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

Diabetic retinopathy is one of the leading causes of vision loss and blindness. Extensive preclinical 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 vascular endothelial growth factor (VEGF)-induced permeability and edema formation after intraocular 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 integrity induced by diabetes contributes to vision loss.

Piezo1 regulates intestinal epithelial function by affecting the tight junction protein claudin-1 via the ROCK pathway.

AIMS: Defective tight junctions (TJs) can induce intestinal epithelial dysfunction, which participates in various diseases such as irritable bowel syndrome. However, the mechanisms of TJ defects remain unclear. Our study revealed the role of Piezo1 in regulating intestinal epithelial function and TJs. MATERIALS AND METHODS: The human colonic adenocarcinoma cell line Caco-2 were cultured on Transwell plate to form an epithelial barrier in vitro, and Piezo1 expression was manipulated using a lentivirus vector. Epithelial function was evaluated by measuring transepithelial electronic resistance (TEER) and 4-kDa FITC-dextran (FD4) transmission. TJ proteins (claudin-1, occludin, ZO-1) were evaluated by RT-PCR, western blot, and immunostaining analysis. Potential signal pathways, including the ROCK and Erk pathways, were detected. Moreover, to explore the regulatory effect of Piezo1 activity on epithelial function, inhibitors (ruthenium red, GsMTx4) and an agonist (Yoda1) were introduced both ex vivo and in vitro. KEY FINDINGS: Alteration of Piezo1 expression altered epithelial function and the expression of the tight junction protein claudin-1. Piezo1 expression regulated phosphorylated ROCK1/2 expression, whereas interference on ROCK1/2 prevented the regulation of claudin-1 by Piezo1. In both Caco-2 monolayer and mouse colon epithelium, Piezo1 activity directly modulated epithelial function and permeability. SIGNIFICANCE: Piezo1 negatively regulates epithelial barrier function by affecting the expression of claudin-1. Such regulation may be achieved partially via the ROCK1/2 pathway. Moreover, activating Piezo1 can induce epithelial dysfunction.

Occludin and tricellulin facilitate formation of anastomosing tight-junction strand network to improve barrier function.

Tight junctions (TJs) are composed of a claudin-based anastomosing network of TJ strands at which plasma membranes of adjacent epithelial cells are closely attached to regulate the paracellular permeability. Although the TJ proteins occludin and tricellulin have been known to be incorporated in the TJ strand network, their molecular functions remain unknown. Here, we established tricellulin/occludin-double knockout (dKO) MDCK II cells using a genome editing technique and evaluated the structure and barrier function of these cells. In freeze-fracture replica electron microscopy, the TJ strands of tricellulin/occludin-dKO cells had fewer branches and were less anastomosed compared with the controls. The paracellular permeability of ions and small tracers was increased in the dKO cells. A single KO of tricellulin or occludin had limited effects on the morphology and permeability of TJs. Mathematical simulation using a simplified TJ strand network model predicted that reduced cross-links in TJ strands lead to increased permeability of ions and small macromolecules. Furthermore, overexpression of occludin increased the complexity of TJ strand network and strengthened barrier function. Taken together, our data suggest that tricellulin and occludin mediate the formation and/or stabilization of TJ-strand branching points and contribute to the maintenance of epithelial barrier integrity.

Occludin endocytosis is involved in the disruption of the intestinal epithelial barrier in a mouse model of alcoholic steatohepatitis.

OBJECTIVE: We aimed to investigate the involvement of the endocytosis of occludin, a key component of tight junction (TJ), in the ethanol-induced disassembly of TJ in a model of alcoholic steatohepatitis. METHODS: Wild-type mice were fed an ethanol-containing or isocaloric liquid diet for 8 weeks and then assessed for liver injury (histopathology and measurement of serum enzymes), gut permeability (in vivo lactulose/mannitol and ex vivo dye leakage assays), intestinal epithelium ultrastructure (transmission electron microscopy), and intestinal occludin localization (immunofluorescence microscopy). The human intestinal epithelial cell line Caco-2 was also analyzed in vitro for the effects of ethanol on the barrier function (transepithelial electrical resistance), occludin localization (immunofluorescence microscopy and Western blotting), and endocytosis pathways (double-labeling immunofluorescence microscopy with selective pathway inhibitors). RESULTS: The ethanol-fed mice developed steatohepatitis and displayed intestinal barrier dysfunction, the disruption of intestinal TJ, and enhanced intestinal endocytosis of occluding compared with the control mice. In the Caco-2 monolayers, ethanol treatment decreased transepithelial electrical resistance, disrupted TJ formation, and enhanced occludin endocytosis in a dose- and time-dependent manner. These deleterious events were reversed by pretreating the Caco-2 cells with a selective pharmacological inhibitor of macropinocytosis, but not with the inhibitors of clathrin or caveolin-mediated endocytic pathways. CONCLUSION: Chronic ethanol exposure may increase intestinal permeability by inducing the micropinocytosis of occludin, resulting in the disruption of intestinal TJ.

Early Porcine Sapovirus Infection Disrupts Tight Junctions and Uses Occludin as a Coreceptor.

The genus Sapovirus belongs to the family Caliciviridae, and its members are common causative agents of severe acute gastroenteritis in both humans and animals. Some caliciviruses are known to use either terminal sialic acids or histo-blood group antigens as attachment factors and/or cell surface proteins, such as CD300lf, CD300ld, and junctional adhesion molecule 1 of tight junctions (TJs), as receptors. However, the roles of TJs and their proteins in sapovirus entry have not been examined. In this study, we found that porcine sapovirus (PSaV) significantly decreased transepithelial electrical resistance and increased paracellular permeability early in infection of LLC-PK cells, suggesting that PSaV dissociates TJs of cells. This led to the interaction between PSaV particles and occludin, which traveled in a complex into late endosomes via Rab5- and Rab7-dependent trafficking. Inhibition of occludin using small interfering RNA (siRNA), a specific antibody, or a dominant-negative mutant significantly blocked the entry of PSaV. Transient expression of occludin in nonpermissive Chinese hamster ovary (CHO) cells conferred susceptibility to PSaV, but only for a limited time. Although claudin-1, another TJ protein, neither directly interacted nor was internalized with PSaV particles, it facilitated PSaV entry and replication in the LLC-PK cells. We conclude that PSaV particles enter LLC-PK cells by binding to occludin as a coreceptor in PSaV-dissociated TJs. PSaV and occludin then form a complex that moves to late endosomes via Rab5- and Rab7-dependent trafficking. In addition, claudin-1 in the TJs opened by PSaV infection facilitates PSaV entry and infection as an entry factor.IMPORTANCE Sapoviruses (SaVs) cause severe acute gastroenteritis in humans and animals. Although they replicate in intestinal epithelial cells, which are tightly sealed by apical-junctional complexes, such as tight junctions (TJs), the mechanisms by which SaVs hijack TJs and their proteins for successful entry and infection remain largely unknown. Here, we demonstrate that porcine SaVs (PSaVs) induce early dissociation of TJs, allowing them to bind to the TJ protein occludin as a functional coreceptor. PSaVs then travel in a complex with occludin into late endosomes through Rab5- and Rab7-dependent trafficking. Claudin-1, another TJ protein, does not directly interact with PSaV but facilitates the entry of PSaV into cells as an entry factor. This work contributes to our understanding of the entry of SaV and other caliciviruses into cells and may aid in the development of efficient and affordable drugs to treat SaV infections.

Hypoxic glioblastoma release exosomal VEGF-A induce the permeability of blood-brain barrier.

Exosomes are nano-vesicles released by tumor cells to modulate extracellular environment. Accumulating evidence revealed that glioblastoma derived exosomes contain multiple pro-angiogenic factors to induce the proliferation of endothelial cells. Here, we investigated the role of GBM-derived exosomes in inducing the permeability of the blood-brain barrier. We found that VEGF-A was over-expressed in hypoxic GBM-derived exosomes, which enhance the permeability of a BBB in vitro model by interrupting the expression of claudin-5 and occludin. In vivo permeability assay showed hypoxic GBM-derived exosomes remained functional in the blood circulation and induced the permeability of BBB.

Occludin induces microvillus formation via phosphorylation of ezrin in a mouse hepatic cell line.

Apical and basolateral cell membranes are separated by tight junctions (TJs). Microvilli are limited to the apical cell membrane. TJs and microvilli are the landmarks for epithelial cell polarity. However, the direct relationship between TJ proteins (TJPs) and the components of microvilli remains unclear. In this study, we investigated whether occludin, which is considered to be a functional TJP, is involved in microvillus formation. In occludin knockout mouse hepatic cells (OcKO cells), the microvillus density was less than that in wild-type (WT) cells and the length of microvilli was short. Immunoreactivity of ezrin was decreased in OcKO cells compared with that in WT cells. Although there was no change in the expression level of ezrin, phosphorylation of ezrin was decreased in OcKO cells. The microvillus density and the length of microvilli were increased in OcKO cells by transfection of full-length mouse occludin and COOH-terminal domains of occludin. These results suggested that occludin induced microvillus formation via phosphorylation of ezrin and that the COOH-terminal domain of occludin, which is localized in non-TJ areas, might be able to induce microvilli formation. Our results provide new insights into the function of occludin.

Effect of local scrotal heating on the expression of tight junction-associated molecule Occludin in boar testes.

The aim of this study was to determine whether local scrotal heating (42°C, for 1 hr) had an effect on the expression of tight junction (TJ)-associated molecule Occludin in boar testes. Adult boars (Landrace, n = 6) were used and randomly divided into two groups (n = 3 each). Three boars were given local scrotal exposure to 42°C for approximately 1 h with a home-made electric blanket of controlled temperature as local scrotal heating group, the other three boars received no heat treatment and were left at standard room temperature as control group. After 6 hr, all boars were castrated and the testes were harvested. qRT-PCR, Western blotting and immunohistochemistry were used to explore the expression and localization of Occludin. qRT-PCR and Western blotting showed that the protein and mRNA levels of Occludin significantly decreased in local scrotal heating group as compared to the control. Furthermore, immunoreactivity staining of Occludin was localized at the sites of the blood-testis barrier (BTB) and formed an almost consecutive and strong immunoreactivity strand in the control, while Occludin was limited to Sertoli cells (SCs) and no obvious immunoreactivity strand was present in local scrotal heating group. These data indicated that local scrotal heating decreased the expression of TJ-associated molecule Occludin, which may be involved in heat-induced spermatogenesis damage.

Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity.

Energetic regulation at the blood-brain barrier is critical for maintaining its integrity, transport capabilities, and brain demands for glucose. However, the underlying mechanisms that regulate these processes are still poorly explored. We recently characterized the protein occludin as a NADH oxidase and demonstrated its influence on the expression and activation of the histone deacetylase SIRT-1. Because SIRT-1 works in concert with AMP-activated protein kinase (AMPK) (AMPK), we investigated the impact of occludin on this metabolic switch. Here we show that in blood-brain barrier pericytes, occludin promotes AMPK expression and activation, influencing the expression of glucose transporters GLUT-1 and GLUT-4, glucose uptake, and ATP content. Furthermore, occludin expression, AMP-dependent protein kinase activity, and glucose uptake were altered under inflammatory (TNFα) and infectious (HIV) conditions. We also show that pericytes share glucose and mitochondria with astrocytes, and that occludin levels modify the ability of pericytes to share those energetic resources. In addition, we demonstrate that murine mitochondria can be transferred from live brain microvessels to energetically impaired human astrocytes, promoting their survival. Our findings demonstrate that occludin plays an important role in blood-brain barrier pericyte metabolism by influencing AMPK protein kinase activity, glucose uptake, ATP production, and by regulating the ability of pericytes to interact metabolically with astrocytes.

Process of tight junction recovery in the injured vocal fold epithelium: Morphological and paracellular permeability analysis.

OBJECTIVES/HYPOTHESIS: The vocal fold epithelium that includes tight junction (TJ)-based barrier function protects underlying connective tissues from external insults. TJs play an important role to control paracellular permeability of not only solutes but also ions, and preserve the vocal fold homeostasis. However, the distribution of TJs and paracellular diffusion barrier across the entire vocal fold epithelium are still unknown. The aim of this study was to identify the distribution of TJs in the vocal fold epithelium and to characterize the recovery process of TJ-based paracellular diffusion barrier in a rat model of vocal fold injury. STUDY DESIGN: Animal experiments with controls. METHODS: Normal and vocal fold-injured rats were used. Larynges were harvested for immunohistochemical examination of TJ proteins. For functional analysis, a tracer permeability assay was performed using EZ-Link Sulfo-NHS-LC-Biotin. RESULTS: TJ proteins occludin and zonula occludens 1 signals were localized to the junctional regions of the most luminal cell layers of the vocal fold epithelium. The injured region had been recovered with epithelium at 5 days postinjury, but the paracellular diffusion barrier assays revealed that biotinylation reagents diffused into the lamina propria at 5 days postinjury, and were blocked at the epithelium at 14 and 28 days postinjury. CONCLUSIONS: It was strongly suggested that TJs in the vocal fold epithelium exist at the junctional regions of the first layer of stratified squamous epithelium. TJ-based paracellular diffusion barrier following vocal fold injury is recovered by 14 days postinjury, and this period corresponds with the time course of structural changes in the regenerating epithelium layer. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E150-E156, 2018.

Tight Junction Proteins Claudin-1 and Occludin Are Important for Cutaneous Wound Healing.

Tight junction (TJ) proteins are known to be involved in proliferation and differentiation. These processes are essential for normal skin wound healing. Here, we investigated the TJ proteins claudin-1 and occludin in ex vivo skin wound healing models and tissue samples of acute and chronic human wounds and observed major differences in localization/expression of these proteins, with chronic wounds often showing a loss of the proteins at the wound margins and/or in the regenerating epidermis. Knockdown experiments in primary human keratinocytes showed that decreased claudin-1 expression resulted in significantly impaired scratch wound healing, with delayed migration and reduced proliferation. Activation of AKT pathway was significantly attenuated after claudin-1 knockdown, and protein levels of extracellular signal-related kinase 1/2 were reduced. For occludin, down-regulation had no impact on wound healing in normal scratch assays, but after subjecting the cells to mechanical stress, which is normally present in wounds, wound healing was impaired. For both proteins we show that most of these actions are independent from the formation of barrier-forming TJ structures, thus demonstrating nonbarrier-related functions of TJ proteins in the skin. However, for claudin-1 effects on scratch wound healing were more pronounced when TJs could form. Together, our findings provide evidence for a role of claudin-1 and occludin in epidermal regeneration with potential clinical importance.

PHD3 Stabilizes the Tight Junction Protein Occludin and Protects Intestinal Epithelial Barrier Function.

Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD.

Tricellulin and its role in the epididymal epithelium of the rat.

Tricellulin is a tight-junction protein present at tricellular tight junctions. It has been suggested that basal cells are implicated in the blood-epididymis barrier. Basal cells express claudins, a component of tight junctions; however, there is no information regarding the potential architecture or regulation of basal cell-principal cell interactions. The present objectives were to determine the expression and localization of tricellulin in rat epididymis in relation to occludin, basal cell-principal cell interactions, and other junctional proteins. Tricellulin levels were similar in all segments of the adult epididymis, and the protein was localized to the apical region of the epithelium. Postnatal development showed that tricellulin levels increased with age and localization changed from cytoplasmic to membrane-bound as a function of age. Colocalization with occludin indicated that both proteins are in the region of the tight junction. In the initial segment, the proteins did not colocalize compared to the epididymis where they were both colocalized. Tricellulin did not colocalize with cytokeratin 5, a marker of basal cells, in any region of the epididymis, including the corpus and cauda epididymidis, where apical projections of basal cells were apparent. Tricellulin knockdown studies using small interfering RNA in rat caput epididymal principal cells resulted in decreased transepithelial resistance and was correlated with decreased levels of Cldn3, Cldn1, and occludin. Tight-junction protein1, also known as ZO-1, and cadherin1 levels were unchanged. This is the first report of tricellulin in the epididymis and on the interaction between tricellulin and other tight-junction proteins.

Hepatic cells derived from induced pluripotent stem cells of pigtail macaques support hepatitis C virus infection.

The narrow species tropism of hepatitis C virus (HCV) limits animal studies. We found that pigtail macaque (Macaca nemestrina) hepatic cells derived from induced pluripotent stem cells support the entire HCV life cycle, although infection efficiency was limited by defects in the HCV cell entry process. This block was overcome by either increasing occludin expression, complementing the cells with human CD81, or infecting them with a strain of HCV with less restricted requirements for CD81. Using this system, we can modify viral and host cell genetics to make pigtail macaques a suitable, clinically relevant model for the study of HCV infection.

Tight junction proteins: from barrier to tumorigenesis.

The tight junction is a multi-protein complex and is the apical most junctional complex in certain epithelial and endothelial cells. A great deal of attention has been devoted to the understanding of these proteins in contributing to the barrier function - that is, regulating the paracellular flux or permeability between adjacent cells. However, tight junction proteins are now recognized as having functions beyond the barrier. The focus of this review is to discuss the barrier function of the tight junction and to summarize the literature with a focus on the role of tight junction proteins in proliferation, transformation, and metastasis.

DNA microarray-based screening of differentially expressed genes related to acute lung injury and functional analysis.

OBJECTIVES: The purpose of this study was to identify differentially expressed genes (DEGs) related to acute lung injury (ALI) induced by sepsis with DNA microarray. MATERIALS AND METHODS: Gene expression profile GSE10474 was downloaded from Gene Expression Omnibus (GEO) database which includes 34 samples, among which 13 patients with ALI + sepsis and 21 patients with sepsis alone. The DEGs were identified between ALI + sepsis and sepsis alone samples using R, which were further analyzed using bioinformatics methods. Firstly, HitPredict was used to search protein-protein interactions of the DEGs. Secondly, WebGestalt was adopted for functional enrichment analysis of genes in the interaction networks. Finally, DNA methylation was analyzed to explain the differential expression. RESULTS: A total of 12 genes were identified as DEGs by comparing chip data from ALI + sepsis samples and those from sepsis alone samples, among which occludin (OCLN) and major histocompatibility complex, class II, DQ beta 1 (HLA-DQB1) had 21 and 6 interactors, respectively. Functional enrichment analysis revealed several significantly over-represented terms: cellular component organization, macromolecular organization and biosynthesis, and response to stimulus. In addition, methylation was found in the promoters of OCLN and HLA-DQB1. CONCLUSIONS: We screened DEGs in septic ALI samples, and several interesting genes were obtained, especially OCLN and HLA-DQB1. They may be developed into marker genes for diagnosis or treatment of ALI.

Contribution of tight junction proteins to ion, macromolecule, and water barrier in keratinocytes.

Tight junctions (TJs) form a selective barrier for ions, water, and macromolecules in simple epithelia. In keratinocytes and epidermis, TJs were shown to be involved in individual barrier functions. The absence of the TJ protein claudin-1 (Cldn1) in mice results in a skin-barrier defect characterized by lethal water loss. However, detailed molecular analyses of the various TJ barriers in keratinocytes and the contribution of distinct TJ proteins are missing. Herein, we discriminate TJ-dependent paracellular resistance from transcellular resistance in cultured keratinocytes using the two-path impedance spectroscopy. We demonstrate that keratinocyte TJs form a barrier for Na(+), Cl(-), and Ca(2+), and contribute to barrier function for water and larger molecules of different size. In addition, knockdown of Cldn1, Cldn4, occludin, and zonula occludens-1 increased paracellular permeabilities for ions and larger molecules, demonstrating that all of these TJ proteins contribute to barrier formation. Remarkably, Cldn1 and Cldn4 are not critical for TJ barrier function for water in submerged keratinocyte cultures. However, Cldn1 influences stratum corneum (SC) proteins important for SC water barrier function, and is crucial for TJ barrier formation for allergen-sized macromolecules.

Tight junction in blood-brain barrier: an overview of structure, regulation, and regulator substances.

Blood-brain barrier (BBB) is a dynamic interference that regulates the nutrition and toxic substance in and out of the central nervous system (CNS), and plays a crucial role in maintaining a stable circumstance of the CNS. Tight junctions among adjacent cells form the basic structure of BBB to limiting paracellular permeability. In the present review, the constituents of tight junction proteins are depicted in detail, together with the regulation of tight junction under stimulation and in pathological conditions. Tight junction modulators are also discussed.

Barriers and more: functions of tight junction proteins in the skin.

Although the existence of tight junction (TJ) structures (or a secondary epidermal barrier) was postulated for a long time, the first description of TJ proteins in the epidermis (occludin, ZO-1, and ZO-2) was only fairly recent. Since then, a wealth of new insights concerning TJs and TJ proteins, including their functional role in the skin, have been gathered. Of special interest is that the epidermis as a multilayered epithelium exhibits a very complex localization pattern of TJ proteins, which results in different compositions of TJ protein complexes in different layers. In this review, we summarize our current knowledge about the role of TJ proteins in the epidermis in barrier function, cell polarity, vesicle trafficking, differentiation, and proliferation. We hypothesize that TJ proteins fulfill TJ structure-dependent and structure-independent functions and that the specific function of a TJ protein may depend on the epidermal layer where it is expressed.