition, an Occludin-ubiquitin chimera disrupted TJs and increased permeability without VEGF. These data demonstrate a novel mechanism of VEGF-induced Occludin phosphorylation and ubiquitination that contributes to TJ trafficking and subsequent vascular permeability.
Shoichiro Tsukita - One of the best experts on this subject based on the ideXlab platform.
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epithelial transport and barrier function in Occludin deficient mice
Biochimica et Biophysica Acta, 2005Co-Authors: Jörg-dieter Schulzke, Joachim Mankertz, Shoichiro Tsukita, Mitinori Saitou, Alfred H. Gitter, S. Spiegel, U. Seidler, Salah Amasheh, Michael FrommAbstract:Abstract Background and Aims This study aimed at functional characterization of the tight junction protein Occludin using the Occludin-deficient mouse model. Methods Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (Re, epithelial resistance). Conductance scanning differentiated transcellular (Gc) and tight junctional conductance (Gtj). The pH-stat technique quantified gastric acid secretion. Results In Occludin+/+ mice, Re was 23±5 Ω cm2 in jejunum, 66±5 Ω cm2 in distal colon and 33±6 Ω cm2 in gastric corpus and was not altered in heterozygotic Occludin+/− or homozygotic Occludin−/− mice. Additionally, [3H]mannitol fluxes were unaltered. In the control colon, Gc and Gtj were 7.6±1.0 and 0.3±0.1 mS/cm2 and not different in Occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in Occludin−/− mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control Rt was 5.8±0.8 kΩ cm2 in urinary bladder and 33±6 Ω cm2 in stomach and not altered in Occludin−/− mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in Occludin−/− mice. Conclusion Epithelial barrier characterization in Occludin-deficiency points against an essential barrier function of Occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like Occludin. A dramatic change in gastric morphology and secretory function indicates that Occludin is involved in gastric epithelial differentiation.
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Epithelial transport and barrier function in Occludin-deficient mice
Biochimica et biophysica acta, 2005Co-Authors: Jörg-dieter Schulzke, Joachim Mankertz, Shoichiro Tsukita, Mitinori Saitou, Alfred H. Gitter, S. Spiegel, U. Seidler, Salah Amasheh, Michael FrommAbstract:This study aimed at functional characterization of the tight junction protein Occludin using the Occludin-deficient mouse model. Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (Re, epithelial resistance). Conductance scanning differentiated transcellular (Gc) and tight junctional conductance (Gtj). The pH-stat technique quantified gastric acid secretion. In Occludin+/+ mice, Re was 23+/-5 Omega cm2 in jejunum, 66+/-5 Omega cm2 in distal colon and 33+/-6 Omega cm2 in gastric corpus and was not altered in heterozygotic Occludin+/- or homozygotic Occludin-/- mice. Additionally, [3H]mannitol fluxes were unaltered. In the control colon, Gc and Gtj were 7.6+/-1.0 and 0.3+/-0.1 mS/cm2 and not different in Occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in Occludin-/- mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control Rt was 5.8+/-0.8 kOmega cm2 in urinary bladder and 33+/-6 Omega cm2 in stomach and not altered in Occludin-/- mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in Occludin-/- mice. Epithelial barrier characterization in Occludin-deficiency points against an essential barrier function of Occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like Occludin. A dramatic change in gastric morphology and secretory function indicates that Occludin is involved in gastric epithelial differentiation.
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regulation of tight junctions during the epithelium mesenchyme transition direct repression of the gene expression of claudins Occludin by snail
Journal of Cell Science, 2003Co-Authors: Junichi Ikenouchi, Mikio Furuse, Miho Matsuda, Shoichiro TsukitaAbstract:Snail is a transcription repressor that plays a central role in the epithelium-mesenchyme transition (EMT), by which epithelial cells lose their polarity. Claudins and Occludin are integral membrane proteins localized at tight junctions, which are responsible for establishing and maintaining epithelial cell polarity. We examined the relationship between Snail and the promoter activity of claudins and Occludin. When Snail was overexpressed in cultured mouse epithelial cells, EMT was induced with concomitant repression of the expression of claudins and Occludin not only at the protein but also at the mRNA level. We then isolated the promoters of genes encoding claudins and Occludin, in which multiple E-boxes were identified. Transfection experiments with various promoter constructs as well as electrophoretic mobility assays revealed that Snail binds directly to the E-boxes of the promoters of claudin/Occludin genes, resulting in complete repression of their promoter activity. Because the gene encoding E-cadherin was also reported to be repressed by Snail, we concluded that EMT was associated with the simultaneous repression of the genes encoding E-cadherin and claudins/Occludin (i.e. the expression of adherens and tight junction adhesion molecules, respectively).
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Complex Phenotype of Mice Lacking Occludin, a Component of Tight Junction Strands
Molecular biology of the cell, 2000Co-Authors: Mitinori Saitou, Jörg-dieter Schulzke, Michael Fromm, Mikio Furuse, Hiroshi Takano, Tetsuo Noda, Hiroyuki Sasaki, Shoichiro TsukitaAbstract:Occludin is an integral membrane protein with four transmembrane domains that is exclusively localized at tight junction (TJ) strands. Here, we describe the generation and analysis of mice carrying a null mutation in the Occludin gene. Occludin -/- mice were born with no gross phenotype in the expected Mendelian ratios, but they showed significant postnatal growth retardation. Occludin -/- males produced no litters with wild-type females, whereas Occludin -/- females produced litters normally when mated with wild-type males but did not suckle them. In Occludin -/- mice, TJs themselves did not appear to be affected morphologically, and the barrier function of intestinal epithelium was normal as far as examined electrophysiologically. However, histological abnormalities were found in several tissues, i.e., chronic inflammation and hyperplasia of the gastric epithelium, calcification in the brain, testicular atrophy, loss of cytoplasmic granules in striated duct cells of the salivary gland, and thinning of the compact bone. These phenotypes suggested that the functions of TJs as well as Occludin are more complex than previously supposed.
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Occludin is concentrated at tight junctions of mouse/rat but not human/guinea pig Sertoli cells in testes
The American journal of physiology, 1998Co-Authors: Seiji Moroi, Mitinori Saitou, Kazushi Fujimoto, Mikio Furuse, Akira Sakakibara, Osamu Yoshida, Shoichiro TsukitaAbstract:Occludin is the only integral membrane protein identified to date as a component of tight junctions (TJs). Here, we examined the distribution and expression of Occludin in murine testis bearing well-developed TJ. In the adult mouse testis, Occludin was concentrated at TJ strands, which are located at the most basal regions of lateral membranes of Sertoli cells. In immunoblotting, Occludin showed a characteristic multiple banding pattern, suggesting that Occludin is highly phosphorylated in the testis. In 1-wk-old mouse testis, Occludin was distributed diffusely at the lateral membranes of Sertoli cells, and even at this stage, highly phosphorylated Occludin was detected. With development, Occludin gradually became concentrated at the most basal regions of Sertoli cells. The same results were obtained in rat, but unexpectedly Occludin was not detected in human or guinea pig Sertoli cells by immunofluorescence microscopy as well as by immunoblotting. Inasmuch as TJs are also well developed in Sertoli cells of these species, we concluded that, at least in the testes of these species, there are some Sertoli cell-specific isoforms of Occludin or other TJ-associated integral membrane proteins that differ from Occludin.
Le Shen - One of the best experts on this subject based on the ideXlab platform.
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inflammation induced Occludin downregulation limits epithelial apoptosis by suppressing caspase 3 expression
Gastroenterology, 2019Co-Authors: Wei Ting Kuo, Le Shen, Li Zuo, Nitesh Shashikanth, Ma. Lora Drizella M. Ong, Juanmin Zha, Karen L. EdelblumAbstract:Background & Aims Epithelial tight junctions are compromised in gastrointestinal disease. Processes that contribute to the resulting barrier loss include endocytic Occludin removal from the tight junction and reduced Occludin expression. Nevertheless, the relatively-normal basal phenotype of Occludin knockout (KO) mice has been taken as evidence that Occludin does not contribute to gastrointestinal barrier function. We asked whether stress could unmask Occludin functions within intestinal epithelia. Methods Wildtype (WT), universal and intestinal epithelial-specific Occludin KO, and villin-EGFP-Occludin transgenic mice as well as WT and Occludin knockdown (KD) Caco-2BBe cell monolayers were challenged with DSS, TNBS, staurosporine, 5-FU, or TNF. Occludin and caspase-3 expression were assessed in patient biopsies. Results Intestinal epithelial Occludin loss limited severity of DSS- and TNBS-induced colitis due to epithelial resistance to apoptosis; activation of both intrinsic and extrinsic apoptotic pathways was blocked in Occludin KO epithelia. Promoter analysis revealed that Occludin enhances CASP3 transcription and, conversely, that Occludin downregulation reduces caspase-3 expression. Analysis of biopsies from Crohn's disease and ulcerative colitis patients and normal controls demonstrated that disease-associated Occludin downregulation was accompanied by and correlated with reduced caspase-3 expression. In vitro, cytokine-induced Occludin downregulation resulted in reduced caspase-3 expression and resistance to intrinsic and extrinsic pathway apoptosis, demonstrating an overall protective effect of inflammation-induced Occludin loss. Conclusions The tight junction protein Occludin regulates apoptosis by enhancing caspase-3 transcription. These data suggest that reduced epithelial caspase-3 expression downstream of Occludin downregulation is a previously-unappreciated anti-apoptotic process that contributes to mucosal homeostasis in inflammatory conditions.
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Inflammation-induced Occludin Downregulation Limits Epithelial Apoptosis by Suppressing Caspase-3 Expression
Gastroenterology, 2019Co-Authors: Wei Ting Kuo, Le Shen, Li Zuo, Nitesh Shashikanth, Ma. Lora Drizella M. Ong, Juanmin Zha, Karen L. Edelblum, Yitang Wang, Yingmin WangAbstract:Epithelial tight junctions are compromised in gastrointestinal disease. Processes that contribute to the resulting barrier loss include endocytic Occludin removal from the tight junction and reduced Occludin expression. Nevertheless, the relatively-normal basal phenotype of Occludin knockout (KO) mice has been taken as evidence that Occludin does not contribute to gastrointestinal barrier function. We asked whether stress could unmask Occludin functions within intestinal epithelia. Wildtype (WT), universal and intestinal epithelial-specific Occludin KO, and villin-EGFP-Occludin transgenic mice as well as WT and Occludin knockdown (KD) Caco-2BBe cell monolayers were challenged with DSS, TNBS, staurosporine, 5-FU, or TNF. Occludin and caspase-3 expression were assessed in patient biopsies. Intestinal epithelial Occludin loss limited severity of DSS- and TNBS-induced colitis due to epithelial resistance to apoptosis; activation of both intrinsic and extrinsic apoptotic pathways was blocked in Occludin KO epithelia. Promoter analysis revealed that Occludin enhances CASP3 transcription and, conversely, that Occludin downregulation reduces caspase-3 expression. Analysis of biopsies from Crohn's disease and ulcerative colitis patients and normal controls demonstrated that disease-associated Occludin downregulation was accompanied by and correlated with reduced caspase-3 expression. In vitro, cytokine-induced Occludin downregulation resulted in reduced caspase-3 expression and resistance to intrinsic and extrinsic pathway apoptosis, demonstrating an overall protective effect of inflammation-induced Occludin loss. The tight junction protein Occludin regulates apoptosis by enhancing caspase-3 transcription. These data suggest that reduced epithelial caspase-3 expression downstream of Occludin downregulation is a previously-unappreciated anti-apoptotic process that contributes to mucosal homeostasis in inflammatory conditions. Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.
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Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux
Molecular biology of the cell, 2013Co-Authors: Mary M. Buschmann, Le Shen, David R Raleigh, Yingmin Wang, Juanmin Zha, Yitang Wang, Harsha E. Rajapakse, Amulya Lingaraju, Elliot Abbott, Erin M. McauleyAbstract:In vitro and in vivo studies implicate Occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of Occludin in these processes, we established intestinal epithelia with stable Occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ~62.5 A. TNF increased paracellular flux of large molecules in Occludin-sufficient, but not Occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil Occludin/ELL domain (OCEL)-deficient enhanced green fluorescent protein (EGFP)-Occludin showed that TNF-induced Occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-Occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-Occludin(ΔOCEL). Further, the free OCEL domain prevented TNF-induced acceleration of Occludin fluorescence recovery, Occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1-binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK-binding region (K485/K488) remained functional. We conclude that OCEL-mediated Occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.
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Occludin s408 phosphorylation regulates tight junction protein interactions and barrier function
Journal of Cell Biology, 2011Co-Authors: David R Raleigh, Emily M Bradford, Eveline E Schneeberger, Licheng Wu, Christopher R Weber, Le Shen, Amanda M Marchiando, Yingmin Wang, Dan Yu, Jerrold R TurnerAbstract:Although the C-terminal cytoplasmic tail of the tight junction protein Occludin is heavily phosphorylated, the functional impact of most individual sites is undefined. Here, we show that inhibition of CK2-mediated Occludin S408 phosphorylation elevates transepithelial resistance by reducing paracellular cation flux. This regulation requires Occludin, claudin-1, claudin-2, and ZO-1. S408 dephosphorylation reduces Occludin exchange, but increases exchange of ZO-1, claudin-1, and claudin-2, thereby causing the mobile fractions of these proteins to converge. Claudin-4 exchange is not affected. ZO-1 domains that mediate interactions with Occludin and claudins are required for increases in claudin-2 exchange, suggesting assembly of a phosphorylation-sensitive protein complex. Consistent with this, binding of claudin-1 and claudin-2, but not claudin-4, to S408A Occludin tail is increased relative to S408D. Finally, CK2 inhibition reversed IL-13–induced, claudin-2–dependent barrier loss. Thus, Occludin S408 dephosphorylation regulates paracellular permeability by remodeling tight junction protein dynamic behavior and intermolecular interactions between Occludin, ZO-1, and select claudins, and may have therapeutic potential in inflammation-associated barrier dysfunction.
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phosphorylation of tyr 398 and tyr 402 in Occludin prevents its interaction with zo 1 and destabilizes its assembly at the tight junctions
Journal of Biological Chemistry, 2009Co-Authors: Bertha C Elias, Le Shen, Jerrold R Turner, Takuya Suzuki, Ankur Seth, Francesco Giorgianni, Gautam Kale, Anjaparavanda P Naren, Dominic M Desiderio, Radhakrishna RaoAbstract:Occludin is phosphorylated on tyrosine residues during the oxidative stress-induced disruption of tight junction, and in vitro phosphorylation of Occludin by c-Src attenuates its binding to ZO-1. In the present study mass spectrometric analyses of C-terminal domain of Occludin identified Tyr-379 and Tyr-383 in chicken Occludin as the phosphorylation sites, which are located in a highly conserved sequence of Occludin, YETDYTT; Tyr-398 and Tyr-402 are the corresponding residues in human Occludin. Deletion of YETDYTT motif abolished the c-Src-mediated phosphorylation of Occludin and the regulation of ZO-1 binding. Y398A and Y402A mutations in human Occludin also abolished the c-Src-mediated phosphorylation and regulation of ZO-1 binding. Y398D/Y402D mutation resulted in a dramatic reduction in ZO-1 binding even in the absence of c-Src. Similar to wild type Occludin, its Y398A/Y402A mutant was localized at the plasma membrane and cell-cell contact sites in Rat-1 cells. However, Y398D/Y402D mutants of Occludin failed to localize at the cell-cell contacts. Calcium-induced reassembly of Y398D/Y402D mutant Occludin in Madin-Darby canine kidney cells was significantly delayed compared with that of wild type Occludin or its T398A/T402A mutant. Furthermore, expression of Y398D/Y402D mutant of Occludin sensitized MDCK cells for hydrogen peroxide-induced barrier disruption. This study reveals a unique motif in the Occludin sequence that is involved in the regulation of ZO-1 binding by reversible phosphorylation of specific Tyr residues.
Mikio Furuse - One of the best experts on this subject based on the ideXlab platform.
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regulation of tight junctions during the epithelium mesenchyme transition direct repression of the gene expression of claudins Occludin by snail
Journal of Cell Science, 2003Co-Authors: Junichi Ikenouchi, Mikio Furuse, Miho Matsuda, Shoichiro TsukitaAbstract:Snail is a transcription repressor that plays a central role in the epithelium-mesenchyme transition (EMT), by which epithelial cells lose their polarity. Claudins and Occludin are integral membrane proteins localized at tight junctions, which are responsible for establishing and maintaining epithelial cell polarity. We examined the relationship between Snail and the promoter activity of claudins and Occludin. When Snail was overexpressed in cultured mouse epithelial cells, EMT was induced with concomitant repression of the expression of claudins and Occludin not only at the protein but also at the mRNA level. We then isolated the promoters of genes encoding claudins and Occludin, in which multiple E-boxes were identified. Transfection experiments with various promoter constructs as well as electrophoretic mobility assays revealed that Snail binds directly to the E-boxes of the promoters of claudin/Occludin genes, resulting in complete repression of their promoter activity. Because the gene encoding E-cadherin was also reported to be repressed by Snail, we concluded that EMT was associated with the simultaneous repression of the genes encoding E-cadherin and claudins/Occludin (i.e. the expression of adherens and tight junction adhesion molecules, respectively).
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Complex Phenotype of Mice Lacking Occludin, a Component of Tight Junction Strands
Molecular biology of the cell, 2000Co-Authors: Mitinori Saitou, Jörg-dieter Schulzke, Michael Fromm, Mikio Furuse, Hiroshi Takano, Tetsuo Noda, Hiroyuki Sasaki, Shoichiro TsukitaAbstract:Occludin is an integral membrane protein with four transmembrane domains that is exclusively localized at tight junction (TJ) strands. Here, we describe the generation and analysis of mice carrying a null mutation in the Occludin gene. Occludin -/- mice were born with no gross phenotype in the expected Mendelian ratios, but they showed significant postnatal growth retardation. Occludin -/- males produced no litters with wild-type females, whereas Occludin -/- females produced litters normally when mated with wild-type males but did not suckle them. In Occludin -/- mice, TJs themselves did not appear to be affected morphologically, and the barrier function of intestinal epithelium was normal as far as examined electrophysiologically. However, histological abnormalities were found in several tissues, i.e., chronic inflammation and hyperplasia of the gastric epithelium, calcification in the brain, testicular atrophy, loss of cytoplasmic granules in striated duct cells of the salivary gland, and thinning of the compact bone. These phenotypes suggested that the functions of TJs as well as Occludin are more complex than previously supposed.
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Occludin is concentrated at tight junctions of mouse/rat but not human/guinea pig Sertoli cells in testes
The American journal of physiology, 1998Co-Authors: Seiji Moroi, Mitinori Saitou, Kazushi Fujimoto, Mikio Furuse, Akira Sakakibara, Osamu Yoshida, Shoichiro TsukitaAbstract:Occludin is the only integral membrane protein identified to date as a component of tight junctions (TJs). Here, we examined the distribution and expression of Occludin in murine testis bearing well-developed TJ. In the adult mouse testis, Occludin was concentrated at TJ strands, which are located at the most basal regions of lateral membranes of Sertoli cells. In immunoblotting, Occludin showed a characteristic multiple banding pattern, suggesting that Occludin is highly phosphorylated in the testis. In 1-wk-old mouse testis, Occludin was distributed diffusely at the lateral membranes of Sertoli cells, and even at this stage, highly phosphorylated Occludin was detected. With development, Occludin gradually became concentrated at the most basal regions of Sertoli cells. The same results were obtained in rat, but unexpectedly Occludin was not detected in human or guinea pig Sertoli cells by immunofluorescence microscopy as well as by immunoblotting. Inasmuch as TJs are also well developed in Sertoli cells of these species, we concluded that, at least in the testes of these species, there are some Sertoli cell-specific isoforms of Occludin or other TJ-associated integral membrane proteins that differ from Occludin.
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Occludin deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions
Journal of Cell Biology, 1998Co-Authors: Mitinori Saitou, M Itoh, Kazushi Fujimoto, Yoshinori Doi, Toyoshi Fujimoto, Mikio Furuse, Hiroshi Takano, Tetsuo Noda, Shoichiro TsukitaAbstract:Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the Occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from Occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and Occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in Occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in Occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without Occludin.
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Occludin as a possible determinant of tight junction permeability in endothelial cells
Journal of Cell Science, 1997Co-Authors: Tetsuaki Hirase, James M Staddon, Yuhko Andoakatsuka, Masahiko Itoh, Shoichiro Tsukita, Mitinori Saitou, Kazushi Fujimoto, Mikio Furuse, Lee L RubinAbstract:Endothelial cells provide a crucial interface between blood and tissue environments. Free diffusion of substances across endothelia is prevented by the endothelial tight junction, the permeability of which varies enormously depending on tissue. Endothelial cells of the blood-brain barrier possess tight junctions of severely limited permeability, whereas those of non-neural tissue are considerably leakier, but the molecular basis for this difference is not clear. Occludin is a major transmembrane protein localizing at the tight junction. In this study, we show, by immunocytochemistry, that Occludin is present at high levels and is distributed continuously at cell-cell contacts in brain endothelial cells. In contrast, endothelial cells of non-neural tissue have a much lower expression of Occludin, which is distributed in a discontinuous fashion at cell-cell contacts. The apparent differences in Occludin expression levels were directly confirmed by immunoblotting. The differences in Occludin protein were reflected at the message level, suggesting transcriptional regulation of expression. We also show that Occludin expression is developmentally regulated, being low in rat brain endothelial cells at postnatal day 8 but clearly detectable at post-natal day 70. Our data indicate that regulation of Occludin expression may be a crucial determinant of the tight junction permeability properties of endothelial cells in different tissues.
Radhakrishna Rao - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation hotspot in the C-terminal domain of Occludin regulates the dynamics of epithelial junctional complexes.
Journal of cell science, 2018Co-Authors: Bhargavi Manda, Takuya Suzuki, Hina Mir, Ruchika Gangwar, Avtar S. Meena, Shrunali Amin, Pradeep Kumar Shukla, Kesha Dalal, Radhakrishna RaoAbstract:The apical junctional complex (AJC), which includes tight junctions (TJs) and adherens junctions (AJs), determines the epithelial polarity, cell-cell adhesion and permeability barrier. An intriguing characteristic of a TJ is the dynamic nature of its multiprotein complex. Occludin is the most mobile TJ protein, but its significance in TJ dynamics is poorly understood. On the basis of phosphorylation sites, we distinguished a sequence in the C-terminal domain of Occludin as a regulatory motif (ORM). Deletion of ORM and expression of a deletion mutant of Occludin in renal and intestinal epithelia reduced the mobility of Occludin at the TJs. ORM deletion attenuated Ca2+ depletion, osmotic stress and hydrogen peroxide-induced disruption of TJs, AJs and the cytoskeleton. The double point mutations T403A/T404A, but not T403D/T404D, in Occludin mimicked the effects of ORM deletion on Occludin mobility and AJC disruption by Ca2+ depletion. Both Y398A/Y402A and Y398D/Y402D double point mutations partially blocked AJC disruption. Expression of a deletion mutant of Occludin attenuated collective cell migration in the renal and intestinal epithelia. Overall, this study reveals the role of ORM and its phosphorylation in Occludin mobility, AJC dynamics and epithelial cell migration.
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Occludin phosphorylation in regulation of epithelial tight junctions.
Annals of the New York Academy of Sciences, 2009Co-Authors: Radhakrishna RaoAbstract:Occludin is the first transmembrane protein of the tight junction to be discovered. While numerous studies emphasized the important role of Occludin in assembly and maintenance of tight junctions, Occludin knockout studies indicated that it was not required for tight junction assembly in different epithelia. However, a detailed characterization of the Occludin knockout mouse concluded that the Occludin gene is indispensable, and plays a complex role in regulation of epithelial tight junctions in different organs. This article describes the role of Occludin phosphorylation in the regulation of its assembly into the tight junctions. Occludin is highly phosphorylated on Ser and Thr residues, while Tyr-phosphorylation is kept at minimum in the intact epithelium. During the disruption of tight junctions by various factors, Occludin undergoes dephosphorylation on Ser/Thr residues and elevated phosphorylation on Tyr residues. The phosphorylation of Occludin on Tyr, Ser, and Thr residues appears to be regulated by the balance between protein kinases such as c-Src, PKCzeta, and PKClambda/iota, and protein phosphatases such as PP2A, PP1, and PTP1B. The precise mechanism of regulation of the tight junction by Occludin phosphorylation is unclear at this time. However, an in vitro study indicated that Tyr-phosphorylation of Occludin C-terminal domain attenuates its interaction with ZO-1. Therefore, phosphorylation of specific Ser/Thr/Tyr residues in Occludin may regulate its interactions with ZO-1 and possibly other tight junction proteins. It is likely that Occludin plays a regulatory role in tight junctions rather than a role in the de novo assembly of tight junctions.
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phosphorylation of tyr 398 and tyr 402 in Occludin prevents its interaction with zo 1 and destabilizes its assembly at the tight junctions
Journal of Biological Chemistry, 2009Co-Authors: Bertha C Elias, Le Shen, Jerrold R Turner, Takuya Suzuki, Ankur Seth, Francesco Giorgianni, Gautam Kale, Anjaparavanda P Naren, Dominic M Desiderio, Radhakrishna RaoAbstract:Occludin is phosphorylated on tyrosine residues during the oxidative stress-induced disruption of tight junction, and in vitro phosphorylation of Occludin by c-Src attenuates its binding to ZO-1. In the present study mass spectrometric analyses of C-terminal domain of Occludin identified Tyr-379 and Tyr-383 in chicken Occludin as the phosphorylation sites, which are located in a highly conserved sequence of Occludin, YETDYTT; Tyr-398 and Tyr-402 are the corresponding residues in human Occludin. Deletion of YETDYTT motif abolished the c-Src-mediated phosphorylation of Occludin and the regulation of ZO-1 binding. Y398A and Y402A mutations in human Occludin also abolished the c-Src-mediated phosphorylation and regulation of ZO-1 binding. Y398D/Y402D mutation resulted in a dramatic reduction in ZO-1 binding even in the absence of c-Src. Similar to wild type Occludin, its Y398A/Y402A mutant was localized at the plasma membrane and cell-cell contact sites in Rat-1 cells. However, Y398D/Y402D mutants of Occludin failed to localize at the cell-cell contacts. Calcium-induced reassembly of Y398D/Y402D mutant Occludin in Madin-Darby canine kidney cells was significantly delayed compared with that of wild type Occludin or its T398A/T402A mutant. Furthermore, expression of Y398D/Y402D mutant of Occludin sensitized MDCK cells for hydrogen peroxide-induced barrier disruption. This study reveals a unique motif in the Occludin sequence that is involved in the regulation of ZO-1 binding by reversible phosphorylation of specific Tyr residues.
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protein phosphatases 2a and 1 interact with Occludin and negatively regulate the assembly of tight junctions in the caco 2 cell monolayer
Journal of Biological Chemistry, 2007Co-Authors: Ankur Seth, Bertha C Elias, Parimal Sheth, Radhakrishna RaoAbstract:Occludin is hyperphosphorylated on Ser and Thr residues in intact epithelial tight junction (TJ); however, the role of this phosphorylation in the assembly of TJ is unclear. The influence of protein phosphatases PP2A and PP1 on the assembly of TJ and phosphorylation of Occludin was evaluated in Caco-2 cells. Protein phosphatase inhibitors and reduced expression of PP2A-Calpha and PP1alpha accelerated the calcium-induced increase in transepithelial electrical resistance and barrier to inulin permeability and also enhanced the junctional organization of Occludin and ZO-1 during TJ assembly. Phosphorylation of Occludin on Thr residues, but not on Ser residues, was dramatically reduced during the disassembly of TJ and was gradually increased during the reassembly. PP2A and PP1 co-immunoprecipitate with Occludin, and this association was reduced during the assembly of TJ. Glutathione S-transferase (GST) pull-down assay using recombinant GST-Occludin demonstrated that cellular PP2A and PP1 bind to the C-terminal tail of Occludin, and these interactions were also reduced during the assembly of TJ. A pairwise binding assay using GST-Occludin and purified PP2A and PP1 demonstrates that PP2A and PP1 directly interacts with the C-terminal tail of Occludin. In vitro incubation of phospho-Occludin with PP2A or PP1 indicated that PP2A dephosphorylates Occludin on phospho-Thr residues, whereas PP1 dephosphorylates it on phospho-Ser. This study shows that PP2A and PP1 directly interact with Occludin and negatively regulate the assembly of TJ by modulating the phosphorylation status of Occludin.
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Protein Phosphatases 2A and 1 Interact with Occludin and Negatively Regulate the Assembly of Tight Junctions in the
2007Co-Authors: Ankur Seth, Bertha C Elias, Parimal Sheth, Radhakrishna RaoAbstract:Occludin is hyperphosphorylated on Ser and Thr residues in intact epithelial tight junction (TJ); however, the role of this phosphorylation in the assembly of TJ is unclear. The influence of protein phosphatases PP2A and PP1 on the assembly of TJ and phosphorylation of Occludin was evaluated in Caco-2 cells. Protein phosphatase inhibitors and reduced expression of PP2A-C and PP1 accelerated the calcium-induced increase in transepithelial electrical resistance and barrier to inulin permeability and also enhanced the junctional organization of Occludin and ZO-1 during TJ assembly. Phosphorylation of Occludin on Thr residues, but not on Ser residues, was dramatically reduced during the disassembly of TJ and was gradually increased during the reassembly. PP2A and PP1 co-immunoprecipitate with Occludin, and this association was reduced during the assembly of TJ. Glutathione S-transferase (GST) pulldown assay using recombinant GST-Occludin demonstrated that cellular PP2A and PP1 bind to the C-terminal tail of Occludin, and these interactions were also reduced during the assembly of TJ. A pairwise binding assay using GST-Occludin and purified PP2A and PP1 demonstrates that PP2A and PP1 directly interacts with the C-terminal tail of Occludin. In vitro incubation of phospho-Occludin with PP2A or PP1 indicated that PP2A dephosphorylates Occludin on phospho-Thr residues, whereas PP1 dephosphorylates it on phospho-Ser. This study shows that PP2A and PP1 directly interact with Occludin and negatively regulate the assembly of TJ by modulating the phosphorylation status of Occludin.
