Tight Junction-associated Protein ZO-1 Research Articles

Mechanobiological Mechanisms Involved in the Regualation of the Blood-Brain Barrier by Fluid Shear Force

To explore the mechanobiological mechanism of fluid shear force (FSF) on the protection, injury, and destruction of the structure and function of the blood-brain barrier (BBB) under normal physiological conditions, ischemic hypoperfusion, and postoperative hyperperfusion conditions. BBB is mainly composed of brain microvascular endothelial cells. Rat brain microvascular endothelial cells (rBMECs) were used as model cells to conduct the investigation. rBMECs were seeded at a density of 1×105 cells/cm2 and incubated for 48 h. FSF was applied to the rBMECs at 0.5, 2, and 20 dyn/cm2, respectively, simulating the stress BBB incurs under low perfusion, normal physiological conditions, and high FSF after bypass grafting when there is cerebral vascular stenosis. In addition, a rBMECs static culture group was set up as the control (no force was applied). Light microscope, scanning electron microscope (SEM), and laser confocal microscope (LSCM) were used to observe the changes in cell morphology and cytoskeleton. Transmission electron microscope (TEM) was used to observe the tight junctions. Immunofluorescence assay was performed to determine changes in the distribution of tight junction-associated proteins claudin-5, occludin, and ZO-1 and adherens junction-associated proteins VE-cadherin and PECAM-1. Western blot was performed to determine the expression levels of tight junction-associated proteins claudin-5, ZO-1, and JAM4, adherens junction-associated protein VE-cadherin, and key proteins in Rho GTPases signaling (Rac1, Cdc42, and RhoA) under FSF at different intensities. Microscopic observation showed that the cytoskeleton exhibited disorderly arrangement and irregular orientation under static culture and low shear force (0.5 dyn/cm2). Under normal physiological shear force (2 dyn/cm2), the cytoskeleton was rearranged in the orientation of the FSF and an effective tight junction structure was observed between cells. Under high shear force (20 dyn/cm2), the intercellular space was enlarged and no effective tight junction structure was observed. Immunofluorescence results showed that, under low shear force, the gap between the cells decreased, but there was also decreased distribution of tight junction-associated proteins and adherens junction-associated proteins at the intercellular junctions. Under normal physiological conditions, the cells were tightly connected and most of the tight junction-associated proteins were concentrated at the intercellular junctions. Under high shear force, the gap between the cells increased significantly and the tight junction and adherens junction structures were disrupted. According to the Western blot results, under low shear force, the expression levels of claudin-5, ZO-1, and VE-cadherin were significantly up-regulated compared with those of the control group (P<0.05). Under normal physiological shear force, claudin-5, ZO-1, JAM4, and VE-cadherin were highly expressed compared with those of the control group (P<0.05). Under high shear force, the expressions of claudin-5, ZO-1, JAM4, and VE-cadherin were significantly down-regulated compared with those of the normal physiological shear force group (P<0.05). Under normal physiological shear force, intercellular expressions of Rho GTPases proteins (Rac1, Cdc42, and RhoA) were up-regulated and were higher than those of the other experimental groups (P<0.05). The expressions of Rho GTPases under low and high shear forces were down-regulated compared with that of the normal physiological shear force group (P<0.05). Under normal physiological conditions, FSF helps maintain the integrity of the BBB structure, while low or high shear force can damage or destroy the BBB structure. The regulation of BBB by FSF is closely related to the expression and distribution of tight junction-associated proteins and adherens junction-associated proteins.

MiR-185-5p / ATG101 axis alleviated intestinal barrier damage in intestinal ischemia reperfusion through autophagy

ObjectiveIntestinal ischemia-reperfusion (II/R) is a common pathological injury in clinic, and the systemic inflammatory response it causes will lead to multiple organ damage and functional failure. miR-185-5p has been reported to be a regulator of inflammatory response and autophagy, but whether it participates in the regulation of autophagy in II/R is still unclear. Therefore, we aimed to explore the mechanism of miR-185-5p regulating intestinal barrier injury in (II/R). MethodsCaco-2 cells was induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to establish II/R model. The superior mesenteric artery of C57BL/6 mice was clamped for 45 min and then subjected to reperfusion for 4 h for the establishment of II/R mice model. miR-185-5p mimic, miR-185-5p inhibitor, pcDNA-autophagy-related 101 (ATG101) were respectively transfected into Caco-2 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to assess miR-185-5p expression. Western blot detected the level of ATG101 and tight junction-associated proteins ZO1, Occludin, E-cadherin, β-catenin, as well as autophagy markers ATG5, ATG12, LC3Ⅰ/Ⅱ, Beclin1 and SQSTM1. Transepithelial electrical resistance (TEER) values was detected by a resistance meter. FITC-Dextran was performed to measure cell permeability. 5-ethynyl-2’-deoxyuridine (EDU) staining measured cell proliferation. Transmission electron microscope was conducted to observe autophagosomes. Hematoxylin & eosin (H&E) staining observed the damage of mice intestinal. Immunohistochemistry (IHC) measured the percentage of ki67 positive cells. TdT-mediated dUTP nick-end labeling (TUNEL) assay assessed cell apoptosis in intestinal tissues of II/R. Dual-luciferase assay verified the targeting relationship between miR-185-5p and ATG101.Results miR-185-5p was overexpressed in OGD/R-induced Caco-2 cells and intestinal tissues of II/R mice. Knocking down miR-185-5p markedly promoted autophagy and TEER values, reduced cell permeability, and alleviated intestinal barrier damage. ATG101 was a target of miR-185-5p, and overexpression of ATG101 promoted autophagy and dampened OGD/R-induced intestinal barrier damage. Overexpression of miR-185-5p reversed the effect of overexpressed ATG101 on OGD/R-induced Caco-2 cells. ConclusionKnockdown of miR-185-5p enhanced autophagy and alleviated II/R intestinal barrier damage by targeting ATG101.

High expression of EphA2 led to secondary injury by destruction of BBB integrity though the ROCK pathway after diffuse axonal injury

Blood-brain barrier (BBB) disruption exacerbates diffuse axonal injury (DAI), but the underlying mechanisms are not fully understood. Inactivation or deletion of erythropoietin-producing hepatoma (EPH) receptor A2 (EphA2) attenuated BBB damage and promoted tight junction formation. In this study, we aimed to investigate the role of EphA2 in the protection of BBB integrity and the relevant mechanisms involved in a rat model of DAI. Blocking activation of the EphA receptor by EphA2-Fc ameliorated axonal injury, cell apoptosis, and glial activation, protected BBB integrity and increased expression of the tight junction-associated proteins ZO-1, claudin-5 and occludin-1. In vitro BBB models established by human brain microvascular endothelial cells (HBMECs) were subjected to oxygen deprivation (OGD). Treatment with EphrinA1, which activates EphA2, exacerbated the OGD-induced destruction of permeability and integrity of the BBB models by reducing the expression of tight junction-associated proteins. However, inhibition of Rho-associated coiled coil-containing protein kinases 1 and 2 (ROCK1 and 2) abrogated all of the effects of EphrinA1 on the BBB models in vitro. In conclusion, we provide evidence that EphA2 plays an important role in the destruction of BBB integrity by decreasing the expression of tight junction proteins through the ROCK pathway.

Mechanism of Fisetin Inhibiting Oxidative Stress to Alleviate the Damage of Blood Brain Barrier, Around the Intracerebral Hemorrhag Lesions

Background: The paper determined the brain water content, blood-brain barrier permeability, oxidative stress-related indicators, expressions of tight junction-associated proteins ZO-1 and Occludin, as well as expressions of ERK, p38, and JNK proteins in cerebral hemorrhage rats. The paper explored whether Fisetin reduced the damage of blood-brain barrier caused by cerebral hemorrhage through inhibiting oxidative stress, and investigated whether MAPK pathway is the main pathway for BBB damage caused by oxidative stress. Material and Methods: Rat ICH model was established. After 72 hours, mNSS score was used to measure the behavior of rats. The water content of brain tissue was determined by dry-wet weight method. Evans blue method was applied to measure the blood-brain barrier permeability. Oxidative stress index was detected by spectrophotometer. The expressions of ZO-1, Occludin, ERK, p38 and JNK proteins in rat brain tissue were determined by Western blot. Results: Fisetin significantly reduced the mNSS score, brain water content, bloodbrain barrier permeability, oxidative stress index and the expressions of ERK, p38 and JNK in rat ICH model, and also significantly increased the antioxidant index of rat ICH model, as well as the expressions of ZO-1 and Occludin. The effect is becoming more significant as the therapeutic dose is increased. Conclusion: Fisetin can inhibit the oxidative stress and alleviate the damage of BBB around the lesions of rat ICH model, thus alleviating secondary brain injury. In the rat ICH model, the expressions of ERK, p38 and JNK in the brain tissue around the lesion are increased and oxidized. The stress response is enhanced, and Fisetin inhibits oxidative stress and the expressions of ERK, p38, and JNK. MAPK is imperative in the process of oxidative stress-induced BBB damage in rat ICH model.

β-Defensin 129 Attenuates Bacterial Endotoxin-Induced Inflammation and Intestinal Epithelial Cell Apoptosis.

Defensins have attracted considerable research interest worldwide because of their potential to serve as a substitute for antibiotics. In this study, we characterized a novel porcine β-defensin (pBD129) and explored its role in alleviating bacterial endotoxin-induced inflammation and intestinal epithelium atrophy. The pBD129 gene was cloned and expressed in Escherichia coli. A recombinant pBD129 protein was also purified. To explore its role in alleviating the endotoxin-induced inflammation, mice, with or without lipopolysaccharide (LPS) challenge were treated by pBD129 at different doses. The recombinant pBD129 showed significant antimicrobial activities against the E. coli and Streptococcus with a minimal inhibitory concentration (MICs) of 32 μg/mL. Hemolytic assays showed that the pBD129 had no detrimental impact on cell viabilities. Interestingly, we found that pBD129 attenuated LPS-induced inflammatory responses by decreasing serum concentrations of inflammatory cytokines, such as the IL-1β, IL-6, and TNF-α (P < 0.05). Moreover, pBD129 elevated the intestinal villus height (P < 0.05) and enhanced the expression and localization of the major tight junction-associated protein ZO-1 in LPS-challenged mice. Additionally, pDB129 at a high dose significantly decreased serum diamine oxidase (DAO) concentration (P < 0.05) and reduced intestinal epithelium cell apoptosis (P < 0.05) in LPS-challenged mice. Importantly, pBD129 elevated the expression level of Bcl-2-associated death promoter (Bcl-2), but down-regulated the expression levels of apoptosis-related genes such as the B-cell lymphoma-2-associated X protein (Bax), BH3-interacting domain death agonist (Bid), cysteinyl aspartate-specific proteinase-3 (Caspase-3), and caspase-9 in the intestinal mucosa (P < 0.05). These results suggested a novel function of the mammalian defensins, and the anti-bacterial and anti-inflammatory properties of pBD129 may allow it a potential substitute for conventionally used antibiotics or drugs.

β-Defensin 129 Attenuates Bacterial Endotoxin-Induced Inflammation and Intestinal Epithelial Cell Apoptosis

Background: Defensins have attracted considerable research interest worldwide because of their potential to serve as a substitute for antibiotics. In this study, we characterized a novel porcine β-defensin (pBD129) and explored its role in alleviating bacterial endotoxin-induced inflammation and intestinal epithelium atrophy. Methods: The pBD129 gene was cloned and expressed in Escherichia coli. A recombinant pBD129 protein was purified. To explore its role in alleviating the endotoxin-induced inflammation, mice with or without lipopolysaccharide (LPS) challenge were treated by the pBD129 at different doses. Results: The recombinant pBD129 showed significant antimicrobial activities against the E. coli and Streptococcus with a minimal inhibitory concentration (MICs) of 32 μg/mL. Hemolytic assays showed that the pBD129 had no detrimental impact on cell viabilities. Interestingly, we found that pBD129 attenuated LPS-induced inflammatory responses by decreasing serum concentrations of inflammatory cytokines such as the IL-1β, IL-6, and TNF-α (P<0.05). Moreover, pBD129 elevated the intestinal villus height (P<0.05) and enhanced the expression and localization of the major tight junction-associated protein ZO-1 in LPS-challenged mice. Additionally, pDB129 at a high dose significantly decreased serum diamine oxidase (DAO) concentration (P<0.05) and reduced intestinal epithelium cell apoptosis (P<0.05) in LPS-challenged mice. Importantly, pBD129 elevated the expression level of Bcl-2-associated death promoter (Bcl-2), but down-regulated the expression levels of apoptosis-related genes such as the B-cell lymphoma-2-associated X protein (Bax), BH3-interacting domain death agonist (Bid), cysteinyl aspartate-specific proteinase-3 (Caspase-3), and caspase-9 in the intestinal mucosa (P<0.05). Conclusions: These results suggested a novel function of the mammalian defensins, and the anti-bacterial and anti-inflammatory properties of pBD129 may allow it a potential substitute for conventionally used antibiotics or drugs. Funding: This study was supported by the key Research and Development program of Sichuan Province (2018NZDZX0005) and the Youth Innovation teams of animal Feed Biotechnology of Sichuan Province (2016TD0028) Declaration of Interest: The authors confirm that this article content has no conflict of interest. Ethical Approval: The animal trial was approved by the Animal Welfare Committee of Sichuan Agricultural University (No. 20180718).

Gut microbiota translocation promotes autoimmune cholangitis

Gut microbiota and bacterial translocation have been implicated as significant contributors to mucosal immune responses and tolerance; alteration of microbial molecules, termed pathogen-associated molecular patterns (PAMP) and bacterial translocation are associated with immune pathology. However, the mechanisms by which dysregulated gut microbiota promotes autoimmunity is unclear. We have taken advantage of a well-characterized murine model of primary biliary cholangitis, dnTGFβRII mice, and an additional unique construct, toll-like receptor 2 (TLR2)-deficient dnTGFβRII mice coined dnTGFβRIITLR2−/− mice to investigate the influences of gut microbiota on autoimmune cholangitis. Firstly, we report that dnTGFβRII mice manifest altered composition of gut microbiota and that alteration of this gut microbiota by administration of antibiotics significantly alleviates T-cell-mediated infiltration and bile duct damage. Second, toll-like receptor 2 (TLR2)-deficient dnTGFβRII mice demonstrate significant exacerbation of autoimmune cholangitis when their epithelial barrier integrity was disrupted. Further, TLR2-deficiency mediates downregulated expression of tight junction-associated protein ZO-1 leading to increased gut permeability and bacterial translocation from gut to liver; use of antibiotics reduces microbiota translocation to liver and also decreases biliary pathology. In conclusion, our data demonstrates the important role of gut microbiota and bacterial translocation in the pathogenesis of murine autoimmune cholangitis.

Tight junctions negatively regulate mechanical forces applied to adherens junctions in vertebrate epithelial tissue.

Epithelia are layers of polarised cells tightly bound to each other by adhesive contacts. Epithelia act as barriers between an organism and its external environment. Understanding how epithelia maintain their essential integrity while remaining sufficiently plastic to allow events such as cytokinesis to take place is a key biological problem. In vertebrates, the remodelling and reinforcement of adherens junctions maintains epithelial integrity during cytokinesis. The involvement of tight junctions in cell division, however, has remained unexplored. Here, we examine the role of tight junctions during cytokinesis in the epithelium of the Xenopus laevis embryo. Depletion of the tight junction-associated proteins ZO-1 and GEF-H1 leads to altered cytokinesis duration and contractile ring geometry. Using a tension biosensor, we show that cytokinesis defects originate from misregulation of tensile forces applied to adherens junctions. Our results reveal that tight junctions regulate mechanical tension applied to adherens junctions, which in turn impacts cytokinesis.This article has an associated First Person interview with the first author of the paper.

Cytosolic TMEM88 promotes invasion and metastasis in lung cancer cells by binding DVLS.

Transmembrane protein 88 (TMEM88) is a transmembrane protein that plays a crucial role in regulating human stem cell differentiation and embryonic development. However, its expression and clinicopathologic significance in human neoplasms is unclear. In this study, the expression and subcellular localizations of TMEM88 were assessed in 214 cases of non-small cell lung cancer (NSCLC). Notably, TMEM88 was highly expressed in the cytosol of ∼60% NSCLC specimens examined. Higher expression of cytosolic TMEM88 in NSCLC correlated significantly with poor differentiation, high TNM stage, lymph node metastasis, and inferior survival. In NSCLC cells displaying membrane-localized TMEM88, we observed an inhibition of canonical Wnt signaling due to interactions of TMEM88 with the Wnt pathway factor Dishevelled (DVLS). In contrast, NSCLC cells with cytosol-localized TMEM88 lacked effects on Wnt signaling. Cytosolic interactions of TMEM88 and DVLS increased the expression of phosphorylated, active forms of p38, GSK3β (Thr390), and Snail, thereby reducing the expression of the tight junction-associated proteins ZO-1 and occludin, effects associated with enhanced invasive and metastatic cell characters. Importantly, attenuating the expression of cytosolic TMEM88 reduced metastatic prowess in xenograft models. Overall, our findings show how mislocalization of TMEM88 to the cytosol in NSCLC cells ablates its Wnt pathway regulatory properties, thereby promoting invasion and metastasis by activating the p38-GSK3β-Snail signaling pathway.

Low-Dose Endothelial Monocyte-Activating Polypeptide-II Increases Blood-Tumor Barrier Permeability by Activating the RhoA/ROCK/PI3K Signaling Pathway.

Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can increase blood-tumor barrier (BTB) permeability via both paracellular and transcellular pathways. In addition, we revealed that the RhoA/Rho kinase (ROCK) signaling pathway is involved in EMAP-II-induced BTB opening. This study further investigated the exact mechanisms by which the RhoA/ROCK signaling pathway affects EMAP-II-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II significantly activated phosphatidylinositol-3-kinase (PI3K) in rat brain microvascular endothelial cells (RBMECs) at 0.75h. Pretreatment with RhoA inhibitor C3 exoenzyme or ROCK inhibitor Y-27632 completely blocked EMAP-II-induced activation of PI3K. PKC-α/β inhibitor GÖ6976 pretreatment caused no change in EMAP-II-induced activation of PI3K. Besides, pretreatment with LY294002, a specific inhibitor of PI3K, did not affect EMAP-II-induced activation of PKC-α/β. Furthermore, LY294002 pretreatment significantly diminished EMAP-II-induced changes in BTB permeability, phosphorylation of myosin light chain and cofilin, expression and distribution of tight junction-associated protein ZO-1, and actin cytoskeleton arrangement in RBMECs. In summary, this study demonstrates that low-dose EMAP-II can increase BTB permeability by activating the RhoA/ROCK/PI3K signaling pathway.

Interaction of human trophoblast cells with gland-like endometrial spheroids: a model system for trophoblast invasion.

Do maternal endometrial epithelial cell (EEC) differentiation and polarity impact the invasive capacity of extravillous trophoblast (EVT) cells during early human implantation? In a three dimensional (3D) confrontation co-culture the invasiveness of the human trophoblast cell line AC-1M88 was inversely correlated with the degree of differentiation and polarization of human endometrial adenocarcinoma cell spheroids. In a previous study desmosomal and adherens junction proteins were shown to spread from a subapically restricted lateral position to the entire lateral membrane in human glandular EECs during the implantation window of the menstrual cycle. Whether this change in EEC junction localization has an impact on the interaction of EVT cells with glandular EECs during early human implantation is not known. A new 3D cell culture system was developed in order to mimic early implantation events in humans. As a model for the invasion of endometrial glands by EVT cells, spheroids of three differently differentiated and polarized endometrial adenocarcinoma cell lines were confronted with an EVT cell line in co-culture experiments. Three human adenocarcinoma EEC lines were chosen for this study because of their differences in differentiation and polarization: HEC-1-A, which is well differentiated and highly polarized, Ishikawa, which is well differentiated and moderately polarized, and RL95-2, which is moderately differentiated and poorly polarized. When the cell lines were grown in reconstituted basement membrane, they formed gland-like, multicellular spheroids. The degree of polarization within the different EEC spheroids was assessed by 3D confocal immunofluorescence microscopy detecting the basal membrane protein integrin α6, the apical tight junction-associated protein ZO-1 and the desmosomal plaque protein desmoplakin 1/2 (Dsp). Cells of the human EVT cell line AC-1M88, which is a fusion cell line of primary EVT cells and choriocarcinoma-derived JEG-3 cells, were added to the different EEC spheroids to examine their interaction. For the analyses of trophoblast-endometrial confrontation sites, HLA-G was used as a specific EVT cell marker. The endometrial HEC-1-A and Ishikawa cells formed gland-like structures in reconstituted basement membrane with apicobasal polarization towards their well-developed internal lumina, while most of the RL95-2 spheroids showed no lumen formation at all. The three EEC lines strongly differed in their apicobasal distribution pattern of Dsp. Ishikawa and HEC-1-A spheroids showed a subapical concentration of Dsp. In contrast, an equal distribution of Dsp was discerned along the entire lateral membranes in RL95-2 spheroids. In 3D confrontation co-cultures the highest invasiveness of AC-1M88 was observed in the poorly polarized RL95-2 spheroids. Human endometrial and trophoblast cell lines were used for this study because of ethical and legal restrictions for implantation studies with human blastocysts and because of limited access to primary human endometrial cells. The presented 3D cell culture system can be used to investigate the contribution of epithelial junctions to trophoblast-endometrial interactions. The identified impact of endometrial differentiation and polarity on the invasiveness of EVT cells improves our understanding of the relevance of endometrial receptivity for early implantation and may contribute to higher success rates in assisted reproductive technology. This work was supported by Grant 146/14, 'START-Program', Medical Faculty, RWTH Aachen University, to V.U.B., by Grant Lec_16_12, 'RWTH Lecturer Award', RWTH Aachen University to I.C.-L. and by the German Research Council (Grant LE 566-20-1). The authors declare no conflict of interest.

Role of cAMP-dependent protein kinase A activity in low-dose endothelial monocyte-activating polypeptide-II-induced opening of blood-tumor barrier.

Our previous studies demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can selectively increase the permeability of blood-tumor barrier (BTB). In addition, low-dose EMAP-II significantly decreases the cyclic adenosine monophosphate (cAMP) concentration and the protein kinase A (PKA) expression level in tumor tissues in the rat C6 glioma model. In this study, an in vitro BTB model was used to investigate the potential role of cAMP/PKA signaling cascade in EMAP-II-induced BTB hyperpermeability. Our data revealed that low-dose EMAP-II (0.05nM) induced a significant decrease in total intracellular cAMP concentration and PKA activity in rat brain microvascular endothelial cells (RBMECs). Pretreatment with forskolin to increase intracellular cAMP nearly completely blocked the EMAP-II-induced decrease in transendothelial electric resistance and increase in horseradish peroxidase flux across the BTB. Similar pretreatment completely prevented the EMAP-II-induced changes in RhoA/Rho kinase activity, expression and distribution of tight junction-associated protein ZO-1, and myosin light chain phosphorylation, as well as actin cytoskeleton arrangement in RBMECs. Pretreatment with 6Bnz-cAMP to activate PKA significantly attenuated these EMAP-II-induced alterations in RBMECs. In summary, our present study demonstrates that the cAMP/PKA signaling cascade works as a crucial signaling pathway in EMAP-II-induced BTB hyperpermeability.

Effect of vitamin D receptor knockout on cornea epithelium wound healing and tight junctions.

Our laboratory previously determined that vitamin D3, the vitamin D receptor (VDR), and 1α hydroxylase are present and active in the eye. In this study, we examined the effects of VDR knockout on wound healing, the tight junction-associated proteins occludin and ZO-1, and tight junction numbers in mouse corneas. Epithelial wounds (2-mm) were made with an agar brush on 4-week-old and 10-week-old wild-type, heterozygous, and VDR knockout mouse corneas. Mice were on a normal or high lactose, Ca(2+), and PO₄(-) diet. Wound-healing area was measured over time. Real-time PCR was used to quantify occludin and ZO-1 message expression. Western blot was used for protein expression. Transmission electron microscopy was used to examine corneal epithelium and endothelium tight junctions. Immunofluorescence was used to examine epithelial ZO-1 distribution. Results showed a decreased healing rate in 10-week-old VDR knockout mice compared with wild-types. Vitamin D receptor knockout mice on the special diet had no difference in healing rate compared with wild-types. Real-time PCR showed decreased expression of occludin and ZO-1 in 10-week-old VDR knockout mice compared with wild-types. Western blot of 10-week-old knockout mouse corneas showed decreased occludin expression compared with wild-types. Transmission electron microscopy showed a significant difference in tight junction numbers in VDR knockouts versus wild-types. Immunofluorescence showed a change in ZO-1 distribution among genotypes. Vitamin D receptor knockout affects mouse corneal epithelium wound healing and tight junction integrity.

Restoration of MAGI-1 expression in human papillomavirus-positive tumor cells induces cell growth arrest and apoptosis.

The cancer-causing high-risk human papillomavirus (HPV) E6 oncoproteins target a number of cellular proteins that contain PDZ domains. However, the role of many of these interactions in either the HPV life cycle or in HPV-induced malignancy remains to be defined. Previous studies had shown that MAGI-1 was one of the most strongly bound PDZ domain-containing substrates of E6, and one consequence of this interaction appeared to facilitate the perturbation of tight junctions (TJs) by E6. In this study, we describe the generation of a mutation, K499E, within the MAGI-1 PDZ1 domain, which is resistant to E6 targeting. This mutant allows restoration of MAGI-1 expression in HPV-positive cells and defines additional activities of MAGI-1 that are overcome as a consequence of the association with E6. The reexpression of MAGI-1 in HPV-positive cells results in an increased recruitment of ZO-1 and PAR3 to sites of cell-cell contact, repression of cell proliferation, and induction of apoptosis. While the K499E mutation does not significantly affect these intrinsic activities of MAGI-1 in HPV-negative cells, its resistance to E6 targeting in an HPV-positive setting results in more cells expressing the mutant MAGI-1 than the wild-type MAGI-1, with a corresponding increase in TJ assembly, induction of apoptosis, and reduction in cell proliferation. These studies provide compelling evidence of a direct role for the perturbation of MAGI-1 function by E6 in the HPV life cycle and in HPV-induced malignancy. It is clear that the targeting of PDZ-containing substrates by E6 is important for the normal viral life cycle and for the progression to malignancy. Nevertheless, which of these PDZ domain-containing proteins is relevant for HPV pathology is still elusive. In a previous study, we provided evidence that MAGI-1 is a sensitive proteolytic substrate for both the HPV-16 and HPV-18 E6 oncoproteins; however, the biological consequences associated with loss of MAGI-1 expression in HPV-positive cervical cancer cells are still poorly understood. Using a mutant MAGI-1, resistant to E6-mediated degradation, we show that its expression in cervical cancer cells promotes membrane recruitment of the tight junction-associated proteins ZO-1 and PAR3, represses cell proliferation, and promotes apoptosis. These findings suggest that E6-mediated inhibition of MAGI-1 function contributes to HPV pathology by perturbing tight junction assembly with concomitant stimulation of proliferation and inhibition of apoptosis.

Effects of Poly (ADP-ribose) Polymerase Inhibitor 3-Aminobenzamide on Blood–Brain Barrier and Dopaminergic Neurons of Rats with Lipopolysaccharide-Induced Parkinson’s Disease

Neuro-inflammation and dysfunction of blood-brain barrier play an important role in the occurrence, development, and neuronal degeneration of Parkinson's disease (PD). Studies have demonstrated that a variety of cytokines such as TNF-α and IL-1β destroy the structure and function of blood-brain barrier. The damage to blood-brain barrier results in death of dopaminergic neurons, while protection of blood-brain barrier slows down the progression of PD. Also, it has been shown that activation of poly (ADP-ribose) polymerase (PARP) plays an important role in causing damage to blood-brain barrier. In addition, the PARP inhibitor 3-AB has been shown to protect blood-brain barrier from damage and has neuroprotective effects. In this study, using a lipopolysaccharide (LPS)-induced PD rat model, we investigated whether 3-AB protects blood-brain barrier and dopaminergic neurons from functional damage. LPS significantly increased Evans blue content in the substantia nigra which peaked at 12 h, while administration of 3-AB significantly inhibited the LPS-induced increase in Evans blue content and also significantly increased the expression of the tight junction-associated proteins claudin-5, occludin and ZO-1. 3-AB also increased the number of tyrosine hydroxylase positive cells and reduced the IL-1β and TNF-α content significantly. According to western blot analysis, 3-AB significantly reduced the p-ERK1/2 expression, while the expression of p-p38MAPK increased. These results suggest that 3-AB protects the blood-brain barrier from functional damage in an LPS-induced PD rat model and dopaminergic neurons are protected from degeneration by upregulation of tight junction-associated proteins. These protective effects of 3-AB may be related to modulation of the ERK1/2 pathway.

CRM197-Induced Blood–Brain Barrier Permeability Increase is Mediated by Upregulation of Caveolin-1 Protein

Cross-reacting material 197 (CRM197), a non-toxin mutant of diphtheria toxin, could act as a diphtheria toxin receptor-specific carrier protein for the targeted delivery of macromolecular substances across the blood-brain barrier (BBB) in vitro. This study was performed to investigate the effects and mechanisms of CRM197 on the permeability of BBB in guinea pigs. Data from the Evans blue extravasation showed that the BBB permeability significantly increased after CRM197 injection in a dose-dependent manner. Transmission electron microscopy indicated CRM197 could induce increased pinocytotic vesicles and vacuoles in brain microvascular endothelial cells. Immunohistochemistry and western blot assay revealed that CRM197 enhanced caveolin-1 protein expression in brain microvessels. The caveolin-1 protein in the membrane fraction of microvessels began to upregulate at 5 min and reached the peak at 10 min after CRM197 treatment, associated by diminished expression of several tight junction-associated proteins ZO-1, occludin, and claudin-5. Thus, our results indicate that the in vivo targeting CRM197 leads to increased BBB permeability via upregulation of caveolin-1 protein, increased pinocytotic vesicles, and redistribution of tight junction-associated proteins in brain microvessels. CRM197 may have a potential application for targeted drug delivery across the BBB.

Selective Translocation of the Bordetella pertussis Adenylate Cyclase Toxin across the Basolateral Membranes of Polarized Epithelial Cells

The catalytic domain of Bordetella pertussis adenylate cyclase toxin (ACT) translocates directly across the plasma membrane of mammalian cells to induce toxicity by the production of cAMP. Here, we use electrophysiology to examine the translocation of toxin into polarized epithelial cells that model the mucosal surfaces of the host. We find that both polarized T84 cell monolayers and human airway epithelial cultures respond to nanomolar concentrations of ACT when applied to basolateral membranes, with little or no response to toxin applied apically. The induction of toxicity is rapid and fully explained by increases in intracellular cAMP, consistent with toxin translocation directly across the basolateral membrane. Intoxication of T84 cells occurs in the absence of CD11b/CD18 or evidence of another specific membrane receptor, and it is not dependent on post-translational acylation of the toxin or on host cell membrane potential, both previously reported to be required for toxin action. Thus, elements of the basolateral membrane render epithelial cells highly sensitive to the entry of ACT in the absence of a specific receptor for toxin binding.

Effects of oral Lactobacillus plantarum on hepatocyte tight junction structure and function in rats with obstructive jaundice

Surgery and infection are prominent risk factors for the development of obstructive cholestasis which in turn is associated with failure of the liver barrier. We studied the effects of oral Lactobacillus plantarum (LP) supplementation on endotoxemia, oxidative stress, apoptosis, and tight junctions of hepatocytes in an experimental model of obstructive jaundice. Fifty male Wistar rats were randomly divided into five groups of 10 each: group I, sham-operated; group II, ligation and division of the common bile duct (BDL); group III, BLD followed by oral LP treatment; group IV, BDL followed by internal biliary drainage (IBD); group V, BDL followed by IBD and oral LP treatment. Hepatocyte apoptosis, plasma reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, and portal blood endotoxin levels were measured and changes in tight junction-associated proteins occludin, claudin-1, claudin-4, and ZO-1 were observed. Compared to the sham-operated group I, significant increases in endotoxemia, apoptosis, and GSSG were observed in group II and significant decreases were observed in group V. Tight junctions were destroyed in group II animals but were not in animals treated with oral LP (groups III and V). An increase in occludin, claudin-1, claudin-4, and ZO-1 mRNA and protein levels were detected in livers in LP-treated animals (group V) compared with group II levels. Oral LP treatment of rats with obstructive jaundice assisted in the return of active hepatic barrier function. These results may lead to treatments to prevent the deleterious effects of obstructive jaundice.

Association of cellular localization of glycogen synthase kinase 3β in the digestive tract with cancer development

Glycogen synthase kinase 3beta (GSK-3beta) is a multifunctional serine/threonine kinase involved in several signaling pathways. Recently, we reported the polarized localization of GSK-3beta on the apical membrane of normal colon epithelium. To investigate the functions of this molecule, we studied stomach and colorectal cancer tissues. In normal simple columnar epithelium, GSK-3beta was localized with tight junction-associated protein ZO-1 in a single line at the apical cell border. GSK-3beta and ZO-1 were localized in the apical regions of tubular adenocarcinoma, similar to their localization in normal epithelium; however, their localization was different at the invasive front of the cancer and was found to be associated with lymphatic invasion. In signet-ring cell carcinoma of the stomach, the expression of these proteins was reduced and dot-like expression was observed in each cell of the signet-ring cell carcinoma. We speculated that GSK-3beta is involved in glandular structure formation and that the non-apical localization of membrane-localized GSK-3beta in tubular adenocarcinoma is associated with cancer development.

Reg I protein in healthy and seminoma human testis.

Regenerating gene (Reg), encodes a secretory protein with growth and differentiation stimulating effects mostly in digestive tissues. Overexpression of Reg proteins and specifically of Reg I, one member of the Reg family, is associated with several human diseases and cancers. In the present study we analyzed the expression of Reg I in normal rodent and human testes where germ cells normally proliferate and differentiate into spermatozoa, and in seminoma testis, the most common cancer of young men. Western blot analyses demonstrated the presence of a specific band at 19 kDa in human and rodent testis extracts. Immunofluorescence and deconvolution microscopy demonstrated that Reg I was present within the seminiferous tubules in both Sertoli and germ cells. By using a Sertoli cell line we demonstrated that Reg I was localized at the plasma membrane even in the absence of contact between neighboring cells and appeared before the tight junction associated protein ZO-1 was revealed at this location. Reg I was strongly expressed in human seminoma testis tissue and in a human tumor germ cell line where the immunoreactive signal was mainly detected at the plasma membrane level. These data showing for the first time the weak presence of Reg I in the normal testis and its strong expression in the testis cancer suggest a potential role of Reg I in normal and neoplastic germ cell proliferation.