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Sample records for claudin 1 3

  1. Rab3Gap1 mediates exocytosis of Claudin-1 and tight junction formation during epidermal barrier acquisition

    PubMed Central

    Youssef, G.; Gerner, L.; Naeem, A.S.; Ralph, O.; Ono, M.; O’Neill, C.A.; O’Shaughnessy, R.F.L.

    2013-01-01

    Epidermal barrier acquisition during late murine gestation is accompanied by an increase in Akt kinase activity and cJun dephosphorlyation. The latter is directed by the Ppp2r2a regulatory subunit of the Pp2a phosphatase. This was accompanied by a change of Claudin-1 localisation to the cell surface and interaction between Occludin and Claudin-1 which are thought to be required for tight junction formation. The aim of this study was to determine the nature of the barrier defect caused by the loss of AKT/Ppp2r2a function. There was a paracellular barrier defect in rat epidermal keratinocytes expressing a Ppp2r2a siRNA. In Ppp2r2a knockdown cells, Claudin-1 was located to the cytoplasm and its expression was increased. Inhibiting cJun phosphorylation restored barrier function and plasma membrane localisation of Claudin-1. Expression of the Rab3 GTPase activating protein, Rab3Gap1, was restored in Ppp2r2a siRNA cells when cJun phosphorylation was inhibited. During normal mouse epidermal development, Claudin-1 plasma membrane localisation and Rab3Gap1 cell surface expression were co-incident with Akt activation in mouse epidermis, strongly suggesting a role of Rab3Gap1 in epidermal barrier acquisition. Supporting this hypothesis, siRNA knockdown of Rab3Gap1 prevented plasma membrane Claudin-1 expression and the formation of a barrier competent epithelium. Replacing Rab3Gap1 in Ppp2r2a knockdown cells was sufficient to rescue Claudin-1 transport to the cell surface. Therefore these data suggest Rab3Gap1 mediated exocytosis of Claudin-1 is an important component of epidermal barrier acquisition during epidermal development. PMID:23685254

  2. Claudin-3 Overexpression Increases the Malignant Potential of Colorectal Cancer Cells: Roles of ERK1/2 and PI3K-Akt as Modulators of EGFR signaling

    PubMed Central

    de Souza, Waldemir F.; Fortunato-Miranda, Natalia; Robbs, Bruno K.; de Araujo, Wallace M.; de-Freitas-Junior, Julio C.; Bastos, Lilian G.; Viola, João P. B.; Morgado-Díaz, José A.

    2013-01-01

    The altered expressions of claudin proteins have been reported during the tumorigenesis of colorectal cancer. However, the molecular mechanisms that regulate these events in this cancer type are poorly understood. Here, we report that epidermal growth factor (EGF) increases the expression of claudin-3 in human colorectal adenocarcinoma HT-29 cells. This increase was related to increased cell migration and the formation of anchorage-dependent and anchorage-independent colonies. We further showed that the ERK1/2 and PI3K-Akt pathways were involved in the regulation of these effects because specific pharmacological inhibition blocked these events. Genetic manipulation of claudin-1 and claudin-3 in HT-29 cells showed that the overexpression of claudin-1 resulted in decreased cell migration; however, migration was not altered in cells that overexpressed claudin-3. Furthermore, the overexpression of claudin-3, but not that of claudin-1, increased the tight junction-related paracellular flux of macromolecules. Additionally, an increased formation of anchorage-dependent and anchorage-independent colonies were observed in cells that overexpressed claudin-3, while no such changes were observed when claudin-1 was overexpressed. Finally, claudin-3 silencing alone despite induce increase proliferation, and the formation of anchoragedependent and -independent colonies, it was able to prevent the EGF-induced increased malignant potential. In conclusion, our results show a novel role for claudin-3 overexpression in promoting the malignant potential of colorectal cancer cells, which is potentially regulated by the EGF-activated ERK1/2 and PI3K-Akt pathways. PMID:24069372

  3. Sequential biphasic changes in claudin1 and claudin4 expression are correlated to colorectal cancer progression and liver metastasis

    PubMed Central

    Georges, Rania; Bergmann, Frank; Hamdi, Hadjar; Zepp, Michael; Eyol, Ergül; Hielscher, Thomas; Berger, Martin R; Adwan, Hassan

    2012-01-01

    Abstract Terminal progression of colorectal cancer (CRC) culminates in liver metastasis. To identify genes that are involved in the metastatic phenotype, cDNA microarrays were used to analyse mRNA expression profiles of colorectal carcinoma (CC)531 rat colon adenocarcinoma cells for changes related to their homing into the liver. Briefly, CC531 cells were intraportally implanted into the liver of Wag-Rij rats and re-isolated after 3, 6, 9, 14 and 21 days. Compared to control CC531 cells, claudin1 and claudin4 were among the ≥8-fold initially down-regulated genes. The co-culture of tumour cells with isolated rat hepatocytes and Kupffer cells did not induce down-regulation of either claudin1 or 4. When the environment effective on circulating tumour cells was simulated by cell culture conditions favouring their adhesion, only claudin4 showed augmented expression. Knockdown of claudin1 and claudin4 mediated by small interfering RNA caused significantly increased migration and decreased clonogenic growth of tumour cells (P < 0.05), but had no effect on their proliferation. These experimental results were paralleled by increased claudin1 and claudin4 expression in human CRC samples in Union for International Cancer Control (UICC) stages I–III, as evaluated by real-time PCR. Increased claudin4 levels were correlated with significantly reduced overall survival (log-rank test, P= 0.018). Further, significantly (P < 0.05) reduced expression of claudin1 and claudin4 was observed in stage IV and liver metastasis by immunohistochemistry. In conclusion, sequential biphasic changes in claudin1 and claudin4 expression occur during the homing of rat CC531 CRC cells to the liver. This modulation is reflected by significant changes in claudin expression in human primary and metastatic CRC. PMID:21388515

  4. Claudin-1, -2 and -3 Are Selectively Expressed in the Epithelia of the Choroid Plexus of the Mouse from Early Development and into Adulthood While Claudin-5 is Restricted to Endothelial Cells.

    PubMed

    Steinemann, Alexandra; Galm, Isabel; Chip, Sophorn; Nitsch, Cordula; Maly, Ireneusz Piotr

    2016-01-01

    A primary function of epithelial and endothelial monolayers is the formation of barriers that separate tissues into functional compartments. Tight junctions (TJs) seal the intercellular space between the single cells of a monolayer. TJs thus contribute importantly to the homeostasis of the cerebrospinal fluid as they help in maintaining the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (CSF). The composition of TJs differs by its localization as well as the stage of development according to its respective function. Claudin-3 is typically present in the epithelia and has been claimed to be a constituent of the BBB. It is, however, notoriously difficult to demonstrate its expression in endothelial cells of the brain vasculature at the morphological level by means of immunohistochemical techniques. Using an improved fixation strategy (4% paraformaldehyde at pH 11, in the presence of EDTA) and the sensitive alkaline phosphatase as a detection system, we show that claudin-3 is present in mouse epithelia from embryonic day 14 onwards. In brain, it is restricted to the anlage of choroid plexus in the ventricles, together with claudin-1 and -2. In adult mice, it is clearly delineating the epithelium of the choroid plexus in the lateral and fourth ventricles. In contrast, in cerebral blood vessels claudin-3 as well as claudin-1 and -2 are absent in cerebral blood vessels during all developmental stages up to adulthood. Rather, the BBB is characterized by the presence of claudin-5, ZO-1 and occludin. Thus, in mice claudin-3 is an important constituent of TJ in the embryonic and in the adult choroid plexus. PMID:26941614

  5. Claudin-1, -2 and -3 Are Selectively Expressed in the Epithelia of the Choroid Plexus of the Mouse from Early Development and into Adulthood While Claudin-5 is Restricted to Endothelial Cells

    PubMed Central

    Steinemann, Alexandra; Galm, Isabel; Chip, Sophorn; Nitsch, Cordula; Maly, Ireneusz Piotr

    2016-01-01

    A primary function of epithelial and endothelial monolayers is the formation of barriers that separate tissues into functional compartments. Tight junctions (TJs) seal the intercellular space between the single cells of a monolayer. TJs thus contribute importantly to the homeostasis of the cerebrospinal fluid as they help in maintaining the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (CSF). The composition of TJs differs by its localization as well as the stage of development according to its respective function. Claudin-3 is typically present in the epithelia and has been claimed to be a constituent of the BBB. It is, however, notoriously difficult to demonstrate its expression in endothelial cells of the brain vasculature at the morphological level by means of immunohistochemical techniques. Using an improved fixation strategy (4% paraformaldehyde at pH 11, in the presence of EDTA) and the sensitive alkaline phosphatase as a detection system, we show that claudin-3 is present in mouse epithelia from embryonic day 14 onwards. In brain, it is restricted to the anlage of choroid plexus in the ventricles, together with claudin-1 and -2. In adult mice, it is clearly delineating the epithelium of the choroid plexus in the lateral and fourth ventricles. In contrast, in cerebral blood vessels claudin-3 as well as claudin-1 and -2 are absent in cerebral blood vessels during all developmental stages up to adulthood. Rather, the BBB is characterized by the presence of claudin-5, ZO-1 and occludin. Thus, in mice claudin-3 is an important constituent of TJ in the embryonic and in the adult choroid plexus. PMID:26941614

  6. Claudin-1, -3, -4 and -7 gene expression analyses in canine prostate carcinoma and mammary tissue derived cell lines.

    PubMed

    Hammer, S C; Nagel, S; Junginger, J; Hewicker-Trautwein, M; Wagner, S; Heisterkamp, A; Ngezahayo, A; Nolte, I; Murua Escobar, H

    2016-01-01

    Claudins (CLDNs) are transmembrane proteins localised in the cell membrane of epithelial cells composing a structural and functional component of the tight junction protein complexes. In canine tumors deregulations of the CLDN expression patterns were described immunohistochemically. Targeting of claudin proteins has further been evaluated to establish novel therapeutic approaches by directed claudin binding. Precondition for the development of claudin targeting approaches in canine cells is the possibility to characterise claudin expression specifically and the availability of claudin positive cell lines. Herein PCR/qPCR assays were established allowing a rapid qualitative and quantitative characterisation of CLDN-1, -3, -4 and -7 gene expression in canine cell lines and tissues. Further commercially available antibodies were used to verify CLDN gene expression on protein level by Western blots. The developed assays were used to analyse six canine cell lines derived from mammary and prostate tissue for their CLDN-1, -3, -4 and -7 expressions. The canine cell line DT08/40 (prostate transitional cell carcinoma) was used for the establishment of specific CLDNs -1, -3, -4 and -7PCR/qPCR. The designed assays were verified by amplicon cloning and sequencing. Gene expressions were verified on protein level by Western blot. Additionally further cell lines were analysed for their CLDN-1, -3, -4 and -7 expression on mRNA and protein level (mammary derived cell lines: MTH53A (non-neoplastic), ZMTH3 (adenoma), MTH52C (carcinoma); prostate derived cell lines: DT08/46 and CT1258 (both adenocarcinoma).The screened cell lines showed expression for the CLDNs as follows: DT08/46 and DT08/40: CLDN-1, -3, -4 and -7 positive; CT1258: CLDN-1, -3, -4 and -7 negative; ZMTH3 and MTH52C: CLDN-1 and -7 positive, CLDN-3 and -4 negative; MTH53A: CLDN-1, -3 and -4 negative, CLDN-7 positive. Western blot analyses reflect the detected CLDN-1, -3, -4 and -7 expressions in the analysed cell

  7. Claudin 1 in Breast Cancer: New Insights

    PubMed Central

    Zhou, Bowen; Moodie, Amanda; Blanchard, Anne A. A.; Leygue, Etienne; Myal, Yvonne

    2015-01-01

    Claudin 1 is a small transmembrane protein responsible for maintaining the barrier function that exists between epithelial cells. A tight junction protein that regulates the paracellular transport of small ions across adjacent cells, claudin 1 maintains cellular polarity and plays a major role in cell-cell communication and epithelial cell homeostasis. Long considered to be a putative tumor suppressor in human breast cancer, new studies suggest a role much more complex. While most invasive breast cancers exhibit a down regulation or absence of claudin 1, some aggressive subtypes that exhibit high claudin 1 levels have now been described. Furthermore, a causal role for claudin 1 in breast cancer progression has recently been demonstrated in some breast cancer cell lines. In this review we highlight new insights into the role of claudin 1 in breast cancer, including its involvement in collective migration and epithelial mesenchymal transition (EMT). PMID:26633531

  8. Claudin 1 in Breast Cancer: New Insights.

    PubMed

    Zhou, Bowen; Moodie, Amanda; Blanchard, Anne A A; Leygue, Etienne; Myal, Yvonne

    2015-01-01

    Claudin 1 is a small transmembrane protein responsible for maintaining the barrier function that exists between epithelial cells. A tight junction protein that regulates the paracellular transport of small ions across adjacent cells, claudin 1 maintains cellular polarity and plays a major role in cell-cell communication and epithelial cell homeostasis. Long considered to be a putative tumor suppressor in human breast cancer, new studies suggest a role much more complex. While most invasive breast cancers exhibit a down regulation or absence of claudin 1, some aggressive subtypes that exhibit high claudin 1 levels have now been described. Furthermore, a causal role for claudin 1 in breast cancer progression has recently been demonstrated in some breast cancer cell lines. In this review we highlight new insights into the role of claudin 1 in breast cancer, including its involvement in collective migration and epithelial mesenchymal transition (EMT). PMID:26633531

  9. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    SciTech Connect

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.; Parkos, Charles A.; Nusrat, Asma

    2010-07-02

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

  10. Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions

    PubMed Central

    Schlingmann, Barbara; Overgaard, Christian E.; Molina, Samuel A.; Lynn, K. Sabrina; Mitchell, Leslie A.; Dorsainvil White, StevenClaude; Mattheyses, Alexa L.; Guidot, David M.; Capaldo, Christopher T.; Koval, Michael

    2016-01-01

    Claudins are tetraspan transmembrane tight-junction proteins that regulate epithelial barriers. In the distal airspaces of the lung, alveolar epithelial tight junctions are crucial to regulate airspace fluid. Chronic alcohol abuse weakens alveolar tight junctions, priming the lung for acute respiratory distress syndrome, a frequently lethal condition caused by airspace flooding. Here we demonstrate that in response to alcohol, increased claudin-5 paradoxically accompanies an increase in paracellular leak and rearrangement of alveolar tight junctions. Claudin-5 is necessary and sufficient to diminish alveolar epithelial barrier function by impairing the ability of claudin-18 to interact with a scaffold protein, zonula occludens 1 (ZO-1), demonstrating that one claudin affects the ability of another claudin to interact with the tight-junction scaffold. Critically, a claudin-5 peptide mimetic reverses the deleterious effects of alcohol on alveolar barrier function. Thus, claudin controlled claudin-scaffold protein interactions are a novel target to regulate tight-junction permeability. PMID:27452368

  11. Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions.

    PubMed

    Schlingmann, Barbara; Overgaard, Christian E; Molina, Samuel A; Lynn, K Sabrina; Mitchell, Leslie A; Dorsainvil White, StevenClaude; Mattheyses, Alexa L; Guidot, David M; Capaldo, Christopher T; Koval, Michael

    2016-01-01

    Claudins are tetraspan transmembrane tight-junction proteins that regulate epithelial barriers. In the distal airspaces of the lung, alveolar epithelial tight junctions are crucial to regulate airspace fluid. Chronic alcohol abuse weakens alveolar tight junctions, priming the lung for acute respiratory distress syndrome, a frequently lethal condition caused by airspace flooding. Here we demonstrate that in response to alcohol, increased claudin-5 paradoxically accompanies an increase in paracellular leak and rearrangement of alveolar tight junctions. Claudin-5 is necessary and sufficient to diminish alveolar epithelial barrier function by impairing the ability of claudin-18 to interact with a scaffold protein, zonula occludens 1 (ZO-1), demonstrating that one claudin affects the ability of another claudin to interact with the tight-junction scaffold. Critically, a claudin-5 peptide mimetic reverses the deleterious effects of alcohol on alveolar barrier function. Thus, claudin controlled claudin-scaffold protein interactions are a novel target to regulate tight-junction permeability. PMID:27452368

  12. Evaluation of a novel human IgG1 anti-claudin3 antibody that specifically recognizes its aberrantly localized antigen in ovarian cancer cells and that is suitable for selective drug delivery

    PubMed Central

    Romani, Chiara; Cocco, Emiliano; Bignotti, Eliana; Moratto, Daniele; Bugatti, Antonella; Todeschini, Paola; Bandiera, Elisabetta; Tassi, Renata; Zanotti, Laura; Pecorelli, Sergio; Sartori, Enrico; Odicino, Franco E.; de Marco, Ario; Davide Santin, Alessandro; Ravaggi, Antonella; Mitola, Stefania

    2015-01-01

    Membrane protein claudin3 has been recently suggested as a marker for biologically aggressive tumors and a possible target for the therapeutic delivery of active anti-cancer compounds. Claudin3-binding molecules such as the Clostridium perfringens enterotoxin (CPE), CPE-related molecules, and murine and chimeric antibodies have shown promising antitumor efficacy in preclinical oncological settings. We first engineered a fully human anti-claudin3 IgG1 antibody (IgGH6) by fusing the human IgG1 Fc-domain to the anti-claudin3 scFvH6 previously isolated from a pre-immune phage display library. The construct was expressed in mammalian cells and specifically targeted claudin3 endogenously expressed on the surface of different human ovarian cancer cell lines. No detectable cross-reactivity with other homologous claudins was observed. The epitope recognized by IgGH6 is located within the minor extracellular domain of claudin3 and becomes accessible only in tumor cells characterized by incomplete junction formation. Confocal microscopy experiments demonstrated that IgGH6 was actively internalized in tumor cells after binding to native claudin3 and co-localized, likely within intracellular vesicles, with the C-CPE peptide. Preliminary results indicate that IgGH6 accumulated in vivo in free claudin3 ovarian carcinoma xenografts. For its selective uptake in tumor cells and its human nature, IgGH6 represents a valuable candidate for antibody-drug conjugate therapeutic applications in ovarian cancer patients. PMID:26416446

  13. Claudin-18 inhibits cell proliferation and motility mediated by inhibition of phosphorylation of PDK1 and Akt in human lung adenocarcinoma A549 cells.

    PubMed

    Shimobaba, Shun; Taga, Saeko; Akizuki, Risa; Hichino, Asami; Endo, Satoshi; Matsunaga, Toshiyuki; Watanabe, Ryo; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Sugatani, Junko; Ikari, Akira

    2016-06-01

    Abnormal expression of claudin subtypes has been reported in various cancers. However, the pathological role of each claudin has not been clarified in detail. Claudin-18 was absent in human non-small cell and small cell lung cancers, although it is expressed in normal lung tissues. Here, we examined the effect of claudin-18 expression on the expression of junctional proteins, cell proliferation, and cell motility using human lung adenocarcinoma A549 cells. Real-time PCR and western blotting showed that exogenous expression of claudin-18 had no effect on the expression of junctional proteins including claudin-1, zonula occludens-1 (ZO-1), occludin, and E-cadherin. Claudin-18 was mainly distributed in cell-cell contact areas concomitant with ZO-1. Cell proliferation was significantly decreased at 48 and 72h after seeding of claudin 18-expressing cells. Claudin-18 suppressed cell motility, whereas it increased cell death in anoikis. Claudin-18 decreased phosphorylated (p)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and p-Akt levels without affecting p-epidermal growth factor receptor and p-phosphatidylinositol-3 kinase (PI3K) levels. Furthermore, claudin-18 was bound with PDK1 and suppressed the nuclear localization of PDK1. We suggest that claudin-18 suppresses the abnormal proliferation and motility of lung epithelial cells mediated by inhibition of the PI3K/PDK1/Akt signaling pathway. PMID:26919807

  14. Clinical significance of serum claudin-1 levels in melanoma patients.

    PubMed

    Tas, Faruk; Bilgin, Elif; Erturk, Kayhan; Duranyildiz, Derya

    2016-08-01

    Claudins are the most important structural and functional components of tight-junction integral membrane proteins. They play roles in major cellular functions including growth and adhesion and are responsible for regulating the paracellular transport of molecules. The objective of this study was to determine the clinical significance of the serum levels of claudin-1, an oldest and important member of the claudin family, in melanoma patients. A total of 98 patients with a pathologically confirmed melanoma were enrolled into this study. Serum claudin-1 concentrations were determined by the solid-phase sandwich enzyme-linked immunosorbent assay method. Age-matched and sex-matched 43 healthy controls were included in the analysis. The median age at diagnosis was 51 years, ranging from 16 to 85 years. The majority of the patients were male (61%) and had axial localized (54%) and metastatic disease (61%). Moreover, most of the patients with metastatic disease had M1c (73%). The baseline serum claudin-1 levels of the melanoma patients were significantly lower than those of control subjects (median values 9.17 vs. 13.82 ng/ml, respectively, P<0.001). However, known clinical variables including age of the patient, sex, site of lesion, histology, lymph node involvement, stage of disease, serum lactate dehydrogenase levels, and response to chemotherapy were not found to be correlated with serum claudin-1 concentrations (P>0.05). Similarly, serum claudin-1 concentration was found to have no prognostic role in survival (P=0.524). In conclusion, serum levels of claudin-1 may have a diagnostic value in melanoma patients. However, its predictive and prognostic value has not been determined. PMID:26886788

  15. Claudin-3, claudin-7, and claudin-10 show different distribution patterns during decidualization and trophoblast invasion in mouse and human.

    PubMed

    Schumann, S; Buck, V U; Classen-Linke, I; Wennemuth, G; Grümmer, R

    2015-12-01

    Implantation of the mammalian embryo requires profound endometrial changes for successful pregnancy, including epithelial-mesenchymal transition of the luminal epithelium and stromal-epithelial transition of the stromal cells resulting in decidualization. Claudins (Cldn) determine the variability in tight junction paracellular permeability and may play a role during these epithelial and decidual changes. We here localized Cldn3, Cldn7 and Cldn10 proteins in the different compartments of murine endometrium up to day 8.5 of pregnancy (dpc) as well as in human endometrium and first trimester decidua. In murine estrous endometrium, luminal and glandular epithelium exhibited Cldn3 and Cldn7, whereas Cldn10 was only detectable in glandular epithelium. At 4.5 dpc, Cldn3 protein shifted to an apical localization, whereas Cldn7 vanished in the epithelium of the implantation chamber. At this stage, there was no stromal signal for Cldn3 and Cldn7, but a strong induction of Cldn10 in the primary decidual zone. Cldn3 proteins emerged at 5.5 dpc spreading considerably from 6.5 dpc onward in the endothelial cells of the decidual blood sinusoids and in the decidual cells of the compact antimesometrial region. In addition to Cldn3, Cldn10 was identified in human endometrial epithelia. Both proteins were not detected in human first trimester decidual cells. Cldn3 was shown in murine trophoblast giant cells as well as in human extravillous trophoblast cells and thus may have an impact on trophoblast invasion in both species. We here showed a specific claudin signature during early decidualization pointing to a role in decidual angiogenesis and regulation of trophoblast invasion. PMID:26340953

  16. Expression of claudin-1 and -11 in immature and mature pheasant (Phasianus colchicus) testes.

    PubMed

    Park, C J; Lee, J E; Oh, Y S; Shim, S; Nah, W H; Choi, K J; Gye, M C

    2011-02-01

    The expression of claudin-1 and -11, tight junctions (TJs) proteins was examined in immature and adult pheasant (Phasianus colchicus) testes. Claudin-1 and -11 cDNA were highly similar to those of human, mice, and chicken. Claudin-1 mRNA and protein (21 kDa) levels in immature testes were higher than those of adult testis. In immature testes until 6 weeks of age, Claudin-1 was found at contacts between adjacent Sertoli cells and between Sertoli cells and germ cells. In adult testis, Claudin-1 was found in early spermatocytes migrating the blood testis barrier (BTB). Blood vessels were positive for claudin-1. Claudin-11 mRNA and protein (21 kDa) increased during adulthood development of testis. In immature testis, Claudin-11 was found in apicolateral contacts between adjacent Sertoli cells, indicating its involvement in cell adhesion in immature testis. In adult testis, strong wavy Claudin-11 immunoreactivity was parallel to basal lamina at the basal part of seminiferous epithelium, indicating that Claudin-11 at the inter-Sertoli TJs may act as a structural element of the BTB. Weak Claudin-1 and -11 immunoreactivity at contacts between Sertoli cells to elongating/elongated spermatids, meiotic germ cells, and basal lamina suggests that they also participate in the cell-cell and cell-extracellular matrix adhesion in pheasant testis. Testosterone increased claudin-11 mRNA in testis organ culture and Sertoli cell primary culture, suggesting positive regulation of claudin-11 gene by androgen in Sertoli cells of pheasant testis. This is the first report on the claudins expression at BTB in avian testis. PMID:21074249

  17. Claudin 1 Expression Levels Affect miRNA Dynamics in Human Basal-Like Breast Cancer Cells.

    PubMed

    Majer, Anna; Blanchard, Anne A; Medina, Sarah; Booth, Stephanie A; Myal, Yvonne

    2016-07-01

    Deemed a putative tumor suppressor in breast cancer, the tight junction protein claudin 1 has now been shown to be highly expressed in the basal-like molecular subtype. Moreover, recent in vitro studies show that claudin 1 can regulate breast cancer cell motility and proliferation. Herein, we investigated whether microRNA (miRNA) dysregulation is associated with alterations in the level of claudin 1. Using next-generation sequencing (NGS), we identified seven miRNAs (miR-9-5p, miR-9-3p, let-7c, miR-127-3p, miR-99a-5p, miR-129-5p, and miR-146a-5p) that were deregulated as a consequence of claudin 1 overexpression in the MDA-MB231 human breast cancer (HBC) cell line. Most of these miRNAs have been associated with tumor suppression in a variety of cancers, including breast cancer. Moreover, through gene expression profiling analysis, we identified epithelial-mesenchymal transition-related genes, including platelet-derived growth factor receptor-beta (PDGFRB) and cadherin 1 (CDH1, E cadherin), whose downregulation correlated with claudin 1 overexpression. Collectively, we show for the first time that in HBC, claudin 1 can alter the dynamics of a number of miRNAs involved in tumor progression. Our data suggest that the dysregulated expression of these miRNAs, in conjunction with the high claudin 1 levels, could serve as a useful biomarker that identifies a subset of tumors within the poorly characterized basal-like subtype of breast cancer. Further studies are warranted to determine the role of these miRNAs in facilitating the function of claudin 1 in breast cancer. PMID:26982264

  18. Production, purification and characterization of recombinant, full-length human claudin-1.

    PubMed

    Bonander, Nicklas; Jamshad, Mohammed; Oberthür, Dominik; Clare, Michelle; Barwell, James; Hu, Ke; Farquhar, Michelle J; Stamataki, Zania; Harris, Helen J; Dierks, Karsten; Dafforn, Timothy R; Betzel, Christian; McKeating, Jane A; Bill, Roslyn M

    2013-01-01

    The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81. PMID:23704991

  19. Sex-Dependent Claudin-1 Expression in the Liver of Euthyroid and Hypothyroid Mice

    PubMed Central

    Zwanziger, Denise; Rakov, Helena; Engels, Kathrin; Moeller, Lars C.; Führer, Dagmar

    2015-01-01

    Background In the liver the tight junction protein claudin-1 plays an important role in bile secretion by maintaining the paracellular barrier of bile canaliculi and the bile duct. A diminished bile excretion has been found in hypothyroid patients, and the prevalence of gallstones is increased in hypothyroidism. This association, however, only applies for men and is in contrast to the well-established female preponderance of biliary disease in the general population. Objectives We hypothesized that hypothyroidism could lead to altered claudin-1 expression in the liver, and that this effect may be sex specific. Methods We characterized claudin-1 expression and localization in livers of euthyroid and hypothyroid male and female C57BL/6NTac mice by real-time PCR, Western blot and immunofluorescence. Results Claudin-1 is expressed in canalicular regions and the bile ducts of the murine liver. Livers of female mice showed lower claudin-1 expression than male livers. In hypothyroid livers, female animals showed an elevated claudin-1 expression, whereas reduced claudin-1 expression was found in male animals compared to the euthyroid controls. Conclusion We demonstrate a correlation between claudin-1 expression and hypothyroidism in the murine liver. Furthermore, a sex-dependent alteration of claudin-1 expression was found. PMID:26601075

  20. Clinical significance of serum claudin-1 and claudin-7 levels in patients with colorectal cancer

    PubMed Central

    KARABULUT, MEHMET; ALIS, HALIL; BAS, KORAY; KARABULUT, SENEM; AFSAR, CIGDEM USUL; OGUZ, HILAL; GUNALDI, MERAL; AKARSU, CEVHER; KONES, OSMAN; AYKAN, NURI FARUK

    2015-01-01

    The present study aimed to investigate the serum levels and clinical relevance of claudin (CLDN) 1 and CLDN7 in patients with colorectal cancer (CRC). A total of 140 patients with a pathologically confirmed diagnosis of CRC were enrolled in this study. The serum levels of CLDN1 and CLDN7 were determined using the solid-phase sandwich ELISA method. A total of 40 healthy age- and gender-matched controls were included in the analysis. The median age of the patients was 60 years (range, 24–84 years). The localization of the tumor in the majority of the patients was the colon (n=81, 58%). Of the 55 metastatic patients who received palliative chemotheraphy, 31% were chemotherapy-responsive. The baseline median serum CLDN1 and CLDN7 levels were significantly lower in non-metastatic and metastatic patients compared with those in healthy controls (CLND1, P=0.008 and 0.002; and CLND7, P=0.002 and 0.002, respectively). Moreover, known clinical variables, including poor performance status and high carcinoembryonic antigen (CEA) levels were found to be associated with lower serum CLDN1 concentrations for all patients (P=0.03 and P=0.03, respectively). High T stage and high CEA levels were found to be correlated with lower serum CLDN7 concentrations for all patients (P=0.04 and 0.03, respectively). A correlation was identified between CLDN1 and CLDN7 levels in non-metastatic and metastatic CRC patients (both P-values <0.001). Our study results did not reveal any statistical significance for serum CLDN1 or CLND7 concentrations regarding progression-free and overall survival rate. Therefore, reduced serum levels of CLDN1 and CLND7 may be useful markers in the differential diagnosis of CRC. PMID:26807230

  1. Biochemical and biophysical analyses of tight junction permeability made of claudin-16 and claudin-19 dimerization

    PubMed Central

    Gong, Yongfeng; Renigunta, Vijayaram; Zhou, Yi; Sunq, Abby; Wang, Jinzhi; Yang, Jing; Renigunta, Aparna; Baker, Lane A.; Hou, Jianghui

    2015-01-01

    The molecular nature of tight junction architecture and permeability is a long-standing mystery. Here, by comprehensive biochemical, biophysical, genetic, and electron microscopic analyses of claudin-16 and -19 interactions—two claudins that play key polygenic roles in fatal human renal disease, FHHNC—we found that 1) claudin-16 and -19 form a stable dimer through cis association of transmembrane domains 3 and 4; 2) mutations disrupting the claudin-16 and -19 cis interaction increase tight junction ultrastructural complexity but reduce tight junction permeability; and 3) no claudin hemichannel or heterotypic channel made of claudin-16 and -19 trans interaction can exist. These principles can be used to artificially alter tight junction permeabilities in various epithelia by manipulating selective claudin interactions. Our study also emphasizes the use of a novel recording approach based on scanning ion conductance microscopy to resolve tight junction permeabilities with submicrometer precision. PMID:26446843

  2. Claudin-3 expression in radiation-exposed rat models: A potential marker for radiation-induced intestinal barrier failure

    SciTech Connect

    Shim, Sehwan; Lee, Jong-geol; Bae, Chang-hwan; Lee, Seung Bum; Jang, Won-Suk; Lee, Sun-Joo; Lee, Seung-Sook; Park, Sunhoo

    2015-01-02

    Highlights: • Irradiation increased intestinal bacterial translocation, accompanied by claudin protein expression in rats. • Neurotensin decreased the bacterial translocation and restored claudin-3 expression. • Claudin-3 can be used as a marker in evaluating radiation induced intestinal injury. - Abstract: The molecular events leading to radiation-induced intestinal barrier failure are not well known. The influence of the expression of claudin proteins in the presence and absence of neurotensin was investigated in radiation-exposed rat intestinal epithelium. Wistar rats were randomly divided into control, irradiation, and irradiation + neurotensin groups, and bacterial translocation to the mesenteric lymph node and expression of claudins were determined. Irradiation led to intestinal barrier failure as demonstrated by significant bacterial translocation. In irradiated terminal ilea, expression of claudin-3 and claudin-4 was significantly decreased, and claudin-2 expression was increased. Administration of neurotensin significantly reduced bacterial translocation and restored the structure of the villi as seen by histologic examination. Among the three subtype of claudins, only claudin-3 expression was restored. These results suggest that the therapeutic effect of neurotensin on the disruption of the intestinal barrier is associated with claudin-3 alteration and that claudin-3 could be used as a marker in evaluating radiation-induced intestinal injury.

  3. Claudin-1 Regulates Intestinal Epithelial Homeostasis through the Modulation of Notch Signaling

    PubMed Central

    Pope, Jillian L.; Bhat, Ajaz. A.; Sharma, Ashok; Ahmad, Rizwan; Krishnan, Moorthy; Washington, Mary K.; Beauchamp, Robert D.; Singh, Amar B.; Dhawan, Punita

    2014-01-01

    Objective Claudin-1 expression is increased and dysregulated in colorectal cancer and causally associates with the dedifferentiation of colonic epithelial cells, cancer progression and metastasis. Here, we have sought to determine the role claudin-1 plays in the regulation of intestinal epithelial homeostasis. Design We have used a novel Villin-claudin-1 transgenic (Cl-1Tg) mouse as model (with intestinal claudin-1 overexpression). Effect of claudin-1 expression upon colonic epithelial differentiation, lineage commitment, and Notch signaling were determined using immunohistochemical, immunoblot and real time PCR analysis. The frequently used mouse model of DSS-colitis was used to model inflammation, injury and repair. Results In Cl-1Tg mice, normal colonocyte differentiation program was disrupted and goblet cell number and muc-2 expressions were significantly downregulated while Notch- and ERK1/2-signaling were upregulated, compared to the wild type (WT)-littermates. Cl-1Tg mice were also susceptible to colonic inflammation and demonstrated impaired recovery and hyperproliferation following the DSS-colitis. Our data further show that claudin-1 regulates Notch-signaling through the regulation of MMP-9 and p-ERK signaling to regulate proliferation and differentiation. Conclusion Claudin-1 helps regulate intestinal epithelial homeostasis through the regulation of Notch-signaling. An upregulated claudin-1 expression induces MMP-9 and p-ERK signaling to activate Notch-signaling, which in turn inhibits the goblet cell differentiation. Decreased goblet cell number decreases muc-2 expression and thus enhances susceptibility to mucosal inflammation. Claudin-1 expression also induces colonic epithelial proliferation in a Notch-dependent manner. Our findings may help understand the role of claudin-1 in the regulation of IBD and CRC. PMID:23766441

  4. Cellular zinc is required for intestinal epithelial barrier maintenance via the regulation of claudin-3 and occludin expression.

    PubMed

    Miyoshi, Yuka; Tanabe, Soichi; Suzuki, Takuya

    2016-07-01

    Intracellular zinc is required for a variety of cell functions, but its precise roles in the maintenance of the intestinal tight junction (TJ) barrier remain unclear. The present study investigated the essential roles of intracellular zinc in the preservation of intestinal TJ integrity and the underlying molecular mechanisms. Depletion of intracellular zinc in both intestinal Caco-2 cells and mouse colons through the application of a cell-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) induced a disruption of the TJ barrier, as indicated by increased FITC-labeled dextran flux and decreased transepithelial electrical resistance. The TPEN-induced TJ disruption is associated with downregulation of two TJ proteins, occludin and claudin-3. Biotinylation of cell surface proteins revealed that the zinc depletion induced the proteolysis of occludin but not claudin-3. Occludin proteolysis was sensitive to the inhibition of calpain activity, and increased calpain activity was observed in the zinc-depleted cells. Although quantitative PCR analysis and promoter reporter assay have demonstrated that the zinc depletion-induced claudin-3 downregulation occurred at transcriptional levels, a site-directed mutation in the egr1 binding site in the claudin-3 promoter sequence induced loss of both the basal promoter activity and the TPEN-induced decreases. Reduced egr1 expression by a specific siRNA also inhibited claudin-3 expression and transepithelial electrical resistance maintenance in cells. This study shows that intracellular zinc has an essential role in the maintenance of the intestinal epithelial TJ barrier through regulation of occludin proteolysis and claudin-3 transcription. PMID:27151944

  5. Checkpoint Kinase 1 Activation Enhances Intestinal Epithelial Barrier Function via Regulation of Claudin-5 Expression

    PubMed Central

    Watari, Akihiro; Hasegawa, Maki; Yagi, Kiyohito; Kondoh, Masuo

    2016-01-01

    Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine. PMID:26727128

  6. Chronic inflammatory demyelinating polyneuropathy: decreased claudin-5 and relocated ZO-1

    PubMed Central

    Kanda, T; Numata, Y; Mizusawa, H

    2004-01-01

    Objectives: To clarify the dynamics of molecules composing the blood–nerve barrier (BNB) in inflammatory neuropathies. Methods: The expression of four tight junction (TJ) proteins—claudin-1, claudin-5, occludin, and ZO-1—was analysed immunohistochemically in sural nerve biopsy specimens obtained from patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Results: Claudin-1 was detected only in perineurial cells, whereas claudin-5 was present in endothelial cells, irrespective of vessel location or size. Occludin and ZO-1 were found in perineurial cells, in addition to some epineurial and endoneurial endothelial cells. In CIDP, percentages of endoneurial small vessels immunoreactive for claudin-5 were significantly decreased, as were ZO-1 immunoreactive endoneurial small vessels, with staining localised to interfaces between cells. Claudin-1 and occludin immunoreactivity did not differ appreciably between the neuropathies examined. Conclusions: The downregulation of claudin-5 and altered localisation of ZO-1 seen in CIDP specimens may indicate that BNB derangement occurs in inflammatory neuropathies. Further investigation of TJ molecules may suggest new treatments based on properties of the BNB. PMID:15090575

  7. Claudin 1 mediates tumor necrosis factor alpha-induced cell migration in human gastric cancer cells

    PubMed Central

    Shiozaki, Atsushi; Shimizu, Hiroki; Ichikawa, Daisuke; Konishi, Hirotaka; Komatsu, Shuhei; Kubota, Takeshi; Fujiwara, Hitoshi; Okamoto, Kazuma; Iitaka, Daisuke; Nakashima, Shingo; Nako, Yoshito; Liu, Mingyao; Otsuji, Eigo

    2014-01-01

    AIM: To investigate the role of claudin 1 in the regulation of genes involved in cell migration and tumor necrosis factor alpha (TNF-α)-induced gene expression in human gastric adenocarcinoma cells. METHODS: Knockdown experiments were conducted with claudin 1 small interfering RNA (siRNA), and the effects on the cell cycle, apoptosis, migration and invasion were analyzed in human gastric adenocarcinoma MKN28 cells. The gene expression profiles of cells were analyzed by microarray and bioinformatics. RESULTS: The knockdown of claudin 1 significantly inhibited cell proliferation, migration and invasion, and increased apoptosis. Microarray analysis identified 245 genes whose expression levels were altered by the knockdown of claudin 1. Pathway analysis showed that the top-ranked molecular and cellular function was the cellular movement related pathway, which involved MMP7, TNF-SF10, TGFBR1, and CCL2. Furthermore, TNF- and nuclear frctor-κB were the top-ranked upstream regulators related to claudin 1. TNF-α treatment increased claudin 1 expression and cell migration in MKN28 cells. Microarray analysis indicated that the depletion of claudin 1 inhibited 80% of the TNF-α-induced mRNA expression changes. Further, TNF-α did not enhance cell migration in the claudin 1 siRNA transfected cells. CONCLUSION: These results suggest that claudin 1 is an important messenger that regulates TNF-α-induced gene expression and migration in gastric cancer cells. A deeper understanding of these cellular processes may be helpful in establishing new therapeutic strategies for gastric cancer. PMID:25548484

  8. Structural characterization of CD81-Claudin-1 hepatitis C virus receptor complexes.

    PubMed

    Bonander, Nicklas; Jamshad, Mohammed; Hu, Ke; Farquhar, Michelle J; Stamataki, Zania; Balfe, Peter; McKeating, Jane A; Bill, Roslyn M

    2011-04-01

    Tetraspanins are thought to exert their biological function(s) by co-ordinating the lateral movement and trafficking of associated molecules into tetraspanin-enriched microdomains. A second four-TM (transmembrane) domain protein family, the Claudin superfamily, is the major structural component of cellular TJs (tight junctions). Although the Claudin family displays low sequence homology and appears to be evolutionarily distinct from the tetraspanins, CD81 and Claudin-1 are critical molecules defining HCV (hepatitis C virus) entry; we recently demonstrated that CD81-Claudin-1 complexes have an essential role in this process. To understand the molecular basis of CD81-Claudin-1 complex formation, we produced and purified milligram quantities of full-length CD81 and Claudin-1, alone and in complex, in both detergent and lipid contexts. Structural characterization of these purified proteins will allow us to define the mechanism(s) underlying virus-cell interactions and aid the design of therapeutic agents targeting early steps in the viral life cycle. PMID:21428935

  9. Fucoidan enhances intestinal barrier function by upregulating the expression of claudin-1

    PubMed Central

    Iraha, Atsushi; Chinen, Hiroshi; Hokama, Akira; Yonashiro, Takumi; Kinjo, Tetsu; Kishimoto, Kazuto; Nakamoto, Manabu; Hirata, Tetsuo; Kinjo, Nagisa; Higa, Futoshi; Tateyama, Masao; Kinjo, Fukunori; Fujita, Jiro

    2013-01-01

    AIM: To evaluate the protective effects of fucoidan on oxidative stress-induced barrier disruption in human intestinal epithelial cells. METHODS: In Caco-2 cell monolayer models, the disruption of barrier function by oxidative stress is mediated by H2O2. The integrity of polarized Caco-2 cell monolayers was determined by measuring the transepithelial resistance (TER) and permeability was estimated by measuring the paracellular transport of FITC-labeled 4-kDa dextran (FD4). The protective effects of fucoidan on epithelial barrier functions on polarized Caco-2 cell monolayers were evaluated by TER and FD4 flux. The expression of tight junction (TJ) proteins was assessed using reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining. RESULTS: Without H2O2 treatment, fucoidan significantly increased the TER compared to control (P < 0.05), indicating a direct enhancement of intestinal epithelial barrier function. Next, H2O2 disrupted the epithelial barrier function in a time-dependent manner. Fucoidan prevented the H2O2-induced destruction in a dose-dependent manner. Fucoidan significantly decreased H2O2-induced FD4 flux (P < 0.01), indicating the prevention of disruption in paracellular permeability. RT-PCR showed that Caco-2 cells endogenously expressed claudin-1 and -2, and occludin and that H2O2 reduced the mRNA expression of these TJ proteins. Treatment with fucoidan attenuated the reduction in the expressions of claudin-1 and claudin-2 but not occludin. Immunofluorescence staining revealed that the expression of claudin-1 was intact and high on the cell surface. H2O2 disrupted the integrity of claudin-1. Treatment with fucoidan dramatically attenuated the expression of claudin-1. CONCLUSION: Fucoidan enhanced intestinal epithelial barrier function by upregulating the expression of claudin-1. Thus, fucoidan may be an appropriate therapy for the treatment of inflammatory bowel diseases. PMID:24023493

  10. IQGAP1 controls tight junction formation through differential regulation of claudin recruitment

    PubMed Central

    Tanos, Barbara E.; Perez Bay, Andres E.; Salvarezza, Susana; Vivanco, Igor; Mellinghoff, Ingo; Osman, Mahasin; Sacks, David B.; Rodriguez-Boulan, Enrique

    2015-01-01

    ABSTRACT IQGAP1 is a scaffolding protein previously implicated in adherens junction formation. However, its role in the establishment or maintenance of tight junctions (TJs) has not been explored. We hypothesized that IQGAP1 could regulate TJ formation by modulating the expression and/or localization of junctional proteins, and we systematically tested this hypothesis in the model Madin-Darby canine kidney (MDCK) cell line. We find that IQGAP1 silencing enhances a transient increase in transepithelial electrical resistance (TER) observed during the early stages of TJ formation (Cereijido et al., 1978). Quantitative microscopy and biochemical experiments suggest that this effect of IQGAP1 on TJ assembly is accounted for by reduced expression and TJ recruitment of claudin 2, and increased TJ recruitment of claudin 4. Furthermore, we show that IQGAP1 also regulates TJ formation through its interactor CDC42, because IQGAP1 knockdown increases the activity of the CDC42 effector JNK and dominant-negative CDC42 prevents the increase in TER caused by IQGAP1 silencing. Hence, we provide evidence that IQGAP1 modulates TJ formation by a twofold mechanism: (1) controlling the expression and recruitment of claudin 2 and recruitment of claudin 4 to the TJ, and (2) transient inhibition of the CDC42–JNK pathway. PMID:25588839

  11. Clearance of persistent hepatitis C virus infection using a claudin-1-targeting monoclonal antibody

    PubMed Central

    Mailly, Laurent; Wilson, Garrick K.; Aubert, Philippe; Duong, François H. T.; Calabrese, Diego; Leboeuf, Céline; Fofana, Isabel; Thumann, Christine; Bandiera, Simonetta; Lütgehetmann, Marc; Volz, Tassilo; Davis, Christopher; Harris, Helen J.; Mee, Christopher J.; Girardi, Erika; Chane-Woon-Ming, Béatrice; Ericsson, Maria; Fletcher, Nicola; Bartenschlager, Ralf; Pessaux, Patrick; Vercauteren, Koen; Meuleman, Philip; Villa, Pascal; Kaderali, Lars; Pfeffer, Sébastien; Heim, Markus H.; Neunlist, Michel; Zeisel, Mirjam B.; Dandri, Maura; McKeating, Jane A.; Robinet, Eric; Baumert, Thomas F.

    2015-01-01

    Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer1. Cell entry of HCV2 and other pathogens3-5 is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver-chimeric mouse model6 we show that a monoclonal antibody specific for TJ protein claudin-17 eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection via host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy. PMID:25798937

  12. Claudin 1 promotes migration and increases sensitivity to tamoxifen and anticancer drugs in luminal-like human breast cancer cells MCF7.

    PubMed

    Zhou, Bowen; Blanchard, Anne; Wang, Nan; Ma, Xiuli; Han, Jihyun; Schroedter, Ingo; Leygue, Etienne; Myal, Yvonne

    2015-01-01

    Downregulation of claudin 1, a critical tight junction protein, has been correlated with increased invasiveness in breast cancer. However, recent studies suggest that claudin 1 contributes to the progression of some molecular subtypes of breast cancer. In this study, claudin 1 promotes migration in luminal-like MCF7 human breast cancer cells and increases their sensitivity to tamoxifen, etoposide, and cisplatin. We also observed an inverse relationship between upregulation of claudin 1 and TGFβ. Collectively, our results suggest that claudin 1 has the potential to be used as a predictive marker for treatment efficacy for specific breast cancer patient subgroups. PMID:26288115

  13. miR-7 and miR-218 epigenetically control tumor suppressor genes RASSF1A and Claudin-6 by targeting HoxB3 in breast cancer

    SciTech Connect

    Li, Qiaoyan; Zhu, Fufan; Chen, Puxiang

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Both miR-7 and miR-218 down-regulates HoxB3 expression by targeting the 3 Prime -UTR of HoxB3 mRNA. Black-Right-Pointing-Pointer A reverse correlation between the levels of endogenous miR-7, miR218 and HoxB3 expression. Black-Right-Pointing-Pointer Epigenetic changes involve in the reactivation of HoxB3. Black-Right-Pointing-Pointer Both miRNAs inhibits the cell cycle and clone formation of breast cancer cells. -- Abstract: Many microRNAs have been implicated as key regulators of cellular growth and differentiation and have been found to dysregulate proliferation in human tumors, including breast cancer. Cancer-linked microRNAs also alter the epigenetic landscape by way of DNA methylation and post-translational modifications of histones. Aberrations in Hox gene expression are important for oncogene or tumor suppressor during abnormal development and malignancy. Although recent studies suggest that HoxB3 is critical in breast cancer, the putative role(s) of microRNAs impinging on HoxB3 is not yet fully understood. In this study, we found that the expression levels of miR-7 and miR-218 were strongly and reversely associated with HoxB3 expression. Stable overexpression of miR-7 and miR-218 was accompanied by reactivation of tumor suppressor genes including RASSF1A and Claudin-6 by means of epigenetic switches in DNA methylation and histone modification, giving rise to inhibition of the cell cycle and clone formation of breast cancer cells. The current study provides a novel link between overexpression of collinear Hox genes and multiple microRNAs in human breast malignancy.

  14. Claudin-2 Knockout by TALEN-Mediated Gene Targeting in MDCK Cells: Claudin-2 Independently Determines the Leaky Property of Tight Junctions in MDCK Cells

    PubMed Central

    Tokuda, Shinsaku; Furuse, Mikio

    2015-01-01

    Tight junctions (TJs) regulate the movements of substances through the paracellular pathway, and claudins are major determinants of TJ permeability. Claudin-2 forms high conductive cation pores in TJs. The suppression of claudin-2 expression by RNA interference in Madin-Darby canine kidney (MDCK) II cells (a low-resistance strain of MDCK cells) was shown to induce a three-fold increase in transepithelial electrical resistance (TER), which, however, was still lower than in high-resistance strains of MDCK cells. Because RNA interference-mediated knockdown is not complete and only reduces gene function, we considered the possibility that the remaining claudin-2 expression in the knockdown study caused the lower TER in claudin-2 knockdown cells. Therefore, we investigated the effects of claudin-2 knockout in MDCK II cells by establishing claudin-2 knockout clones using transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout. Surprisingly, claudin-2 knockout increased TER by more than 50-fold in MDCK II cells, and TER values in these cells (3000–4000 Ω·cm2) were comparable to those in the high-resistance strains of MDCK cells. Claudin-2 re-expression restored the TER of claudin-2 knockout cells dependent upon claudin-2 protein levels. In addition, we investigated the localization of claudin-1, -2, -3, -4, and -7 at TJs between control MDCK cells and their respective knockout cells using their TALENs. Claudin-2 and -7 were less efficiently localized at TJs between control and their knockout cells. Our results indicate that claudin-2 independently determines the ‘leaky’ property of TJs in MDCK II cells and suggest the importance of knockout analysis in cultured cells. PMID:25781928

  15. CXCR4 Antagonist AMD3100 Modulates Claudin Expression and Intestinal Barrier Function in Experimental Colitis

    PubMed Central

    Xia, Xian-Ming; Wang, Fang-Yu; Zhou, Ju; Hu, Kai-Feng; Li, Su-Wen; Zou, Bing-Bing

    2011-01-01

    Ulcerative colitis is a gastrointestinal disorder characterized by local inflammation and impaired epithelial barrier. Previous studies demonstrated that CXC chemokine receptor 4 (CXCR4) antagonists could reduce colonic inflammation and mucosal damage in dextran sulfate sodium (DSS)-induced colitis. Whether CXCR4 antagonist has action on intestinal barrier and the possible mechanism, is largely undefined. In the present study, the experimental colitis was induced by administration of 5% DSS for 7 days, and CXCR4 antagonist AMD3100 was administered intraperitoneally once daily during the study period. For in vitro study, HT-29/B6 colonic cells were treated with cytokines or AMD3100 for 24 h until assay. DSS-induced colitis was characterized by morphologic changes in mice. In AMD3100-treated mice, epithelial destruction, inflammatory infiltration, and submucosal edema were markedly reduced, and the disease activity index was also significantly decreased. Increased intestinal permeability in DSS-induced colitis was also significantly reduced by AMD3100. The expressions of colonic claudin-1, claudin-3, claudin-5, claudin-7 and claudin-8 were markedly decreased after DSS administration, whereas colonic claudin-2 expression was significantly decreased. Treatment with AMD3100 prevented all these changes. However, AMD3100 had no influence on claudin-3, claudin-5, claudin-7 and claudin-8 expression in HT-29/B6 cells. Cytokines as TNF-α, IL-6, and IFN-γ increased apoptosis and monolayer permeability, inhibited the wound-healing and the claudin-3, claudin-7 and claudin-8 expression in HT-29/B6 cells. We suggest that AMD3100 acted on colonic claudin expression and intestinal barrier function, at least partly, in a cytokine-dependent pathway. PMID:22073304

  16. The Value of HBME-1 and Claudin-1 Expression Profile in the Distinction of BRAF-Like and RAS-Like Phenotypes in Papillary Thyroid Carcinoma.

    PubMed

    Gucer, Hasan; Bagci, Pelin; Bedir, Recep; Sehitoglu, Ibrahim; Mete, Ozgur

    2016-09-01

    This study compared the expression profile of HBME-1 and claudin-1 in 90 papillary thyroid carcinomas (PTCs) with respect to the tumor architecture and invasive growth as reflected in 46 BRAF-like, 31 non-invasive RAS, and 13 invasive RAS-like phenotypes. Individual tumors were given an expression score (max 300) by multiplying the percent positive tumor cells by the intensity score (range 0-3). The higher expression of HBME-1 and claudin-1 distinguished BRAF-like phenotype from RAS-like phenotype. The same correlation was also retained for both markers when comparing BRAF-like phenotype with non-invasive and invasive RAS-like phenotypes. The expression scores and positivity rates for both markers did not yield any statistical difference among BRAF-like PTCs. Except the higher positivity rate of HBME-1, invasive RAS-like tumors were not statistically different than their non-invasive counterparts with respect to the positivity rate of claudin-1 and the expression scores of both markers. A central lymph node dissection or selective lymph node sampling was available in 20 specimens. The absence of claudin-1 expression has not been a feature of lymph node metastasis in this series. Despite the limited number of nodal sampling, BRAF-like phenotype and claudin-1 positivity status have been considered the best determinants of positive predictive value and negative predictive value in the prediction of lymph node metastasis among variables, respectively. Adoption of the simplified architectural classification approach to PTCs showed distinct biomarker expression profile in this series; however, immunohistochemistry for HBME-1 and claudin-1 does not seem to be useful in the distinction of invasive RAS-like PTCs from their non-invasive counterparts. Given the overlapping molecular signatures within the RAS-like phenotype, further studies with additional biomarkers are still needed to identify distinct protein expression signatures of non-invasive RAS-like phenotype as this

  17. Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood-brain barrier damage in early ischemic stroke stage.

    PubMed

    Liu, Jie; Jin, Xinchun; Liu, Ke J; Liu, Wenlan

    2012-02-29

    Blood-brain barrier (BBB) disruption occurs early enough to be within the thrombolytic time window, and this early ischemic BBB damage is closely associated with hemorrhagic transformation and thus emerging as a promising target for reducing the hemorrhagic complications of thrombolytic stroke therapy. However, the mechanisms underlying early ischemic BBB damage remain poorly understood. Here, we investigated the early molecular events of ischemic BBB damage using in vitro oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery occlusion (MCAO) models. Exposure of bEND3 monolayer to OGD for 2 h significantly increased its permeability to FITC-labeled dextran and promoted the secretion of metalloproteinase-2 and -9 (MMP-2/9) and cytosolic translocation of caveolin-1 (Cav-1). This same OGD treatment also led to rapid degradation of tight junction protein occludin and dissociation of claudin-5 from the cytoskeleton, which contributed to OGD-induced endothelial barrier disruption. Using selective MMP-2/9 inhibitor SB-3CT (2-[[(4-phenoxyphenyl)sulfonyl]methyl]-thiirane) or their neutralizing antibodies or Cav-1 siRNA, we found that MMP-2 was the major enzyme mediating OGD-induced occludin degradation, while Cav-1 was responsible for claudin-5 redistribution. The interaction between Cav-1 and claudin-5 was further confirmed by coimmunoprecipitation. Consistent with these in vitro findings, we observed fluorescence tracer extravasation, increased gelatinolytic activity, and elevated interstitial MMP-2 levels in ischemic subcortical tissue after 2 h MCAO. Moreover, occludin protein loss and claudin-5 redistribution were detected in ischemic cerebromicrovessels. These data indicate that cerebral ischemia initiates two rapid parallel processes, MMP-2-mediated occludin degradation and Cav-1-mediated claudin-5 redistribution, to cause BBB disruption at early stroke stages relevant to acute thrombolysis. PMID:22378877

  18. Claudin-3 and occludin tissue content in the glands of colonic mucosa with and without a fecal stream.

    PubMed

    Martinez, Carlos Augusto Real; de Campos, Fabio Guilherme Caserta Maryssael; de Carvalho, Viviel Rodrigo José; de Castro Ferreira, Caroline; Rodrigues, Murilo Rocha; Sato, Daniela Tiemi; Pereira, José Aires

    2015-04-01

    The synthesis of the proteins of the apical tight junctions (TJs) depends on a continuous supply of short-chain fatty acids (SCFAs) in colonic epithelium. No studies have evaluated the tissue contents of the TJs proteins in colon segments devoid of a fecal stream. To evaluate the contents of claudin-3 and occludin in the glands of colonic mucosa devoid of a fecal stream. Forty-five rats underwent a diversion of the fecal stream via a left side colostomy and distal mucous fistula. Three groups of 15 animals each were sacrificed at 6, 12 or 18 weeks after surgery. The presence and severity of colitis were defined by histology and inflammation grading scales, respectively. The expression of claudin-3 and occludin were evaluated by immunohistochemistry, and their contents were evaluated by computer-assisted image analysis. Mann-Whitney and Kruskal-Wallis tests were used to evaluate the results at a significance level of 5% (p < 0.05). The colonic epithelium without a fecal stream had a higher degree of inflammation. Colonic glands without a fecal stream showed a reduction in claudin-3 content independent of the time and reduction in occludin content after 12 weeks of intestinal exclusion. The content of claudin-3 and occludin were mainly reduced at the apical surfaces of the colon glands, whereas segments retaining the fecal stream were maintained. The content of claudin-3 was not reduced with time, although the levels of occludin were reduced after 6 weeks and did not vary thereafter. Deficiencies in SCFAs decreased the content of claudin-3 and occludin in colonic glands with the areas of worst inflammation, confirming the importance of an adequate supply of SCFAs in maintaining the integrity of TJ proteins. PMID:25649016

  19. The Cap1claudin-4 regulatory pathway is important for renal chloride reabsorption and blood pressure regulation

    PubMed Central

    Gong, Yongfeng; Yu, Miao; Yang, Jing; Gonzales, Ernie; Perez, Ronaldo; Hou, Mingli; Tripathi, Piyush; Hering-Smith, Kathleen S.; Hamm, L. Lee; Hou, Jianghui

    2014-01-01

    The paracellular pathway through the tight junction provides an important route for transepithelial chloride reabsorption in the kidney, which regulates extracellular salt content and blood pressure. Defects in paracellular chloride reabsorption may in theory cause deregulation of blood pressure. However, there is no evidence to prove this theory or to demonstrate the in vivo role of the paracellular pathway in renal chloride handling. Here, using a tissue-specific KO approach, we have revealed a chloride transport pathway in the kidney that requires the tight junction molecule claudin-4. The collecting duct-specific claudin-4 KO animals developed hypotension, hypochloremia, and metabolic alkalosis due to profound renal wasting of chloride. The claudin-4–mediated chloride conductance can be regulated endogenously by a protease—channel-activating protease 1 (cap1). Mechanistically, cap1 regulates claudin-4 intercellular interaction and membrane stability. A putative cap1 cleavage site has been identified in the second extracellular loop of claudin-4, mutation of which abolished its regulation by cap1. The cap1 effects on paracellular chloride permeation can be extended to other proteases such as trypsin, suggesting a general mechanism may also exist for proteases to regulate the tight junction permeabilities. Together, we have discovered a theory that paracellular chloride permeability is physiologically regulated and essential to renal salt homeostasis and blood pressure control. PMID:25157135

  20. Functional ESCRT machinery is required for constitutive recycling of claudin-1 and maintenance of polarity in vertebrate epithelial cells.

    PubMed

    Dukes, Joseph D; Fish, Laura; Richardson, Judith D; Blaikley, Elizabeth; Burns, Samir; Caunt, Christopher J; Chalmers, Andrew D; Whitley, Paul

    2011-09-01

    Genetic screens in Drosophila have identified regulators of endocytic trafficking as neoplastic tumor suppressor genes. For example, Drosophila endosomal sorting complex required for transport (ESCRT) mutants lose epithelial polarity and show increased cell proliferation, suggesting that ESCRT proteins could function as tumor suppressors. In this study, we show for the for the first time to our knowledge that ESCRT proteins are required to maintain polarity in mammalian epithelial cells. Inhibition of ESCRT function caused the tight junction protein claudin-1 to accumulate in intracellular vesicles. In contrast E-cadherin and occludin localization was unaffected. We investigated the cause of this accumulation and show that claudin-1 is constitutively recycled in kidney, colon, and lung epithelial cells, identifying claudin-1 recycling as a newly described feature of diverse epithelial cell types. This recycling requires ESCRT function, explaining the accumulation of intracellular claudin-1 when ESCRT function is inhibited. We further demonstrate that small interfering RNA knockdown of the ESCRT protein Tsg101 causes epithelial monolayers to lose their polarized organization and interferes with the establishment of a normal epithelial permeability barrier. ESCRT knockdown also reduces the formation of correctly polarized three-dimensional cysts. Thus, in mammalian epithelial cells, ESCRT function is required for claudin-1 trafficking and for epithelial cell polarity, supporting the hypothesis that ESCRT proteins function as tumor suppressors. PMID:21757541

  1. Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

    SciTech Connect

    Imamura, Masafumi; Kojima, Takashi . E-mail: ktakashi@sapmed.ac.jp; Lan, Mengdong; Son, Seiichi; Murata, Masaki; Osanai, Makoto; Chiba, Hideki; Hirata, Koichi; Sawada, Norimasa

    2007-05-15

    In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.

  2. Redox-Sensitive Structure and Function of the First Extracellular Loop of the Cell–Cell Contact Protein Claudin-1: Lessons from Molecular Structure to Animals

    PubMed Central

    Dabrowski, Sebastian; Staat, Christian; Zwanziger, Denise; Sauer, Reine-Solange; Bellmann, Christian; Günther, Ramona; Krause, Eberhard; Haseloff, Reiner Fritz; Rittner, Heike

    2015-01-01

    Abstract The paracellular cleft within epithelia/endothelia is sealed by tight junction (TJ) proteins. Their extracellular loops (ECLs) are assumed to control paracellular permeability and are targets of pathogenes. We demonstrated that claudin-1 is crucial for paracellular tightening. Its ECL1 is essential for the sealing and contains two cysteines conserved throughout all claudins. Aims: We prove the hypothesis that this cysteine motif forms a redox-sensitive intramolecular disulfide bridge and, hence, the claudin-1-ECL1 constitutes a functional structure which is associated to ECLs of this and other TJ proteins. Results: The structure and function of claudin-1-ECL1 was elucidated by investigating sequences of this ECL as synthetic peptides, C1C2, and as recombinant proteins, and exhibited a β-sheet binding surface flanked by an α-helix. These sequences bound to different claudins, their ECL1, and peptides with nanomolar binding constants. C-terminally truncated C1C2 (-4aaC) opened cellular barriers and the perineurium. Recombinant ECL1 formed oligomers, and bound to claudin-1 expressing cells. Oligomerization and claudin association were abolished by reducing agents, indicating intraloop disulfide bridging and redox sensitivity. Innovation: The structural and functional model based on our in vitro and in vivo investigations suggested that claudin-1-ECL1 constitutes a functional and ECL-binding β-sheet, stabilized by a shielded and redox-sensitive disulfide bond. Conclusion: Since the β-sheet represents a consensus sequence of claudins and further junctional proteins, a general structural feature is implied. Therefore, our model is of general relevance for the TJ assembly in normal and pathological conditions. C1C2-4aaC is a new drug enhancer that is used to improve pharmacological treatment through tissue barriers. Antioxid. Redox Signal. 22, 1–14. PMID:24988310

  3. Claudin heterogeneity and control of lung tight junctions.

    PubMed

    Koval, Michael

    2013-01-01

    Lung epithelial cells interconnected by tight junctions provide a barrier to the free diffusion of solutes into airspaces. Transmembrane tight junction proteins known as claudins are essential for epithelial barrier function. Claudins are regulated through interactions with each other that are coordinated with other transmembrane tight junction proteins and cytosolic scaffold proteins. Of the 14 claudins expressed by the alveolar epithelium, claudin-3, claudin-4, and claudin-18 are the most prominent; each confers unique properties to alveolar barrier function. In particular, a protective role for claudin-4 in preventing lung injury has emerged. By contrast, lung diseases that affect claudin expression and impair barrier function, including alcoholic lung syndrome and sepsis, prime the lung for pulmonary edema. Thus, approaches to restore and/or augment lung claudin expression provide potential targets for promoting healthy barrier function. PMID:23072447

  4. Expression of the ALS-causing variant hSOD1G93A leads to an impaired integrity and altered regulation of claudin-5 expression in an in vitro blood–spinal cord barrier model

    PubMed Central

    Meister, Sabrina; Storck, Steffen E; Hameister, Erik; Behl, Christian; Weggen, Sascha; Clement, Albrecht M; Pietrzik, Claus U

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to the loss of primary and secondary motor neurons. Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene are associated with familial ALS and to date numerous hypotheses for ALS pathology exist including impairment of the blood–spinal cord barrier. In transgenic mice carrying mutated SOD1 genes, a disrupted blood–spinal cord barrier as well as decreased levels of tight junction (TJ) proteins ZO-1, occludin, and claudin-5 were detected. Here, we examined TJ protein levels and barrier function of primary blood–spinal cord barrier endothelial cells of presymptomatic hSOD1G93A mice and bEnd.3 cells stably expressing hSOD1G93A. In both cellular systems, we observed reduced claudin-5 levels and a decreased transendothelial resistance (TER) as well as an increased apparent permeability. Analysis of the β-catenin/AKT/forkhead box protein O1 (FoxO1) pathway and the FoxO1-regulated activity of the claudin-5 promoter revealed a repression of the claudin-5 gene expression in hSOD1G93A cells, which was depended on the phosphorylation status of FoxO1. These results strongly indicate that mutated SOD1 affects the expression and localization of TJ proteins leading to impaired integrity and breakdown of the blood–spinal cord barrier. PMID:25853911

  5. Claudins and pathogenesis of viral infection.

    PubMed

    Tawar, Rajiv G; Colpitts, Che C; Lupberger, Joachim; El-Saghire, Hussein; Zeisel, Mirjam B; Baumert, Thomas F

    2015-06-01

    Since their discovery, tremendous progress has been made in our understanding of the roles of claudins in tight junction physiology. In addition, interactions between claudins and other cellular proteins have highlighted their novel roles in cell physiology. Moreover, the importance of claudins is becoming apparent in the pathophysiology of several diseases, including viral infections. Notable is the discovery of CLDN1 as an essential host factor for hepatitis C virus (HCV) entry, which led to detailed characterization of CLDN1 and its association with tetraspanin CD81 for the initiation of HCV infection. CLDN1 has also been shown to facilitate dengue virus entry. Furthermore, owing to the roles of claudins in forming anatomical barriers, several viruses have been shown to alter claudin expression at the tight junction. This review summarizes the role of claudins in viral infection, with particular emphasis on HCV. PMID:25960372

  6. HIF-dependent regulation of claudin-1 is central to intestinal epithelial tight junction integrity

    PubMed Central

    Saeedi, Bejan J.; Kao, Daniel J.; Kitzenberg, David A.; Dobrinskikh, Evgenia; Schwisow, Kayla D.; Masterson, Joanne C.; Kendrick, Agnieszka A.; Kelly, Caleb J.; Bayless, Amanda J.; Kominsky, Douglas J.; Campbell, Eric L.; Kuhn, Kristine A.; Furuta, Glenn T.; Colgan, Sean P.; Glover, Louise E.

    2015-01-01

    Intestinal epithelial cells (IECs) are exposed to profound fluctuations in oxygen tension and have evolved adaptive transcriptional responses to a low-oxygen environment. These adaptations are mediated primarily through the hypoxia-inducible factor (HIF) complex. Given the central role of the IEC in barrier function, we sought to determine whether HIF influenced epithelial tight junction (TJ) structure and function. Initial studies revealed that short hairpin RNA–mediated depletion of the HIF1β in T84 cells resulted in profound defects in barrier and nonuniform, undulating TJ morphology. Global HIF1α chromatin immunoprecipitation (ChIP) analysis identified claudin-1 (CLDN1) as a prominent HIF target gene. Analysis of HIF1β-deficient IEC revealed significantly reduced levels of CLDN1. Overexpression of CLDN1 in HIF1β-deficient cells resulted in resolution of morphological abnormalities and restoration of barrier function. ChIP and site-directed mutagenesis revealed prominent hypoxia response elements in the CLDN1 promoter region. Subsequent in vivo analysis revealed the importance of HIF-mediated CLDN1 expression during experimental colitis. These results identify a critical link between HIF and specific tight junction function, providing important insight into mechanisms of HIF-regulated epithelial homeostasis. PMID:25904334

  7. HIF-dependent regulation of claudin-1 is central to intestinal epithelial tight junction integrity.

    PubMed

    Saeedi, Bejan J; Kao, Daniel J; Kitzenberg, David A; Dobrinskikh, Evgenia; Schwisow, Kayla D; Masterson, Joanne C; Kendrick, Agnieszka A; Kelly, Caleb J; Bayless, Amanda J; Kominsky, Douglas J; Campbell, Eric L; Kuhn, Kristine A; Furuta, Glenn T; Colgan, Sean P; Glover, Louise E

    2015-06-15

    Intestinal epithelial cells (IECs) are exposed to profound fluctuations in oxygen tension and have evolved adaptive transcriptional responses to a low-oxygen environment. These adaptations are mediated primarily through the hypoxia-inducible factor (HIF) complex. Given the central role of the IEC in barrier function, we sought to determine whether HIF influenced epithelial tight junction (TJ) structure and function. Initial studies revealed that short hairpin RNA-mediated depletion of the HIF1β in T84 cells resulted in profound defects in barrier and nonuniform, undulating TJ morphology. Global HIF1α chromatin immunoprecipitation (ChIP) analysis identified claudin-1 (CLDN1) as a prominent HIF target gene. Analysis of HIF1β-deficient IEC revealed significantly reduced levels of CLDN1. Overexpression of CLDN1 in HIF1β-deficient cells resulted in resolution of morphological abnormalities and restoration of barrier function. ChIP and site-directed mutagenesis revealed prominent hypoxia response elements in the CLDN1 promoter region. Subsequent in vivo analysis revealed the importance of HIF-mediated CLDN1 expression during experimental colitis. These results identify a critical link between HIF and specific tight junction function, providing important insight into mechanisms of HIF-regulated epithelial homeostasis. PMID:25904334

  8. Dose-dependent role of claudin-1 in vivo in orchestrating features of atopic dermatitis.

    PubMed

    Tokumasu, Reitaro; Yamaga, Kosuke; Yamazaki, Yuji; Murota, Hiroyuki; Suzuki, Koya; Tamura, Atsushi; Bando, Kana; Furuta, Yasuhide; Katayama, Ichiro; Tsukita, Sachiko

    2016-07-12

    Atopic dermatitis (AD) is a chronic inflammatory skin disease in humans. It was recently noted that the characteristics of epidermal barrier functions critically influence the pathological features of AD. Evidence suggests that claudin-1 (CLDN1), a major component of tight junctions (TJs) in the epidermis, plays a key role in human AD, but the mechanism underlying this role is poorly understood. One of the main challenges in studying CLDN1's effects is that Cldn1 knock-out mice cannot survive beyond 1 d after birth, due to lethal dehydration. Here, we established a series of mouse lines that express Cldn1 at various levels and used these mice to study Cldn1's effects in vivo. Notably, we discovered a dose-dependent effect of Cldn1's expression in orchestrating features of AD. In our experimental model, epithelial barrier functions and morphological changes in the skin varied exponentially with the decrease in Cldn1 expression level. At low Cldn1 expression levels, mice exhibited morphological features of AD and an innate immune response that included neutrophil and macrophage recruitment to the skin. These phenotypes were especially apparent in the infant stages and lessened as the mice became adults, depending on the expression level of Cldn1 Still, these adult mice with improved phenotypes showed an enhanced hapten-induced contact hypersensitivity response compared with WT mice. Furthermore, we revealed a relationship between macrophage recruitment and CLDN1 levels in human AD patients. Our findings collectively suggest that CLDN1 regulates the pathogenesis, severity, and natural course of human AD. PMID:27342862

  9. Discovery of anti-claudin-1 antibodies as candidate therapeutics against hepatitis C virus.

    PubMed

    Yamashita, Mayo; Iida, Manami; Tada, Minoru; Shirasago, Yoshitaka; Fukasawa, Masayoshi; Nagase, Shotaro; Watari, Akihiro; Ishii-Watabe, Akiko; Yagi, Kiyohito; Kondoh, Masuo

    2015-04-01

    Claudin-1 (CLDN1), a known host factor for hepatitis C virus (HCV) entry and cell-to-cell transmission, is a target molecule for inhibiting HCV infection. We previously developed four clones of mouse anti-CLDN1 monoclonal antibody (mAb) that prevented HCV infection in vitro. Two of these mAbs showed the highest antiviral activity. Here, we optimized the anti-CLDN1 mAbs as candidates for therapeutics by protein engineering. Although Fab fragments of the mAbs prevented in vitro HCV infection, their inhibitory effects were much weaker than those of the whole mAbs. In contrast, human chimeric IgG1 mAbs generated by grafting the variable domains of the mouse mAb light and heavy chains inhibited in vitro HCV infection as efficiently as the parental mouse mAbs. However, the chimeric IgG1 mAbs activated Fcγ receptor, suggesting that cytotoxicity against mAb-bound CLDN1-expressing cells occurred through the induction of antibody-dependent cellular cytotoxicity (ADCC). To avoid ADCC-induced side effects, we prepared human chimeric IgG4 mAbs. The chimeric IgG4 mAbs did not activate Fcγ receptor or induce ADCC, but they prevented in vitro HCV infection as efficiently as did the parental mouse mAbs. These findings indicate that the IgG4 form of human chimeric anti-CLDN1 mAb may be a candidate molecule for clinically applicable HCV therapy. PMID:25628391

  10. Fluid reabsorption in proximal convoluted tubules of mice with gene deletions of claudin-2 and/or aquaporin1

    PubMed Central

    Huang, Yuning; Mizel, Diane

    2013-01-01

    Deletions of claudin-2 (Cldn2) and aquaporin1 (AQP1) reduce proximal fluid reabsorption (PFR) by about 30% and 50%, respectively. Experiments were done to replicate these observations and to determine in AQP1/claudin-2 double knockout mice (DKO) if the effects of deletions of these established water pores are additive. PFR was determined in inactin/ketamine-anesthetized mice by free-flow micropuncture using single-nephron I125-iothalamate (io) clearance. Animal means of PFR [% of glomerular filtration rate (GFR)] derived from TF/Piothalamate ratios in 12 mice in each of four groups [wild type (WT), Cldn2−/−, AQP1−/−, and DKO) were 45.8 ± 0.85 (51 tubules), 35.4 ± 1 (54 tubules; P < 0.01 vs. WT), 36.8 ± 1 (63 tubules; P < 0.05 vs. WT), and 33.9 ± 1.4 (69 tubules; P < 0.01 vs. WT). Kidney and single-nephron GFRs (SNGFR) were significantly reduced in all mutant strains. The direct relationship between PFR and SNGFR was maintained in mutant mice, but the slope of this relationship was reduced in the absence of Cldn2 and/or AQP1. Transtubular osmotic pressure differences were not different between WT and Cldn2−/− mice, but markedly increased in DKO. In conclusion, the deletion of Cldn2, AQP1, or of both Cldn2 and AQP1 reduces PFR by 22.7%, 19.6%, and 26%, respectively. Our data are consistent with an up to 25% paracellular contribution to PFR. The reduced osmotic water permeability caused by absence of AQP1 augments luminal hypotonicity. Aided by a fall in filtered load, the capacity of non-AQP1-dependent transcellular reabsorption is sufficient to maintain PFR without AQP1 and claudin-2 at 75% of control. PMID:24049145

  11. Autocrine CSF1R signaling mediates switching between invasion and proliferation downstream of TGFβ in claudin-low breast tumor cells

    PubMed Central

    Patsialou, Antonia; Wang, Yarong; Pignatelli, Jeanine; Chen, Xiaoming; Entenberg, David; Oktay, Maja; Condeelis, John S.

    2014-01-01

    Patient data suggest that colony stimulating factor-1 (CSF1) and its receptor (CSF1R) play critical roles during breast cancer progression. We have previously shown that in human breast tumors expressing both CSF1 and CSF1R, invasion in vivo is dependent both on a paracrine interaction with tumor-associated macrophages and an autocrine regulation of CSF1R in the tumor cells themselves. Although the role of the paracrine interaction between tumor cells and macrophages has been extensively studied, very little is known about the mechanism by which the autocrine CSF1R signaling contributes to tumor progression. We show here that breast cancer patients of the claudin-low subtype have significantly increased expression of CSF1R. Using a panel of breast cancer cells lines, we confirm that CSF1R expression is elevated and regulated by TGFβ specifically in claudin-low cell lines. Abrogation of autocrine CSF1R signaling in MDA-MB-231 xenografts (a claudin-low cell line) leads to increased tumor size by enhanced proliferation, but significantly reduced invasion, dissemination and metastasis. Indeed, we show that proliferation and invasion are oppositely regulated by CSF1R downstream of TGFβ only in claudin-low cells lines. Intravital multiphoton imaging revealed that inhibition of CSF1R in the tumor cells leads to decreased in vivo motility and a more cohesive morphology. We show that, both in vitro and in vivo, CSF1R inhibition results in a reversal of claudin-low marker expression by significant upregulation of luminal keratins and tight junction proteins such as claudins. Finally, we show that artificial overexpression of claudins in MDA-MB-231 cells is sufficient to tip the cells from an invasive state to a proliferative state. Our results suggest that autocrine CSF1R signaling is essential in maintaining low claudin expression and that it mediates a switch between the proliferative and the invasive state in claudin-low tumor cells downstream of TGFβ. PMID:25088194

  12. Sirolimus and cyclosporine A alter barrier function in renal proximal tubular cells through stimulation of ERK1/2 signaling and claudin-1 expression.

    PubMed

    Martin-Martin, Natalia; Ryan, Gavin; McMorrow, Tara; Ryan, Michael P

    2010-03-01

    Alteration of the tight junction complex in renal epithelial cells can affect renal barrier function and perturb normal kidney homeostasis. The immunosuppressant drugs cyclosporine A (CsA) and sirolimus (SRL) used in combination demonstrated beneficial effects in organ transplantation but this combination can also result in increased adverse effects. We previously showed that CsA treatment alone caused an alteration of the tight junction complex, resulting in changes in transepithelial permeability in Madin-Darby canine kidney distal tubular/collecting duct cells. The potential effect of SRL on transepithelial permeability in kidney cells is unknown. In this study, subcytotoxic doses of SRL or CsA were found to decrease the paracellular permeability of the porcine proximal tubular epithelial cells, LLC-PK1 cell monolayers, which was detected as an increase in transepithelial electrical resistance (TER). The cotreatment with SRL and CsA was found to increase TER in a synergistic manner. CsA treatment increased total cellular expression and membrane localization of the tight junction protein claudin-1 and this further increased with the combination of SRL/CsA. SRL and CsA treatment alone or in combination stimulated the phosphorylation of ERK1/2. The MEK-ERK1/2 pathway inhibitor, U0126, reduced the SRL, CsA, and CsA/SRL-induced increase in TER. U0126 also reduced the CsA and CsA/SRL-induced increase in the membrane localization of claudin-1. Alterations in claudin-2 and claudin-4 were also detected. However, the results suggest that the modulation in expression and localization of claudin-1 appears to be pivotal in the SRL- and CsA-induced modulation of the epithelial barrier function and that modulation is regulated by ERK1/2 signaling pathway. PMID:19955189

  13. Hailey-Hailey disease and tight junctions: Claudins 1 and 4 are regulated by ATP2C1 gene encoding Ca(2+) /Mn(2+) ATPase SPCA1 in cultured keratinocytes.

    PubMed

    Raiko, Laura; Siljamäki, Elina; Mahoney, Mỹ G; Putaala, Heli; Suominen, Erkki; Peltonen, Juha; Peltonen, Sirkku

    2012-08-01

    Mutations in the ATP2C1 gene encoding Ca(2+) /Mn(2+) ATPase SPCA1 cause Hailey-Hailey disease (HHD, OMIM 16960). HHD is characterized by epidermal acantholysis. We attempted to model HHD using normal keratinocytes, in which the SPCA1 mRNA was down-regulated with the small inhibitory RNA (siRNA) method. SiRNA inhibition significantly down-regulated the SPCA1 mRNA, as demonstrated by qPCR, and decreased the SPCA1 protein beyond detectable level, as shown by Western analysis. The expression of selected desmosomal, adherens and tight junction (TJ) proteins was then studied in the SPCA1-deficient and control keratinocytes cultured in low (0.06 mm) or high (1.2 mm) calcium concentration. The mRNA and protein levels of most TJ components were up-regulated in non-treated control keratinocyte cultures upon switch from low to high calcium concentration. In contrast, SPCA1-deficient keratinocytes displayed high levels of TJ proteins claudins 1 and 4 even in low calcium. ZO-1 did not, however, follow similar expression patterns. Protein levels of occludin, beta-catenin, E-cadherin, desmoplakin, desmogleins 1-3, desmocollin 2/desmocollin 3 and plakoglobin did not show marked changes in SPCA1-deficient keratinocytes. Indirect immunofluorescence labelling revealed delayed translocation of desmoplakin and desmoglein 3 in desmosomes and increased intracellular pools of TJ and desmosomal components in SPCA1-inhibited keratinocytes. The results show that SPCA1 regulates the levels of claudins 1 and 4, but does not affect desmosomal protein levels, indicating that TJ proteins are differently regulated. The results also suggest a potential role for claudins in HHD. PMID:22639968

  14. Hailey-Hailey disease and tight junctions: Claudins 1 and 4 are regulated by ATP2C1 gene encoding Ca2+/Mn2+ ATPase SPCA1 in cultured keratinocytes

    PubMed Central

    Raiko, Laura; Siljamäki, Elina; Mahoney, Mỹ G.; Putaala, Heli; Suominen, Erkki; Peltonen, Juha; Peltonen, Sirkku

    2013-01-01

    Mutations in the ATP2C1 gene encoding Ca2+/Mn2+ ATPase SPCA1 cause Hailey-Hailey disease (HHD, OMIM 16960). HHD is characterized by epidermal acantholysis. We attempted to model HHD using normal keratinocytes in which the SPCA1 mRNA was down-regulated with the small inhibitory RNA (siRNA) method. SiRNA inhibition significantly down-regulated the SPCA1 mRNA, as demonstrated by qPCR, and decreased the SPCA1 protein beyond detectable level, as shown by western analysis. The expression of selected desmosomal, adherens and tight junction (TJ) proteins was then studied in the SPCA1-deficient and control keratinocytes cultured in low (0.06 mM) or high (1.2 mM) calcium concentration. The mRNA and protein levels of most TJ components were up-regulated in non-treated control keratinocyte cultures upon switch from low to high calcium concentration. In contrast, SPCA1-deficient keratinocytes displayed high levels of TJ proteins claudin 1 and 4 even in low calcium. ZO-1 did not however follow similar expression patterns. Protein levels of occludin, betacatenin, E-cadherin, desmoplakin, desmogleins 1-3, desmocollin 2/3 and plakoglobin did not show marked changes in SPCA1 deficient keratinocytes. Indirect immunofluorescence labeling revealed delayed translocation of desmoplakin and desmoglein 3 in desmosomes, and increased intracellular pools of TJ and desmosomal components in SPCA1 inhibited keratinocytes. The results show that SPCA1 regulates the levels of claudins 1 and 4, but does not affect desmosomal protein levels, indicating that TJ proteins are differently regulated. The results also suggest a potential role for claudins in HHD. PMID:22639968

  15. Build them up and break them down: Tight junctions of cell lines expressing typical hepatocyte polarity with a varied repertoire of claudins.

    PubMed

    Grosse, Brigitte; Degrouard, Jeril; Jaillard, Danielle; Cassio, Doris

    2013-10-01

    Tight junctions (TJs) of cells expressing simple epithelial polarity have been extensively studied, but less is known about TJs of cells expressing complex polarity. In this paper we analyzed, TJs of four different lines, that form bile canaliculi (BC) and express typical hepatocyte polarity; WIF-B9, 11-3, Can 3-1, Can 10. Striking differences were observed in claudin expression. None of the cell lines produced claudin-1. WIF-B9 and 11-3 expressed only claudin-2 while Can 3-1 and Can 10 expressed claudin-2,-3,-4,-5. TJs of these two classes of lines differed in their ultra-stucture, paracellular permeability, and robustness. Lines expressing a large claudin repertoire, especially Can 10, had complex and efficient TJs, that were maintained when cells were depleted in calcium. Inversely, TJs of WIF-B9 and 11-3 were leaky, permissive and dismantled by calcium depletion. Interestingly, we found that during the polarization process, TJ proteins expressed by all lines were sequentially settled in a specific order: first occludin, ZO-1 and cingulin, then JAM-A and ZO-2, finally claudin-2. Claudins expressed only in Can lines were also sequentially settled: claudin-3 was the first settled. Inhibition of claudin-3 expression delayed BC formation in Can10 and induced the expression of simple epithelial polarity. These results highlight the role of claudins in the settlement and the efficiency of TJs in lines expressing typical hepatocyte polarity. Can 10 seems to be the most promising of these lines because of its claudin repertoire near that of hepatocytes and its capacity to form extended tubular BC sealed by efficient TJs. PMID:24665408

  16. Differential Expression of Claudin Family Members during Osteoblast and Osteoclast Differentiation: Cldn-1 Is a Novel Positive Regulator of Osteoblastogenesis

    PubMed Central

    Alshbool, Fatima Z.; Mohan, Subburaman

    2014-01-01

    Claudins (Cldns), a family of 27 transmembrane proteins, represent major components of tight junctions. Aside from functioning as tight junctions, Cldns have emerging roles as regulators of cell proliferation and differentiation. While Cldns are known to be expressed and have important functions in various tissues, their expression and function in bone cells is ill-defined. In this study, the expression of Cldns was examined during osteoblast and osteoclast differentiation. The expression of Cldn-1, -7, -11, and -15 was downregulated during early stages of osteoclast differentiation, whereas Cldn-6 was upregulated. Moreover, the expression of several Cldns increased 3–7 fold in fully differentiated osteoclasts. As for osteoblasts, the expression of several Cldns was found to increase more than 10-fold during differentiation, with some peaking at early, and others at late stages. By contrast, only expression of Cldn-12, and -15 decreased during osteoblast differentiation. In subsequent studies, we focused on the role of Cldn-1 in osteoblasts as its expression was increased by more than 10 fold during osteoblast differentiation and was found to be regulated by multiple osteoregulatory agents including IGF-1 and Wnt3a. We evaluated the consequence of lentiviral shRNA-mediated knockdown of Cldn-1 on osteoblast proliferation and differentiation using MC3T3-E1 mouse osteoblasts. Cldn-1 knockdown caused a significant reduction in MC3T3-E1 cell proliferation and ALP activity. Accordingly, expression levels of cyclinD1 and ALP mRNA levels were reduced in Cldn-1 shRNA knockdown cells. We next determined if Cldn-1 regulates the expression of Runx-2 and osterix, master transcription factors of osteoblast differentiation, and found that their levels were reduced significantly as a consequence of Cldn-1 knockdown. Moreover, knocking down Cldn-1 reduced β-catenin level. In conclusion, the expression of Cldn family members during bone cell differentiation is complex and

  17. Stanniocalicin 2 Suppresses Breast Cancer Cell Migration and Invasion via the PKC/Claudin-1-Mediated Signaling

    PubMed Central

    Yang, Lina; Yu, Xiaoli; Yang, Zhaozhi; Deng, Yun; Meng, Jiao; Feng, Yan; Guo, Xiaomao; Yang, Gong

    2015-01-01

    Stanniocalcin (STC), a glycoprotein hormone, is expressed in a wide variety of tissues to regulate Ca2+ and PO4- homeostasis. STC2, a member of STC family, has been reported to be associated with tumor development. In this study, we investigated whether the expression of STC2 is associated with migration and invasion of breast cancer cells. We found that breast cancer cell line 231 HM transfected with STC2 shRNA displayed high motility, fibroblast morphology, and enhanced cell migration and invasion. Introduction of STC2 in 231 cells reduced cell migration and invasion. In response to irradiation, silencing of STC2 in 231 HM cells reduced apoptosis, whereas overexpression of STC2 in 231 cells promoted apoptosis, compared with in control cells. Mechanistic study showed that STC2 negatively regulated PKC to control the expression of Claudin-1, which subsequently induced the expressions of EMT-related factors including ZEB1, ZO-1, Slug, Twist, and MMP9. Suppression of PKC activity by using a PKC inhibitor (Go 6983) restored the normal motility of STC2-silenced cells. Furthermore, in vivo animal assay showed that STC2 inhibited tumorigenesis and metastasis of breast cancer cells. Collectively, these results indicate that STC2 may inhibit EMT at least partially through the PKC/Claudin-1-mediated signaling in human breast cancer cells. Thus, STC2 may be exploited as a biomarker for metastasis and targeted therapy in human breast cancer. PMID:25830567

  18. Claudins and the kidney.

    PubMed

    Hou, Jianghui; Rajagopal, Madhumitha; Yu, Alan S L

    2013-01-01

    Claudins are tight junction membrane proteins that regulate paracellular permeability of renal epithelia to small ions, solutes, and water. Claudins interact within the cell membrane and between neighboring cells to form tight junction strands and constitute both the paracellular barrier and the pore. The first extracellular domain of claudins is thought to be the pore-lining domain and contains the determinants of charge selectivity. Multiple claudins are expressed in different nephron segments; such differential expression likely determines the permeability properties of each segment. Recent evidence has identified claudin-2 as constituting the cation-reabsorptive pathway in the proximal tubule; claudin-14, -16, and -19 as forming a complex that regulates calcium transport in the thick ascending limb of the loop of Henle; and claudin-4, -7, and -8 as determinants of collecting duct chloride permeability. Mutations in claudin-16 and -19 cause familial hypercalciuric hypomagnesemia with nephrocalcinosis. The roles of other claudins in kidney diseases remain to be fully elucidated. PMID:23140368

  19. Claudin and ovarian cancer

    PubMed Central

    Bose, Chinmoy K.; Mukhopadhyay, Ashis

    2010-01-01

    Claudins are a family of proteins and the most important component of the tight junction. They constitute a paracellular barrier that controls the flow of molecules in the intercellular space of an epithelium. Although it seems that claudin should be down regulated in cancer cell, some claudins are, in fact highly elevated in various human cancers, including ovarian cancer. Whereas the functional significance of claudin overexpression in ovarian carcinoma is unclear, these proteins are important for migration, invasion, and survival of ovarian cancer cells. They clearly represent a general pathway in tumorigenesis, are a novel marker for ovarian cancer and may become a target for therapy or diagnosis of this disease. PMID:24591894

  20. Claudin and ovarian cancer.

    PubMed

    Bose, Chinmoy K; Mukhopadhyay, Ashis

    2010-01-01

    Claudins are a family of proteins and the most important component of the tight junction. They constitute a paracellular barrier that controls the flow of molecules in the intercellular space of an epithelium. Although it seems that claudin should be down regulated in cancer cell, some claudins are, in fact highly elevated in various human cancers, including ovarian cancer. Whereas the functional significance of claudin overexpression in ovarian carcinoma is unclear, these proteins are important for migration, invasion, and survival of ovarian cancer cells. They clearly represent a general pathway in tumorigenesis, are a novel marker for ovarian cancer and may become a target for therapy or diagnosis of this disease. PMID:24591894

  1. Dehydroepiandrosterone Sulfate Stimulates Expression of Blood-Testis-Barrier Proteins Claudin-3 and -5 and Tight Junction Formation via a Gnα11-Coupled Receptor in Sertoli Cells.

    PubMed

    Papadopoulos, Dimitrios; Dietze, Raimund; Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2016-01-01

    Dehydroepiandrosterone sulfate (DHEAS) is a circulating sulfated steroid considered to be a pro-androgen in mammalian physiology. Here we show that at a physiological concentration (1 μM), DHEAS induces the phosphorylation of the kinase Erk1/2 and of the transcription factors CREB and ATF-1 in the murine Sertoli cell line TM4. This signaling cascade stimulates the expression of the tight junction (TJ) proteins claudin-3 and claudin-5. As a consequence of the increased expression, tight junction connections between neighboring Sertoli cells are augmented, as demonstrated by measurements of transepithelial resistance. Phosphorylation of Erk1/2, CREB, or ATF-1 is not affected by the presence of the steroid sulfatase inhibitor STX64. Erk1/2 phosphorylation was not observed when dehydroepiandrosterone (DHEA) was used instead of DHEAS. Abrogation of androgen receptor (AR) expression by siRNA did not affect DHEAS-stimulated Erk1/2 phosphorylation, nor did it change DHEAS-induced stimulation of claudin-3 and claudin-5 expression. All of the above indicate that desulfation and conversion of DHEAS into a different steroid hormone is not required to trigger the DHEAS-induced signaling cascade. All activating effects of DHEAS, however, are abolished when the expression of the G-protein Gnα11 is suppressed by siRNA, including claudin-3 and -5 expression and TJ formation between neighboring Sertoli cells as indicated by reduced transepithelial resistance. Taken together, these results are consistent with the effects of DHEAS being mediated through a membrane-bound G-protein-coupled receptor interacting with Gnα11 in a signaling pathway that resembles the non-classical signaling pathways of steroid hormones. Considering the fact that DHEAS is produced in reproductive organs, these findings also suggest that DHEAS, by acting as an autonomous steroid hormone and influencing the formation and dynamics of the TJ at the blood-testis barrier, might play a crucial role for the

  2. Dehydroepiandrosterone Sulfate Stimulates Expression of Blood-Testis-Barrier Proteins Claudin-3 and -5 and Tight Junction Formation via a Gnα11-Coupled Receptor in Sertoli Cells

    PubMed Central

    Papadopoulos, Dimitrios; Dietze, Raimund; Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2016-01-01

    Dehydroepiandrosterone sulfate (DHEAS) is a circulating sulfated steroid considered to be a pro-androgen in mammalian physiology. Here we show that at a physiological concentration (1 μM), DHEAS induces the phosphorylation of the kinase Erk1/2 and of the transcription factors CREB and ATF-1 in the murine Sertoli cell line TM4. This signaling cascade stimulates the expression of the tight junction (TJ) proteins claudin-3 and claudin-5. As a consequence of the increased expression, tight junction connections between neighboring Sertoli cells are augmented, as demonstrated by measurements of transepithelial resistance. Phosphorylation of Erk1/2, CREB, or ATF-1 is not affected by the presence of the steroid sulfatase inhibitor STX64. Erk1/2 phosphorylation was not observed when dehydroepiandrosterone (DHEA) was used instead of DHEAS. Abrogation of androgen receptor (AR) expression by siRNA did not affect DHEAS-stimulated Erk1/2 phosphorylation, nor did it change DHEAS-induced stimulation of claudin-3 and claudin-5 expression. All of the above indicate that desulfation and conversion of DHEAS into a different steroid hormone is not required to trigger the DHEAS-induced signaling cascade. All activating effects of DHEAS, however, are abolished when the expression of the G-protein Gnα11 is suppressed by siRNA, including claudin-3 and -5 expression and TJ formation between neighboring Sertoli cells as indicated by reduced transepithelial resistance. Taken together, these results are consistent with the effects of DHEAS being mediated through a membrane-bound G-protein-coupled receptor interacting with Gnα11 in a signaling pathway that resembles the non-classical signaling pathways of steroid hormones. Considering the fact that DHEAS is produced in reproductive organs, these findings also suggest that DHEAS, by acting as an autonomous steroid hormone and influencing the formation and dynamics of the TJ at the blood-testis barrier, might play a crucial role for the

  3. Monoclonal Antibodies against Extracellular Domains of Claudin-1 Block Hepatitis C Virus Infection in a Mouse Model

    PubMed Central

    Nagase, Shotaro; Shirasago, Yoshitaka; Iida, Manami; Yamashita, Mayo; Endo, Kohki; Yagi, Kiyohito; Suzuki, Tetsuro; Wakita, Takaji; Hanada, Kentaro; Kuniyasu, Hiroki

    2015-01-01

    ABSTRACT Hepatitis C virus (HCV) entry into host cells is a complex process requiring multiple host factors, including claudin-1 (CLDN1). Safe and effective therapeutic entry inhibitors need to be developed. We isolated a human hepatic Huh7.5.1-derived cell mutant that is nonpermissive to HCV, and comparative microarray analysis showed that the mutant was CLDN1 defective. Four hybridomas were obtained, which produced monoclonal antibodies (MAbs) that interacted with the parental Huh7.5.1 cell but not with the CLDN1-defective mutant. All MAbs produced by these hybridomas specifically bound to human CLDN1 with a very high affinity and prevented HCV infection of Huh7.5.1 cells in a dose-dependent manner, without apparent cytotoxicity. Two selected MAbs also inhibited HCV infection of human liver-chimeric mice without significant adverse effects. CLDN1 may be a potential target to prevent HCV infection in vivo. Anti-CLDN1 MAbs may hence be promising candidates as novel anti-HCV agents. IMPORTANCE Safe and effective therapeutic entry inhibitors against hepatitis C virus (HCV) are very useful for combination therapies with other anti-HCV drugs, such as direct-acting antivirals. In this study, we first showed an effective strategy for developing functional monoclonal antibodies (MAbs) against extracellular domains of a multimembrane-spanning target protein, claudin-1 (CLDN1), by using parental cells expressing the intact target membrane protein and target-defective cells. The established MAbs against CLDN1, which had a very high affinity for intact CLDN1, efficiently inhibited in vitro and in vivo HCV infections. These anti-CLDN1 MAbs are promising leads for novel entry inhibitors against HCV. PMID:25673725

  4. Expression of claudin-5, claudin-7 and occludin in oral squamous cell carcinoma and their clinico-pathological significance

    PubMed Central

    Phattarataratip, Ekarat

    2016-01-01

    Background Claudin and occludin are the important tight junctions protein in human. The downregulation or upregulation of claudins and occludin might have a role in cancer development. The objective of this study was to investigate the expression of claudin-5, claudin-7 and occludin in oral squamous cell carcinoma (OSCC) and their relationships with the prognostically-related clinico-pathologic features. Material and Methods Standard indirect immunohistochemical technique using anti-claudin-5, anti-claudin-7 and anti-occludin was performed in formalin-fixed paraffin-embedded tissue sections of 66 OSCC samples from Faculty of Dentistry, Chulalongkorn University. The positive cases were divided into 2 groups, the low expression group (cases with less than 50% of positive cancer cells) and the high expression group for statistical analysis. Categorical analysis of the clinico-pathologic parameters together with univariate analysis using the Kaplan-Meier method and the log rank test were performed. Results There were 22 male and 23 female patients enrolled in this study, with a mean age of 65.82+12.10 years. The claudin-5 immunoreactivity was observed in 26.6% of cases. The positive immunoreactivity of claudin-7 is more noted (93.3%). Only 4 cases showed occludin immunoreactivity (8.9%) and all of them show positivity less than 25% of cancer cells. Only loss of claudin-7 expression was associated with the high pathologic grade, advanced TNM staging, large tumor size, the presence of microscopic perineural, vascular invasions and regional lymph node involvement. There is a tendency towards the association of the higher claudin-7 expression and a longer survival time (P=0.012). Conclusions The results showed expression of claudin-5, claudin-7 and low expression of occludin in OSCC. Only claudin-7 expression showed impact on clinic-pathological parameter of OSCC. Key words:Claudin, occludin, oral squamous cell carcinoma, tight junctions, oral cancer. PMID:27398181

  5. Cortisol differentially alters claudin isoforms in cultured puffer fish gill epithelia.

    PubMed

    Bui, Phuong; Bagherie-Lachidan, Mazdak; Kelly, Scott P

    2010-04-12

    A primary cultured gill epithelium from the puffer fish Tetraodon nigroviridis was developed to examine the corticosteroid regulation of claudin isoform mRNA abundance in fish gills. Preparations were composed of polygonal epithelial cells exhibiting concentric apical microridges and zonula occludens-1 immunoreactivity along cell margins. No evidence was found to indicate the presence of Na(+)-K(+)-ATPase-immunoreactive or mitochondria-rich cells in cultured preparations. Therefore, epithelia appear to be composed of gill pavement cells (PVCs) only. An RT-PCR profile of 12 salinity responsive gill claudin tight junction (TJ) proteins (Tncldn3a, -3c, -6, -8d, -10d, -10e, -11a, -23b, -27a, -27c, -32a, and -33b) revealed the absence of Tncldn6, -10d and -10e in cultured epithelia, suggesting that these isoforms are not associated with gill PVCs. Cortisol treatment of cultured epithelia dose-dependently increased or decreased mRNA abundance of select claudin isoforms. Transcript abundance of several claudin isoforms was unaffected by cortisol treatment. These data provide evidence for the cell specific distribution of claudins in fish gills and suggest that heterogeneous alterations in the abundance of select claudin isoforms contribute to the corticosteroid regulation of gill permeability. PMID:19969041

  6. PKCι interacts with Rab14 and modulates epithelial barrier function through regulation of claudin-2 levels

    PubMed Central

    Lu, Ruifeng; Dalgalan, Dogukan; Mandell, Edward K.; Parker, Sara S.; Ghosh, Sourav; Wilson, Jean M.

    2015-01-01

    PKCι is essential for the establishment of epithelial polarity and the normal assembly of tight junctions. We find that PKCι knockdown does not compromise the steady-state distribution of most tight junction proteins but results in increased transepithelial resistance (TER) and decreased paracellular permeability. Analysis of the levels of tight junction components demonstrates that claudin-2 protein levels are decreased. However, other tight junction proteins, such as claudin-1, ZO-1, and occludin, are unchanged. Incubation with an aPKC pseudosubstrate recapitulates the phenotype of PKCι knockdown, including increased TER and decreased levels of claudin-2. In addition, overexpression of PKCι results in increased claudin-2 levels. ELISA and coimmunoprecipitation show that the TGN/endosomal small GTPase Rab14 and PKCι interact directly. Immunolabeling shows that PKCι and Rab14 colocalize in both intracellular puncta and at the plasma membrane and that Rab14 expression is required for normal PKCι distribution in cysts in 3D culture. We showed previously that knockdown of Rab14 results in increased TER and decreased claudin-2. Our results suggest that Rab14 and aPKC interact to regulate trafficking of claudin-2 out of the lysosome-directed pathway. PMID:25694446

  7. Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody.

    PubMed

    Mailly, Laurent; Xiao, Fei; Lupberger, Joachim; Wilson, Garrick K; Aubert, Philippe; Duong, François H T; Calabrese, Diego; Leboeuf, Céline; Fofana, Isabel; Thumann, Christine; Bandiera, Simonetta; Lütgehetmann, Marc; Volz, Tassilo; Davis, Christopher; Harris, Helen J; Mee, Christopher J; Girardi, Erika; Chane-Woon-Ming, Béatrice; Ericsson, Maria; Fletcher, Nicola; Bartenschlager, Ralf; Pessaux, Patrick; Vercauteren, Koen; Meuleman, Philip; Villa, Pascal; Kaderali, Lars; Pfeffer, Sébastien; Heim, Markus H; Neunlist, Michel; Zeisel, Mirjam B; Dandri, Maura; McKeating, Jane A; Robinet, Eric; Baumert, Thomas F

    2015-05-01

    Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer. Cell entry of HCV and other pathogens is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver-chimeric mouse model, we show that a monoclonal antibody specific for the TJ protein claudin-1 (ref. 7) eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection by means of host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy. PMID:25798937

  8. Generation and characterization of a human-mouse chimeric antibody against the extracellular domain of claudin-1 for cancer therapy using a mouse model.

    PubMed

    Hashimoto, Yosuke; Tada, Minoru; Iida, Manami; Nagase, Shotaro; Hata, Tomoyuki; Watari, Akihiro; Okada, Yoshiaki; Doi, Takefumi; Fukasawa, Masayoshi; Yagi, Kiyohito; Kondoh, Masuo

    2016-08-12

    Claudin-1 (CLDN-1), an integral transmembrane protein, is an attractive target for drug absorption, prevention of infection, and cancer therapy. Previously, we generated mouse anti-CLDN-1 monoclonal antibodies (mAbs) and found that they enhanced epidermal absorption of a drug and prevented hepatitis C virus infection in human hepatocytes. Here, we investigated anti-tumor activity of a human-mouse chimeric IgG1, xi-3A2, from one of the anti-CLDN-1 mAbs, clone 3A2. Xi-3A2 accumulated in the tumor tissues in mice bearing with human CLDN-1-expressing tumor cells. Xi-3A2 activated Fcγ receptor IIIa-expressing reporter cells in the presence of human CLDN-1-expressing cells, suggesting xi-3A2 has a potential to exhibit antibody-dependent cellular cytotoxicity against CLDN-1 expressing tumor cells. We also constructed a mutant xi-3A2 antibody with Gly, Ser, and Ile substituted with Ala, Asp, and Arg at positions 236, 239, and 332 of the Fc domain. This mutant antibody showed greater activation of Fcγ receptor IIIa and in vivo anti-tumor activity in mice bearing human CLDN-1-expressing tumors than xi-3A2 did. These findings indicate that the G236A/S239D/I332E mutant of xi-3A2 might be a promising lead for tumor therapy. PMID:27286708

  9. Tongxinluo Reverses the Hypoxia-suppressed Claudin-9 in Cardiac Microvascular Endothelial Cells

    PubMed Central

    Liu, Kun; Wang, Xiu-Juan; Li, Yan-Ning; Li, Bin; Qi, Jin-Sheng; Zhang, Jing; Wang, Yu

    2016-01-01

    Background: Claudin-5, claudin-9, and claudin-11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeability, which is the initiating process and pathological basis of cardiovascular disease. Although tongxinluo (TXL) has satisfactory antianginal effects, whether and how it modulates claudin-5, claudin-9, and claudin-11 in hypoxia-stimulated human cardiac microvascular endothelial cells (HCMECs) have not been reported. Methods: In this study, HCMECs were stimulated with CoCl2 to mimic hypoxia and treated with TXL. First, the messenger RNA (mRNA) expression of claudin-5, claudin-9, and claudin-11 was confirmed. Then, the protein content and distribution of claudin-9, as well as cell morphological changes were evaluated after TXL treatment. Furthermore, the distribution and content histone H3K9 acetylation (H3K9ac) in the claudin-9 gene promoter, which guarantees transcriptional activation, were examined to explore the underlying mechanism, by which TXL up-regulates claudin-9 in hypoxia-stimulated HCMECs. Results: We found that hypoxia-suppressed claudin-9 gene expression in HCMECs (F = 7.244; P = 0.011) and the hypoxia-suppressed claudin-9 could be reversed by TXL (F = 61.911; P = 0.000), which was verified by its protein content changes (F = 29.142; P = 0.000). Moreover, high-dose TXL promoted the cytomembrane localization of claudin-9 in hypoxia-stimulated HCMECs, with attenuation of cell injury. Furthermore, high-dose TXL elevated the hypoxia-inhibited H3K9ac in the claudin-9 gene promoter (F = 37.766; P = 0.000), activating claudin-9 transcription. Conclusions: The results manifested that TXL reversed the hypoxia-suppressed claudin-9 by elevating H3K9ac in its gene promoter, playing protective roles in HCMECs. PMID:26879018

  10. Mode of action of claudin peptidomimetics in the transient opening of cellular tight junction barriers.

    PubMed

    Staat, Christian; Coisne, Caroline; Dabrowski, Sebastian; Stamatovic, Svetlana M; Andjelkovic, Anuska V; Wolburg, Hartwig; Engelhardt, Britta; Blasig, Ingolf E

    2015-06-01

    In epithelial/endothelial barriers, claudins form tight junctions, seal the paracellular cleft, and limit the uptake of solutes and drugs. The peptidomimetic C1C2 from the C-terminal half of claudin-1's first extracellular loop increases drug delivery through epithelial claudin-1 barriers. However, its molecular and structural mode of action remains unknown. In the present study, >100 μM C1C2 caused paracellular opening of various barriers with different claudin compositions, ranging from epithelial to endothelial cells, preferentially modulating claudin-1 and claudin-5. After 6 h incubation, C1C2 reversibly increased the permeability to molecules of different sizes; this was accompanied by redistribution of claudins and occludin from junctions to cytosol. Internalization of C1C2 in epithelial cells depended on claudin-1 expression and clathrin pathway, whereby most C1C2 was retained in recyclosomes >2 h. In freeze-fracture electron microscopy, C1C2 changed claudin-1 tight junction strands to a more parallel arrangement and claudin-5 strands from E-face to P-face association - drastic and novel effects. In conclusion, C1C2 is largely recycled in the presence of a claudin, which explains the delayed onset of barrier and junction loss, the high peptide concentration required and the long-lasting effect. Epithelial/endothelial barriers are specifically modulated via claudin-1/claudin-5, which can be targeted to improve drug delivery. PMID:25907035

  11. STAT3 signaling is activated preferentially in tumor-initiating cells in claudin-low models of human breast cancer.

    PubMed

    Wei, Wei; Tweardy, David J; Zhang, Mei; Zhang, Xiaomei; Landua, John; Petrovic, Ivana; Bu, Wen; Roarty, Kevin; Hilsenbeck, Susan G; Rosen, Jeffrey M; Lewis, Michael T

    2014-10-01

    In breast cancer, a subset of tumor-initiating cells (TIC) or "cancer stem cells" are thought to be responsible for tumor maintenance, treatment resistance, and disease recurrence. While current breast cancer stem cell markers (e.g., CD44(high) /CD24(low/neg) , ALDH positive) have allowed enrichment for such cells, they are not universally expressed and may actually identify distinct TIC subpopulations in the same tumor. Thus, additional markers of functional stem cells are needed. The STAT3 pathway is a critical regulator of the function of normal stem cells, and evidence is accumulating for its important role in breast cancer stem cells. However, due to the lack of a method for separating live cells based on their level of STAT3 activity, it remains unknown whether STAT3 functions in the cancer stem cells themselves, or in surrounding niche cells, or in both. To approach this question, we constructed a series of lentiviral fluorescent (enhanced green fluorescent protein, EGFP) reporters that enabled flow cytometric enrichment of cells differing in STAT3-mediated transcriptional activity, as well as in vivo/in situ localization of STAT3 responsive cells. Using in vivo claudin-low cell line xenograft models of human breast cancer, we found that STAT3 signaling reporter activity (EGFP(+) ) is associated with a subpopulation of cancer cells enriched for mammosphere-forming efficiency, as well as TIC function in limiting dilution transplantation assays compared to negative or unsorted populations. Our results support STAT3 signaling activity as another functional marker for human breast cancer stem cells thus making it an attractive therapeutic target for stem-cell-directed therapy in some breast cancer subtypes. PMID:24891218

  12. Cardiotonic steroid ouabain stimulates expression of blood-testis barrier proteins claudin-1 and -11 and formation of tight junctions in Sertoli cells.

    PubMed

    Dietze, Raimund; Shihan, Mazen; Stammler, Angelika; Konrad, Lutz; Scheiner-Bobis, Georgios

    2015-04-15

    The interaction of ouabain with the sodium pump induces signalling cascades resembling those triggered by hormone/receptor interactions. In the rat Sertoli cell line 93RS2, ouabain at low concentrations stimulates the c-Src/c-Raf/Erk1/2 signalling cascade via its interaction with the α4 isoform of the sodium pump expressed in these cells, leading to the activation of the transcription factor CREB. As a result of this signalling sequence, ouabain stimulates expression of claudin-1 and claudin-11, which are also controlled by a CRE promoter. Both of these proteins are known to be essential constituents of tight junctions (TJ) between Sertoli cells, and as a result of the ouabain-induced signalling TJ formation between neighbouring Sertoli cells is significantly enhanced by the steroid. Thus, ouabain-treated cell monolayers display higher transepithelial resistance and reduced free diffusion of FITC-coupled dextran in tracer diffusion assays. Taking into consideration that the formation of TJ is indispensable for the maintenance of the blood-testis barrier (BTB) and therefore for male fertility, the actions of ouabain described here and the fact that this and other related cardiotonic steroids (CTS) are produced endogenously suggest a direct influence of ouabain/sodium pump interactions on the maintenance of the BTB and thereby an effect on male fertility. Since claudin-1 and claudin-11 are also present in other blood-tissue barriers, one can speculate that ouabain and perhaps other CTS influence the dynamics of these barriers as well. PMID:25666991

  13. Comparable mRNA expression of inflammatory markers but lower claudin-1 mRNA levels in foreskin tissue of HSV-2 seropositive versus seronegative asymptomatic Kenyan young men

    PubMed Central

    Röhl, Maria; Tjernlund, Annelie; Mehta, Supriya D; Pettersson, Pernilla; Bailey, Robert C; Broliden, Kristina

    2015-01-01

    Objectives Skin biopsies from local sites of herpes simplex virus 2 (HSV-2)-induced ulcers can show infiltrates of inflammatory cells several months after macroscopic healing. We hypothesise that foreskin tissue samples of asymptomatic HSV-2 seropositive men had remaining signs of inflammation at the molecular level. Even in the absence of clinical lesions, genital inflammation may contribute to increased HIV susceptibility on sexual exposure to the virus. Setting Foreskin tissue samples were collected from men undergoing elective circumcision in Kisumu, Kenya. Participants The foreskin tissue samples (n=86) were stratified into study groups based on HSV-2 serology and assessed for mRNA expression of inflammatory markers. Markers of interest were further assessed by immunohistochemical staining within the tissue samples. Results The two study groups had comparable levels of all molecular markers (CD3, CD4, CD8, CD69, CCR5, HLA-DR, Langerin, DC-SIGN, Mannose Receptor 1, IL-1, IL-6, TNF-α, β7, IgA, IFN-α, CCL5, E-cadherin, ZO-1 and occludin), except for lower mRNA levels of the epithelial junction protein claudin-1 in the HSV-2 seropositive group (p=0.008). Although mRNA levels of claudin-1 were lower in HSV-2 seropositive individuals, the corresponding protein could be visualised in the foreskin epithelium of all samples tested. Conclusions Whereas no general inflammation was demonstrated in the foreskin of asymptomatic HSV-2 seropositive individuals, a decreased expression of claudin-1 indicates a less robust genital epithelial barrier. An intact epithelial barrier is essential for blocking mucosal entry of genital infections, including HIV. PMID:25694458

  14. Atelectrauma disrupts pulmonary epithelial barrier integrity and alters the distribution of tight junction proteins ZO-1 and claudin 4.

    PubMed

    Jacob, Anne-Marie; Gaver, Donald P

    2012-11-01

    Mechanical ventilation inevitably exposes the delicate tissues of the airways and alveoli to abnormal mechanical stresses that can induce pulmonary edema and exacerbate conditions such as acute respiratory distress syndrome. The goal of our research is to characterize the cellular trauma caused by the transient abnormal fluid mechanical stresses that arise when air is forced into a liquid-occluded airway (i.e., atelectrauma). Using a fluid-filled, parallel-plate flow chamber to model the "airway reopening" process, our in vitro study examined consequent increases in pulmonary epithelial plasma membrane rupture, paracellular permeability, and disruption of the tight junction (TJ) proteins zonula occludens-1 and claudin-4. Computational analysis predicts the normal and tangential surface stresses that develop between the basolateral epithelial membrane and underlying substrate due to the interfacial stresses acting on the apical cell membrane. These simulations demonstrate that decreasing the velocity of reopening causes a significant increase in basolateral surface stresses, particularly in the region between neighboring cells where TJs concentrate. Likewise, pulmonary epithelial wounding, paracellular permeability, and TJ protein disruption were significantly greater following slower reopening. This study thus demonstrates that maintaining a higher velocity of reopening, which reduces the damaging fluid stresses acting on the airway wall, decreases the mechanical stresses on the basolateral cell surface while protecting cells from plasma membrane rupture and promoting barrier integrity. PMID:22898551

  15. Atelectrauma disrupts pulmonary epithelial barrier integrity and alters the distribution of tight junction proteins ZO-1 and claudin 4

    PubMed Central

    Jacob, Anne-Marie

    2012-01-01

    Mechanical ventilation inevitably exposes the delicate tissues of the airways and alveoli to abnormal mechanical stresses that can induce pulmonary edema and exacerbate conditions such as acute respiratory distress syndrome. The goal of our research is to characterize the cellular trauma caused by the transient abnormal fluid mechanical stresses that arise when air is forced into a liquid-occluded airway (i.e., atelectrauma). Using a fluid-filled, parallel-plate flow chamber to model the “airway reopening” process, our in vitro study examined consequent increases in pulmonary epithelial plasma membrane rupture, paracellular permeability, and disruption of the tight junction (TJ) proteins zonula occludens-1 and claudin-4. Computational analysis predicts the normal and tangential surface stresses that develop between the basolateral epithelial membrane and underlying substrate due to the interfacial stresses acting on the apical cell membrane. These simulations demonstrate that decreasing the velocity of reopening causes a significant increase in basolateral surface stresses, particularly in the region between neighboring cells where TJs concentrate. Likewise, pulmonary epithelial wounding, paracellular permeability, and TJ protein disruption were significantly greater following slower reopening. This study thus demonstrates that maintaining a higher velocity of reopening, which reduces the damaging fluid stresses acting on the airway wall, decreases the mechanical stresses on the basolateral cell surface while protecting cells from plasma membrane rupture and promoting barrier integrity. PMID:22898551

  16. Claudin-1 Expression Is Elevated in Colorectal Cancer Precursor Lesions Harboring the BRAF V600E Mutation12

    PubMed Central

    Caruso, Maria; Fung, Kim Y.C.; Moore, James; Brierley, Gemma V.; Cosgrove, Leah J.; Thomas, Michelle; Cheetham, Glenice; Brook, Emma; Fraser, Louise M.; Tin, Teresa; Tran, Ha; Ruszkiewicz, Andrew

    2014-01-01

    BACKGROUND: Sessile serrated adenomas/polyps (SSA/P) are now recognised precursors of colorectal cancer (CRC) including cancers harbouring somatic BRAF (V600E) mutations. While the morphological diagnostic criteria of SSA/P have been established, distinguishing between small/early SSA/P and microvesicular hyperplastic polyps (MVHP) is challenging and may not be possible in routine practice. METHODS: Gene expression profiling of MVHP (n=5, all BRAF V600E wild-type) and SSA/P (n=5, all BRAF V600E mutant) samples was performed. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and immunohistochemical analysis was performed to verify the expression of claudin 1 (CLDN1) in MVHP and SSA/P. RESULTS: Gene expression profiling studies conducted between MVHP and SSA/P identified CLDN1 as the most statistically significant differentially expressed gene (p<0.05). Validation with qRT-PCR confirmed an up-regulation of CLDN1 in BRAF V600E mutant polyps regardless of polyp type (p<0.0005). Immunohistochemical analysis of CLDN1 expression in BRAF V600E mutant SSA/Ps (n=53) and MVHPs (n=111) and BRAF wild-type MVHPs (n=58), demonstrated a strong correlation between CLDN1 expression and the BRAF V600E mutation in both SSA/P and MVHP samples when compared to wild-type polyps (p<0.0001). CONCLUSION: This study demonstrates an up regulation of CLDN1 protein in serrated colorectal polyps including MVHP harbouring the BRAF V600E mutation. Our results demonstrated an apparent heterogeneity on the molecular level within the MVHP group and suggest that MVHP with somatic BRAF V600E mutation and up-regulated expression of CLDN1 are closely related to SSA/P and may in fact represent a continuous spectrum of the same neoplastic process within the serrated pathway of colorectal carcinogenesis. PMID:24954356

  17. Claudins in intestines

    PubMed Central

    Lu, Zhe; Ding, Lei; Lu, Qun; Chen, Yan-Hua

    2013-01-01

    Intestines are organs that not only digest food and absorb nutrients, but also provide a defense barrier against pathogens and noxious agents ingested. Tight junctions (TJs) are the most apical component of the junctional complex, providing one form of cell-cell adhesion in enterocytes and playing a critical role in regulating paracellular barrier permeability. Alteration of TJs leads to a number of pathophysiological diseases causing malabsorption of nutrition and intestinal structure disruption, which may even contribute to systemic organ failure. Claudins are the major structural and functional components of TJs with at least 24 members in mammals. Claudins have distinct charge-selectivity, either by tightening the paracellular pathway or functioning as paracellular channels, regulating ions and small molecules passing through the paracellular pathway. In this review, we have discussed the functions of claudin family members, their distribution and localization in the intestinal tract of mammals, their alterations in intestine-related diseases and chemicals/agents that regulate the expression and localization of claudins as well as the intestinal permeability, which provide a therapeutic view for treating intestinal diseases. PMID:24478939

  18. Generation of specific monoclonal antibodies against the extracellular loops of human claudin-3 by immunizing mice with target-expressing cells.

    PubMed

    Ando, Hiroshi; Suzuki, Masayo; Kato-Nakano, Mariko; Kawamoto, Shinobu; Misaka, Hirofumi; Kimoto, Naoya; Furuya, Akiko; Nakamura, Kazuyasu

    2015-01-01

    Human claudin-3 (CLDN3) is a tetraspanin transmembrane protein of tight junction structures and is known to be over-expressed in some malignant tumors. Although a specific monoclonal antibody (MAb) against the extracellular domains of CLDN3 would be a valuable tool, generation of such MAbs has been regarded as difficult using traditional hybridoma techniques, because of the conserved sequence homology of CLDN3s among various species. In addition, high sequence similarity is shared among claudin family members, and potential cross-reactivity of MAb should be evaluated carefully. To overcome these difficulties, we generated CLDN3-expressing Chinese hamster ovary and Sf9 cells to use an immunogens and performed cell-based screening to eliminate cross-reactive antibodies. As a result, we generated MAbs that recognized the extracellular loops of CLDN3 but not those of CLDN4, 5, 6, or 9. Further in vitro studies suggested that the isolated MAbs possessed the desired binding properties for the detection or targeting of CLDN3. PMID:25744656

  19. Omega-3-Acid Ethyl Esters Block the Protumorigenic Effects of Obesity in Mouse Models of Postmenopausal Basal-like and Claudin-Low Breast Cancer.

    PubMed

    Ford, Nikki A; Rossi, Emily L; Barnett, Kelsey; Yang, Peiying; Bowers, Laura W; Hidaka, Brandon H; Kimler, Bruce F; Carlson, Susan E; Shureiqi, Imad; deGraffenried, Linda A; Fabian, Carol J; Hursting, Stephen D

    2015-09-01

    Obesity induces chronic inflammation and is an established risk and progression factor for triple-negative breast cancers, including basal-like (BL) and claudin-low (CL) subtypes. We tested the effects of dietary supplementation with ethyl esters of the marine-derived anti-inflammatory omega-3 fatty acids eicosapentaenoic and docosahexaenoic acid (EPA+DHA; Lovaza) on growth of murine BL and CL mammary tumors. Female ovariectomized C57BL/6 mice were fed a control diet or a diet-induced obesity (DIO) diet with or without EPA+DHA (0.025%, resulting in blood levels of EPA and DHA comparable with women taking Lovaza 4 g/d) for 6 weeks. All mice were then orthotopically injected with Wnt-1 cells (a BL tumor cell suspension derived from MMTV-Wnt-1 transgenic mouse mammary tumors) or M-Wnt cells (a CL tumor cell line cloned from the Wnt-1 tumor cell suspension). Mice were killed when tumors were 1 cm in diameter. EPA+DHA supplementation did not significantly affect Wnt-1 or M-Wnt mammary tumor growth in normoweight control mice. However, EPA+DHA supplementation in DIO mice reduced growth of Wnt-1 and M-Wnt tumors; reduced leptin:adiponectin ratio and proinflammatory eicosanoids in the serum; improved insulin sensitivity; and decreased tumoral expression of COX-2 and phospho-p65. Thus, EPA+DHA supplementation in mouse models of postmenopausal BL and CL breast cancer offsets many of the protumorigenic effects of obesity. These preclinical findings, in combination with results from parallel biomarker studies in women, suggest that EPA+DHA supplementation may reduce the burden of BL and CL breast cancer in obese women. PMID:26100521

  20. Omega-3-Acid Ethyl Esters Block the Pro-tumorigenic Effects of Obesity in Mouse Models of Postmenopausal Basal-Like and Claudin-Low Breast Cancer

    PubMed Central

    Ford, Nikki A.; Rossi, Emily L.; Barnett, Kelsey; Yang, Peiying; Bowers, Laura W.; Hidaka, Brandon H.; Kimler, Bruce F.; Carlson, Susan E.; Shureiqi, Imad; deGraffenried, Linda A.; Fabian, Carol J.; Hursting, Stephen D.

    2015-01-01

    Obesity induces chronic inflammation and is an established risk and progression factor for triple-negative breast cancers, including basal-like (BL) and claudin-low (CL) subtypes. We tested the effects of dietary supplementation with ethyl esters of the marine-derived anti-inflammatory omega-3 fatty acids eicosapentaenoic and docosahexaenoic acid (EPA+DHA; Lovaza®) on growth of murine BL and CL mammary tumors. Female ovariectomized C57BL/6 mice were fed a control diet or a diet-induced obesity (DIO) diet with or without EPA+DHA (0.025%, resulting in blood levels of EPA and DHA comparable to women taking Lovaza 4 g/day) for 6 weeks. All mice were then orthotopically injected with Wnt-1 cells (a BL tumor cell suspension derived from MMTV-Wnt-1 transgenic mouse mammary tumors) or M-Wnt cells (a CL tumor cell line cloned from the Wnt-1 tumor cell suspension). Mice were killed when tumors were 1 cm in diameter. EPA+DHA supplementation did not significantly impact Wnt-1 or M-Wnt mammary tumor growth in normoweight control mice. However, EPA+DHA supplementation in DIO mice reduced growth of Wnt-1 and M-Wnt tumors; reduced leptin:adiponectin ratio and pro-inflammatory eicosanoids in the serum; improved insulin sensitivity; and decreased tumoral expression of cyclooxygenase-2 and phospho-p65. Thus, EPA+DHA supplementation in mouse models of postmenopausal BL and CL breast cancer offsets many of the pro-tumorigenic effects of obesity. These preclinical findings, in combination with results from parallel biomarker studies in women, suggest EPA+DHA supplementation may reduce the burden of BL and CL breast cancer in obese women. PMID:26100521

  1. Claudin-7 expression induces mesenchymal to epithelial transformation (MET) to inhibit colon tumorigenesis.

    PubMed

    Bhat, A A; Pope, J L; Smith, J J; Ahmad, R; Chen, X; Washington, M K; Beauchamp, R D; Singh, A B; Dhawan, P

    2015-08-27

    In normal colon, claudin-7 is one of the highly expressed claudin proteins and its knockdown in mice results in altered epithelial cell homeostasis and neonatal death. Notably, dysregulation of the epithelial homeostasis potentiates oncogenic transformation and growth. However, the role of claudin-7 in the regulation of colon tumorigenesis remains poorly understood. Using a large colorectal cancer (CRC) patient database and mouse models of colon cancer, we found claudin-7 expression to be significantly downregulated in cancer samples. Most notably, forced claudin-7 expression in poorly differentiated and highly metastatic SW620 colon cancer cells induced epithelial characteristics and inhibited their growth in soft agar and tumor growth in vivo. By contrast, knockdown of claudin-7 in HT-29 or DLD-1 cells induced epithelial-to-mesenchymal transition (EMT), colony formation, xenograft-tumor growth in athymic mice and invasion. Importantly, a claudin-7 signature gene profile generated by overlapping the DEGs (differentially expressed genes in a high-throughput transcriptome analysis using claudin-7-manipulated cells) with human claudin-7 signature genes identified high-risk CRC patients. Furthermore, Rab25, a colon cancer suppressor and regulator of the polarized cell trafficking constituted one of the highly upregulated DEGs in claudin-7 overexpressing cells. Notably, silencing of Rab25 expression counteracted the effects of claudin-7 expression and not only increased proliferation and cell invasion but also increased the expression of p-Src and mitogen-activated protein kinase-extracellular signal-regulated kinase 1/2 that were suppressed upon claudin-7 overexpression. Of interest, CRC cell lines, which exhibited decreased claudin-7 expression, also exhibited promoter DNA hypermethylation, a modification associated with transcriptional silencing. Taken together, our data demonstrate a previously undescribed role of claudin-7 as a colon cancer suppressor and suggest

  2. Salmonella Infection Upregulates the Leaky Protein Claudin-2 in Intestinal Epithelial Cells

    PubMed Central

    Zhang, Yong-guo; Wu, Shaoping; Xia, Yinglin; Sun, Jun

    2013-01-01

    Background Tight junctions seal the space between adjacent epithelial cells. Mounting evidence suggests that tight junction proteins play a key role in the pathogenesis of human disease. Claudin is a member of the tight junction protein family, which has 24 members in humans. To regulate cellular function, claudins interact structurally and functionally with membrane and scaffolding proteins via their cytoplasmic domain. In particular, claudin-2 is known to be a leaky protein that contributes to inflammatory bowel disease and colon cancer. However, the involvement of claudin-2 in bacterial infection in the intestine remains unknown. Methods/Principal Findings We hypothesized that Salmonella elevates the leaky protein claudin-2 for its own benefit to facilitate bacterial invasion in the colon. Using a Salmonella-colitis mouse model and cultured colonic epithelial cells, we found that pathogenic Salmonella colonization significantly increases the levels of claudin-2 protein and mRNA in the intestine, but not that of claudin-3 or claudin-7 in the colon, in a time-dependent manner. Immunostaining studies showed that the claudin-2 expression along the crypt-villous axis postinfection. In vitro, Salmonella stimulated claudin-2 expression in the human intestinal epithelial cell lines SKCO15 and HT29C19A. Further analysis by siRNA knockdown revealed that claudin-2 is associated with the Salmonella-induced elevation of cell permeability. Epithelial cells with claudin-2 knockdown had significantly less internalized Salmonella than control cells with normal claudin-2 expression. Inhibitor assays demonstrated that this regulation is mediated through activation of the EGFR pathway and the downstream protein JNK. Conclusion/Significance We have shown that Salmonella targets the tight junction protein claudin-2 to facilitate bacterial invasion. We speculate that this disruption of barrier function contributes to a new mechanism by which bacteria interact with their host cells and

  3. The metastatic microenvironment: Claudin-1 suppresses the malignant phenotype of melanoma brain metastasis.

    PubMed

    Izraely, Sivan; Sagi-Assif, Orit; Klein, Anat; Meshel, Tsipi; Ben-Menachem, Shlomit; Zaritsky, Assaf; Ehrlich, Marcelo; Prieto, Victor G; Bar-Eli, Menashe; Pirker, Christine; Berger, Walter; Nahmias, Clara; Couraud, Pierre-Olivier; Hoon, Dave S B; Witz, Isaac P

    2015-03-15

    Brain metastases occur frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. Identification of molecular determinants regulating melanoma brain metastasis would advance the development of prevention and therapy strategies for this disease. Gene expression profiles of cutaneous and brain-metastasizing melanoma variants from three xenograft tumor models established in our laboratory revealed that expression of tight junction component CLDN1 was lower in the brain-metastasizing variants than in cutaneous variants from the same melanoma. The objective of our study was to determine the significance of CLDN1 downregulation/loss in metastatic melanoma and its role in melanoma brain metastasis. An immunohistochemical analysis of human cells of the melanocyte lineage indicated a significant CLDN1 downregulation in metastatic melanomas. Transduction of melanoma brain metastatic cells expressing low levels of CLDN1 with a CLDN1 retrovirus suppressed their metastatic phenotype. CLDN1-overexpressing melanoma cells expressed a lower ability to migrate and adhere to extracellular matrix, reduced tumor aggressiveness in nude mice and, most importantly, eliminated the formation of micrometastases in the brain. In sharp contrast, the ability of the CLDN1-overexpressing cells to form lung micrometastases was not impaired. CLDN1-mediated interactions between these cells and brain endothelial cells constitute the mechanism underlying these results. Taken together, we demonstrated that downregulation or loss of CLDN1 supports the formation of melanoma brain metastasis, and that CLDN1 expression could be a useful prognostic predictor for melanoma patients with a high risk of brain metastasis. PMID:25046141

  4. Insights into the C-terminal Peptide Binding Specificity of the PDZ Domain of Neuronal Nitric-oxide Synthase: CHARACTERIZATION OF THE INTERACTION WITH THE TIGHT JUNCTION PROTEIN CLAUDIN-3.

    PubMed

    Merino-Gracia, Javier; Costas-Insua, Carlos; Canales, María Ángeles; Rodríguez-Crespo, Ignacio

    2016-05-27

    Neuronal nitric-oxide synthase, unlike its endothelial and inducible counterparts, displays a PDZ (PSD-95/Dlg/ZO-1) domain located at its N terminus involved in subcellular targeting. The C termini of various cellular proteins insert within the binding groove of this PDZ domain and determine the subcellular distribution of neuronal NOS (nNOS). The molecular mechanisms underlying these interactions are poorly understood because the PDZ domain of nNOS can apparently exhibit class I, class II, and class III binding specificity. In addition, it has been recently suggested that the PDZ domain of nNOS binds with very low affinity to the C termini of target proteins, and a necessary simultaneous lateral interaction must take place for binding to occur. We describe herein that the PDZ domain of nNOS can behave as a bona fide class III PDZ domain and bind to C-terminal sequences with acidic residues at the P-2 position with low micromolar binding constants. Binding to C-terminal sequences with a hydrophobic residue at the P-2 position plus an acidic residue at the P-3 position (class II) can also occur, although interactions involving residues extending up to the P-7 position mediate this type of binding. This promiscuous behavior also extends to its association to class I sequences, which must display a Glu residue at P-3 and a Thr residue at P-2 By means of site-directed mutagenesis and NMR spectroscopy, we have been able to identify the residues involved in each specific type of binding and rationalize the mechanisms used to recognize binding partners. Finally, we have analyzed the high affinity association of the PDZ domain of nNOS to claudin-3 and claudin-14, two tight junction tetraspan membrane proteins that are essential components of the paracellular barrier. PMID:27030110

  5. Effects of lactadherin on plasma D-lactic acid and small intestinal MUC2 and claudin-1 expression levels in rats with rotavirus-induced diarrhea

    PubMed Central

    XU, RUI; LEI, YI-HUI; SHI, JUN; ZHOU, YI-JUN; CHEN, YING-WEI; HE, ZHEN-JUAN

    2016-01-01

    The aim of the present study was to investigate the effects of lactadherin on plasma D-lactic acid and small intestinal mucin (MUC) 2 and claudin-1 expression levels in rats with diarrhea induced by rotavirus (RV) infection. A total of 75 seven-day-old healthy Sprague-Dawley rats were randomly divided into the following five groups: Control (C), RV infection (RVI), lactadherin before rotavirus infection (LBRI), lactadherin after rotavirus infection (LARI), and blank (B). On day 4 of artificial feeding, the rats in groups RVI, LBRI and LARI were intragastric administered 1×106 PFU RV; whereas the rats in groups C and B were intragastrically administered an equal volume of maintenance solution from the RV supernatant and normal saline, respectively. In the LBRI and LARI groups, rats received daily intragastric administration of 0.25 mg lactadherin for three days prior to and following infection with RV, respectively. The course of diarrheal symptoms was observed in each group and samples were collected on days 1, 4, and 7 post-infection in order to determine the mucosal morphology, plasma D-lactic acid levels and the expression levels of MUC2 and the intracellular junction protein, claudin-1, in the small intestine. On day 4 post-infection, the rats in group RVI demonstrated severely damaged small intestines and typical diarrheal characteristics, as detected by light microscopy; whereas rats in groups LBRI and LARI demonstrated intact small intestinal villi with partial vacuolation of epithelial cells and changes in the position of their nuclei. Electron microscopy demonstrated that the rats in the RVI group had sparse, shortened, disordered intestinal microvilli and widened intercellular junctions; whereas those in groups LBRI and LARI had long intestinal microvilli sparser compared with groups B and C and slightly widened intercellular junctions. Plasma D-lactic acid levels were increased in groups RVI, LBRI and LARI, as compared with groups B and C, and the

  6. Changes in the Expression and Distribution of Claudins, Increased Epithelial Apoptosis, and a Mannan-Binding Lectin-Associated Immune Response Lead to Barrier Dysfunction in Dextran Sodium Sulfate-Induced Rat Colitis

    PubMed Central

    Yuan, Bosi; Zhou, Shuping; Lu, Youke; Liu, Jiong; Jin, Xinxin; Wan, Haijun; Wang, Fangyu

    2015-01-01

    Background/Aims This animal study aimed to define the underlying cellular mechanisms of intestinal barrier dysfunction. Methods Rats were fed 4% with dextran sodium sulfate (DSS) to induce experimental colitis. We analyzed the sugars in 24-hour urine output by high pressure liquid chromatography. The expression of claudins, mannan-binding lectin (MBL), and MBL-associated serine proteases 2 (MASP-2) were detected in the colonic mucosa by immunohistochemistry; and apoptotic cells in the colonic epithelium were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method assay. Results The lactulose and sucralose excretion levels in the urine of rats with DSS-induced colitis were significantly higher than those in the control rats. Mannitol excretion was lower and lactulose/mannitol ratios and sucralose/mannitol ratios were significantly increased compared with those in the control group (p<0.05). Compared with the controls, the expression of sealing claudins (claudin 3, claudin 5, and claudin 8) was significantly decreased, but that of claudin 1 was increased. The expression of pore-forming claudin 2 was upregulated and claudin 7 was downregulated in DSS-induced colitis. The epithelial apoptotic ratio was 2.8%±1.2% in controls and was significantly increased to 7.2%±1.2% in DSS-induced colitis. The expression of MBL and MASP-2 in the intestinal mucosa showed intense staining in controls, whereas there was weak staining in the rats with colitis. Conclusions There was increased intestinal permeability in DSS-induced colitis. Changes in the expression and distribution of claudins, increased epithelial apoptosis, and the MASP-2-induced immune response impaired the intestinal epithelium and contributed to high intestinal permeability. PMID:25717051

  7. Role of regenerating gene I in claudin expression and barrier function in the small intestine.

    PubMed

    Kitayama, Yoshitaka; Fukui, Hirokazu; Hara, Ken; Eda, Hirotsugu; Kodani, Mio; Yang, Mo; Sun, Chao; Yamagishi, Hidetsugu; Tomita, Toshihiko; Oshima, Tadayuki; Watari, Jiro; Takasawa, Shin; Miwa, Hiroto

    2016-07-01

    We have recently shown that loss of the regenerating gene (Reg) I causes susceptibility to nonsteroidal anti-inflammatory drug-induced gastrointestinal damage. However, the mechanism by which Reg I plays a protective role against this pathophysiological condition is unclear. Here, we investigated whether Reg I plays roles in the induction of tight junction proteins and mucosal barrier function in the small intestine. The small-intestinal permeability was evaluated in Reg I-deficient mice by FITC-dextran and transepithelial electrical resistance (TEER) assay. The effect of REG Iα on TEER, claudins expression, and intracellular signaling was examined using Caco2 cells in vitro. Small-intestinal expression of claudins 3 and 4 was investigated in Reg I-deficient mice in vivo. REG I deficiency significantly decreased the expression of claudin 3 in the small-intestinal epithelium. When mice were treated with indomethacin, the serum level of FITC-dextran in Reg I knockout mice was significantly higher than that in wild-type (WT) mice. The level of small-intestinal TEER was significantly decreased in Reg I knockout mice compared with WT mice under normal condition. REG Iα stimulation significantly enhanced the level of TEER in Caco2 cells. Treatment with REG Iα enhanced the expression of claudins 3 and 4 and promoted Sp1, Akt, and ERK phosphorylation in Caco2 cells, whereas these effects were attenuated by treatment with anti-REG Iα antibody. Reg I may play a role in the maintenance of mucosal barrier function by inducing tight junction proteins such as claudins 3 and 4. PMID:27055226

  8. Claudins in teleost fishes

    PubMed Central

    Kolosov, Dennis; Bui, Phuong; Chasiotis, Helen; Kelly, Scott P

    2013-01-01

    Teleost fishes are a large and diverse animal group that represent close to 50% of all described vertebrate species. This review consolidates what is known about the claudin (Cldn) family of tight junction (TJ) proteins in teleosts. Cldns are transmembrane proteins of the vertebrate epithelial/endothelial TJ complex that largely determine TJ permeability. Cldns achieve this by expressing barrier or pore forming properties and by exhibiting distinct tissue distribution patterns. So far, ~63 genes encoding for Cldn TJ proteins have been reported in 16 teleost species. Collectively, cldns (or Cldns) are found in a broad array of teleost fish tissues, but select genes exhibit restricted expression patterns. Evidence to date strongly supports the view that Cldns play a vital role in the embryonic development of teleost fishes and in the physiology of tissues and organ systems studied thus far. PMID:24665402

  9. Claudin-16 affects transcellular Cl− secretion in MDCK cells

    PubMed Central

    Günzel, Dorothee; Amasheh, Salah; Pfaffenbach, Sandra; Richter, Jan F; Kausalya, P Jaya; Hunziker, Walter; Fromm, Michael

    2009-01-01

    Claudin-16 (paracellin-1) is a tight junction protein localized mainly in the thick ascending limb of Henle's loop and also in the distal nephron. Its defect causes familial hypomagnesaemia with hypercalciuria and nephrocalcinosis. This had been taken as an indication that claudin-16 conveys paracellular Mg2+ and Ca2+ transport; however, evidence is still conflicting. We studied paracellular ion permeabilties as well as effects of claudin-16 on the driving forces for passive ion movement. MDCK-C7 cells were stably transfected with wild-type (wt) and mutant (R146T, T233R) claudin-16. Results indicated that paracellular permeability to Mg2+ but not to Ca2+ is increased in cells transfected with wt compared to mutant claudin-16 and control cells. Increased basolateral Mg2+ concentration activated a transcellular Cl− current which was greatly enhanced in cells transfected with wt and T233R claudin-16, as compared to R146T claudin-16-transfected or control cells. This current was triggered by the basolateral calcium-sensing receptor causing Ca2+ release from internal stores, thus activating apical Ca2+-sensitive Cl− channels and basolateral Ca2+-sensitive K+ channels. Immunohistochemical data suggest that the Cl− channel involved is bestrophin. We conclude that claudin-16 itself possesses only moderate paracellular Mg2+ permeability but governs transcellular Cl− currents by interaction with apical Ca2+-activated Cl− channels, presumably bestrophin. As the transepithelial voltage generated by such a current alters the driving force for all ions, this may be the major mechanism to regulate Mg2+ and Ca2+ absorption in the kidney. PMID:19528248

  10. Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5.

    PubMed

    Protze, Jonas; Eichner, Miriam; Piontek, Anna; Dinter, Stefan; Rossa, Jan; Blecharz, Kinga Grażyna; Vajkoczy, Peter; Piontek, Joerg; Krause, Gerd

    2015-04-01

    Clostridium perfringens enterotoxin (CPE) binds to distinct claudins (Clds), which regulate paracellular barrier functions in endo- and epithelia. The C-terminal domain (cCPE) has the potential for selective claudin modulation, since it only binds to a subset of claudins, e.g., Cld3 and Cld4 (cCPE receptors). Cld5 (non-CPE receptor) is a main constituent in tight junctions (TJ) of the blood-brain barrier. We aimed to reveal claudin recognition mechanisms of cCPE and to create a basis for a Cld5-binder. By utilizing structure-based interaction models, mutagenesis and assays of cCPE-binding to the TJ-free cell line HEK293, transfected with human Cld1 and murine Cld5, we showed how cCPE-binding to Cld1 and Cld5 is prevented by two residues in extracellular loop 2 of Cld1 (Asn(150) and Thr(153)) and Cld5 (Asp(149) and Thr(151)). Binding to Cld5 is especially attenuated by the lack of a bulky hydrophobic residue like leucine at position 151. By downsizing the binding pocket and compensating for the lack of this leucine residue, we created a novel cCPE-variant; cCPEY306W/S313H binds Cld5 with nanomolar affinity (K d 33 ± 10 nM). Finally, the effective binding to endogenously Cld5-expressing blood-brain barrier model cells (murine microvascular endothelial cEND cell line) suggests cCPEY306W/S313H as basis for Cld5-specific modulation to improve paracellular drug delivery, or to target claudin overexpressing tumors. PMID:25342221

  11. One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

    PubMed Central

    Sasso, Emanuele; Paciello, Rolando; D'Auria, Francesco; Riccio, Gennaro; Froechlich, Guendalina; Cortese, Riccardo; Nicosia, Alfredo; De Lorenzo, Claudia; Zambrano, Nicola

    2015-01-01

    Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications. PMID:26649313

  12. Highly Conserved Testicular Localization of Claudin-11 in Normal and Impaired Spermatogenesis.

    PubMed

    Stammler, Angelika; Lüftner, Benjamin Udo; Kliesch, Sabine; Weidner, Wolfgang; Bergmann, Martin; Middendorff, Ralf; Konrad, Lutz

    2016-01-01

    In this study we tested expression of tight junction proteins in human, mouse and rat and analyzed the localization of claudin-11 in testis of patients with normal and impaired spermatogenesis. Recent concepts generated in mice suggest that the stage-specifically expressed claudin-3 acts as a basal barrier, sealing the seminiferous epithelium during migration of spermatocytes. Corresponding mechanisms have never been demonstrated in humans. Testicular biopsies (n = 103) from five distinct groups were analyzed: normal spermatogenesis (NSP, n = 28), hypospermatogenesis (Hyp, n = 24), maturation arrest at the level of primary spermatocytes (MA, n = 24), Sertoli cell only syndrome (SCO, n = 19), and spermatogonial arrest (SGA, n = 8). Protein expression of claudin-3, -11 and occludin was analyzed. Human, mice and rat testis robustly express claudin-11 protein. Occludin was detected in mouse and rat and claudin-3 was found only in mice. Thus, we selected claudin-11 for further analysis of localization. In NSP, claudin-11 is located at Sertoli-Sertoli junctions and in Sertoli cell contacts towards spermatogonia. Typically, claudin-11 patches do not reach the basal membrane, unless flanked by the Sertoli cell body or patches between two Sertoli cell bodies. The amount of basal claudin-11 patches was found to be increased in impaired spermatogenesis. Only claudin-11 is expressed in all three species examined. The claudin-11 pattern is robust in man with impaired spermatogenesis, but the proportion of localization is altered in SCO and MA. We conclude that claudin-11 might represent the essential component of the BTB in human. PMID:27486954

  13. Highly Conserved Testicular Localization of Claudin-11 in Normal and Impaired Spermatogenesis

    PubMed Central

    Stammler, Angelika; Lüftner, Benjamin Udo; Kliesch, Sabine; Weidner, Wolfgang; Bergmann, Martin; Middendorff, Ralf; Konrad, Lutz

    2016-01-01

    In this study we tested expression of tight junction proteins in human, mouse and rat and analyzed the localization of claudin-11 in testis of patients with normal and impaired spermatogenesis. Recent concepts generated in mice suggest that the stage-specifically expressed claudin-3 acts as a basal barrier, sealing the seminiferous epithelium during migration of spermatocytes. Corresponding mechanisms have never been demonstrated in humans. Testicular biopsies (n = 103) from five distinct groups were analyzed: normal spermatogenesis (NSP, n = 28), hypospermatogenesis (Hyp, n = 24), maturation arrest at the level of primary spermatocytes (MA, n = 24), Sertoli cell only syndrome (SCO, n = 19), and spermatogonial arrest (SGA, n = 8). Protein expression of claudin-3, -11 and occludin was analyzed. Human, mice and rat testis robustly express claudin-11 protein. Occludin was detected in mouse and rat and claudin-3 was found only in mice. Thus, we selected claudin-11 for further analysis of localization. In NSP, claudin-11 is located at Sertoli-Sertoli junctions and in Sertoli cell contacts towards spermatogonia. Typically, claudin-11 patches do not reach the basal membrane, unless flanked by the Sertoli cell body or patches between two Sertoli cell bodies. The amount of basal claudin-11 patches was found to be increased in impaired spermatogenesis. Only claudin-11 is expressed in all three species examined. The claudin-11 pattern is robust in man with impaired spermatogenesis, but the proportion of localization is altered in SCO and MA. We conclude that claudin-11 might represent the essential component of the BTB in human. PMID:27486954

  14. Loss of EP2 receptor subtype in colonic cells compromise epithelial barrier integrity by altering claudin-4.

    PubMed

    Lejeune, Manigandan; Moreau, France; Chadee, Kris

    2014-01-01

    Prostaglandin E2 (PGE2) is a bioactive lipid mediator that exerts its biological function through interaction with four different subtypes of E-Prostanoid receptor namely EP1, EP2, EP3 and EP4. It has been known that EP2 receptor is differentially over-expressed in the epithelia of inflamed human colonic mucosa. However, the significance of the differential expression in altering epithelial barrier function is not known. In this study, we used Caco-2 cells expressing EP2 receptor, either high (EP2S) or low (EP2A), as a model epithelia and determined the barrier function of these cell monolayers by measuring the trans epithelial resistance (TER). Basal TER of EP2A (but not EP2S) monolayer was significantly lower suggesting a loss of colonic epithelial barrier integrity. In comparison, the TER of wild type Caco-2 was decreased in response to an EP2 receptor specific antagonist (AH-6809) indicating an important role for EP2 receptor in the maintenance of epithelial barrier function. The decrease TER in EP2A monolayer corresponded with a significant loss of the tight junction (TJ) protein claudin-4 without affecting other major TJ proteins. Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells. Surprisingly, alteration in claudin-4 was not transcriptionally regulated in EP2A cells but rather undergoes increased proteosomal degradation. Moreover, among the TER compromising cytokines examined (IL-8, IL-1β, TNF-α, IFN-γ) only IFN-γ was significantly up regulated in EP2A cells. However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4. We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4. PMID:25396731

  15. Placental claudin expression and its regulation by endogenous sex steroid hormones.

    PubMed

    Ahn, Changhwan; Yang, Hyun; Lee, Dongoh; An, Beum-soo; Jeung, Eui-Bae

    2015-08-01

    Tight junctions (TJs) form continuous intercellular contacts controlling the paracellular transportation across the cell-to-cell junction. TJ components include the peripheral protein zonula occludens-1 (ZO-1), junctional adhesion molecules (JAMs), and integral proteins such as occludin and claudins. Among the junction proteins, claudins play a major role in regulation of paracellular electrolyte transportation. This study explores the expression and distribution of tight junctions and their regulation during pregnancy. To study the regulation of claudin family, we examined expression of mouse placental tight junction proteins, including claudin-1 to -24, with real-time PCR and Western blotting and distribution of tight junction proteins with immunohistochemistry. Pregnant C57/BL6 mice were used in this study. The pregnant mice were divided into three groups depending on pregnant day (on days 12, 16, and 20 of gestation). Regarding the transcription levels, claudin-1, claudin-2, claudin-4, and claudin-5 expression levels were relatively high compared to other claudin family in all periods of pregnancy. Claudin-4 and 5 expressions, which reduce ion permeability, were increased over a period of time. However, claudin-2 expression, that is the responsive protein for a decrease in paracellular conductance, was decreased. Following this modulation of expression during mid-term pregnancy, we identified endogenous hormonal modulation of claudin family using estrogen receptor antagonist ICI 182,780 and progesterone receptor antagonist RU-486. After administration of ICI and RU-486, expression of claudin-4 mRNA and protein was increased. In addition, immunohistochemistry was performed to identify their localization for inferring permeability in placenta. Due to the function of claudins as effectors of ion transport at the end of regulatory pathways, they must be transducing proteins that modulate the function of claudins and thus link the physiologic inputs to the final

  16. Distinctive immunostaining of claudin-4 in spiradenomas.

    PubMed

    Yiğit, Nuri; Çelik, Ertuğrul; Yavan, İbrahim; Günal, Armağan; Kurt, Bülent; Karslıoğlu, Yıldırım; Öngürü, Önder; Özcan, Ayhan

    2016-02-01

    The intercellular bridges are essential structures in maintaining the histologic organization of the epithelium, while providing a very efficient way to exchange molecules between cells and transduction of the cell-to-cell and matrix-to-cell signals. Derangement in those important structures' physical integrity and/or function, which can be assessed by the presence or absence of several intercellular bridge proteins including claudin-4, E-cadherin, and β-catenin, was found to be related to several phenomena in the path to the neoplastic transformation. However, these proteins have not been studied in the wide variety of the skin neoplasms, in detail. Herein, we immunohistochemically assessed the expression patterns of these 3 intercellular bridge proteins on a total of 86 epidermal and eccrine adnexal tumors including basal cell carcinoma, squamous cell carcinoma, poroma, spiradenoma, syringoma, and hidradenoma. We observed a selective and distinct claudin-4 expression in the ductal-type cells of all cases of spiradenomas. Similarly, in the poromas, syringomas, and hidradenomas, claudin-4 was only positive in the luminal cells of microcystic structures, although not as conspicuous as in the spiradenomas. On the other hand, E-cadherin and β-catenin were positive in almost all types of the tumors, in a way which was not contributory to differentiate from each other. In conclusion, we think that claudin-4 can be helpful at least in making a reliable differential diagnosis of spiradenoma when overlapping morphologic features do not allow to further subclassification in the overwhelming variety of the adnexal tumors. PMID:26616722

  17. Poly(I:C) Induces Human Lung Endothelial Barrier Dysfunction by Disrupting Tight Junction Expression of Claudin-5

    PubMed Central

    Huang, Li-Yun; Stuart, Christine; Takeda, Kazuyo; D’Agnillo, Felice; Golding, Basil

    2016-01-01

    Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA (dsRNA) commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular endothelial cells. Poly(I:C) stimulated IL-6, IL-8, TNFα, and IFNβ production in conjunction with the activation of NF-κB and IRF3 confirming the Poly(I:C)-responsiveness of these cells. Poly(I:C) increased endothelial monolayer permeability with a corresponding dose- and time-dependent decrease in the expression of claudin-5, a transmembrane tight junction protein and reduction of CLDN5 mRNA levels. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of cytoplasmic claudin-5 with lysosomal-associated membrane protein 1. Chloroquine and Bay11-7082, inhibitors of TLR3 and NF-κB signaling, respectively, protected against the loss of claudin-5. Together, these findings provide new insight on how dsRNA-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies. PMID:27504984

  18. Bovine Colostrum Increases Pore-Forming Claudin-2 Protein Expression but Paradoxically Not Ion Permeability Possibly by a Change of the Intestinal Cytokine Milieu

    PubMed Central

    Maletzki, Claudia; Lamprecht, Georg

    2013-01-01

    An impaired intestinal barrier function is involved in the pathogenesis of inflammatory bowel disease (IBD). Several nutritional factors are supposed to be effective in IBD treatment but scientific data about the effects on the intestinal integrity remain scarce. Bovine colostrum was shown to exert beneficial effects in DSS-induced murine colitis, and the present study was undertaken to explore the underlying molecular mechanisms. Western blot revealed increased claudin-2 expression in the distal ileum of healthy mice after feeding with colostrum for 14 days, whereas other tight junction proteins (claudin-3, 4, 10, 15) remained unchanged. The colostrum-induced claudin-2 induction was confirmed in differentiated Caco-2 cells after culture with colostrum for 48 h. Paradoxically, the elevation of claudin-2, which forms a cation-selective pore, was neither accompanied by increased ion permeability nor impaired barrier function. In an in situ perfusion model, 1 h exposure of the colonic mucosa to colostrum induced significantly increased mRNA levels of barrier-strengthening cytokine transforming growth factor-β, while interleukine-2, interleukine-6, interleukine-10, interleukine-13, and tumor-necrosis factor-α remained unchanged. Thus, modulation of the intestinal transforming growth factor-β expression might have compensated the claudin-2 increase and contributed to the observed barrier strengthening effects of colostrum in vivo and in vitro. PMID:23717570

  19. Claudin-7-positive synchronous spontaneous intrahepatic cholangiocarcinoma, adenocarcinoma and adenomas of the gallbladder in a Bearded dragon (Pogona vitticeps).

    PubMed

    Jakab, Csaba; Rusvai, Miklós; Szabó, Zoltán; Gálfi, Péter; Marosán, Miklós; Kulka, Janina; Gál, János

    2011-03-01

    In this study, synchronous spontaneous, independent liver and gallbladder tumours were detected in a Bearded dragon (Pogona vitticeps). The multiple tumours consisted of intrahepatic cholangiocarcinoma as well as in situ adenocarcinoma and two adenomas of the gallbladder. The biliary epithelial cells and the cholangiocarcinoma showed membranous cross-immunoreactivity for claudin-7. The gallbladder epithelial cells, its adenoma and adenocarcinoma showed basolateral cross-reactivity for claudin-7. We think that the humanised anti-claudin-7 antibody is a good marker for the detection of different primary cholangiocellular and gallbladder tumours in Bearded dragons. The cholangiocytes, the cholangiocarcinoma, the endothelial cells of the liver and the epithelial cells and gallbladder tumours all showed claudin-5 cross-reactivity. The humanised anti-cytokeratin AE1-AE3 antibody showed cross-reactivity in the biliary epithelial cells, cholangiocarcinoma cells, epithelial cells and tumour cells of the gallbladder. It seems that this humanised antibody is a useful epithelial marker for the different neoplastic lesions of epithelial cells in reptiles. The humanised anti-α-smooth muscle actin (α-SMA) antibody showed intense cross-reactivity in the smooth muscle cells of the hepatic vessels and in the muscle layer of the gallbladder. The portal myofibroblasts, the endothelial cells of the sinusoids and the stromal cells of the cholangiocarcinoma and gallbladder tumours were positive for α-SMA. The antibovine anti-vimentin and humanised anti-Ki-67 antibodies did not show crossreactivity in the different samples from the Bearded dragon. PMID:21354945

  20. Structural constraints for the binding of short peptides to claudin-4 revealed by surface plasmon resonance.

    PubMed

    Ling, Jun; Liao, Hailing; Clark, Robin; Wong, Mandy Sze Man; Lo, David D

    2008-11-01

    Claudin family transmembrane proteins play an important role in tight junction structure and function in epithelial cells. Among the 24 isoforms identified in mice and humans, claudin-4 and -3 serve as the receptor for Clostridium perfringens enterotoxin (Cpe). The second extracellular loop (Ecl2) of claudin-4 is responsible for the binding to the C-terminal 30 amino acids of Cpe (Cpe30). To define the structural constraints for the claudin-4/Cpe30 interaction, a surface plasmon resonance (SPR) method was developed. GST fusions with claudin-4 revealed that Ecl2 with the downstream transmembrane domain of claudin-4 reconstituted the basic structural requirement for optimal binding activity to Cpe30, with affinity in the nanomolar range. Two 12-mer peptides selected by phage display against claudin-4-transfected CHO cells and a 12-mer Cpe mutant peptide also showed significant affinity for claudin-4 with this SPR assay, suggesting that a short peptide can establish stable contact with Ecl2 with nanomolar affinity. Alignment of these short peptides unveiled a common Ecl2 binding motif: 3 or 4)Y(Y/X)(L/I)XX>. Whereas the short peptides bound native claudin-4 on transfected CHO cells in pull-down assays, only the larger Cpe30 peptide affected trans-epithelial electrical resistance (TER) in peptide-treated Caco-2BBe monolayers. Importantly, Cpe30 retained its binding to claudin-4 when fused to the C terminus of influenza hemagglutinin, demonstrating that its binding activity can be maintained in a different biochemical context. These studies may help in the design of assays for membrane receptor interactions with soluble ligands, and in applying new targeting ligands to delivering attached "cargo" proteins. PMID:18782762

  1. Evidence for a role of claudin 2 as a proximal tubular stress responsive paracellular water channel

    SciTech Connect

    Wilmes, Anja Aschauer, Lydia; Limonciel, Alice; Pfaller, Walter; Jennings, Paul

    2014-09-01

    Claudins are the major proteins of the tight junctions and the composition of claudin subtypes is decisive for the selective permeability of the paracellular route and thus tissue specific function. Their regulation is complex and subject to interference by several factors, including oxidative stress. Here we show that exposure of cultured human proximal tubule cells (RPTEC/TERT1) to the immunosuppressive drug cyclosporine A (CsA) induces an increase in transepithelial electrical resistance (TEER), a decrease in dome formation (on solid growth supports) and a decrease in water transport (on microporous growth supports). In addition, CsA induced a dramatic decrease in the mRNA for the pore forming claudins -2 and -10, and the main subunits of the Na{sup +}/K{sup +} ATPase. Knock down of claudin 2 by shRNA had no discernable effect on TEER or dome formation but severely attenuated apical to basolateral water reabsorption when cultured on microporous filters. Generation of an osmotic gradient in the basolateral compartment rescued water transport in claudin 2 knock down cells. Inhibition of Na{sup +}/K{sup +} ATPase with ouabain prevented dome formation in both cell types. Taken together these results provide strong evidence that dome formation is primarily due to transcellular water transport following a solute osmotic gradient. However, in RPTEC/TERT1 cells cultured on filters under iso-osmotic conditions, water transport is primarily paracellular, most likely due to local increases in osmolarity in the intercellular space. In conclusion, this study provides strong evidence that claudin 2 is involved in paracellular water transport and that claudin 2 expression is sensitive to compound induced cellular stress. - Highlights: • Cyclosporine A increased TEER and decreased water transport in RPTEC/TERT1 cells. • Claudins 2 and 10 were decreased in response to cyclosporine A. • Knock down of claudin 2 inhibited water transport in proximal tubular cells. • We

  2. Claudin immunolocalization in neonatal mouse epithelial tissues.

    PubMed

    Troy, Tammy-Claire; Arabzadeh, Azadeh; Yerlikaya, Seda; Turksen, Kursad

    2007-11-01

    Emerging evidence supports the notion that claudins (Cldns) are dynamically regulated under normal conditions to respond to the selective permeability requirements of various tissues, and that their expression is developmentally controlled. We describe the localization of those Cldns that we have previously demonstrated to be functionally important in epidermal differentiation and the formation of the epidermal permeability barrier, e.g., Cldn1, Cldn6, Cldn11, and Cldn18, and the presence of Cldn3 and Cldn5 in various neonatal mouse epithelia including the epidermis, nail, oral mucosa, tongue, and stomach. Cldn1 is localized in the differentiated and/or undifferentiated compartments of the epidermis and nail and in the dorsal surface of the tongue and glandular compartment of the stomach but is absent from the oral mucosa and the keratinized compartment of the stomach. Cldn3 is present in the basal cells of the nail matrix and both compartments of the murine stomach but not in the epidermis, oral mucosa, or tongue. Cldn5 is found in the glandular compartment of the stomach but not in the epidermis, nail unit, oral mucosa, forestomach, and tongue. Cldn6, Cldn11, and Cldn18 occur in the differentiating suprabasal compartment of the epidermis, nail, and oral mucosa and in the dorsal and ventral surfaces of the tongue and the keratinized squamous epithelium of the stomach. The simple columnar epithelium of the glandular stomach stains for Cldn18 and reveals a non-membranous pattern for Cldn6 and Cldn11 expression. Our results demonstrate differential Cldn protein profiles in various epithelial tissues and their differentiation stages. Although the molecular mechanisms regulating Cldn expression are unknown, elucidation of their differential localization patterns in tissues with diverse permeability requirements should provide a better understanding of the role of tight junctions in tissue function. PMID:17828607

  3. A Key Claudin Extracellular Loop Domain is Critical for Epithelial Barrier Integrity

    PubMed Central

    Mrsny, Randall J.; Brown, G. Thomas; Gerner-Smidt, Kirsten; Buret, Andre G.; Meddings, Jon B.; Quan, Clifford; Koval, Michael; Nusrat, Asma

    2008-01-01

    Intercellular tight junctions (TJs) regulate epithelial barrier properties. Claudins are major structural constituents of TJs and belong to a large family of tetra-spanning membrane proteins that have two predicted extracellular loops (ELs). Given that claudin-1 is widely expressed in epithelia, we further defined the role of its EL domains in determining TJ function. The effects of several claudin-1 EL mimetic peptides on epithelial barrier structure and function were examined. Incubation of model human intestinal epithelial cells with a 27-amino acid peptide corresponding to a portion of the first EL domain (Cldn-153–80) reversibly interfered with epithelial barrier function by inducing the rearrangement of key TJ proteins: occludin, claudin-1, junctional adhesion molecule-A, and zonula occludens-1. Cldn-153–80 associated with both claudin-1 and occludin, suggesting both the direct interference with the ability of these proteins to assemble into functional TJs and their close interaction under physiological conditions. These effects were specific for Cldn-153–80, because peptides corresponding to other claudin-1 EL domains failed to influence TJ function. Furthermore, the oral administration of Cldn-153–80 to rats increased paracellular gastric permeability. Thus, the identification of a critical claudin-1 EL motif, Cldn-153–80, capable of regulating TJ structure and function, offers a useful adjunct to treatments that require drug delivery across an epithelial barrier. PMID:18349130

  4. Claudin Proteins And Neuronal Function.

    PubMed

    Devaux, Jérôme; Fykkolodziej, Bozena; Gow, Alexander

    2010-01-01

    The identification and characterization of the claudin family of tight junction (TJ) proteins in the late 1990s ushered in a new era for research into the molecular and cellular biology of intercellular junctions. Since that time, TJs have been studied in the contexts of many diseases including deafness, male infertility, cancer, bacterial invasion and liver and kidney disorders. In this review, we consider the role of claudins in the nervous system focusing on the mechanisms by which TJs in glial cells are involved in neuronal function. Electrophysiological evidence suggests that claudins may operate in the central nervous system (CNS) in a manner similar to polarized epithelia. We also evaluate hypotheses that TJs are the gatekeepers of an immune-privileged myelin compartment and that TJs emerged during evolution to form major adhesive forces within the myelin sheath. Finally, we consider the implications of CNS myelin TJs in the contexts of behavioral disorders (schizophrenia) and demyelinating/hypomyelinating diseases (multiple sclerosis and the leukodystrophies), and explore evidence of a possible mechanism governing affective disorder symptoms in patients with white matter abnormalities. PMID:25013353

  5. Claudin-4 Overexpression in Epithelial Ovarian Cancer Is Associated with Hypomethylation and Is a Potential Target for Modulation of Tight Junction Barrier Function Using a C-Terminal Fragment of Clostridium perfringens Enterotoxin1

    PubMed Central

    Litkouhi, Babak; Kwong, Joseph; Lo, Chun-Min; Smedley, James G; McClane, Bruce A; Aponte, Margarita; Gao, Zhijian; Sarno, Jennifer L; Hinners, Jennifer; Welch, William R; Berkowitz, Ross S; Mok, Samuel C; Garner, Elizabeth I O

    2007-01-01

    Background Claudin-4, a tight junction (TJ) protein and receptor for the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is overexpressed in epithelial ovarian cancer (EOC). Previous research suggests DNA methylation is a mechanism for claudin-4 overexpression in cancer and that C-CPE acts as an absorption-enhancing agent in claudin-4-expressing cells. We sought to correlate claudin-4 overexpression in EOC with clinical outcomes and TJ barrier function, investigate DNA methylation as a mechanism for overexpression, and evaluate the effect of C-CPE on the TJ. Methods Claudin-4 expression in EOC was quantified and correlated with clinical outcomes. Claudin-4 methylation status was determined, and claudin-4-negative cell lines were treated with a demethylating agent. Electric cell-substrate impedance sensing was used to calculate junctional (paracellular) resistance (Rb) in EOC cells after claudin-4 silencing and after C-CPE treatment. Results Claudin-4 overexpression in EOC does not correlate with survival or other clinical endpoints and is associated with hypomethylation. Claudin-4 overexpression correlates with Rb and C-CPE treatment of EOC cells significantly decreased Rb in a dose- and claudin-4-dependent noncytotoxic manner. Conclusions C-CPE treatment of EOC cells leads to altered TJ function. Further research is needed to determine the potential clinical applications of C-CPE in EOC drug delivery strategies. PMID:17460774

  6. Mycotoxins modify the barrier function of Caco-2 cells through differential gene expression of specific claudin isoforms: Protective effect of illite mineral clay.

    PubMed

    Romero, Alejandro; Ares, Irma; Ramos, Eva; Castellano, Víctor; Martínez, Marta; Martínez-Larrañaga, María-Rosa; Anadón, Arturo; Martínez, María-Aránzazu

    2016-04-15

    Aflatoxin B1 (AFB1), fumonisin B1 (FB1), ochratoxin A (OTA) and T-2 toxin (T2) are mycotoxins that commonly contaminate the food chain and cause various toxicological effects. Their global occurrence is regarded as an important risk factor for human and animal health. In this study, the results demonstrate that, in human Caco-2 cells, AFB1, FB1, OTA and T2 origin cytotoxic effects, determining cell viability through MTT assay and LDH leakage, and decrease trans-epithelial electrical resistance (TEER). The decrease in barrier properties is concomitant with a reduction in the expression levels of the tight junction constituents claudin-3, claudin-4 and occludin. The protective effect of mineral clays (diosmectite, montmorillonite and illite) on alterations in cell viability and epithelial barrier function induced by the mycotoxins was also evaluated. Illite was the best clay to prevent the mycotoxin effects. Illite plus mycotoxin co-treatment completely abolished AFB1 and FB1-induced cytotoxicity. Also, the decreases in the gene expression of claudins and the reduction of TEER induced by mycotoxins were reversed by the illite plus mycotoxin co-treatment. In conclusion, these results demonstrated that mycotoxins AFB1, FB1, T2 and OTA disrupt the intestinal barrier permeability by a mechanism involving reduction of claudin isoform expressions, and illite counteracts this disruption. PMID:27153755

  7. Claudins: Gatekeepers of lung epithelial function.

    PubMed

    Schlingmann, Barbara; Molina, Samuel A; Koval, Michael

    2015-06-01

    The lung must maintain a proper barrier between airspaces and fluid filled tissues in order to maintain lung fluid balance. Central to maintaining lung fluid balance are epithelial cells which create a barrier to water and solutes. The barrier function of these cells is mainly provided by tight junction proteins known as claudins. Epithelial barrier function varies depending on the different needs within the segments of the respiratory tree. In the lower airways, fluid is required to maintain mucociliary clearance, whereas in the terminal alveolar airspaces a thin layer of surfactant enriched fluid lowers surface tension to prevent airspace collapse and is critical for gas exchange. As the epithelial cells within the segments of the respiratory tree differ, the composition of claudins found in these epithelial cells is also different. Among these differences is claudin-18 which is uniquely expressed by the alveolar epithelial cells. Other claudins, notably claudin-4 and claudin-7, are more ubiquitously expressed throughout the respiratory epithelium. Claudin-5 is expressed by both pulmonary epithelial and endothelial cells. Based on in vitro and in vivo model systems and histologic analysis of lungs from human patients, roles for specific claudins in maintaining barrier function and protecting the lung from the effects of acute injury and disease are being identified. One surprising finding is that claudin-18 and claudin-4 control lung cell phenotype and inflammation beyond simply maintaining a selective paracellular permeability barrier. This suggests claudins have more nuanced roles for the control of airway and alveolar physiology in the healthy and diseased lung. PMID:25951797

  8. Urinary Claudin-2 Measurements as a Predictor of Necrotizing Enterocolitis: A Pilot Study

    PubMed Central

    P Blackwood M.D., Brian; R Wood B.S., Douglas; Y Yuan B.S., Carrie; D Nicolas, Joseph; Griffiths M.D., Anne; Mestan M.D., Karen; J Hunter M.D., Catherine

    2015-01-01

    Background: Necrotizing Enterocolitis (NEC) affects 5-10% of NICU patients where initially patients may have only nonspecific clinical findings. A noninvasive tool for detection would aid in diagnosis. Increased urinary claudins have been associated with active adult inflammatory bowel disease. Methods: Institutional Review Board approval was obtained. Neonatal intestinal tissue samples were obtained from patients with and without NEC. Immunofluorescence analysis of claudin-2 was performed on the intestinal tissue. Thirty two urine samples were collected from 6 NICU patients. Proteins were extracted and urinary claudin-2 expression was measured using Western Blot Analysis. All sample concentrations were normalized to urinary creatinine. Differences were analyzed with ANOVA or Student’s T-test. Findings were correlated to the patient’s clinical status. Results: Neonatal intestinal immunofluorescence analysis revealed that claudin-2 is present in healthy intestinal epithelium and is decreased in NEC intestinal tissue (p=0.0001). Of the six patients evaluated, three patients had NEC. All 3 patients with NEC had spikes in urinary claudin-2 levels (p<0.001, p<0.001, p 0.0598 respectively). Spikes did not appear to correlate with other etiologies of neonatal sepsis, medication use or need for mechanical ventilation. Levels during active NEC were almost twice that of NEC-free periods (p<0.0001). Conclusion: A tool for early detection would facilitate early intervention and potential prevention of severe NEC. Preliminary findings indicate that urinary claudin-2 may represent a potential biomarker for NEC worth further investigation. PMID:26500853

  9. Claudins and the Kidney Volume 75: Annual Review of Physiology

    PubMed Central

    Hou, Jianghui; Rajagopal, Madhumitha; Yu, Alan S. L.

    2013-01-01

    Claudins are tight junction membrane proteins that regulate paracellular permeability of renal epithelia to small ions, solutes and water. Claudins interact within the cell membrane and between neighboring cells to form tight junction strands and constitute both the paracellular barrier and pore. The first extracellular domain of claudins is thought to be the pore-lining domain and contains the determinants of charge selectivity. Multiple claudins are expressed in different nephron segments and this likely determines the permeability properties of each segment. Recent evidence has identified claudin-2 as constituting the cation-reabsorptive pathway in the proximal tubule, claudin-14, -16 and -19 as forming a complex that regulates calcium transport in the thick ascending limb of the loop of Henle, and claudin-4, -7 and -8 as determinants of collecting duct choride permeability. Mutations in claudin-16 and -19 cause familial hypercalciuric hypomagnesemia. The roles of other claudins in kidney diseases remain to be fully elucidated. PMID:23140368

  10. Leukocyte Gene Expression and Plasma Concentration in Multiple Sclerosis: Alteration of Transforming Growth Factor-βs, Claudin-11, and Matrix Metalloproteinase-2.

    PubMed

    Hassanzadeh, Gholamreza; Hosseini Quchani, Samaneh; Sahraian, Mohammad Ali; Abolhassani, Farid; Sadighi Gilani, Mohammad Ali; Dehghan Tarzjani, Masoomeh; Atoof, Fatemeh

    2016-08-01

    Multiple sclerosis is a neurodegenerative disease characterized by the present of leukocytes in the brain tissue and subsequently the formation of sclerotic plaques. Leukocytes penetration into the blood-brain barrier is related to several factors, such as, the conversion of leukocyte gene expression or plasma characteristics. In this frame, we explore alteration of matrix metalloproteinase-2 (MMP-2), transforming growth factor beta (TGF-β) family, and Claudin-11 (as a main myelin structural protein) in leukocytes and blood plasma of multiple sclerosis patients compared to the normal group. Blood samples were collected from thirteen men affected by MS and fifteen healthy men. Leukocyte gene expression was measured using real-time PCR and plasma parameters were examined by ELISA. The results of this study showed that the gene expression of Claudin-11 was significantly higher in MS group compared with normal. Interestingly, the MMP-2 pattern was similar to Claudin-11 and correlated positively with it. It was observed that, although the expressions of TGF-β1 and TGF-β2 are down-regulated in the leukocytes of subjects with MS, they showed higher levels of these cytokines in blood plasma. The plasma level of TGF-β3 in MS patients was higher than normal and correlated with Claudin-11 concentration. In conclusion, the aberrant pattern of Claudin-11, TGF-βs family, and MMP-2 expression in leukocytes of the MS patients was observed in this study. Moreover, the plasma levels of TGF-βs family increased in the MS group. The findings of this study provide clues for further investigations to assay MS pathogenesis. PMID:26768647

  11. Targeted colonic claudin-2 expression renders resistance to epithelial injury, induces immune suppression, and protects from colitis.

    PubMed

    Ahmad, R; Chaturvedi, R; Olivares-Villagómez, D; Habib, T; Asim, M; Shivesh, P; Polk, D B; Wilson, K T; Washington, M K; Van Kaer, L; Dhawan, P; Singh, A B

    2014-11-01

    Expression of claudin-2, a tight junction protein, is highly upregulated during inflammatory bowel disease (IBD) and, due to its association with epithelial permeability, has been postulated to promote inflammation. Notably, claudin-2 has also been implicated in the regulation of intestinal epithelial proliferation. However, precise role of claudin-2 in regulating colonic homeostasis remains unclear. Here, we demonstrate, using Villin-Claudin-2 transgenic mice, that increased colonic claudin-2 expression augments mucosal permeability as well as colon and crypt length. Most notably, despite leaky colon, Cl-2TG mice were significantly protected against experimental colitis. Importantly, claudin-2 expression increased colonocyte proliferation and provided protection against colitis-induced colonocyte death in a PI-3Kinase/Bcl-2-dependent manner. However, Cl-2TG mice also demonstrated marked suppression of colitis-induced increases in immune activation and associated signaling, suggesting immune tolerance. Accordingly, colons from naive Cl-2TG mice harbored significantly increased numbers of regulatory (CD4(+)Foxp3(+)) T cells than WT littermates. Furthermore, macrophages isolated from Cl-2TG mouse colon exhibited immune anergy. Importantly, these immunosuppressive changes were associated with increased synthesis of the immunoregulatory cytokine TGF-β by colonic epithelial cells in Cl-2TG mice compared with WT littermates. Taken together, our findings reveal a critical albeit complex role of claudin-2 in intestinal homeostasis by regulating epithelial permeability, inflammation and proliferation and suggest novel therapeutic opportunities. PMID:24670427

  12. Effects of dietary administering chitosan on growth performance, jejunal morphology, jejunal mucosal sIgA, occludin, claudin-1 and TLR4 expression in weaned piglets challenged by enterotoxigenic Escherichia coli.

    PubMed

    Xiao, Dingfu; Tang, Zhiru; Yin, Yulong; Zhang, Bin; Hu, Xionggui; Feng, Zemeng; Wang, Jinquan

    2013-11-01

    This study was conducted to investigate how chitosan (COS) affects intestinal mucosal barrier function and to further explain mechanisms of COS on growth performance. Thirty piglets, weaned at 21 days of age, were challenged with enterotoxigenic Escherichia coli during preliminary trial period. Three groups of Piglets in individual pens were fed a corn-soybean meal diet containing no addition, 50 mg/kg chlortetracycline, or 300 mg/kg COS for 21 days. Jejunal morphology and histology were analyzed under light microscope. The concentrations of occludin proteins were determined by western blot. Immunohistochemistry assays were used to determine secretory immunoglobulin (sIgA) level. Real-time PCR was used to detect Toll-like receptor 4 (TLR4) and Claudin-1 in jejunal mucosa. Feeding COS or chlortetracycline reduced (P<0.05) feed conversion ratio. Villus length, villus length/crypt depth, and goblet cells, were increased (P<0.05), but villus width and crypt depth were decreased (P<0.05) in both COS and chlortetracycline groups. Intraepithelial lymphocytes were higher (P<0.05) in the COS group than both chlortetracycline and control groups. Occludin protein expression was increased (P<0.01) in the COS group, but was decreased (P<0.05) in the chlortetracycline group. Expression of sIgA protein was higher (P<0.05) in the COS group than both control and chlortetracycline groups, however TLR4 mRNA expression was decreased (P<0.05) in both COS and chlortetracycline groups. There was no difference in expression of claudin-1 among the three groups. In conclusion, chitosan and the antibiotic have similar effects in promoting piglet growth and reducing intestinal inflammation, but different effects on intestinal mucosal barrier function. This indicates that chitosan can replace chlortetracycline as a feed additive for piglets. PMID:24007779

  13. Overexpression of claudin-4 may be involved in endometrial tumorigenesis

    PubMed Central

    PAN, XIAO-YU; LI, XUE; CHE, YAN-CI; LI, HONG-YAN; LI, XIN; ZHANG, YUN; YANG, XIN

    2013-01-01

    To clarify the role of claudin-4 in endometrial tumorigenesis and to explore whether claudin-4 could be a potentially useful agent in the treatment of endometrial carcinoma, the expression of claudin-4 in endometrial carcinoma was investigated. The relationship between therapy with anti-neoplastic agents and the expression of claudin-4 was also analyzed using an endometrial carcinoma xenograft model. The expression of claudin-4 in endometrial endometrioid adenocarcinoma (EEC) and normal human endometrial tissue was determined using immunohistochemistry and real-time PCR. Ninety female BALB/c nu/nu mice were transplanted with Ishikawa endometrial cancer cells. The mice were divided into three groups with different intraperitoneal treatments: cisplatin, paclitaxel or saline solution. After the observation period tumors were extracted and stained with monoclonal antibody against claudin-4. The mRNA expression of claudin-4 was also detected using real-time PCR. Expression of claudin-4 was significantly increased at both protein and mRNA levels in the EEC group compared with the group of normal cyclic endometrium. In the study of Ishikawa xenografts, no significant changes in tumor volume and claudin-4 expression were shown in the paclitaxel group compared with the control group. A significant reduction of tumor growth and a significant decrease in claudin-4 expression were observed in the cisplatin group. These results demonstrate that claudin-4 is strongly expressed in EEC. Claudin-4 is a useful biomarker in the treatment of patients with endometrial carcinoma. PMID:23599806

  14. Tumor necrosis factor-α induces a biphasic change in claudin-2 expression in tubular epithelial cells: role in barrier functions.

    PubMed

    Amoozadeh, Yasaman; Dan, Qinghong; Xiao, Jenny; Waheed, Faiza; Szászi, Katalin

    2015-07-01

    The inflammatory cytokine tumor necrosis factor-α (TNF-α) is a pathogenic factor in acute and chronic kidney disease. TNF-α is known to alter expression of epithelial tight junction (TJ) proteins; however, the underlying mechanisms and the impact of this effect on epithelial functions remain poorly defined. Here we describe a novel biphasic effect of TNF-α on TJ protein expression. In LLC-PK1 tubular cells, short-term (1-6 h) TNF-α treatment selectively elevated the expression of the channel-forming TJ protein claudin-2. In contrast, prolonged (>8 h) TNF-α treatment caused a marked downregulation in claudin-2 and an increase in claudin-1, -4, and -7. The early increase and the late decrease in claudin-2 expression involved distinct mechanisms. TNF-α slowed claudin-2 degradation through ERK, causing the early increase. This increase was also mediated by the EGF receptor and RhoA and Rho kinase. In contrast, prolonged TNF-α treatment reduced claudin-2 mRNA levels and promoter activity independent from these signaling pathways. Electric Cell-substrate Impedance Sensing measurements revealed that TNF-α also exerted a biphasic effect on transepithelial resistance (TER) with an initial decrease and a late increase. Thus there was a good temporal correlation between TNF-α-induced claudin-2 protein and TER changes. Indeed, silencing experiments showed that the late TER increase was at least in part caused by reduced claudin-2 expression. Surprisingly, however, claudin-2 silencing did not prevent the early TER drop. Taken together, the TNF-α-induced changes in claudin-2 levels might contribute to TER changes and could also play a role in newly described functions of claudin-2 such as proliferation regulation. PMID:25948735

  15. Tumor necrosis factor-α induces a biphasic change in claudin-2 expression in tubular epithelial cells: role in barrier functions

    PubMed Central

    Amoozadeh, Yasaman; Dan, Qinghong; Xiao, Jenny; Waheed, Faiza

    2015-01-01

    The inflammatory cytokine tumor necrosis factor-α (TNF-α) is a pathogenic factor in acute and chronic kidney disease. TNF-α is known to alter expression of epithelial tight junction (TJ) proteins; however, the underlying mechanisms and the impact of this effect on epithelial functions remain poorly defined. Here we describe a novel biphasic effect of TNF-α on TJ protein expression. In LLC-PK1 tubular cells, short-term (1–6 h) TNF-α treatment selectively elevated the expression of the channel-forming TJ protein claudin-2. In contrast, prolonged (>8 h) TNF-α treatment caused a marked downregulation in claudin-2 and an increase in claudin-1, -4, and -7. The early increase and the late decrease in claudin-2 expression involved distinct mechanisms. TNF-α slowed claudin-2 degradation through ERK, causing the early increase. This increase was also mediated by the EGF receptor and RhoA and Rho kinase. In contrast, prolonged TNF-α treatment reduced claudin-2 mRNA levels and promoter activity independent from these signaling pathways. Electric Cell-substrate Impedance Sensing measurements revealed that TNF-α also exerted a biphasic effect on transepithelial resistance (TER) with an initial decrease and a late increase. Thus there was a good temporal correlation between TNF-α-induced claudin-2 protein and TER changes. Indeed, silencing experiments showed that the late TER increase was at least in part caused by reduced claudin-2 expression. Surprisingly, however, claudin-2 silencing did not prevent the early TER drop. Taken together, the TNF-α-induced changes in claudin-2 levels might contribute to TER changes and could also play a role in newly described functions of claudin-2 such as proliferation regulation. PMID:25948735

  16. Transforming growth factor-β, a whey protein component, strengthens the intestinal barrier by upregulating claudin-4 in HT-29/B6 cells.

    PubMed

    Hering, Nina A; Andres, Susanne; Fromm, Anja; van Tol, Eric A; Amasheh, Maren; Mankertz, Joachim; Fromm, Michael; Schulzke, Joerg D

    2011-05-01

    TGFβ (isoforms 1-3) has barrier-protective effects in the intestine. The mechanisms involved in regulating tight junction protein expression are poorly understood. The aim of this study was to elucidate TGFβ-dependent protective effects with special attention to promoter regulation of tight junction proteins using the HT-29/B6 cell model. In addition, the effects of whey protein concentrate 1 (WPC1), a natural source of TGFβ in human nutrition, were examined. For this purpose, the claudin-4 promoter was cloned and tested for its activity. It exhibited transactivation in response to TGFβ1, which was intensified when Smad-4 was cotransfected, indicating a Smad-4-dependent regulatory component. Shortening and mutation of the promoter altered and attenuated this effect. WPC1 induced an increase in the claudin-4 protein level and resistance of HT-29/B6 cell monolayers. Anti-TGFβ(1-3) antibodies blocked these whey protein effects, suggesting that a main part of this function was mediated through TGFβ. This effect was observed on intact monolayers as well as when barrier function was impaired by preexposure to IFNγ. In conclusion, TGFβ1 affects claudin-4 gene expression via Smad-4-dependent and -independent transcriptional regulation, resulting in barrier protection, a cytokine effect that is also found in whey protein concentrates used in enteral nutrition. PMID:21430244

  17. Mutations in the Tight-Junction Gene Claudin 19 (CLDN19) Are Associated with Renal Magnesium Wasting, Renal Failure, and Severe Ocular Involvement

    PubMed Central

    Konrad, Martin; Schaller, André; Seelow, Dominik; Pandey, Amit V.; Waldegger, Siegfried; Lesslauer, Annegret; Vitzthum, Helga; Suzuki, Yoshiro; Luk, John M.; Becker, Christian; Schlingmann, Karl P.; Schmid, Marcel; Rodriguez-Soriano, Juan; Ariceta, Gema; Cano, Francisco; Enriquez, Ricardo; Jüppner, Harald; Bakkaloglu, Sevcan A.; Hediger, Matthias A.; Gallati, Sabina; Neuhauss, Stephan C. F.; Nürnberg, Peter; Weber, Stefanie

    2006-01-01

    Claudins are major components of tight junctions and contribute to the epithelial-barrier function by restricting free diffusion of solutes through the paracellular pathway. We have mapped a new locus for recessive renal magnesium loss on chromosome 1p34.2 and have identified mutations in CLDN19, a member of the claudin multigene family, in patients affected by hypomagnesemia, renal failure, and severe ocular abnormalities. CLDN19 encodes the tight-junction protein claudin-19, and we demonstrate high expression of CLDN19 in renal tubules and the retina. The identified mutations interfere severely with either cell-membrane trafficking or the assembly of the claudin-19 protein. The identification of CLDN19 mutations in patients with chronic renal failure and severe visual impairment supports the fundamental role of claudin-19 for normal renal tubular function and undisturbed organization and development of the retina. PMID:17033971

  18. Claudin-10 is required for relay of left-right patterning cues from Hensen's node to the lateral plate mesoderm.

    PubMed

    Collins, Michelle M; Baumholtz, Amanda I; Simard, Annie; Gregory, Mary; Cyr, Daniel G; Ryan, Aimee K

    2015-05-15

    Species-specific symmetry-breaking events at the left-right organizer (LRO) drive an evolutionarily-conserved cascade of gene expression in the lateral plate mesoderm that is required for the asymmetric positioning of organs within the body cavity. The mechanisms underlying the transfer of the left and right laterality information from the LRO to the lateral plate mesoderm are poorly understood. Here, we investigate the role of Claudin-10, a tight junction protein, in facilitating the transfer of left-right identity from the LRO to the lateral plate mesoderm. Claudin-10 is asymmetrically expressed on the right side of the chick LRO, Hensen's node. Gain- and loss-of-function studies demonstrated that right-sided expression of Claudin-10 is essential for normal rightward heart tube looping, the first morphological asymmetry during organogenesis. Manipulation of Claudin-10 expression did not perturb asymmetric gene expression at Hensen's node, but did disrupt asymmetric gene expression in the lateral plate mesoderm. Bilateral expression of Claudin-10 at Hensen's node prevented expression of Nodal, Lefty-2 and Pitx2c in the left lateral plate mesoderm, while morpholino knockdown of Claudin-10 inhibited expression of Snail1 in the right lateral plate mesoderm. We also determined that amino acids that are predicted to affect ion selectivity and protein interactions that bridge Claudin-10 to the actin cytoskeleton were essential for its left-right patterning function. Collectively, our data demonstrate a novel role for Claudin-10 during the transmission of laterality information from Hensen's node to both the left and right sides of the embryo and demonstrate that tight junctions have a critical role during the relay of left-right patterning cues from Hensen's node to the lateral plate mesoderm. PMID:25744724

  19. Decreased expression of VE-cadherin and claudin-5 and increased phosphorylation of VE-cadherin in vascular endothelium in nasal polyps.

    PubMed

    Yukitatsu, Yoriko; Hata, Masaki; Yamanegi, Koji; Yamada, Naoko; Ohyama, Hideki; Nakasho, Keiji; Kojima, Yusuke; Oka, Hideki; Tsuzuki, Kenzo; Sakagami, Masafumi; Terada, Nobuyuki

    2013-06-01

    VE-cadherin and claudin-5 are major components of adherens and tight junctions of vascular endothelial cells and a decrease in their expression and an increase in the tyrosine-phosphorylation of VE-cadherin are associated with an increase in endothelial paracellular permeability. To clarify the mechanism underlying the development of edema in nasal polyps, we studied these molecules in polyp microvessels. Normal inferior turbinate mucosal tissues and nasal polyps from patients treated with or without glucocorticoid were stained for VE-cadherin or claudin-5 and CD31 by a double-immunofluorescence method and the immunofluorescence intensities were graded 1-3 with increasing intensity. To correct for differences in fluorescence intensity attributable to a different endothelial area being exposed in a section or to the thickness of a section, the relative immunofluorescence intensity was estimated by dividing the grade of VE-cadherin or claudin-5 by that of CD31 in each microvessel. Tyrosine-phosphorylation of VE-cadherin was examined by Western blot analysis. The relative intensities of VE-cadherin and claudin-5 in the CD31-positive microvessels significantly decreased in the following order; inferior turbinate mucosa, treated polyps and untreated polyps. The ratio of tyrosine-phosphorylated VE-cadherin to VE-cadherin was significantly higher in untreated polyps than in the inferior turbinate mucosa and treated polyps, between which no significant difference in the ratio was seen. Thus, in nasal polyps, the barrier function of endothelial adherens and tight junctions is weakened, although glucocorticoid treatment improves this weakened barrier function. PMID:23474739

  20. A spontaneous metastasis model reveals the significance of claudin-9 overexpression in lung cancer metastasis.

    PubMed

    Sharma, Rajesh K; Chheda, Zinal S; Das Purkayastha, Biswa Pratim; Gomez-Gutierrez, Jorge G; Jala, Venkatakrishna R; Haribabu, Bodduluri

    2016-03-01

    Metastasis causes most cancer related mortality but the mechanisms governing metastatic dissemination are poorly defined. Metastasis involves egression of cancer cells from the primary tumors, their survival in circulation and colonization at the secondary sites. Cancer cell egression from the primary tumor is the least defined process of metastasis as experimental metastasis models directly seed cancer cells in circulation, thus bypassing this crucial step. Here, we developed a spontaneous metastasis model that retains the egression step of metastasis. By repeated in vivo passaging of the poorly metastatic Lewis lung carcinoma (3LL) cells, we generated a cell line (p-3LL) that readily metastasizes to lungs and liver from subcutaneous (s.c.) tumors. Interestingly, when injected intravenously, 3LL and p-3LL cells showed a similar frequency of metastasis. This suggests enhanced egression of p-3LL cells may underlie the enhanced metastatic spread from primary tumors. Microarray analysis of 3LL and p-3LL cells as well as the primary tumors derived from these cells revealed altered expression of several genes including significant upregulation of a tight junction protein, claudin-9. Increased expression of claudin-9 was confirmed in both p-3LL cells and tumors derived from these cells. Knockdown of claudin-9 expression in p-3LL cells by si-RNA significantly reduced their motility, invasiveness in vitro and metastasis in vivo. Conversely, transient overexpression of claudin-9 in 3LL cells enhanced their motility. These results suggest an essential role for claudin-9 in promoting lung cancer metastasis. PMID:26669782

  1. The degradation of airway tight junction protein under acidic conditions is probably mediated by transient receptor potential vanilloid 1 receptor

    PubMed Central

    Xu, Rui; Li, Qi; Zhou, Jia; Zhou, Xiang-dong; Perelman, Juliy M.; Kolosov, Victor P.

    2013-01-01

    Acidic airway microenvironment is one of the representative pathophysiological features of chronic inflammatory respiratory diseases. Epithelial barrier function is maintained by TJs (tight junctions), which act as the first physical barrier against the inhaled substances and pathogens of airway. As previous studies described, acid stress caused impaired epithelial barriers and led the hyperpermeability of epithelium. However, the specific mechanism is still unclear. We have showed previously the existence of TRPV (transient receptor potential vanilloid) 1 channel in airway epithelium, as well as its activation by acidic stress in 16HBE cells. In this study, we explored the acidic stress on airway barrier function and TJ proteins in vitro with 16HBE cell lines. Airway epithelial barrier function was determined by measuring by TER (trans-epithelial electrical resistance). TJ-related protein [claudin-1, claudin-3, claudin-4, claudin-5, claudin-7 and ZO-1 (zonula occluden 1)] expression was examined by western blotting of insoluble fractions of cell extraction. The localization of TJ proteins were visualized by immunofluorescent staining. Interestingly, stimulation by pH 6.0 for 8 h slightly increased the epithelial resistance in 16HBE cells insignificantly. However, higher concentration of hydrochloric acid (lower than pH 5.0) did reduce the airway epithelial TER of 16HBE cells. The decline of epithelial barrier function induced by acidic stress exhibited a TRPV1-[Ca2+]i-dependent pathway. Of the TJ proteins, claudin-3 and claudin-4 seemed to be sensitive to acidic stress. The degradation of claudin-3 and claudin-4 induced by acidic stress could be attenuated by the specific TRPV1 blocker or intracellular Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. PMID:24073800

  2. The density of small tight junction pores varies among cell types and is increased by expression of claudin-2.

    PubMed

    Van Itallie, Christina M; Holmes, Jennifer; Bridges, Arlene; Gookin, Jody L; Coccaro, Maria R; Proctor, William; Colegio, Oscar R; Anderson, James M

    2008-02-01

    Epithelial tight junctions contain size- and charge-selective pores that control the paracellular movement of charged and noncharged solutes. Claudins influence the charge selectivity and electrical resistance of junctions, but there is no direct evidence describing pore composition or whether pore size or density differs among cell types. To characterize paracellular pores independent of influences from charge selectivity, we profiled the ;apparent permeabilities' (P(app)) of a continuous series of noncharged polyethylene glycols (PEGs) across monolayers of five different epithelial cell lines and porcine ileum. We also characterized P(app) of high and low electrical resistance MDCK cell monolayers expressing heterologous claudins. P(app) profiling confirms that the paracellular barrier to noncharged solutes can be modeled as two distinct pathways: high-capacity small pores and a size-independent pathway allowing flux of larger solutes. All cell lines and ileum share a pore aperture of radius 4 A. Using P(app) of a PEG of radius 3.5 A to report the relative pore number provides the novel insight that pore density along the junction varies among cell types and is not necessarily related to electrical resistance. Expression of claudin-2 results in a selective increase in pore number but not size and has no effect on the permeability of PEGs that are larger than the pores; however, neither knockdown of claudin-2 nor overexpression of several other claudins altered either the number of small pores or their size. We speculate that permeability of all small solutes is proportional to pore number but that small electrolytes are subject to further selectivity by the profile of claudins expressed, explaining the dissociation between the P(app) for noncharged solutes and electrical resistance. Although claudins are likely to be components of the small pores, other factors might regulate pore number. PMID:18198187

  3. Claudin gene expression patterns do not associate with interspecific differences in paracellular nutrient absorption.

    PubMed

    Price, Edwin R; Rott, Katherine H; Caviedes-Vidal, Enrique; Karasov, William H

    2016-01-01

    Bats exhibit higher paracellular absorption of glucose-sized molecules than non-flying mammals, a phenomenon that may be driven by higher permeability of the intestinal tight junctions. The various claudins, occludin, and other proteins making up the tight junctions are thought to determine their permeability properties. Here we show that absorption of the paracellular probe l-arabinose is higher in a bat (Eptesicus fuscus) than in a vole (Microtus pennsylvanicus) or a hedgehog (Atelerix albiventris). Furthermore, histological measurements demonstrated that hedgehogs have many more enterocytes in their intestines, suggesting that bats cannot have higher absorption of arabinose simply by having more tight junctions. We therefore investigated the mRNA levels of several claudins and occludin, because these proteins may affect permeability of tight junctions to macronutrients. To assess the expression levels of claudins per tight junction, we normalized the mRNA levels of the claudins to the constitutively expressed tight junction protein ZO-1, and combined these with measurements previously made in a bat and a rodent to determine if there were among-species differences. Although expression ratios of several genes varied among species, there was not a consistent difference between bats and non-flyers in the expression ratio of any particular gene. Protein expression patterns may differ from mRNA expression patterns, and might better explain differences among species in arabinose absorption. PMID:26393434

  4. Emodin promoted pancreatic claudin-5 and occludin expression in experimental acute pancreatitis rats

    PubMed Central

    Xia, Xian-Ming; Li, Bang-Ku; Xing, Shi-Mei; Ruan, Hai-Ling

    2012-01-01

    AIM: To investigate the effect of emodin on pancreatic claudin-5 and occludin expression, and pancreatic paracellular permeability in acute pancreatitis (AP). METHODS: Experimental pancreatitis was induced by retrograde injection of 5% sodium taurocholate into the biliopancreatic duct. Emodin was injected via the external jugular vein 0 or 6 h after induction of AP. Rats from sham operation and AP groups were injected with normal saline at the same time. Samples of pancreas were obtained 6 or 12 h after drug administration. Pancreatic morphology was examined with hematoxylin and eosin staining. Pancreatic edema was estimated by measuring tissue water content. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 level were measured by enzyme-linked immunosorbent assay. Pancreatic paracellular permeability was assessed by tissue dye extravasation. Expression of pancreatic claudin-5 and occludin was examined by immunohistology, quantitative real-time reverse transcriptase polymerase chain reaction and western blotting. RESULTS: Pancreatic TNF-α and IL-6 levels, wet/dry ratio, dye extravasation, and histological score were significantly elevated at 3, 6 and 12 h following sodium taurocholate infusion; treatment with emodin prevented these changes at all time points. Immunostaining of claudin-5 and occludin was detected in rat pancreas, which was distributed in pancreatic acinar cells, ductal cells and vascular endothelial cells, respectively. Sodium taurocholate infusion significantly decreased pancreatic claudin-5 and occludin mRNA and protein levels at 3, 6 and 12 h, and that could be promoted by intravenous administration of emodin at all time points. CONCLUSION: These results demonstrate that emodin could promote pancreatic claudin-5 and occludin expression, and reduce pancreatic paracellular permeability. PMID:22563203

  5. Claudins, dietary milk proteins, and intestinal barrier regulation.

    PubMed

    Kotler, Belinda M; Kerstetter, Jane E; Insogna, Karl L

    2013-01-01

    The family of claudin proteins plays an important role in regulating the intestinal barrier by modulating the permeability of tight junctions. The impact of dietary protein on claudin biology has not been studied extensively. Whey proteins have been reported to improve intestinal barrier function, but their mechanism of action is not clear. Recent studies, however, have demonstrated increased intestinal claudin expression in response to milk protein components. Reviewed here are new findings suggesting that whey-protein-derived transforming growth factor β transcriptionally upregulates claudin-4 expression via a Smad-4-dependent pathway. These and other data, including limited clinical studies, are summarized below and, in the aggregate, suggest a therapeutic role for whey protein in diseases of intestinal barrier dysfunction, perhaps, in part, by regulating claudin expression. PMID:23282252

  6. Musashi-2 (MSI2) supports TGF-β signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis

    PubMed Central

    Kudinov, Alexander E.; Nikonova, Anna S.; Beck, Tim N.; Ahn, Young-Ho; Liu, Xin; Martinez, Cathleen F.; Schultz, Fred A.; Reynolds, Samuel; Yang, Dong-Hua; Cai, Kathy Q.; Yaghmour, Khaled M.; Baker, Karmel A.; Egleston, Brian L.; Nicolas, Emmanuelle; Chikwem, Adaeze; Andrianov, Gregory; Singh, Shelly; Borghaei, Hossein; Serebriiskii, Ilya G.; Gibbons, Don L.; Kurie, Jonathan M.; Golemis, Erica A.; Boumber, Yanis

    2016-01-01

    Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of KrasLA1/+;P53R172HΔG/+ (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial–mesenchymal transition, including the TGF-β receptor 1 (TGFβR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFβRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial–mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFβR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness. PMID:27274057

  7. Musashi-2 (MSI2) supports TGF-β signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis.

    PubMed

    Kudinov, Alexander E; Deneka, Alexander; Nikonova, Anna S; Beck, Tim N; Ahn, Young-Ho; Liu, Xin; Martinez, Cathleen F; Schultz, Fred A; Reynolds, Samuel; Yang, Dong-Hua; Cai, Kathy Q; Yaghmour, Khaled M; Baker, Karmel A; Egleston, Brian L; Nicolas, Emmanuelle; Chikwem, Adaeze; Andrianov, Gregory; Singh, Shelly; Borghaei, Hossein; Serebriiskii, Ilya G; Gibbons, Don L; Kurie, Jonathan M; Golemis, Erica A; Boumber, Yanis

    2016-06-21

    Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of Kras(LA1/+);P53(R172HΔG/+) (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial-mesenchymal transition, including the TGF-β receptor 1 (TGFβR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFβRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial-mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFβR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness. PMID:27274057

  8. Possible angiogenic roles for claudin-4 in ovarian cancer

    PubMed Central

    Li, Jianghong; Chigurupati, Srinivasulu; Agarwal, Rachana; Mughal, Mohamed R.; Mattson, Mark P.; Becker, Kevin G.; Wood, William H.; Zhang, Yongqing; Morin, Patrice J.

    2009-01-01

    Claudin proteins are frequently overexpressed in various tumors such as breast, prostate and ovarian cancer. While their functions in cancer have not been completely elucidated, roles in survival, adhesion, and invasion have been suggested. In order to clarify the roles of claudins in ovarian cancer, we have performed gene expression profiling of ovarian surface epithelial cells overexpressing claudin-4 and compared the expression patterns to the parental, non-expressing cells. Claudin-4 expression leads to the differential expression of several genes, including many that have previously been implicated in angiogenesis. In particular, angiogenic cytokines, such as IL-8, were found elevated while genes of the angiostatic interferon pathway were found down-regulated. In vitro assays show that claudin-4-expressing cells produce factors that can stimulate angiogenesis as measured by tube formation and migration in HUVEC cells. In addition, an in vivo mouse dorsal skinfold assay confirms that cells expressing claudin-4 secrete factors that can mediate angiogenesis in the dorsal skin of mice. Our data suggest a novel function for claudin-4 in cancer and provide an additional rationale for its common overexpression in human tumors. PMID:19657234

  9. Claudin Family of Proteins and Cancer: An Overview

    PubMed Central

    Singh, Amar B.; Sharma, Ashok; Dhawan, Punita

    2010-01-01

    Tight junctions are the apical cell-cell adhesion that regulate paracellular permeability and are critical for epithelial cell polarity. Molecular architecture of tight junction has been studied extensively, which has confirmed that claudin family of proteins is integral component of tight junction. Loss of cell-cell adhesion is central to the cellular transformation and acquisition of metastatic potential; however, the role of claudin family of proteins play in a series of pathophysiological events, including human carcinoma development, is only now beginning to be understood. Several claudin mouse knockout models have been generated and the diversity of phenotypes observed clearly demonstrates their important roles in the maintenance of tissue integrity in various organs and suggest that claudins also participate in cellular contexts other than tight junctions. The mechanisms of claudin regulation and their exact roles in normal physiology and disease are being elucidated, but much work remains to be done. In this review, we have discussed the conceptual framework concerning claudins and their potential implication in cancer. We predict that next several years will likely witness a boom in our understanding of the potential role of claudins in the regulation of tumorigenesis, which may, in turn, provide new approaches for the targeted therapy. PMID:20671913

  10. Metformin attenuates transforming growth factor beta (TGF-β) mediated oncogenesis in mesenchymal stem-like/claudin-low triple negative breast cancer.

    PubMed

    Wahdan-Alaswad, Reema; Harrell, J Chuck; Fan, Zeying; Edgerton, Susan M; Liu, Bolin; Thor, Ann D

    2016-01-01

    Mesenchymal stem-like/claudin-low (MSL/CL) breast cancers are highly aggressive, express low cell-cell adhesion cluster containing claudins (CLDN3/CLDN4/CLDN7) with enrichment of epithelial-to-mesenchymal transition (EMT), immunomodulatory, and transforming growth factor-β (TGF-β) genes. We examined the biological, molecular and prognostic impact of TGF-β upregulation and/or inhibition using in vivo and in vitro methods. Using publically available breast cancer gene expression databases, we show that upregulation and enrichment of a TGF-β gene signature is most frequent in MSL/CL breast cancers and is associated with a worse outcome. Using several MSL/CL breast cancer cell lines, we show that TGF-β elicits significant increases in cellular proliferation, migration, invasion, and motility, whereas these effects can be abrogated by a specific inhibitor against TGF-β receptor I and the anti-diabetic agent metformin, alone or in combination. Prior reports from our lab show that TNBC is exquisitely sensitive to metformin treatment. Mechanistically, metformin blocks endogenous activation of Smad2 and Smad3 and dampens TGF-β-mediated activation of Smad2, Smad3, and ID1 both at the transcriptional and translational level. We report the use of ID1 and ID3 as clinical surrogate markers, where high expression of these TGF-β target genes was correlated to poor prognosis in claudin-low patients. Given TGF-β's role in tumorigenesis and immunomodulation, blockade of this pathway using direct kinase inhibitors or more broadly acting inhibitors may dampen or abolish pro-carcinogenic and metastatic signaling in patients with MCL/CL TNBC. Metformin therapy (with or without other agents) may be a heretofore unrecognized approach to reduce the oncogenic activities associated with TGF-β mediated oncogenesis. PMID:26919310

  11. Claudin-2-dependent paracellular channels are dynamically gated

    PubMed Central

    Weber, Christopher R; Liang, Guo Hua; Wang, Yitang; Das, Sudipto; Shen, Le; Yu, Alan S L; Nelson, Deborah J; Turner, Jerrold R

    2015-01-01

    Intercellular tight junctions form selectively permeable barriers that seal the paracellular space. Trans-tight junction flux has been measured across large epithelial surfaces, but conductance across individual channels has never been measured. We report a novel trans-tight junction patch clamp technique that detects flux across individual claudin-2 channels within the tight junction of cultured canine renal tubule or human intestinal epithelial monolayers. In both cells, claudin-2 channels display conductances of ~90 pS. The channels are gated, strictly dependent on claudin-2 expression, and display size- and charge-selectivity typical of claudin-2. Kinetic analyses indicate one open and two distinct closed states. Conductance is symmetrical and reversible, characteristic of a passive, paracellular process, and blocked by reduced temperature or site-directed mutagenesis and chemical derivatization of the claudin-2 pore. We conclude that claudin-2 forms gated paracellular channels and speculate that modulation of tight junction channel gating kinetics may be an unappreciated mechanism of barrier regulation. DOI: http://dx.doi.org/10.7554/eLife.09906.001 PMID:26568313

  12. Systems Proteomics View of the Endogenous Human Claudin Protein Family.

    PubMed

    Liu, Fei; Koval, Michael; Ranganathan, Shoba; Fanayan, Susan; Hancock, William S; Lundberg, Emma K; Beavis, Ronald C; Lane, Lydie; Duek, Paula; McQuade, Leon; Kelleher, Neil L; Baker, Mark S

    2016-02-01

    Claudins are the major transmembrane protein components of tight junctions in human endothelia and epithelia. Tissue-specific expression of claudin members suggests that this protein family is not only essential for sustaining the role of tight junctions in cell permeability control but also vital in organizing cell contact signaling by protein-protein interactions. How this protein family is collectively processed and regulated is key to understanding the role of junctional proteins in preserving cell identity and tissue integrity. The focus of this review is to first provide a brief overview of the functional context, on the basis of the extensive body of claudin biology research that has been thoroughly reviewed, for endogenous human claudin members and then ascertain existing and future proteomics techniques that may be applicable to systematically characterizing the chemical forms and interacting protein partners of this protein family in human. The ability to elucidate claudin-based signaling networks may provide new insight into cell development and differentiation programs that are crucial to tissue stability and manipulation. PMID:26680015

  13. Proteomic Analysis of Proteins Surrounding Occludin and Claudin-4 Reveals Their Proximity to Signaling and Trafficking Networks

    PubMed Central

    Fredriksson, Karin; Van Itallie, Christina M.; Aponte, Angel; Gucek, Marjan; Tietgens, Amber J.; Anderson, James M.

    2015-01-01

    Tight junctions are complex membrane structures that regulate paracellular movement of material across epithelia and play a role in cell polarity, signaling and cytoskeletal organization. In order to expand knowledge of the tight junction proteome, we used biotin ligase (BioID) fused to occludin and claudin-4 to biotinylate their proximal proteins in cultured MDCK II epithelial cells. We then purified the biotinylated proteins on streptavidin resin and identified them by mass spectrometry. Proteins were ranked by relative abundance of recovery by mass spectrometry, placed in functional categories, and compared not only among the N- and C- termini of occludin and the N-terminus of claudin-4, but also with our published inventory of proteins proximal to the adherens junction protein E-cadherin and the tight junction protein ZO-1. When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins. Apart from already known tight junction- proteins, occludin and claudin-4 proximal proteins were enriched in signaling and trafficking proteins, especially endocytic trafficking proteins. However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex. Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure. PMID:25789658

  14. Renal dysplasia unrelated to claudin-16 deficiency in Japanese Black cattle.

    PubMed

    Sugiyama, A; Ozaki, K; Miyazaki; Tanabe, Y; Takeuchi, T; Narama, I

    2007-07-01

    Renal lesions of the type usually found in claudin-16 (CL-16) defective Japanese Black cattle (homozygous for CL-16 deficiency) were identified in six animals of this breed, aged 28-59 months, which were either heterozygous for CL-16 deficiency (type 1) or normal, as judged by a DNA-based test associated with the CL-16 gene. Histopathologically, all six cases showed elongated focal lesions which ran through the cortex to terminate in the outer zone of the medulla. The lesions contained components that included: (1) immature tubules, (2) small irregularly shaped tubules with thickening of the basement membrane, (3) mesenchymal cells in an increased interstitium, (4) small atrophic glomeruli, and (5) immature glomeruli. The glomeruli were noticeably reduced in number, and large glomeruli with an increased number of mesangial cells were observed throughout the entire cortical area. Cystic dilation of tubules and flattening of the epithelium were noted in all areas of the kidney. Histopathologically, the renal lesions in the six cases were indistinguishable from those reported previously in cattle homozygous for CL-16 deficiency. These findings demonstrate that such renal lesions in Japanese Black cattle are not necessarily associated with homozygous deletion of the CL-16 gene. PMID:17537453

  15. Clinicopathological significance of claudin 4 expression in gastric carcinoma: a systematic review and meta-analysis

    PubMed Central

    Chen, Xiaowan; Zhao, Junhua; Li, Ailin; Gao, Peng; Sun, Jingxu; Song, Yongxi; Liu, Jingjing; Chen, Ping; Wang, Zhenning

    2016-01-01

    Background The prognostic significance of claudin 4 (CLDN4) in patients with gastric cancer (GC) is controversial. This meta-analysis aims to assess the correlation between CLDN4 expression and clinicopathological characteristics and assess the prognostic significance of CLDN4 in GC. Methods We searched the PubMed and Embase databases. We performed the meta-analysis with odds ratio (OR), hazard ratio (HR), and 95% confidence interval (CI) as effect values. Results Fourteen studies containing 2,106 patients with GC were analyzed. The overall analysis showed that CLDN4 expression was associated with increasing pT category, tumor size, and lymph node metastasis in patients with GC (pT3–T4 vs pT1–T2: OR =1.56, 95% CI =1.13–2.16; P<0.01; large tumor size vs small tumor size: OR =1.64, 95% CI =1.15–2.34; P<0.01; positive lymph node metastasis vs negative lymph node metastasis: OR =1.49, 95% CI =1.12–1.97; P<0.01). CLDN4 expression was associated with histological differentiation (differentiated type vs undifferentiated type: OR =2.90, 95% CI =1.32–6.37; P=0.01; Lauren intestinal type vs diffuse type: OR =3.51, 95% CI =1.48–8.28; P<0.01). CLDN4 expression was also strongly associated with sex and age. This meta-analysis found no significant association between CLDN4 expression and prognosis for overall survival in patients with GC (HR =0.74, 95% CI =0.43–1.27; P=0.28). Conclusion Present study indicates that aberrant CLDN4 expression plays an important role in the clinicopathological characteristics of GC. PMID:27313466

  16. Claudins and the Modulation of Tight Junction Permeability

    PubMed Central

    Günzel, Dorothee

    2013-01-01

    Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. This review summarizes our current knowledge of this large protein family and discusses recent advances in our understanding of their structure and physiological functions. PMID:23589827

  17. Cooling treatment transiently increases the permeability of brain capillary endothelial cells through translocation of claudin-5.

    PubMed

    Inamura, Akinori; Adachi, Yasuhiro; Inoue, Takao; He, Yeting; Tokuda, Nobuko; Nawata, Takashi; Shirao, Satoshi; Nomura, Sadahiro; Fujii, Masami; Ikeda, Eiji; Owada, Yuji; Suzuki, Michiyasu

    2013-08-01

    The blood-brain-barrier (BBB) is formed by different cell types, of which brain microvascular endothelial cells are major structural constituents. The goal of this study was to examine the effects of cooling on the permeability of the BBB with reference to tight junction formation of brain microendothelial cells. The sensorimotor cortex above the dura mater in adult male Wistar rats was focally cooled to a temperature of 5 °C for 1 h, then immunostaining for immunoglobulin G (IgG) was performed to evaluate the permeability of the BBB. Permeability produced by cooling was also evaluated in cultured murine brain endothelial cells (bEnd3) based on measurement of trans-epithelial electric resistance (TEER). Immunocytochemistry and Western blotting of proteins associated with tight junctions in bEnd3 were performed to determine protein distribution before and after cooling. After focal cooling of the rat brain cortex, diffuse immunostaining for IgG was observed primarily around the small vasculature and in the extracellular spaces of parenchyma of the cortex. In cultured bEnd3, TEER significantly decreased during cooling (15 °C) and recovered to normal levels after rewarming to 37 °C. Immunocytochemistry and Western blotting showed that claudin-5, a critical regulatory protein for tight junctions, was translocated from the membrane to the cytoplasm after cooling in cultured bEnd3 cells. These results suggest that focal brain cooling may open the BBB transiently through an effect on tight junctions of brain microendothelial cells, and that therapeutically this approach may allow control of BBB function and drug delivery through the BBB. PMID:23653089

  18. Non-classical testosterone signaling mediated through ZIP9 stimulates claudin expression and tight junction formation in Sertoli cells.

    PubMed

    Bulldan, Ahmed; Dietze, Raimund; Shihan, Mazen; Scheiner-Bobis, Georgios

    2016-08-01

    In the classical signaling pathway, testosterone regulates gene expression by activating the cytosolic/nuclear androgen receptor. In the non-classical pathway, testosterone activates cytosolic signaling cascades that are normally triggered by growth factors. The nature of the receptor involved in this signaling pathway is a source of controversy. In the Sertoli cell line 93RS2, which lacks the classical AR, we determined that testosterone stimulates the non-classical signaling pathway, characterized by the phosphorylation of Erk1/2 and transcription factors CREB and ATF-1. We also demonstrated that testosterone increases the expression of the tight junction (TJ) proteins claudin-1 and claudin-5. Both of these proteins are known to be essential constituents of TJs between Sertoli cells, and as a consequence of their increased expression transepithelial resistance across Sertoli cell monolayers is increased. ZIP9 is a Zn(2+)transporter that was recently shown to be a membrane-bound testosterone receptor. Silencing its expression in 93RS2 Sertoli cells by siRNA completely prevents Erk1/2, CREB, and ATF-1 phosphorylation as well the stimulation of claudin-1 and -5 expression and TJ formation between neighboring cells. The study presented here demonstrates for the first time that in Sertoli cells testosterone acts through the receptor ZIP9 to trigger the non-classical signaling cascade, resulting in increased claudin expression and TJ formation. Since TJ formation is a prerequisite for the maintenance of the blood-testis barrier, the testosterone/ZIP9 effects might be significant for male physiology. Further assessment of these interactions will help to supplement our knowledge concerning the mechanism by which testosterone plays a role in male fertility. PMID:27164415

  19. Adaptive evolution of tight junction protein claudin-14 in echolocating whales.

    PubMed

    Xu, Huihui; Liu, Yang; He, Guimei; Rossiter, Stephen J; Zhang, Shuyi

    2013-11-10

    Toothed whales and bats have independently evolved specialized ultrasonic hearing for echolocation. Recent findings have suggested that several genes including Prestin, Tmc1, Pjvk and KCNQ4 appear to have undergone molecular adaptations associated with the evolution of this ultrasonic hearing in mammals. Here we studied the hearing gene Cldn14, which encodes the claudin-14 protein and is a member of tight junction proteins that functions in the organ of Corti in the inner ear to maintain a cationic gradient between endolymph and perilymph. Particular mutations in human claudin-14 give rise to non-syndromic deafness, suggesting an essential role in hearing. Our results uncovered two bursts of positive selection, one in the ancestral branch of all toothed whales and a second in the branch leading to the delphinid, phocoenid and ziphiid whales. These two branches are the same as those previously reported to show positive selection in the Prestin gene. Furthermore, as with Prestin, the estimated hearing frequencies of whales significantly correlate with numbers of branch-wise non-synonymous substitutions in Cldn14, but not with synonymous changes. However, in contrast to Prestin, we found no evidence of positive selection in bats. Our findings from Cldn14, and comparisons with Prestin, strongly implicate multiple loci in the acquisition of echolocation in cetaceans, but also highlight possible differences in the evolutionary route to echolocation taken by whales and bats. PMID:23965379

  20. The food contaminant deoxynivalenol, decreases intestinal barrier permeability and reduces claudin expression

    SciTech Connect

    Pinton, Philippe; Nougayrede, Jean-Philippe; Del Rio, Juan-Carlos; Moreno, Carolina; Marin, Daniela E.; Ferrier, Laurent; Bracarense, Ana-Paula; Kolf-Clauw, Martine; Oswald, Isabelle P.

    2009-05-15

    'The gastrointestinal tract represents the first barrier against food contaminants as well as the first target for these toxicants. Deoxynivalenol (DON) is a mycotoxin that commonly contaminates cereals and causes various toxicological effects. Through consumption of contaminated cereals and cereal products, human and pigs are exposed to this mycotoxin. Using in vitro, ex vivo and in vivo approaches, we investigated the effects of DON on the intestinal epithelium. We demonstrated that, in intestinal epithelial cell lines from porcine (IPEC-1) or human (Caco-2) origin, DON decreases trans-epithelial electrical resistance (TEER) and increases in a time and dose-dependent manner the paracellular permeability to 4 kDa dextran and to pathogenic Escherichia coli across intestinal cell monolayers. In pig explants treated with DON, we also observed an increased permeability of intestinal tissue. These alterations of barrier function were associated with a specific reduction in the expression of claudins, which was also seen in vivo in the jejunum of piglets exposed to DON-contaminated feed. In conclusion, DON alters claudin expression and decreases the barrier function of the intestinal epithelium. Considering that high levels of DON may be present in food or feed, consumption of DON-contaminated food/feed may induce intestinal damage and has consequences for human and animal health.

  1. Tumor Necrosis Factor Disrupts Claudin-5 Endothelial Tight Junction Barriers in Two Distinct NF-κB-Dependent Phases

    PubMed Central

    Clark, Paul R.; Kim, Richard K.; Pober, Jordan S.; Kluger, Martin S.

    2015-01-01

    Capillary leak in severe sepsis involves disruption of endothelial cell tight junctions. We modeled this process by TNF treatment of cultured human dermal microvascular endothelial cell (HDMEC) monolayers, which unlike human umbilical vein endothelial cells form claudin-5-dependent tight junctions and a high-resistance permeability barrier. Continuous monitoring with electrical cell-substrate impedance sensing revealed that TNF disrupts tight junction-dependent HDMEC barriers in discrete steps: an ~5% increase in transendothelial electrical resistance over 40 minutes; a decrease to ~10% below basal levels over 2 hours (phase 1 leak); an interphase plateau of 1 hour; and a major fall in transendothelial electrical resistance to < 70% of basal levels by 8–10 hours (phase 2 leak), with EC50 values of TNF for phase 1 and 2 leak of ~30 and ~150 pg/ml, respectively. TNF leak is reversible and independent of cell death. Leak correlates with disruption of continuous claudin-5 immunofluorescence staining, myosin light chain phosphorylation and loss of claudin-5 co-localization with cortical actin. All these responses require NF-κB signaling, shown by inhibition with Bay 11 or overexpression of IκB super-repressor, and are blocked by H-1152 or Y-27632, selective inhibitors of Rho-associated kinase that do not block other NF-κB-dependent responses. siRNA combined knockdown of Rho-associated kinase-1 and -2 also prevents myosin light chain phosphorylation, loss of claudin-5/actin co-localization, claudin-5 reorganization and reduces phase 1 leak. However, unlike H-1152 and Y-27632, combined Rho-associated kinase-1/2 siRNA knockdown does not reduce the magnitude of phase 2 leak, suggesting that H-1152 and Y-27632 have targets beyond Rho-associated kinases that regulate endothelial barrier function. We conclude that TNF disrupts TJs in HDMECs in two distinct NF-κB-dependent steps, the first involving Rho-associated kinase and the second likely to involve an as yet

  2. Tight junction protein claudin-6 inhibits growth and induces the apoptosis of cervical carcinoma cells in vitro and in vivo.

    PubMed

    Zhang, Xiaowei; Ruan, Yang; Li, Yanru; Lin, Dongjing; Quan, Chengshi

    2015-05-01

    Claudin-6, a member of claudin family integral membrane proteins, has recently been reported to be a tumor suppressor for breast cancer. However, whether it plays a role in other types of cancer remains unclear. In the present study, we showed that the expression of claudin-6 is down-regulated in cervical carcinoma tissues as revealed by immunohistochemistry. Through over-expressing claudin-6 in HeLa and C33A cervical carcinoma cells, we found that claudin-6 is localized at plasma membrane and it increases transepithelial electrical resistance of the cells. Gain of claudin-6 expression suppresses cell proliferation, colony formation in vitro, and tumor growth in vivo. The effects are accompanied and potentially caused by promotion of tumor cell apoptosis. Taken together, these results suggest that claudin-6 may function as a tumor suppressor and loss of claudin-6 contributes to enhanced tumorigenic properties of cervical carcinoma cells. PMID:25822939

  3. Claudin-20 promotes an aggressive phenotype in human breast cancer cells

    PubMed Central

    Martin, Tracey A; Lane, Jane; Ozupek, Hulya; Jiang, Wen G

    2013-01-01

    Claudin-20 is a member of the Claudin family of transmembrane proteins located in the tight junction (TJ) of cells of epithelial origin. Due to the increasing evidence supporting the role of TJ proteins in preventing tumor cell metastatic behavior, this study sought to evaluate the distribution of Claudin-20 in human breast cancer and the effect of Claudin-20 overexpression in human breast cancer cells. Q-PCR data from breast cancer primary tumors (n = 114) and matched background tissue (n = 30) showed that high claudin-20 expression was correlated with poor survival of patients with breast cancer (p = 0.022). Following transformation of the breast cancer cell lines MDA-MB-231 and MCF7 with a Claudin-20 expression construct functional assays were performed to ascertain changes in cell behavior. Claudin-20 transformed cells showed significantly increased invasion (p < 0.005) and were significantly less adhesive than wild type cells (p < 0.05). There was no effect on growth (either in vitro or in vivo) for either cell line. Overexpression of Claudin-20 resulted in reduced transepithelial resistance (induced by the motogen HGF at 25 ng/ml, p = 0.0007). Interestingly, this was not mirrored by paracellular permeability, as overexpression of Claudin-20 caused a decrease in permeability. The introduction of Claudin-20 into human breast cancer cells resulted in breast cancer cells with an aggressive phenotype and reduced trans-epithelial resistance. There was no corresponding decrease in paracellular permeability, indicating that this Claudin has a differential function in epithelial TJ. This provides further insight into the importance of correctly functioning TJ in preventing the progression of human breast cancer. PMID:24665404

  4. Connexins, E-cadherin, Claudin-7 and β-catenin transiently form junctional nexuses during the post-natal mammary gland development.

    PubMed

    Dianati, Elham; Poiraud, Jérémy; Weber-Ouellette, Anne; Plante, Isabelle

    2016-08-01

    Gap junctions are intercellular channels made of connexins (Cxs) that allow direct communication between adjacent cells. Modulation of Cxs has been associated with abnormal development and function of the mammary gland and breast cancer. However, the mechanisms underlying their expression during normal mammary gland are not yet known. Cxs interact with components of tight and adherens junctions. Thus, we hypothesized that the expression levels of Cxs vary during mammary gland development and are regulated through stage-dependent interactions with members of the tight and adherens junctions. Our specific objectives were to: 1) determine the expression of Cxs and tight and adherens junction proteins throughout development and 2) characterize Cxs interactions with components of tight and adherens junctions. Murine mammary glands were sampled at various developmental stages (pre-pubescent to post-weaning). RT-qPCR and western-blot analyses demonstrated differential expression patterns for all gap (Cx43, Cx32, Cx26, Cx30), tight (Claudin-1, -3, -4, -7) and adherens (β-catenin, E- and P-cadherins) junctions throughout development. Interestingly, co-immunoprecipitation demonstrated interactions between these different types of junctions. Cx30 interacted with Cx26 just at the late pregnancy stage. While Cx43 showed a persistent interaction with β-catenin from virginity to post-weaning, its interactions with E-cadherin and Claudin-7 were transient. Cx32 interacted with Cx26, E-cadherin and β-catenin during lactation. Immunofluorescence results confirmed the existence of a junctional nexus that remodeled during mammary gland development. Together, our results confirm that the expression levels of Cxs vary concomitantly and that Cxs form junctional nexuses with tight and adherens junctions, suggesting the existence of common regulatory pathways. PMID:27291930

  5. Activation of the Ca2+-sensing receptor increases renal claudin-14 expression and urinary Ca2+ excretion

    PubMed Central

    Dimke, Henrik; Desai, Prajakta; Borovac, Jelena; Lau, Alyssa; Pan, Wanling; Alexander, R. Todd

    2016-01-01

    Kidney stones are a prevalent clinical condition imposing a large economic burden on the health-care system. Hypercalciuria remains the major risk factor for development of a Ca2+-containing stone. The kidney’s ability to alter Ca2+ excretion in response to changes in serum Ca2+ is in part mediated by the Ca2+-sensing receptor (CaSR). Recent studies revealed renal claudin-14 (Cldn14) expression localized to the thick ascending limb (TAL) and its expression to be regulated via the CaSR. We find that Cldn14 expression is increased by high dietary Ca2+ intake and by elevated serum Ca2+ levels induced by prolonged 1,25-dihydroxyvitamin D3 administration. Consistent with this, activation of the CaSR in vivo via administration of the calcimimetic cinacalcet hydrochloride led to a 40-fold increase in Cldn14 mRNA. Moreover, overexpression of Cldn14 in two separate cell culture models decreased paracellular Ca2+ flux by preferentially decreasing cation permeability, thereby increasing transepithelial resistance. These data support the existence of a mechanism whereby activation of the CaSR in the TAL increases Cldn14 expression, which in turn blocks the paracellular reabsorption of Ca2+. This molecular mechanism likely facilitates renal Ca2+ losses in response to elevated serum Ca2+. Moreover, dys-regulation of the newly described CaSR-Cldn14 axis likely contributes to the development of hypercalciuria and kidney stones. PMID:23283989

  6. HNF4α Regulates Claudin-7 Protein Expression during Intestinal Epithelial Differentiation

    PubMed Central

    Farkas, Attila E.; Hilgarth, Roland S.; Capaldo, Christopher T.; Gerner-Smidt, Christian; Powell, Doris R.; Vertino, Paula M.; Koval, Michael; Parkos, Charles A.; Nusrat, Asma

    2016-01-01

    The intestinal epithelium is a dynamic barrier that maintains the distinct environments of intestinal tissue and lumen. Epithelial barrier function is defined principally by tight junctions, which, in turn, depend on the regulated expression of claudin family proteins. Claudins are expressed differentially during intestinal epithelial cell (IEC) differentiation. However, regulatory mechanisms governing claudin expression during epithelial differentiation are incompletely understood. We investigated the molecular mechanisms regulating claudin-7 during IEC differentiation. Claudin-7 expression is increased as epithelial cells differentiate along the intestinal crypt–luminal axis. By using model IECs we observed increased claudin-7 mRNA and nascent heteronuclear RNA levels during differentiation. A screen for potential regulators of the CLDN7 gene during IEC differentiation was performed using a transcription factor/DNA binding array, CLDN7 luciferase reporters, and in silico promoter analysis. We identified hepatocyte nuclear factor 4α as a regulatory factor that bound endogenous CLDN7 promoter in differentiating IECs and stimulated CLDN7 promoter activity. These findings support a role of hepatocyte nuclear factor 4α in controlling claudin-7 expression during IEC differentiation. PMID:26216285

  7. Lyn modulates Claudin-2 expression and is a therapeutic target for breast cancer liver metastasis

    PubMed Central

    Tabariès, Sébastien; Annis, Matthew G.; Hsu, Brian E.; Tam, Christine E.; Savage, Paul; Park, Morag; Siegel, Peter M.

    2015-01-01

    Claudin-2 enhances breast cancer liver metastasis and promotes the development of colorectal cancers. The objective of our current study is to define the regulatory mechanisms controlling Claudin-2 expression in breast cancer cells. We evaluated the effect of several Src Family Kinase (SFK) inhibitors or knockdown of individual SFK members on Claudin-2 expression in breast cancer cells. We also assessed the potential effects of pan-SFK and SFK-selective inhibitors on the formation of breast cancer liver metastases. This study reveals that pan inhibition of SFK signaling pathways significantly elevated Claudin-2 expression levels in breast cancer cells. In addition, our data demonstrate that pan-SFK inhibitors can enhance breast cancer metastasis to the liver. Knockdown of individual SFK members reveals that loss of Yes or Fyn induces Claudin-2 expression; whereas, diminished Lyn levels impairs Claudin-2 expression in breast cancer cells. The Lyn-selective kinase inhibitor, Bafetinib (INNO-406), acts to reduce Claudin-2 expression and suppress breast cancer liver metastasis. Our findings may have major clinical implications and advise against the treatment of breast cancer patients with broad-acting SFK inhibitors and support the use of Lyn-specific inhibitors. PMID:25823815

  8. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer

    PubMed Central

    2010-01-01

    Introduction In breast cancer, gene expression analyses have defined five tumor subtypes (luminal A, luminal B, HER2-enriched, basal-like and claudin-low), each of which has unique biologic and prognostic features. Here, we comprehensively characterize the recently identified claudin-low tumor subtype. Methods The clinical, pathological and biological features of claudin-low tumors were compared to the other tumor subtypes using an updated human tumor database and multiple independent data sets. These main features of claudin-low tumors were also evaluated in a panel of breast cancer cell lines and genetically engineered mouse models. Results Claudin-low tumors are characterized by the low to absent expression of luminal differentiation markers, high enrichment for epithelial-to-mesenchymal transition markers, immune response genes and cancer stem cell-like features. Clinically, the majority of claudin-low tumors are poor prognosis estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and epidermal growth factor receptor 2 (HER2)-negative (triple negative) invasive ductal carcinomas with a high frequency of metaplastic and medullary differentiation. They also have a response rate to standard preoperative chemotherapy that is intermediate between that of basal-like and luminal tumors. Interestingly, we show that a group of highly utilized breast cancer cell lines, and several genetically engineered mouse models, express the claudin-low phenotype. Finally, we confirm that a prognostically relevant differentiation hierarchy exists across all breast cancers in which the claudin-low subtype most closely resembles the mammary epithelial stem cell. Conclusions These results should help to improve our understanding of the biologic heterogeneity of breast cancer and provide tools for the further evaluation of the unique biology of claudin-low tumors and cell lines. PMID:20813035

  9. β-1,3/1,6-Glucan alleviated intestinal mucosal barrier impairment of broiler chickens challenged with Salmonella enterica serovar Typhimurium.

    PubMed

    Shao, Yujing; Guo, Yuming; Wang, Zhong

    2013-07-01

    This study investigated the protective effect of β-1,3/1,6-glucan on gut morphology, intestinal epithelial tight junctions, and bacterial translocation of broiler chickens challenged with Salmonella enterica serovar Typhimurium. Ninety Salmonella-free Arbor Acre male broiler chickens were randomly divided into 3 groups: negative control group (NC), Salmonella Typhimurium-infected positive group (PC), and the Salmonella Typhimurium-infected group with dietary 100 mg/kg of β-1,3/1,6-glucan supplementation (T) to determine the effect of β-1,3/1,6-glucan on intestinal barrier function. Salmonella Typhimurium challenge alone significantly decreased villus height (P < 0.001), villus height/crypt depth ratio (P < 0.05), and the number of goblet cells (P < 0.001) in the jejunum at 14 d postinfection (dpi), but significantly increased the number of intestinal secretory IgA (sIgA)-expressing cells at 14 dpi (P < 0.01) and total sIgA levels in the jejunum at 7 (P < 0.05) and 14 dpi (P < 0.01) compared with the unchallenged birds (NC). Dietary β-1,3/1,6-glucan supplementation not only significantly increased villus height, villus height/crypt depth ratio, and the number of goblet cells (P < 0.01), but also increased the number of sIgA-expressing cells (P < 0.05) and sIgA content in the jejunum at 14 dpi (P < 0.01) in birds challenged with Salmonella Typhimurium in comparison with Salmonella Typhimurium challenge alone. β-1,3/1,6-Glucan addition had significant inhibitory effects (P < 0.05) on cecal Salmonella colonization levels and liver Salmonella invasion of the Salmonella Typhimurium-infected birds compared with the PC group. Intestinal tight junction proteins claudin-1, claudin-4, and occludin mRNA expression in the jejunum at 14 dpi was significantly decreased by Salmonella Typhimurium challenge alone (P < 0.01) compared with that of the NC group, whereas β-1,3/1,6-glucan supplementation significantly increased claudin-1 and occludin mRNA expression (P < 0.01) at

  10. Claudin-6, -10d and -10e contribute to seawater acclimation in the euryhaline puffer fish Tetraodon nigroviridis.

    PubMed

    Bui, Phuong; Kelly, Scott P

    2014-05-15

    Expression profiles of claudin-6, -10d and -10e in the euryhaline teleost fish Tetraodon nigroviridis revealed claudin-6 in brain, eye, gill and skin tissue, while claudin-10d and -10e were found in brain, gill and skin only. In fishes, the gill and skin are important tissue barriers that interface directly with surrounding water, but these organs generally function differently in osmoregulation. Therefore, roles for gill and skin claudin-6, -10d and -10e in the osmoregulatory strategies of T. nigroviridis were investigated. In the gill epithelium, claudin-6, -10d and -10e co-localized with Na(+)-K(+)-ATPase immunoreactive (NKA-ir) ionocytes, and differences in sub-cellular localization could be observed in hypoosmotic (freshwater, FW) versus hyperosmotic (seawater, SW) environments. Claudin-10d and -10e abundance increased in the gills of fish acclimated to SW versus FW, while claudin-6 abundance decreased in the gills of fish acclimated to SW. Taken together with our knowledge of claudin-6 and -10 function in other vertebrates, data support the idea that in SW-acclimated T. nigroviridis, these claudins are abundant in gill ionocytes, where they contribute to the formation of a Na(+) shunt and 'leaky' epithelium, both of which are characteristic of salt-secreting SW fish gills. Skin claudin-10d and -10e abundance also increased in fish acclimated to SW versus those in FW, but so did claudin-6. In skin, claudin-6 was found to co-localize with NKA-ir cells, but claudin-10d and -10e did not. This study provides direct evidence that the gill epithelium contains salinity-responsive tight junction proteins that are abundant primarily in ionocytes. These same proteins also appear to play a role in the osmoregulatory physiology of the epidermis. PMID:24526724

  11. Value of claudin-4 immunostaining in the diagnosis of mesothelioma.

    PubMed

    Ordóñez, Nelson G

    2013-05-01

    Claudin-4 (CL-4) is a tight junction-associated protein that is expressed in most epithelial cells but absent in mesothelial cells. The purpose of this study is to evaluate the utility of CL-4 immunostaining for assisting in the differential diagnosis of mesothelioma. Sixty mesotheliomas (40 epithelioid, 10 biphasic, and 10 sarcomatoid), 185 carcinomas of different origins that can potentially be confused with mesotheliomas, 37 soft-tissue sarcomas, and 5 melanomas were investigated for CL-4 expression. All 60 mesotheliomas were CL-4 negative. In contrast, 169 (91%) of 185 carcinomas expressed this marker. Five of 8 desmoplastic small round cell tumors and the epithelial component of all 5 biphasic synovial sarcomas were CL-4 positive, whereas none of the remaining soft-tissue sarcomas or melanomas expressed this marker. It is concluded that CL-4 is a highly specific and sensitive immunohistochemical marker for assisting in distinguishing epithelioid mesotheliomas from metastatic carcinomas to the serosal membranes. PMID:23596113

  12. Claudin-2 as a mediator of leaky gut barrier during intestinal inflammation.

    PubMed

    Luettig, J; Rosenthal, R; Barmeyer, C; Schulzke, J D

    2015-01-01

    The epithelial tight junction determines the paracellular water and ion movement in the intestine and also prevents uptake of larger molecules, including antigens, in an uncontrolled manner. Claudin-2, one of the 27 mammalian claudins regulating that barrier function, forms a paracellular channel for small cations and water. It is typically expressed in leaky epithelia like proximal nephron and small intestine and provides a major pathway for the paracellular transport of sodium, potassium, and fluid. In intestinal inflammation (Crohn's disease, ulcerative colitis), immune-mediated diseases (celiac disease), and infections (HIV enteropathy), claudin-2 is upregulated in small and large intestine and contributes to diarrhea via a leak flux mechanism. In parallel to that upregulation, other epithelial and tight junctional features are altered and the luminal uptake of antigenic macromolecules is enhanced, for which claudin-2 may be partially responsible through induction of tight junction strand discontinuities. PMID:25838982

  13. Probing effects of pH change on dynamic response of Claudin-2 mediated adhesion using single molecule force spectroscopy.

    PubMed

    Lim, Tong Seng; Vedula, Sri Ram Krishna; Hui, Shi; Kausalya, P Jaya; Hunziker, Walter; Lim, Chwee Teck

    2008-08-15

    Claudins belong to a large family of transmembrane proteins that localize at tight junctions (TJs) where they play a central role in regulating paracellular transport of solutes and nutrients across epithelial monolayers. Their ability to regulate the paracellular pathway is highly influenced by changes in extracellular pH. However, the effect of changes in pH on the strength and kinetics of claudin mediated adhesion is poorly understood. Using atomic force microscopy, we characterized the kinetic properties of homophilic trans-interactions between full length recombinant GST tagged Claudin-2 (Cldn2) under different pH conditions. In measurements covering three orders of magnitude change in force loading rate of 10(2)-10(4) pN/s, the Cldn2/Cldn2 force spectrum (i.e., unbinding force versus loading rate) revealed a fast and a slow loading regime that characterized a steep inner activation barrier and a wide outer activation barrier throughout pH range of 4.5-8. Comparing to the neutral condition (pH 6.9), differences in the inner energy barriers for the dissociation of Cldn2/Cldn2 mediated interactions at acidic and alkaline environments were found to be <0.65 k(B)T, which is much lower than the outer dissociation energy barrier (>1.37 k(B)T). The relatively stable interaction of Cldn2/Cldn2 in neutral environment suggests that electrostatic interactions may contribute to the overall adhesion strength of Cldn2 interactions. Our results provide an insight into the changes in the inter-molecular forces and adhesion kinetics of Cldn2 mediated interactions in acidic, neutral and alkaline environments. PMID:18602630

  14. Probing effects of pH change on dynamic response of Claudin-2 mediated adhesion using single molecule force spectroscopy

    SciTech Connect

    Lim, Tong Seng; Vedula, Sri Ram Krishna; Hui Shi; Kausalya, P. Jaya; Hunziker, Walter; Lim, Chwee Teck

    2008-08-15

    Claudins belong to a large family of transmembrane proteins that localize at tight junctions (TJs) where they play a central role in regulating paracellular transport of solutes and nutrients across epithelial monolayers. Their ability to regulate the paracellular pathway is highly influenced by changes in extracellular pH. However, the effect of changes in pH on the strength and kinetics of claudin mediated adhesion is poorly understood. Using atomic force microscopy, we characterized the kinetic properties of homophilic trans-interactions between full length recombinant GST tagged Claudin-2 (Cldn2) under different pH conditions. In measurements covering three orders of magnitude change in force loading rate of 10{sup 2}-10{sup 4} pN/s, the Cldn2/Cldn2 force spectrum (i.e., unbinding force versus loading rate) revealed a fast and a slow loading regime that characterized a steep inner activation barrier and a wide outer activation barrier throughout pH range of 4.5-8. Comparing to the neutral condition (pH 6.9), differences in the inner energy barriers for the dissociation of Cldn2/Cldn2 mediated interactions at acidic and alkaline environments were found to be < 0.65 k{sub B}T, which is much lower than the outer dissociation energy barrier (> 1.37 k{sub B}T). The relatively stable interaction of Cldn2/Cldn2 in neutral environment suggests that electrostatic interactions may contribute to the overall adhesion strength of Cldn2 interactions. Our results provide an insight into the changes in the inter-molecular forces and adhesion kinetics of Cldn2 mediated interactions in acidic, neutral and alkaline environments.

  15. Claudin-4 expression in gastric cancer cells enhances the invasion and is associated with the increased level of matrix metalloproteinase-2 and -9 expression

    PubMed Central

    HWANG, TSANN-LONG; CHANGCHIEN, TZU-TSUNG; WANG, CHEE-CHAN; WU, CHI-MING

    2014-01-01

    Claudin-4 is a member of a large family of transmembrane proteins known as claudins, which are essential for the formation and maintenance of tight junctions. Our previous studies have revealed that claudin-4 proteins are overexpressed in metastatic gastric cancer. To clarify the roles of claudin-4 in gastric cancer metastasis, human gastric adenocarcinoma (AGS) cells constitutively expressing wild-type claudin-4 were generated. Expression of claudin-4 in AGS cells was found to increase cell invasion and migration, as measured by Boyden invasion chamber assays. Moreover, the claudin-4-expressing AGS cells were found to have increased matrix metalloproteinase (MMP)-2 and -9 expression, indicating that claudin-mediated increased invasion may be mediated through the activation of the MMP protein. Overall, the results suggest that claudin-4 overexpression may promote gastric cancer metastasis through the increased invasion of gastric cancer cells. PMID:25120725

  16. Claudins in a primary cultured puffer fish (Tetraodon nigroviridis) gill epithelium model alter in response to acute seawater exposure.

    PubMed

    Bui, Phuong; Kelly, Scott P

    2015-11-01

    Gill epithelium permeability and qualitative/quantitative aspects of gill claudin (cldn) tight junction (TJ) protein transcriptomics were examined with a primary cultured model gill epithelium developed using euryhaline puffer fish (Tetraodon nigroviridis) gills. The model was prepared using seawater-acclimated fish gills and was cultured on permeable cell culture filter supports. The model is composed of 1-2 confluent layers of gill pavement cells (PVCs), with the outer layer exhibiting prominent apical surface microridges and TJs between adjacent cells. During development of electrophysiological characteristics, the model exhibits a sigmoidal increase in transpithelial resistance (TER) and plateaus around 30 kΩcm(2). At this point paracellular movement of [(3)H]polyethylene glycol (PEG) 4000 was low at ~1.75 cm s(-1)×10(-7). When exposed to apical seawater (SW) epithelia exhibit a marked decrease in TER while PEG flux remained unchanged for at least 6 h. In association with this, transcript encoding cldn TJ proteins cldn3c, -23b, -27a, -27c, -32a and -33b increased during the first 6 h while cldn11a decreased. This suggests that these proteins are involved in maintaining barrier properties between gill PVCs of SW fishes. Gill cldn mRNA abundance also altered 6 and 12 h following abrupt SW exposure of puffer fish, but in a manner that differed qualitatively and quantitatively from the cultured model. This most likely reflects the cellular heterogeneity of whole tissue and/or the contribution of the endocrine system in intact fish. The current study provides insight into the physiological and transcriptomic response of euryhaline fish gill cells to a hyperosmotic environment. PMID:26239219

  17. Aberrantly activated claudin 6 and 18.2 as potential therapy targets in non-small-cell lung cancer.

    PubMed

    Micke, Patrick; Mattsson, Johanna Sofia Margareta; Edlund, Karolina; Lohr, Miriam; Jirström, Karin; Berglund, Anders; Botling, Johan; Rahnenfuehrer, Jörg; Marincevic, Millaray; Pontén, Fredrik; Ekman, Simon; Hengstler, Jan; Wöll, Stefan; Sahin, Ugur; Türeci, Ozlem

    2014-11-01

    Claudins (CLDNs) are central components of tight junctions that regulate epithelial-cell barrier function and polarity. Altered CLDN expression patterns have been demonstrated in numerous cancer types and lineage-specific CLDNs have been proposed as therapy targets. The objective of this study was to assess which fraction of patients with non-small-cell lung cancer (NSCLC) express CLDN6 and CLDN18 isoform 2 (CLDN18.2). Protein expression of CLDN6 and CLDN18.2 was examined by immunohistochemistry on a tissue microarray (n = 355) and transcript levels were supportively determined based on gene expression microarray data from fresh-frozen NSCLC tissues (n = 196). Both were analyzed with regard to frequency, distribution and association with clinical parameters. Immunohistochemical analysis of tissue sections revealed distinct membranous positivity of CLDN6 (6.5%) and CLDN18.2 (3.7%) proteins in virtually non-overlapping subgroups of adenocarcinomas and large-cell carcinomas. Pneumocytes and bronchial epithelial cells were consistently negative. Corresponding to the protein expression, in subsets of non-squamous lung carcinoma high mRNA levels of CLDN6 (7-16%) and total CLDN18 (5-12%) were observed. Protein expression correlated well with total mRNA expression of the corresponding gene (rho = 0.4-0.8). CLDN18.2 positive tumors were enriched among slowly proliferating, thyroid transcription factor 1 (TTF-1)-negative adenocarcinomas, suggesting that isoform-specific CLDN expression may delineate a specific subtype. Noteworthy, high CLDN6 protein expression was associated with worse prognosis in lung adenocarcinoma in the univariate [hazard ratio (HR): 1.8; p = 0.03] and multivariate COX regression model (HR: 1.9; p = 0.02). These findings encourage further clinical exploration of targeting ectopically activated CLDN expression as a valuable treatment concept in NSCLC. PMID:24710653

  18. Low claudin-6 expression correlates with poor prognosis in patients with non-small cell lung cancer

    PubMed Central

    Wang, Qiang; Zhang, Yan; Zhang, Tao; Han, Zhi-Gang; Shan, Li

    2015-01-01

    Objective Claudins are found in junctional complexes mediating cell adhesion and are involved in the attachment of tight junctions to the underlying cytoskeleton. Abnormal claudin-6 expression has been observed for a variety of malignant solid tumors, but the expression of claudin-6 in non-small cell lung cancer (NSCLC) has not yet been characterized. Methods Immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and western blot analysis were used to quantify claudin-6 expression in 123 cases of NSCLC and non-cancerous adjacent tissue. We analyzed the relationship between claudin-6 expression and clinicopathological features of NSCLC. The Kaplan–Meier method was used to analyze postoperative survival rates, and the log-rank test was used to assess differences in survival rates. The Cox regression model was used to perform multivariate analysis. Results Claudin-6 expression was low for 61 of 123 (49.6%) NSCLC tissue samples and for 33 of 123 (26.8%) normal adjacent tissue samples. RT-PCR and western blot analyses confirmed the immunohistochemistry results. Claudin-6 expression was associated with lymph node metastasis (P<0.001) and TNM stage (P=0.007). Kaplan–Meier analysis indicated that patients with low claudin-6 expression had significantly lower survival rates than those with high claudin-6 expression. Multivariate analysis suggested that low claudin-6 expression was an independent indicator of prognosis in NSCLC patients. Conclusion Low claudin-6 expression is an independent prognostic biomarker that indicates a worse prognosis in patients with NSCLC. PMID:26261421

  19. Emerging Multifunctional Roles of Claudin Tight Junction Proteins in Bone

    PubMed Central

    Alshbool, Fatima. Z.

    2014-01-01

    The imbalance between bone formation and resorption during bone remodeling has been documented to be a major factor in the pathogenesis of osteoporosis. Recent evidence suggests a significant role for the tight junction proteins, Claudins (Cldns), in the regulation of bone remodeling processes. In terms of function, whereas Cldns act “canonically” as key determinants of paracellular permeability, there is considerable recent evidence to suggest that Cldns also participate in cell signaling, ie, a “noncanonical function”. To this end, Cldns have been shown to regulate cell proliferation, differentiation, and gene expression in a variety of cell types. The present review will discuss Cldns' structure, their expression profile, regulation of expression, and their canonical and non- canonical functions in general with special emphasis on bone cells. In order to shed light on the noncanonical functions of Cldns in bone, we will highlight the role of Cldn-18 in regulating bone resorption and osteoclast differentiation. Collectively, we hope to provide a framework for guiding future research on understanding how Cldns modulate osteoblast and osteoclast function and overall bone homeostasis. Such studies should provide valuable insights into the pathogenesis of osteoporosis, and may highlight Cldns as novel targets for the diagnosis and therapeutic management of osteoporosis. PMID:24758302

  20. Establishment of a neuroepithelial barrier by Claudin5a is essential for zebrafish brain ventricular lumen expansion

    PubMed Central

    Zhang, Jingjing; Piontek, Jörg; Wolburg, Hartwig; Piehl, Christian; Liss, Martin; Otten, Cécile; Christ, Annabel; Willnow, Thomas E.; Blasig, Ingolf E.; Abdelilah-Seyfried, Salim

    2010-01-01

    Lumen expansion driven by hydrostatic pressure occurs during many morphogenetic processes. Although it is well established that members of the Claudin family of transmembrane tight junction proteins determine paracellular tightness within epithelial/endothelial barrier systems, functional evidence for their role in the morphogenesis of lumenized organs has been scarce. Here, we identify Claudin5a as a core component of an early cerebral-ventricular barrier system that is required for ventricular lumen expansion in the zebrafish embryonic brain before the establishment of the embryonic blood–brain barrier. Loss of Claudin5a or expression of a tight junction-opening Claudin5a mutant reduces brain ventricular volume expansion without disrupting the polarized organization of the neuroepithelium. Perfusion experiments with the electron-dense small molecule lanthanum nitrate reveal that paracellular tightness of the cerebral-ventricular barrier decreases upon loss of Claudin5a. Genetic analyses show that the apical neuroepithelial localization of Claudin5a depends on epithelial cell polarity and provide evidence for concerted activities between Claudin5a and Na+,K+-ATPase during luminal expansion of brain ventricles. These data establish an essential role of a barrier-forming Claudin in ventricular lumen expansion, thereby contributing to brain morphogenesis. PMID:20080584

  1. Presence of claudins mRNA in the brain. Selective modulation of expression by kindling epilepsy.

    PubMed

    Lamas, Mónica; González-Mariscal, Lorenza; Gutiérrez, Rafael

    2002-08-15

    In the central nervous system, the junctional types that establish and maintain tissue architecture include gap junctions, for cytoplasmic connectivity, and tight junctions, for paracellular and/or cell polarity barriers. Connexins are the integral membrane proteins of gap junctions, whereas occludin and members of the multigene family of claudins form tight junctions. In the brain, there are no transendothelial pathways, as continuous tight junctions are present between the endothelial cells. Thus, they provide a continuous cellular barrier between the blood and the insterstitial fluid. However, several brain pathologies, including epilepsy, are known to alter the permeability of the blood-brain barrier and to cause edema. Therefore, since claudins, as constitutive proteins of tight junctions are likely candidates for modulation under pathological states, we explored their normal pattern of expression in the brain and its modulation by seizures. We found that several members of this family are normally expressed in the hippocampus and cortex. Interestingly, claudin-7 is expressed in the hippocampus but not in the cortex. On the other hand, the expression of claudin-8 is selectively down-regulated in the hippocampus as kindling evolves. These results link for the first time the modulation of expression of a tight junction protein to abnormal neuronal synchronization that could probably be reflected in permeability changes of the blood-brain barrier or edema. PMID:12225881

  2. Microelectrode bioimpedance analysis distinguishes basal and claudin-low subtypes of triple negative breast cancer cells.

    PubMed

    Srinivasaraghavan, Vaishnavi; Strobl, Jeannine; Agah, Masoud

    2015-08-01

    Triple negative breast cancer (TNBC) is highly aggressive and has a poor prognosis when compared to other molecular subtypes. In particular, the claudin-low subtype of TNBC exhibits tumor-initiating/cancer stem cell like properties. Here, we seek to find new biomarkers to discriminate different forms of TNBC by characterizing their bioimpedance. A customized bioimpedance sensor with four identical branched microelectrodes with branch widths adjusted to accommodate spreading of individual cells was fabricated on silicon and pyrex/glass substrates. Cell analyses were performed on the silicon devices which showed somewhat improved inter-electrode and intra-device reliability. We performed detailed analysis of the bioimpedance spectra of four TNBC cell lines, comparing the peak magnitude, peak frequency and peak phase angle between claudin-low TNBC subtype represented by MDA-MB-231 and Hs578T with that of two basal cells types, the TNBC MDA-MB-468, and an immortalized non-malignant basal breast cell line, MCF-10A. The claudin-low TNBC cell lines showed significantly higher peak frequencies and peak phase angles than the properties might be useful in distinguishing the clinically significant claudin-low subtype of TNBC. PMID:26216474

  3. Par3 regulates invasion of pancreatic cancer cells via interaction with Tiam1.

    PubMed

    Guo, Xingjun; Wang, Min; Zhao, Yan; Wang, Xin; Shen, Ming; Zhu, Feng; Shi, Chengjian; Xu, Meng; Li, Xu; Peng, Feng; Zhang, Hang; Feng, Yechen; Xie, Yu; Xu, Xiaodong; Jia, Wei; He, Ruizhi; Jiang, Jianxin; Hu, Jun; Tian, Rui; Qin, Renyi

    2016-08-01

    The conserved polarity complex, which comprises partitioning-defective proteins Par3, Par6, and the atypical protein kinase C, affects various cell-polarization events, including assembly of tight junctions. Control of tight junction assembly is closely related to invasion and migration potential. However, as the importance of conserved polarity complexes in regulating pancreatic cancer invasion and metastasis is unclear, we investigated their role and mechanism in pancreatic cancers. We first detect that the key protein of the conserved polarity complex finds that only Par3 is down-regulated in pancreatic cancer tissues while Par6 and aPKC show no difference. What is more, Par3 tissues level was significantly and positively associated with patient overall survival. Knocking-down Par3 promotes pancreatic cancer cells invasion and migration. And Par3 requires interaction with Tiam1 to affect tight junction assembly, and then affect invasion and migration of pancreatic cancer cells. Then, we find that tight junction marker protein ZO-1 and claudin-1 are down-regulated in pancreatic cancer tissues. And the relationship of the expression of Par3 and ZO-1 in pancreatic cancer tissue is linear correlation. We establish liver metastasis model of human pancreatic cancer cells in Balb/c nude mice and find that knocking down Par3 promotes invasion and metastasis and disturbs tight junction assembly in vivo. Taken together, these results suggest that the Par3 regulates invasion and metastasis in pancreatic cancers by controlling tight junction assembly. PMID:26084985

  4. Claudin-11 and occludin are major contributors to Sertoli cell tight junction function, in vitro.

    PubMed

    McCabe, Mark J; Foo, Caroline Fh; Dinger, Marcel E; Smooker, Peter M; Stanton, Peter G

    2016-01-01

    The Sertoli cell tight junction (TJ) is the key component of the blood-testis barrier, where it sequesters developing germ cells undergoing spermatogenesis within the seminiferous tubules. Hormonally regulated claudin-11 is a critical transmembrane protein involved in barrier function and its murine knockout results in infertility. We aimed to assess quantitatively the significance of the contribution of claudin-11 to TJ function, in vitro, using siRNA-mediated gene silencing. We also conducted an analysis of the contribution of occludin, another intrinsic transmembrane protein of the TJ. Silencing of claudin-11 and/or occludin was conducted using siRNA in an immature rat Sertoli cell culture model. Transepithelial electrical resistance was used to assess quantitatively TJ function throughout the culture. Two days after siRNA treatment, cells were fixed for immunocytochemical localization of junction proteins or lyzed for RT-PCR assessment of mRNA expression. Silencing of claudin-11, occludin, or both resulted in significant decreases in TJ function of 55% (P < 0.01), 51% (P < 0.01), and 62% (P < 0.01), respectively. Data were concomitant with significant decreases in mRNA expression and marked reductions in the localization of targeted proteins to the Sertoli cell TJ. We provide quantitative evidence that claudin-11 contributes significantly (P < 0.01) to Sertoli cell TJ function in vitro. Interestingly, occludin, which is hormonally regulated but not implicated in infertility until late adulthood, is also a significant (P < 0.01) contributor to barrier function. Our data are consistent with in vivo studies that clearly demonstrate a role for these proteins in maintaining normal TJ barrier structure and function. PMID:26585695

  5. Claudin-11 and occludin are major contributors to Sertoli cell tight junction function, in vitro

    PubMed Central

    McCabe, Mark J; Foo, Caroline FH; Dinger, Marcel E; Smooker, Peter M; Stanton, Peter G

    2016-01-01

    The Sertoli cell tight junction (TJ) is the key component of the blood-testis barrier, where it sequesters developing germ cells undergoing spermatogenesis within the seminiferous tubules. Hormonally regulated claudin-11 is a critical transmembrane protein involved in barrier function and its murine knockout results in infertility. We aimed to assess quantitatively the significance of the contribution of claudin-11 to TJ function, in vitro, using siRNA-mediated gene silencing. We also conducted an analysis of the contribution of occludin, another intrinsic transmembrane protein of the TJ. Silencing of claudin-11 and/or occludin was conducted using siRNA in an immature rat Sertoli cell culture model. Transepithelial electrical resistance was used to assess quantitatively TJ function throughout the culture. Two days after siRNA treatment, cells were fixed for immunocytochemical localization of junction proteins or lyzed for RT-PCR assessment of mRNA expression. Silencing of claudin-11, occludin, or both resulted in significant decreases in TJ function of 55% (P < 0.01), 51% (P < 0.01), and 62% (P < 0.01), respectively. Data were concomitant with significant decreases in mRNA expression and marked reductions in the localization of targeted proteins to the Sertoli cell TJ. We provide quantitative evidence that claudin-11 contributes significantly (P < 0.01) to Sertoli cell TJ function in vitro. Interestingly, occludin, which is hormonally regulated but not implicated in infertility until late adulthood, is also a significant (P < 0.01) contributor to barrier function. Our data are consistent with in vivo studies that clearly demonstrate a role for these proteins in maintaining normal TJ barrier structure and function. PMID:26585695

  6. Synthesis of 1,3,3-trinitroazetidine

    DOEpatents

    Hiskey, M.A.; Coburn, M.D.

    1994-08-09

    A process of preparing 1,3,3-trinitroazetidine includes forming a 5-hydroxymethyl-5-nitro-1-alkyltetrahydro-1,3-oxazine, e.g., reacting a 1,3,5-trialkyl hexahydrotriazine and tris(hydroxymethyl)nitromethane, ring opening said 5-hydroxymethyl-5-nitro-1-alkyltetrahydro-1,3-oxazine to form a 3-alkylamino-2-hydroxymethyl-2-nitro-1-propanol salt, ring closing said 3-alkylamino-2-hydroxymethyl-2-nitro-1-propanol salt to form a 3-hydroxymethyl-3-nitro-1-alkylazetidine salt, nitrating said 3-hydroxymethyl-3-nitro-1-alkylazetidine salt to form a 1-alkyl-3,3-dinitroazetidine, and converting said 1-alkyl-3,3-dinitroazetidine into 1,3,3-trinitroazetidine is disclosed. 1 fig.

  7. Synthesis of 1,3,3-trinitroazetidine

    DOEpatents

    Hiskey, Michael A.; Coburn, Michael D.

    1994-01-01

    A process of preparing 1,3,3-trinitroazetidine including forming a 5-hydroxymethyl-5-nitro-1-alkyltetrahydro-1,3-oxazine, e.g., reacting a 1,3,5-trialkyl hexahydrotriazine and tris(hydroxymethyl)nitromethane, ring opening said 5-hydroxymethyl-5-nitro-1-alkyltetrahydro-1,3-oxazine to form a 3-alkylamino-2-hydroxymethyl-2-nitro-1-propanol salt, ring closing said 3-alkylamino-2-hydroxymethyl-2-nitro-1-propanol salt to form a 3-hydroxymethyl-3-nitro-1-alkylazetidine salt, nitrating said 3-hydroxymethyl-3-nitro-1-alkylazetidine salt to form a 1-alkyl-3,3-dinitroazetidine, and converting said 1-alkyl-3,3-dinitroazetidine into 1,3,3-trinitroazetidine is disclosed.

  8. Role of TGF-β-induced Claudin-4 expression through c-Jun signaling in non-small cell lung cancer.

    PubMed

    Rachakonda, Girish; Vu, Trung; Jin, Lin; Samanta, Debangshu; Datta, Pran K

    2016-10-01

    Claudin-4 has been identified as an integral member of tight junctions and has been found to be upregulated in various types of cancers especially in metastatic cancers. However, the molecular mechanism of the upregulation of Claudin-4 and its role in lung tumorigenesis are unknown. The aim of the present study is to investigate the role of Claudin-4 on migration and tumorigenicity of lung cancer cells and to examine the regulatory effects of TGF-β on Claudin-4 expression. We have observed that TGF-β induces the expression of Claudin-4 dramatically in lung cell lines in a time dependent manner. TGF-β-induced Smad signaling is important for enhancing Claudin-4 mRNA level through inducing its promoter activity. Treatment with curcumin, a c-Jun inhibitor, or stable knockdown of c-Jun abrogates TGF-β-induced Claudin-4 expression suggesting an involvement of the c-Jun pathway. Notably, TGF-β-induced Claudin-4 expression through c-Jun pathway plays a role in TGF-β-mediated motility and tumorigenicity of these cells. In support of these observations, we have uncovered that Claudin-4 is upregulated in 14 of 24 (58%) lung tumors when compared with normal lung tissue. This is the first study to show how TGF-β regulates the expression of Claudin-4 through c-Jun signaling and how this pathway contributes to the migratory and tumorigenic phenotype of lung tumor cells. PMID:27424491

  9. Expression of claudins -2 and -4 and cingulin is coordinated with the start of stratification and differentiation in corneal epithelial cells: retinoic acid reversibly disrupts epithelial barrier

    PubMed Central

    Ortiz-Melo, María Teresa; Sánchez-Guzmán, Erika; González-Robles, Arturo; Valdés, Jesús; Gómez-Flores, Eber; Castro-Muñozledo, Federico

    2013-01-01

    Summary Although tight junctions (TJ) have been extensively studied in simple epithelial cells, it is still unknown whether their organization is coupled to cell differentiation in stratified epithelia. We studied the expression of TJ in RCE1(5T5) cells, an in vitro model which mimics the sequential steps of rabbit corneal epithelial differentiation. RCE1(5T5) cells expressed TJ components which were assembled once cells constituted differentiated epithelia, as suggested by the increase of transepithelial electrical resistance (TER) which followed a similar kinetic to the expression of the early differentiation marker Pax-6. TJ were functional as indicated by the establishment of an epithelial barrier nonpermeable to ruthenium red or a biotin tracer. In immunostaining experiments, TJ were located at the superficial cells from the suprabasal layers; Western blot and RT-PCR suggested that TJ were composed of claudins (cldn) -1, -2, -4, cingulin (cgn), occludin (ocln) and ZO-1. Semi-quantitative RT-PCR and TER measurements showed that TJ became organized when cells began to form a 3–5 layers stratified epithelium; TER increased once cells reached confluence, with a time course comparable to the raise in the expression of cgn, cldn-2 and -4. Nevertheless, cldn-1, -2, ZO-1 and ocln were present in the cells from the beginning of cultivation, suggesting that TER increases mainly depend on TJ assembly. While EGF increased epithelial barrier strength, retinoic acid disrupted it, increasing paracellular flux about 2-fold; this effect was concentration dependent and completely reversible. Our results suggest that TJ assembly is tightly linked to the expression of corneal epithelial terminal phenotype. PMID:23429425

  10. 3.1 Introduction

    NASA Astrophysics Data System (ADS)

    Kramer, H.-M.

    This document is part of Subvolume A 'Fundamentals and Data in Radiobiology, Radiation Biophysics, Dosimetry and Medical Radiological Protection' of Volume 7 'Medical Radiological Physics' of Landolt-Börnstein - Group VIII 'Advanced Materials and Technologies'. It contains the Section '3.1 Introduction' of the Chapter '3 Dosimetry in Diagnostic Radiology and Radiotherapy'.

  11. 1,3-Dichlorobenzene

    Integrated Risk Information System (IRIS)

    1,3 - Dichlorobenzene ; CASRN 541 - 73 - 1 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinog

  12. Functional dynamics of claudin expression in Japanese medaka (Oryzias latipes): Response to environmental salinity.

    PubMed

    Bossus, Maryline C; Madsen, Steffen S; Tipsmark, Christian K

    2015-09-01

    Salinity regulation of 13 claudin paralogs was investigated in osmoregulatory organs of euryhaline Japanese medaka. They were identified by blast-search in the medaka genome database based on representation in osmoregulatory organs of other teleosts. Our hypothesis was that, because of their sequence similarities to mammalian orthologs previously characterized as barrier- and ion-selective channel-forming proteins, these paralogs would respond to salinity according to expected modulation of osmoregulatory function. Cldn10c, -10d, -10e, -10f, -27a, -28a, -28b and -30c had 4- to 100-fold higher expression in gill than other examined organs. Two splice variants of cldn10b were predominantly expressed in kidney, while cldn15a, -15b and -25 were found mainly in intestine. In gills, cldn27a, -28a, -28b and -30c did not change between fresh water (FW) and seawater (SW)-acclimated fish, while cldn10c, -10d, -10e, and -10f were most abundant in SW. Short-term SW transfer induced up-regulation of cldn10 gill paralogs after 1 day, decrease in cldn28b and no difference for cldn27a, -28a and -30c. The reverse pattern was observed after FW transfer of SW medaka. Intestinal cldn15a and -25 did not differ between FW and SW fish. However, cldn15b was 10-fold higher in FW than SW, suggesting a role in functional modulation of the intestine related to water and salt transport. In kidney, cldn10bs were elevated in SW fish, suggesting a role in paracellular ion transport in the marine nephron. Based on in silico analysis, most gill Cldn10s were predicted to form cation pores, whereas Cldn27a, 28a, 28b and 30c may increase epithelial resistance. PMID:25957710

  13. 1,3-Dichloropropene

    Integrated Risk Information System (IRIS)

    1,3 - Dichloropropene ( DCP ) ; CASRN 542 - 75 - 6 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Non

  14. 1,3-Butadiene

    Integrated Risk Information System (IRIS)

    1,3 - Butadiene ; CASRN 106 - 99 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic E

  15. Structure-Function Studies of Claudin Extracellular Domains by Cysteine-scanning Mutagenesis*

    PubMed Central

    Angelow, Susanne; Yu, Alan S. L.

    2009-01-01

    Claudins form size- and charge-selective pores in the tight junction that control the paracellular flux of inorganic ions and small molecules. However, the structural basis for ion selectivity of paracellular pores is poorly understood. Here we applied cysteine scanning to map the paracellular pathway of ion permeation across claudin-2-transfected Madin-Darby canine kidney type I cells. Four potential pore-lining amino acid residues in the first extracellular loop were mutated to cysteine and screened for their accessibility to thiol-reactive reagents. All mutants were functional except D65C, which formed dimers by intermolecular disulfide bonding, leading to a loss of charge and size selectivity. This suggests that claudin-2 pores are multimeric and that Asp65 lies close to a protein-protein interface. Methanethiosulfonate reagents of different size and charge and the organic mercury derivate, p-(chloromercuri)benzenesulfonic acid, significantly decreased paracellular ion permeation across I66C-transfected cells by a mechanism that suggests steric blocking of the pore. The conductance of wild-type claudin-2 and the other cysteine mutants was only weakly affected. The rate of reaction with I66C decreased dramatically with increasing size of the reagent, suggesting that Ile66 is buried deep within a narrow segment of the pore with its side group facing into the lumen. Furthermore, labeling with N-biotinoylaminoethyl methanethiosulfonate showed that I66C was weakly reactive, whereas Y35C was strongly reactive, suggesting that Tyr35 is located at the protein surface outside of the pore. PMID:19690347

  16. Claudin-2 Expression Levels in Ulcerative Colitis: Development and Validation of an In-Situ Hybridisation Assay for Therapeutic Studies.

    PubMed

    Randall, Kevin; Henderson, Neil; Reens, Jaimini; Eckersley, Sonia; Nyström, Ann-Christin; South, Marie C; Balendran, Clare A; Böttcher, Gerhard; Hughes, Glen; Price, Sally A

    2016-01-01

    Ulcerative colitis is a chronic inflammatory disease affecting the colon and is characterized by epithelial damage and barrier dysfunction. Upregulation of the tight junction protein claudin-2 by cytokines is hypothesized to contribute to the dysregulation of the epithelial barrier. New therapeutic agents which block the action of cytokines are being investigated in patients with ulcerative colitis. In order to understand the potential of these therapies, it is important to have reliable assays that can assess downstream endpoints that reflect drug mechanism of action. The aim of the current study was therefore to establish & validate an assay to reproducibly assess the expression and distribution of claudin-2 in human colon biopsy samples. Initially, the potential to measure claudin-2 protein by immunohistochemistry (IHC) was investigated. To identify suitable reagents to develop an IHC assay, pre-established criteria were used to screen five commercial antibodies by Western blotting, immunofluorescence and immunohistochemistry on claudin-2 positive and negative cells and healthy and ulcerative colitis colon tissue. Despite some of these antibodies specifically detecting claudin-2 using some of these techniques, none of the antibodies showed the expected specific staining pattern in formalin fixed human colon samples. As an alternative method to detect claudin-2 expression and distribution in formalin fixed biopsy sections, an in situ hybridization assay was developed. This assay underwent a novel tiered approach of validation to establish that it was fit-for-purpose, and suitable for clinical deployment. In addition, to understand the possible relationship of claudin-2 in the context of disease severity, expression was compared to the Geboes score. Overall, the microscopical Geboes score correlated with the claudin-2 biomarker score for samples that retained crypt morphology; samples with the highest Geboes score were not specifically distinguished, probably due

  17. Claudin-4 undergoes age-dependent change in cellular localization on pig jejunal villous epithelial cells, independent of bacterial colonization.

    PubMed

    Pasternak, J Alex; Kent-Dennis, Coral; Van Kessel, Andrew G; Wilson, Heather L

    2015-01-01

    Newborn piglets are immunologically naïve and must receive passive immunity via colostrum within 24 hours to survive. Mechanisms by which the newborn piglet gut facilitates uptake of colostral cells, antibodies, and proteins may include FcRn and pIgR receptor-mediated endocytosis and paracellular transport between tight junctions (TJs). In the present study, FcRn gene (FCGRT) was minimally expressed in 6-week-old gut and newborn jejunum but it was expressed at significantly higher levels in the ileum of newborn piglets. pIgR was highly expressed in the jejunum and ileum of 6-week-old animals but only minimally in neonatal gut. Immunohistochemical analysis showed that Claudin-5 localized to blood vessel endothelial cells. Claudin-4 was strongly localized to the apical aspect of jejunal epithelial cells for the first 2 days of life after which it was redistributed to the lateral surface between adjacent enterocytes. Claudin-4 was localized to ileal lateral surfaces within 24 hours after birth indicating regional and temporal differences. Tissue from gnotobiotic piglets showed that commensal microbiota did not influence Claudin-4 surface localization on jejunal or ileal enterocytes. Regulation of TJs by Claudin-4 surface localization requires further investigation. Understanding the factors that regulate gut barrier maturation may yield protective strategies against infectious diseases. PMID:25948883

  18. Myofibroblast keratinocyte growth factor reduces tight junctional integrity and increases claudin-2 levels in polarized Caco-2 cells

    PubMed Central

    Kim, Tae Il; Poulin, Emily J.; Blask, Elliot; Bukhalid, Raghida; Whitehead, Robert H.; Franklin, Jeffrey L.; Coffey, Robert J.

    2013-01-01

    The colonic epithelium is composed of a polarized monolayer sheathed by a layer of pericryptal myofibroblasts (PCMFs). We mimicked these cellular compartments in vitro to assess the effects of paracrine-acting PCMF-derived factors on tight junction (TJ) integrity, as measured by transepithelial electrical resistance (TER). Co-culture with 18Co PCMFs, or basolateral administration of 18Co conditioned medium (CM), significantly reduced TER of polarized Caco-2 cells. Amongst candidate paracrine factors, only keratinocyte growth factor (KGF) reduced Caco-2 TER; basolateral KGF treatment led to time- and concentration-dependent increases in claudin-2 levels. We also demonstrate amphiregulin (AREG), produced largely by Caco-2 cells, increased claudin-2 levels, leading to epidermal growth factor receptor-mediated TER reduction. We propose that colonic epithelial TJ integrity can be modulated by paracrine KGF and autocrine AREG through increased claudin-2 levels. KGF-regulated claudin-2 induction may have implications for inflammatory bowel disease, where both KGF and claudin-2 are upregulated. PMID:22946653

  19. Claudin-4 Undergoes Age-Dependent Change in Cellular Localization on Pig Jejunal Villous Epithelial Cells, Independent of Bacterial Colonization

    PubMed Central

    Van Kessel, Andrew G.; Wilson, Heather L.

    2015-01-01

    Newborn piglets are immunologically naïve and must receive passive immunity via colostrum within 24 hours to survive. Mechanisms by which the newborn piglet gut facilitates uptake of colostral cells, antibodies, and proteins may include FcRn and pIgR receptor-mediated endocytosis and paracellular transport between tight junctions (TJs). In the present study, FcRn gene (FCGRT) was minimally expressed in 6-week-old gut and newborn jejunum but it was expressed at significantly higher levels in the ileum of newborn piglets. pIgR was highly expressed in the jejunum and ileum of 6-week-old animals but only minimally in neonatal gut. Immunohistochemical analysis showed that Claudin-5 localized to blood vessel endothelial cells. Claudin-4 was strongly localized to the apical aspect of jejunal epithelial cells for the first 2 days of life after which it was redistributed to the lateral surface between adjacent enterocytes. Claudin-4 was localized to ileal lateral surfaces within 24 hours after birth indicating regional and temporal differences. Tissue from gnotobiotic piglets showed that commensal microbiota did not influence Claudin-4 surface localization on jejunal or ileal enterocytes. Regulation of TJs by Claudin-4 surface localization requires further investigation. Understanding the factors that regulate gut barrier maturation may yield protective strategies against infectious diseases. PMID:25948883

  20. 1 CFR 3.1 - Information services.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 1 General Provisions 1 2012-01-01 2012-01-01 false Information services. 3.1 Section 3.1 General Provisions ADMINISTRATIVE COMMITTEE OF THE FEDERAL REGISTER GENERAL SERVICES TO THE PUBLIC § 3.1 Information services. Except in cases where the time required would be excessive, information concerning...

  1. 1 CFR 3.1 - Information services.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 1 General Provisions 1 2011-01-01 2011-01-01 false Information services. 3.1 Section 3.1 General Provisions ADMINISTRATIVE COMMITTEE OF THE FEDERAL REGISTER GENERAL SERVICES TO THE PUBLIC § 3.1 Information services. Except in cases where the time required would be excessive, information concerning...

  2. 1 CFR 3.1 - Information services.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 1 General Provisions 1 2013-01-01 2012-01-01 true Information services. 3.1 Section 3.1 General Provisions ADMINISTRATIVE COMMITTEE OF THE FEDERAL REGISTER GENERAL SERVICES TO THE PUBLIC § 3.1 Information services. Except in cases where the time required would be excessive, information concerning...

  3. 1 CFR 3.1 - Information services.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 1 General Provisions 1 2010-01-01 2010-01-01 false Information services. 3.1 Section 3.1 General Provisions ADMINISTRATIVE COMMITTEE OF THE FEDERAL REGISTER GENERAL SERVICES TO THE PUBLIC § 3.1 Information services. Except in cases where the time required would be excessive, information concerning...

  4. 1 CFR 3.1 - Information services.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 1 General Provisions 1 2014-01-01 2012-01-01 true Information services. 3.1 Section 3.1 General Provisions ADMINISTRATIVE COMMITTEE OF THE FEDERAL REGISTER GENERAL SERVICES TO THE PUBLIC § 3.1 Information services. Except in cases where the time required would be excessive, information concerning...

  5. The targeted overexpression of a Claudin mutant in the epidermis of transgenic mice elicits striking epidermal and hair follicle abnormalities.

    PubMed

    Troy, Tammy-Claire; Turksen, Kursad

    2007-06-01

    Skin is one of the largest organs of the body, and is formed during development through a highly orchestrated process involving mesenchymal-epithelial interactions, cell commitment, and terminal differentiation. It protects against microorganism invasion and UV irradiation, inhibits water loss, regulates body temperature, and is an important part of the immune system. Using transgenic mouse technology, we have demonstrated that Claudin (Cldn)-containing tight junctions (TJs) are intricately involved in cell signaling during epidermal differentiation and that an epidermal suprabasal overexpression of Cldn6 results in a perturbed epidermal terminal differentiation program with distinct phenotypic abnormalities. To delineate the role of the Cldn cytoplasmic tail domain in epidermal differentiation, we engineered transgenic mice targeting the overexpression of a Cldn6 cytoplasmic tail-truncation mutant in the epidermis. Transgenic mice were characterized by a lethal barrier dysfunction in addition to the existence of hyperproliferative squamous invaginations/cysts replacing hair follicles. Immunohistochemical analysis revealed an epidermal cytoplasmic accumulation of Cldn6, Cldn11, Cldn12, and Cldn18, downregulation of Cldn1 and aberrant expression of various classical markers of epidermal differentiation; namely the basal keratins as well as K1, involucrin, loricrin, and filaggrin. Collectively these studies suggest an important role for Cldns in epidermal/hair follicle differentiation programs likely involving cross talk to signaling pathways (e.g., Notch) directing cell fate selection and differentiation. PMID:17914196

  6. DRMV1.3

    SciTech Connect

    Baldonado, Esther; Barber, William; Beiriger, Judy; Bivens, Hugh; Coles, Dwight; Detry, Richard; Fellig, Daniel; Humphreys, Steven; Jette, Morris; johnson, Wilbur; Kanipe, Frances; kleban, Stephen; Lauer, Lois; Lemen, Ellen; Moore, Pat; Rhea, Ron; Rheinheimer, Randal; Roehrig, Mary; vandewart, Ruthe; Warrant, Marilyn

    2002-03-18

    The DRM software provides advanced grid services. It is built on top of and uses a modified version of the Globus Toolkit. Globus has been modified in two ways: (1) to interface with ASCI specific local resource managers and (2) to use DOE approved Kerberos V5 for authentication between Globus components. Note: The DRM software package includes the changes to the Globus Toolkit in order to use the Kerberos security. However, the package does not include any Kerberos libraries or a necessary patch for the Kerberos. The advanced grid services include: (2) desktop tools that allow users to submit, monitor, and control jobs on both local and remote resources without a single remote login, (2) workflow management that supports serial job dependencies, file staging, and standard output and standard error redirection, (3) brokering and resource discovery that locates hardware and software resources on behalf of the user, selecting the best resources available, and (4) Web-based monioring that allows users to access resource and job status from all three nuclear weapons laboratories from a single web page.

  7. DRMV1.3

    Energy Science and Technology Software Center (ESTSC)

    2002-03-18

    The DRM software provides advanced grid services. It is built on top of and uses a modified version of the Globus Toolkit. Globus has been modified in two ways: (1) to interface with ASCI specific local resource managers and (2) to use DOE approved Kerberos V5 for authentication between Globus components. Note: The DRM software package includes the changes to the Globus Toolkit in order to use the Kerberos security. However, the package does notmore » include any Kerberos libraries or a necessary patch for the Kerberos. The advanced grid services include: (2) desktop tools that allow users to submit, monitor, and control jobs on both local and remote resources without a single remote login, (2) workflow management that supports serial job dependencies, file staging, and standard output and standard error redirection, (3) brokering and resource discovery that locates hardware and software resources on behalf of the user, selecting the best resources available, and (4) Web-based monioring that allows users to access resource and job status from all three nuclear weapons laboratories from a single web page.« less

  8. Influenza Infects Lung Microvascular Endothelium Leading to Microvascular Leak: Role of Apoptosis and Claudin-5

    PubMed Central

    Armstrong, Susan M.; Wang, Changsen; Tigdi, Jayesh; Si, Xiaoe; Dumpit, Carlo; Charles, Steffany; Gamage, Asela; Moraes, Theo J.; Lee, Warren L.

    2012-01-01

    Severe influenza infections are complicated by acute lung injury, a syndrome of pulmonary microvascular leak. The pathogenesis of this complication is unclear. We hypothesized that human influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed by spectrofluorimetry and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. We found that both clinical and laboratory strains of human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced degradation of the tight junction protein claudin-5; the adherens junction protein VE-cadherin and the actin cytoskeleton were unaffected. Over-expression of claudin-5 was sufficient to prevent replication-deficient virus-induced permeability. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dose-dependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Thus, we describe two distinct mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza. PMID:23115643

  9. Cysteine mutagenesis to study the structure of claudin-2 paracellular pores.

    PubMed

    Angelow, Susanne; Yu, Alan S L

    2009-05-01

    The structure and transport mechanism of paracellular pores are only poorly understood. Here we describe for the first time how the substituted cysteine accessibility method (SCAM), previously developed to study transmembrane transport, can be applied to analyze the pathway of paracellular ion permeation. Using stable transfected Madin Darby canine kidney type I cells, induced to express claudin-2, we show that paracellular cation transport can be blocked by sulfhydryl-specific methanethiosulfonate (MTS) and that SCAM can be used to identify residues that line paracellular pores. PMID:19538299

  10. Claudin-16 Deficiency Impairs Tight Junction Function in Ameloblasts, Leading to Abnormal Enamel Formation.

    PubMed

    Bardet, Claire; Courson, Frédéric; Wu, Yong; Khaddam, Mayssam; Salmon, Benjamin; Ribes, Sandy; Thumfart, Julia; Yamaguti, Paulo M; Rochefort, Gael Y; Figueres, Marie-Lucile; Breiderhoff, Tilman; Garcia-Castaño, Alejandro; Vallée, Benoit; Le Denmat, Dominique; Baroukh, Brigitte; Guilbert, Thomas; Schmitt, Alain; Massé, Jean-Marc; Bazin, Dominique; Lorenz, Georg; Morawietz, Maria; Hou, Jianghui; Carvalho-Lobato, Patricia; Manzanares, Maria Cristina; Fricain, Jean-Christophe; Talmud, Deborah; Demontis, Renato; Neves, Francisco; Zenaty, Delphine; Berdal, Ariane; Kiesow, Andreas; Petzold, Matthias; Menashi, Suzanne; Linglart, Agnes; Acevedo, Ana Carolina; Vargas-Poussou, Rosa; Müller, Dominik; Houillier, Pascal; Chaussain, Catherine

    2016-03-01

    Claudin-16 protein (CLDN16) is a component of tight junctions (TJ) with a restrictive distribution so far demonstrated mainly in the kidney. Here, we demonstrate the expression of CLDN16 also in the tooth germ and show that claudin-16 gene (CLDN16) mutations result in amelogenesis imperfecta (AI) in the 5 studied patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). To investigate the role of CLDN16 in tooth formation, we studied a murine model of FHHNC and showed that CLDN16 deficiency led to altered secretory ameloblast TJ structure, lowering of extracellular pH in the forming enamel matrix, and abnormal enamel matrix protein processing, resulting in an enamel phenotype closely resembling human AI. This study unravels an association of FHHNC owing to CLDN16 mutations with AI, which is directly related to the loss of function of CLDN16 during amelogenesis. Overall, this study indicates for the first time the importance of a TJ protein in tooth formation and underlines the need to establish a specific dental follow-up for these patients. PMID:26426912

  11. Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b

    PubMed Central

    Cheung, Isla D.; Bagnat, Michel; Ma, Taylur P.; Datta, Anirban; Evason, Kimberley; Moore, John C.; Lawson, Nathan; Mostov, Keith E.; Moens, Cecilia B.; Stainier, Didier Y.R.

    2011-01-01

    The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis. PMID:22020048

  12. Glucocorticoids Regulate Tight Junction Permeability of Lung Epithelia by Modulating Claudin 8.

    PubMed

    Kielgast, Felix; Schmidt, Hanna; Braubach, Peter; Winkelmann, Veronika E; Thompson, Kristin E; Frick, Manfred; Dietl, Paul; Wittekindt, Oliver H

    2016-05-01

    The lung epithelium constitutes a selective barrier that separates the airways from the aqueous interstitial compartment. Regulated barrier function controls water and ion transport across the epithelium and is essential for maintaining lung function. Tight junctions (TJs) seal the epithelial barrier and determine the paracellular transport. The properties of TJs depend especially on their claudin composition. Steroids are potent drugs used to treat a variety of airway diseases. Therefore, we addressed whether steroid hormones directly act on TJ properties in lung epithelia. Primary human tracheal epithelial cells and NCI-H441 cells, both cultivated under air-liquid interface conditions, were used as epithelial cell models. Our results demonstrate that glucocorticoids, but not mineralocorticoids, decreased paracellular permeability and shifted the ion permselectivity of TJs toward Cl(-). Glucocorticoids up-regulated claudin 8 (cldn8) expression via glucocorticoid receptors. Silencing experiments revealed that cldn8 is necessary to recruit occludin at the TJs. Immunohistochemistry on human lung tissue showed that cldn8 is specifically expressed in resorptive epithelia of the conducting and respiratory airways but not in the alveolar epithelium. We conclude that glucocorticoids enhance lung epithelia barrier function and increase paracellular Cl(-) selectivity via modulation of cldn8-dependent recruitment of occludin at the TJs. This mode of glucocorticoid action on lung epithelia might be beneficial to patients who suffer from impaired lung barrier function in various diseased conditions. PMID:26473470

  13. Changes in the expression profiles of claudins during gonocyte differentiation and in seminomas.

    PubMed

    Manku, G; Hueso, A; Brimo, F; Chan, P; Gonzalez-Peramato, P; Jabado, N; Gayden, T; Bourgey, M; Riazalhosseini, Y; Culty, M

    2016-01-01

    Testicular germ cell tumors (TGCTs) are the most common type of cancer in young men and their incidence has been steadily increasing for the past decades. TGCTs and their precursor carcinoma in situ (CIS) are thought to arise from the deficient differentiation of gonocytes, precursors of spermatogonial stem cells. However, the mechanisms relating failed gonocyte differentiation to CIS formation remain unknown. The goal of this study was to uncover genes regulated during gonocyte development that would show abnormal patterns of expression in testicular tumors, as prospective links between failed gonocyte development and TGCT. To identify common gene and protein signatures between gonocytes and seminomas, we first performed gene expression analyses of transitional rat gonocytes, spermatogonia, human normal testicular, and TGCT specimens. Gene expression arrays, pathway analysis, and quantitative real-time PCR analysis identified cell adhesion molecules as a functional gene category including genes downregulated during gonocyte differentiation and highly expressed in seminomas. In particular, the mRNA and protein expressions of claudins 6 and 7 were found to decrease during gonocyte transition to spermatogonia, and to be abnormally elevated in seminomas. The dynamic changes in these genes suggest that they may play important physiological roles during gonocyte development. Moreover, our findings support the idea that TGCTs arise from a disruption of gonocyte differentiation, and position claudins as interesting genes to further study in relation to testicular cancer. PMID:26588606

  14. Kinetic mixing effect in the 3 -3 -1 -1 model

    NASA Astrophysics Data System (ADS)

    Dong, P. V.; Si, D. T.

    2016-06-01

    We show that the mixing effect of the neutral gauge bosons in the 3 -3 -1 -1 model comes from two sources. The first one is due to the 3 -3 -1 -1 gauge symmetry breaking as usual, whereas the second one results from the kinetic mixing between the gauge bosons of U (1 )X and U (1 )N groups, which are used to determine the electric charge and baryon minus lepton numbers, respectively. Such mixings modify the ρ -parameter and the known couplings of Z with fermions. The constraints that arise from flavor-changing neutral currents due to the gauge boson mixings and nonuniversal fermion generations are also given.

  15. Specific binding of a mutated fragment of Clostridium perfringens enterotoxin to endothelial claudin-5 and its modulation of cerebral vascular permeability.

    PubMed

    Liao, Zhuangbin; Yang, Zhenguo; Piontek, Anna; Eichner, Miriam; Krause, Gerd; Li, Longxuan; Piontek, Joerg; Zhang, Jingjing

    2016-07-01

    The vertebrate blood-brain barrier (BBB) creates an obstacle for central nervous system-related drug delivery. Claudin-5 (Cldn5), expressed in large quantities in BBB, plays a vital role in restricting BBB permeability. The C-terminal domain of Clostridium perfringens enterotoxin (cCPE) has been verified as binding to a subset of claudins (Cldns). The Cldn5-binding cCPE194-319 variant cCPEY306W/S313H was applied in this study to investigate its ability to modulate the permeability of zebrafish larval BBB. In vitro results showed that cCPEY306W/S313H is able to bind specifically to Cldn5 in murine brain vascular endothelial (bEnd.3) cells, and is transported along with Cldn5 from the cell membrane to the cytoplasm, which in turn results in a reduction in transendothelial electrical resistance (TEER). Conversely, this effect can be reversed by removal of cCPEY306W/S313H. In an in vivo experiment, this study estimates the capability of cCPEY306W/S313H to modulate Cldn5 using a rhodamine B-Dextran dye diffusion assay in zebrafish larval BBB. The results show that cCPEY306W/S313H co-localized with Cldn5 in zebrafish cerebral vascular cells and modulated BBB permeability, resulting in dye leakage. Taken together, this study suggests that cCPEY306W/S313H has the capability - both in vitro and in vivo - to modulate BBB permeability temporarily by specific binding to Cldn5. PMID:27095710

  16. A 1,3-Dihydro-1,3-azaborine Debuts

    PubMed Central

    Xu, Senmiao; Zakharov, Lev N.

    2011-01-01

    We present the first synthesis and characterization of a 1,3-dihydro-1,3-azaborine, a long-sought BN isostere of benzene. 1,3-Dihydro-1,3-azaborine is a stable structural motif with considerable aromatic character as evidenced by structural analysis and its reaction chemistry. Single crystal X-ray analysis indicates bonding consistent with significant electron delocalization. 1,3-Dihydro-1,3-azaborines also undergo nucleophilic substitutions at boron and electrophilic aromatic substitution reactions. In view of the versatility and impact of aromatic compounds in the biomedical field and in materials science, the present study further expands the available chemical space of arenes via BN/CC isosterism. PMID:22091703

  17. 3-3-1-1 model for dark matter

    NASA Astrophysics Data System (ADS)

    Dong, P. V.; Tham, T. D.; Hung, H. T.

    2013-06-01

    We show that the SU(3)C⊗SU(3)L⊗U(1)X (3-3-1) model of strong and electroweak interactions can naturally accommodate an extra U(1)N symmetry behaving as a gauge symmetry. The resulting theory based on SU(3)C⊗SU(3)L⊗U(1)X⊗U(1)N (3-3-1-1) gauge symmetry realizes B-L=-(2/3)T8+N as a charge of SU(3)L⊗U(1)N. Consequently, a residual symmetry, W parity, resulting from broken B-L, similarly to R-parity in supersymmetry, is always conserved and may be unbroken. There is a specific fermion content recently studied in which all new particles that have wrong lepton numbers are odd under W parity, while the standard model particles are even. Therefore, the lightest wrong-lepton particle (LWP) responsible for dark matter is naturally stabilized. We explicitly show that the non-Hermitian neutral gauge boson (X0) as the LWP cannot be dark matter. However, the LWP as a new neutral fermion (NR) can be dark matter if its mass is in the range 1.9TeV≤mNR≤2.5TeV, provided that the new neutral gauge boson (Z') mass satisfies 2.2TeV≤mZ'≤2.5TeV. Moreover, the scalar dark matter candidate (H'≃η3) that has traditionally been studied is only stabilized by W parity. All the unwanted interactions and vacuums that are often encountered in the 3-3-1 model are naturally suppressed. And, the standing issues on tree-level flavor changing neutral currents and CPT violation also disappear.

  18. Claudin 11 inter-sertoli tight junctions in the testis of the korean soft-shelled turtle (Pelodiscus maackii).

    PubMed

    Park, Chan Jin; Ha, Cheol Min; Lee, Jae Eun; Gye, Myung Chan

    2015-04-01

    Expression of claudin 11 (CLDN11), a tight junction (TJ) protein, was examined in the Korean soft-shelled turtle (Pelodiscus maackii) testis. Spermatogenesis began during the breeding season and peaked at the end of the breeding season. Spermiation started in summer and peaked in autumn. The deduced amino acid sequence of P. maackii CLDN11 was similar to those of avian and mammalian species. During the nonbreeding season when spermatogenesis and testosterone production were active, testicular Cldn11 levels were high. In the seminiferous epithelium, strong, wavy CLDN11 strands parallel to the basement membrane delaminate the spermatogonia, and early spermatocytes are in the open compartment. Otherwise, CLDN11 was found beneath the early spermatocytes and in the Sertoli cell cytoplasm. Punctate zonula occludens 1 (ZO-1) immunoreactivity was found within the CLDN11 strands parallel to the basement membrane or at the outermost periphery of the seminiferous epithelium close to the basal lamina. During the breeding season, when circulating testosterone levels and spermatogenic activity was low, testicular CLDN11 level was lower than those during the nonbreeding season. CLDN11 was found at apicolateral contact sites between adjacent Sertoli cells devoid of the postmeiotic germ cells. At this time, lanthanum tracer diffused to the adluminal compartment of seminiferous epithelium. In cultured testis tissues, testosterone propionate significantly increased the level of Cldn11 mRNA. In P. maackii testis, CLDN11 participates in the development of the blood-testis barrier (BTB), where the CLDN11 expression was coupled with spermatogenic activity and circulating androgen levels, indicating the conserved nature of TJs expressing CLDN11 at the BTB in amniotes. PMID:25761591

  19. The simplest 3-3-1 model

    NASA Astrophysics Data System (ADS)

    Hue, Le Tho; Ninh, Le Duc

    2016-03-01

    A simple extension of the Standard Model (SM), based on the gauge group SU(3)C ⊗SU(3)L ⊗U(1)Y with Y being the hypercharge, is considered. We show that, by imposing an approximate global SU(2)L ×SU(2)R custodial symmetry at the SM energy scale, the Z - Z‧ mixing is absent at tree level and the value of the ρ parameter can be kept close to one. Tree-level flavor-changing neutral currents (FCNCs) are also reduced to three particles, namely Z‧, a CP-odd Higgs and a CP-even Higgs. The model predicts new leptons with electric charges of ±1/2e and new quarks with ±1/6e charges as well as new gauge and scalar bosons with ±1/2e charges. Electric charge conservation requires that one of them must be stable. Their masses are unfortunately free parameters.

  20. Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model

    SciTech Connect

    Carette, Diane; Perrard, Marie-Hélène; Prisant, Nadia; Gilleron, Jérome; Pointis, Georges; Segretain, Dominique; Durand, Philippe

    2013-04-01

    Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 μg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood–testis barrier dynamic is postulated. - Highlights: ► Influence of Cr(VI) on the Sertoli cell barrier and on junctional proteins ► Use of cultured seminiferous tubules in bicameral chambers ► Low concentrations of Cr(VI) (10 μg/l) altered the trans-epithelial resistance. ► Cr(VI) did not alter claudin-11 and N-cadherin. ► Cr(VI) delocalized connexin 43 from the membrane to the cytoplasm of Sertoli cells.

  1. Vought XF3U-1

    NASA Technical Reports Server (NTRS)

    1933-01-01

    Vought XF3U-1: The Vought XF3U-1 was tested in Langley's 30 x 60 Full Scale Tunnel in 1933. The XF3U-1 was built in response to a Navy specification for a two-seat fighter. This aircraft was later used as an engine testbed. The design was revised for a scout bomber requirement and in this form saw service as the SBU.

  2. Claudin-based barrier differentiation in the colonic epithelial crypt niche involves Hopx/Klf4 and Tcf7l2/Hnf4-α cascades.

    PubMed

    Lili, Loukia N; Farkas, Attila E; Gerner-Smidt, Christian; Overgaard, Christian E; Moreno, Carlos S; Parkos, Charles A; Capaldo, Christopher T; Nusrat, Asma

    2016-01-01

    Colonic enterocytes form a rapidly renewing epithelium and barrier to luminal antigens. During renewal, coordinated expression of the claudin family of genes is vital to maintain the epithelial barrier. Disruption of this process contributes to barrier compromise and mucosal inflammatory diseases. However, little is known about the regulation of this critical aspect of epithelial cell differentiation. In order to identify claudin regulatory factors we utilized high-throughput gene microarrays and correlation analyses. We identified complex expression gradients for the transcription factors Hopx, Hnf4a, Klf4 and Tcf7l2, as well as 12 claudins, during differentiation. In vitro confirmatory methods identified 2 pathways that stimulate claudin expression; Hopx/Klf4 activation of Cldn4, 7 and 15, and Tcf7l2/Hnf4a up-regulation of Cldn23. Chromatin immunoprecipitation confirmed a Tcf7l2/Hnf4a/Claudin23 cascade. Furthermore, Hnf4a conditional knockout mice fail to induce Cldn23 during colonocyte differentiation. In conclusion, we report a comprehensive screen of colonic claudin gene expression and discover spatiotemporal Hopx/Klf4 and Tcf7l2/Hnf4a signaling as stimulators of colonic epithelial barrier differentiation. PMID:27583195

  3. Matrix Metalloproteinase-9 Leads to Claudin-5 Degradation via the NF-κB Pathway in BALB/c Mice with Eosinophilic Meningoencephalitis Caused by Angiostrongylus cantonensis

    PubMed Central

    Chiu, Ping-Sung; Lai, Shih-Chan

    2013-01-01

    The epithelial barrier regulates the movement of ions, macromolecules, immune cells and pathogens. The objective of this study was to investigate the role of the matrix metalloproteinase (MMP)-9 in the degradation of tight junction protein during infection with rat nematode lungworm Angiostrongylus cantonensis. The results showed that phosphorylation of IκB and NF-κB was increased in mice with eosinophilic meningoencephalitis. Treatment with MG132 reduced the phosphorylation of NF-κB and the activity of MMP-9, indicating upregulation of MMP-9 through the NF-κB signaling pathway. Claudin-5 was reduced in the brain but elevated in the cerebrospinal fluid (CSF), implying that A. cantonensis infection caused tight junction breakdown and led to claudin-5 release into the CSF. Degradation of claudin-5 coincided with alteration of the blood-CSF barrier permeability and treatment with the MMP inhibitor GM6001 attenuated the degradation of claudin-5. These results suggested that degradation of claudin-5 was caused by MMP-9 in angiostrongyliasis meningoencephalitis. Claudin-5 could be used for the pathophysiologic evaluation of the blood-CSF barrier breakdown and tight junction disruption after infection with A. cantonensis. PMID:23505411

  4. Vought XSB3U-1

    NASA Technical Reports Server (NTRS)

    1938-01-01

    Vought XSB3U-1: This Navy biplane scout bomber was the first biplane built by Vought with retractable gear. Note that his XSB3U-1 has a test boom off the left upper wing. The bomber was at Langley for NACA's investigation of tail loads.

  5. Boeing F3B-1

    NASA Technical Reports Server (NTRS)

    1930-01-01

    Boeing F3B-1: While most Boeing F3B-1s served aboard the U. S. Navy aircraft carriers Lexington and Saratoga, this example flew in NACA hands at the Langley Memorial Aeronautical Laboratory in the late 1920's. Also known as the Boeing Model 77, the aircraft was powered by a Pratt & Whitney Wasp radial engine.

  6. Characterization of the first-in-class T-cell-engaging bispecific single-chain antibody for targeted immunotherapy of solid tumors expressing the oncofetal protein claudin 6

    PubMed Central

    Stadler, Christiane R.; Bähr-Mahmud, Hayat; Plum, Laura M.; Schmoldt, Kathrin; Kölsch, Anne C.; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    abstract The fetal tight junction molecule claudin 6 (CLDN6) is virtually absent from any normal tissue, whereas it is aberrantly and frequently expressed in various cancers of high medical need. We engineered 6PHU3, a T-cell-engaging bispecific single chain molecule (bi-(scFv)2) with anti-CD3/anti-CLDN6 specificities, and characterized its pharmacodynamic properties. Our data show that upon engagement by 6PHU3, T cells strongly upregulate cytotoxicity and activation markers, proliferate and acquire an effector phenotype. 6PHU3 exerts potent killing of cancer cells in vitro with EC50 values in the pg/mL range. Subcutaneous xenograft tumors in NSG mice engrafted with human PBMCs are eradicated by 6PHU3 treatment and survival of mice is significantly prolonged. Tumors of 6PHU3-treated mice are strongly infiltrated with activated CD4+, CD8+ T cells and TEM type cells but not Tregs and display a general activation of a mostly inflammatory phenotype. These effects are only observed upon bispecific but not monospecific engagement of 6PHU3. Together with the exceptionally cancer cell selective expression of the oncofetal tumor marker CLDN6, this provides a safeguard with regard to toxicity. In summary, our data shows that the concept of T-cell redirection combined with that of highly selective targeting of CLDN6-positive solid tumors is effective. Thus, exploring 6PHU3 for clinical therapy is warranted. PMID:27141353

  7. Prognostic Significance of Lymphatic Vessel Density Detected by D2-40 and Its Relation to Claudin-4 Expression in Prostatic Adenocarcinoma.

    PubMed

    Radi, Dina A; Abd-Elazeem, Marwa A

    2016-05-01

    Background Lymphovascular invasion is an important pathway of metastatic spread and regional lymph node metastasis is the major prognostic factor in prostatic adenocarcinoma. D2-40 is used to identify the lymphatic vessels and to assess the lymphatic vessel density (LVD). Expression of claudin-4 may be related to invasion and progression of carcinoma cells in several primary tumors. Aim To evaluate intra- and peritumoral LVD through immunohistochemical expression of D2-40 in relation to claudin-4 expression and clinicopathological parameters in prostatic adenocarcinoma. Materials and Methods Immunohistochemical staining procedure was performed on 53 paraffin-embedded blocks of radical prostatectomy specimens for prostatic adenocarcinoma using anti D2-40 and claudin-4 antibodies. Sections were evaluated for mean LVD in intratumoral and peritumoral tissues assessed by D2-40 expression. Results LVD in intratumoral tissues was significantly lower compared with peritumoral areas (P = .0001). Peritumoral mean LVD was significantly higher in cases with lymphovascular invasion (P = .041) and in cases with positive lymph node metastasis (P = .003) than intratumoral mean LVD. High claudin-4 expression was significantly correlated with high tumor grade (P = .0001), lymphovascular invasion (P = .006), and positive lymph node metastasis (P = .004). High claudin-4 expression was significantly associated with increased mean LVD in peritumoral tissues. Conclusion Increased peritumoral mean LVD in prostatic adenocarcinoma is associated with lymphovascular invasion and positive lymph node metastasis. High claudin-4 expression is associated with high tumor grade, lymphocascular invasion, positive lymph node metastasis, and high mean peritumoral LVD suggesting that D2-40 and claudin-4 may represent different mechanisms of lymphatic vessel invasion with both biomarkers is related to poor prognosis. PMID:26464161

  8. Claudin 28b and F-actin are involved in rainbow trout gill pavement cell tight junction remodeling under osmotic stress.

    PubMed

    Sandbichler, Adolf Michael; Egg, Margit; Schwerte, Thorsten; Pelster, Bernd

    2011-05-01

    Permeability of rainbow trout gill pavement cells cultured on permeable supports (single seeded inserts) changes upon exposure to freshwater or treatment with cortisol. The molecular components of this change are largely unknown, but tight junctions that regulate the paracellular pathway are prime candidates in this adaptational process. Using differential display polymerase chain reaction we found a set of 17 differentially regulated genes in trout pavement cells that had been exposed to freshwater apically for 24 h. Five genes were related to the cell-cell contact. One of these genes was isolated and identified as encoding claudin 28b, an integral component of the tight junction. Immunohistochemical reactivity to claudin 28b protein was concentrated in a circumferential ring colocalized to the cortical F-actin ring. To study the contribution of this isoform to changes in transepithelial resistance and Phenol Red diffusion under apical hypo-or hyperosmotic exposure we quantified the fluorescence signal of this claudin isoform in immunohistochemical stainings together with the fluorescence of phalloidin-probed F-actin. Upon hypo-osmotic stress claudin 28b fluorescence and epithelial tightness remained stable. Under hyperosmotic stress, the presence of claudin 28b at the junction significantly decreased, and epithelial tightness was severely reduced. Cortical F-actin fluorescence increased upon hypo-osmotic stress, whereas hyperosmotic stress led to a separation of cortical F-actin rings and the number of apical crypt-like pores increased. Addition of cortisol to the basolateral medium attenuated cortical F-actin separation and pore formation during hyperosmotic stress and reduced claudin 28b in junctions except after recovery of cells from exposure to freshwater. Our results showed that short-term salinity stress response in cultured trout gill cells was dependent on a dynamic remodeling of tight junctions, which involves claudin 28b and the supporting F-actin ring

  9. Preparation of 1,1'-dinitro-3,3'-azo-1,2,4-triazole. [1,1'-dinitro-3,3'-azo-1,2,4-triazole

    DOEpatents

    Lee, K.Y.

    1985-03-05

    A new high density composition of matter, 1,1'-dinitro-3,3'-azo-1,2,4-triazole, has been synthesized using inexpensive, commonly available compounds. This compound has been found to be an explosive, and its use as a propellant is anticipated. 1 fig., 1 tab.

  10. Tight Junction Proteins in Human Schwann Cell Autotypic Junctions

    PubMed Central

    Alanne, Maria H.; Pummi, Kati; Heape, Anthony M.; Grènman, Reidar; Peltonen, Juha; Peltonen, Sirkku

    2009-01-01

    Tight junctions (TJs) form physical barriers in various tissues and regulate paracellular transport of ions, water, and molecules. Myelinating Schwann cells form highly organized structures, including compact myelin, nodes of Ranvier, paranodal regions, Schmidt-Lanterman incisures, periaxonal cytoplasmic collars, and mesaxons. Autotypic TJs are formed in non-compacted myelin compartments between adjacent membrane lamellae of the same Schwann cell. Using indirect immunofluorescence and RT-PCR, we analyzed the expression of adherens junction (E-cadherin) and TJ [claudins, zonula occludens (ZO)-1, occludin] components in human peripheral nerve endoneurium, showing clear differences with published rodent profiles. Adult nerve paranodal regions contained E-cadherin, claudin-1, claudin-2, and ZO-1. Schmidt-Lanterman incisures contained E-cadherin, claudin-1, claudin-2, claudin-3, claudin-5, ZO-1, and occludin. Mesaxons contained E-cadherin, claudin-1, claudin-2, claudin-3, ZO-1, and occludin. None of the proteins studied were associated with nodal inter-Schwann cell junctions. Fetal nerve expression of claudin-1, claudin-3, ZO-1, and occludin was predominantly punctate, with a mesaxonal labeling pattern, but paranodal (ZO-1, claudin-3) and Schmidt-Lanterman incisure (claudins-1 and -3) expression profiles typical of compact myelin were visible by gestational week 37. The clear differences observed between human and published rodent nerve profiles emphasize the importance of human studies when translating the results of animal models to human diseases. (J Histochem Cytochem 57:523–529, 2009) PMID:19153196

  11. Water and ion permeability of a claudin model: A computational study.

    PubMed

    Laghaei, Rozita; Yu, Alan S L; Coalson, Rob D

    2016-03-01

    At present, the three-dimensional structure of the multimeric paracellular claudin pore is unknown. Using extant biophysical data concerning the size of the pore and permeation of water and cations through it, two three-dimensional models of the pore are constructed in silico. Molecular Dynamics (MD) calculations are then performed to compute water and sodium ion permeation fluxes under the influence of applied hydrostatic pressure. Comparison to experiment is made, under the assumption that the hydrostatic pressure applied in the simulations is equivalent to osmotic pressure induced in experimental measurements of water/ion permeability. One model, in which pore-lining charged is distributed evenly over a selectivity filter section 10-16 Å in length, is found to be generally consistent with experimental data concerning the dependence of water and ion permeation on channel pore diameter, pore length, and the sign and magnitude of pore lining charge. The molecular coupling mechanism between water and ion flow under conditions where hydrostatic pressure is applied is computationally elucidated. Proteins 2016; 84:305-315. © 2016 Wiley Periodicals, Inc. PMID:26650625

  12. 40 CFR 721.533 - Propane, 1,1,1,3,3-pentachloro-.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.533 Propane, 1,1,1,3,3-pentachloro-. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as propane, 1,1,1,3,3-pentachloro- (PMN...

  13. 40 CFR 721.533 - Propane, 1,1,1,3,3-pentachloro-.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.533 Propane, 1,1,1,3,3-pentachloro-. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as propane, 1,1,1,3,3-pentachloro- (PMN...

  14. 1,2,3-Trichloropropane

    Integrated Risk Information System (IRIS)

    1,2,3 - Trichloropropane ; CASRN 96 - 18 - 4 Human health assessment information on a chemical substance is included in IRIS only after a comprehensive review of toxicity data by U.S . EPA health scientists from several program offices , regional offices , and the Office of Research and Development

  15. 1,3,5-Trinitrobenzene

    Integrated Risk Information System (IRIS)

    1,3,5 - Trinitrobenzene ; CASRN 99 - 35 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcino

  16. 1 of 3 pregnancies unwanted.

    PubMed

    Sweeney, C

    1992-01-01

    UNICEF's 1992 State of the World's Children Report observes that family planning is one of the most effective and least expensive ways of improving quality of life on earth. If all women were able to determine the size and spacing of their families, the report notes, there would be 1.3 billion fewer people in the world by the year 2025. A recent World Fertility Survey underscored the demand for family planning, pointing out that 1/3 of all pregnancies in the world are unwanted. Increased access to family planning services could go a long way toward decreasing maternal and child mortality and improving the quality of life for women and children. Family planning could save the lives of up to 1/3 of the 1/2 million women who die every year from pregnancy and birth related causes. Family planning could also prevent many of the 50,000 illegal abortions which are performed around the world every day, and could decrease the incidence of high risk pregnancies involving women under 18, over 35, or who already have more than 3 children. The report says that women who practice birth control have more time for education, earning income, child care, and leisure. And as parents are able to devote more time and resources to less children, the quality of child care rises. Unfortunately, as UNICEF executive director James P. Grant notes, the benefits of family planning have been obscured by controversy. But as recent experience has demonstrated, family planning can be promoted and practices in a way that is sensitive to the religion and culture of almost any society. The failure to realize the potential of family planning, the report says, is one of the greatest mistakes of our time. PMID:12343548

  17. Interferon-gamma increased epithelial barrier function via upregulating claudin-7 expression in human submandibular gland duct epithelium.

    PubMed

    Abe, Ayumi; Takano, Kenichi; Kojima, Takashi; Nomura, Kazuaki; Kakuki, Takuya; Kaneko, Yakuto; Yamamoto, Motohisa; Takahashi, Hiroki; Himi, Tetsuo

    2016-06-01

    Tight junctions (TJs) are necessary for salivary gland function and may serve as indicators of salivary gland epithelial dysfunction. IgG4-related disease (IgG4-RD) is a newly recognized fibro-inflammatory condition which disrupts the TJ associated epithelial barrier. The salivary glands are one of the most frequently involved organs in IgG4-RD, however, changes of the TJ associated epithelial barrier in salivary gland duct epithelium is poorly understood. Here, we investigated the regulation and function of TJs in human submandibular gland ductal epithelial cells (HSDECs) in normal and IgG4-RD. We examined submandibular gland (SMG) tissue from eight control individuals and 22 patients with IgG4-RD and established an HSDEC culture system. Immunohistochemistry, immunocytochemistry, western blotting, and measurement of transepithelial electrical resistance (TER) were performed. Claudin-4, claudin-7, occludin, and JAM-A were expressed at the apical side of the duct epithelium in submandibular gland (SMG) tissue and at the cell borders in HSDECs of normal and IgG4-RD. The expression and distribution of TJs in SMG tissue were not different in control individuals and patients with IgG4-RD in vivo and in vitro. Although interferon-gamma (IFNγ) generally disrupts the integrity and function of TJs, as manifested by decreased epithelial barrier function, IFNγ markedly increased the epithelial barrier function of HSDECs via upregulation of claudin-7 expression in HSDECs from patients with IgG4-RD. This is the first report showing an IFNγ-dependent increase in epithelial barrier function in the salivary gland duct epithelium. Our results provide insights into the functional significance of TJs in salivary gland duct epithelium in physiological and pathological conditions, including IgG4-RD. PMID:26956365

  18. Regulation of gill claudin paralogs by salinity, cortisol and prolactin in Mozambique tilapia (Oreochromis mossambicus).

    PubMed

    Tipsmark, Christian K; Breves, Jason P; Rabeneck, D Brett; Trubitt, Rebecca T; Lerner, Darren T; Grau, E Gordon

    2016-09-01

    In euryhaline teleosts, reorganization of gill tight junctions during salinity acclimation involves dynamic expression of specific claudin (Cldn) paralogs. We identified four transcripts encoding Cldn tight junction proteins in the tilapia gill transcriptome: cldn10c, cldn10e, cldn28a and cldn30. A tissue distribution experiment found cldn10c and cldn10e expression levels in the gill to be 100-fold higher than any other tissues examined. cldn28a and cldn30 levels in the gill were 10-fold greater than levels in other tissues. Expression of these genes in Mozambique tilapia was examined during acclimation to fresh water (FW), seawater (SW), and in response to hormone treatments. Transfer of tilapia from FW to SW elevated cldn10c and cldn10e, while cldn28a and cldn30 were stimulated following transfer from SW to FW. In hypophysectomized tilapia transferred to FW, pituitary extirpation induced reduced expression of cldn10c, cldn10e and cldn28a; these effects were mitigated equally by either prolactin or cortisol replacement. In vitro experiments with gill filaments showed that cortisol stimulated expression of all four cldns examined, suggesting a direct action of cortisol in situ. Our data indicate that elevated cldn10c and cldn10e expression is important during acclimation of tilapia to SW possibly by conferring ion specific paracellular permeability. On the other hand, expression of cldn28a and cldn30 appears to contribute to reorganization of branchial epithelium during FW acclimation. Hormone treatment experiments showed that particular FW- and SW-induced cldns are controlled by cortisol and prolactin. PMID:27210417

  19. 10 CFR 960.3-1-3 - Regionality.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 4 2012-01-01 2012-01-01 false Regionality. 960.3-1-3 Section 960.3-1-3 Energy DEPARTMENT OF ENERGY GENERAL GUIDELINES FOR THE PRELIMINARY SCREENING OF POTENTIAL SITES FOR A NUCLEAR WASTE REPOSITORY Implementation Guidelines § 960.3-1-3 Regionality. In making site recommendations for...

  20. 10 CFR 960.3-1-3 - Regionality.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 4 2014-01-01 2014-01-01 false Regionality. 960.3-1-3 Section 960.3-1-3 Energy DEPARTMENT OF ENERGY GENERAL GUIDELINES FOR THE PRELIMINARY SCREENING OF POTENTIAL SITES FOR A NUCLEAR WASTE REPOSITORY Implementation Guidelines § 960.3-1-3 Regionality. In making site recommendations for...

  1. 10 CFR 960.3-1-3 - Regionality.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 4 2013-01-01 2013-01-01 false Regionality. 960.3-1-3 Section 960.3-1-3 Energy DEPARTMENT OF ENERGY GENERAL GUIDELINES FOR THE PRELIMINARY SCREENING OF POTENTIAL SITES FOR A NUCLEAR WASTE REPOSITORY Implementation Guidelines § 960.3-1-3 Regionality. In making site recommendations for...

  2. The Noncompetitive AMPAR Antagonist Perampanel Abrogates Brain Endothelial Cell Permeability in Response to Ischemia: Involvement of Claudin-5.

    PubMed

    Lv, Jian-Meng; Guo, Xiao-Min; Chen, Bo; Lei, Qi; Pan, Ya-Juan; Yang, Qian

    2016-07-01

    The blood-brain barrier (BBB) is formed by brain endothelial cells, and decreased BBB integrity contributes to vasogenic cerebral edema and increased mortality after stroke. In the present study, we investigated the protective effect of perampanel, an orally active noncompetitive AMPA receptor antagonist, on BBB permeability in an in vitro ischemia model in murine brain endothelial cells (mBECs). The results showed that perampanel significantly attenuated oxygen glucose deprivation (OGD)-induced loss of cell viability, release of lactate dehydrogenase, and apoptotic cell death in a dose-dependent manner. Perampanel treatment did not alter the expression and surface distribution of various glutamate receptors. Furthermore, the results of calcium imaging showed that perampanel had no effect on OGD-induced increase in intracellular Ca(2+) concentrations. Treatment with perampanel markedly reduced the paracellular permeability of mBECs after OGD in different time points, as measured by transepithelial electrical resistance assay. In addition, the expression of claudin-5 at protein level, but not at mRNA level, was increased by perampanel treatment after OGD. Knockdown of claudin-5 partially prevented perampanel-induced protection in cell viability and BBB integrity in OGD-injured mBECs. These data show that the noncompetitive AMPA receptor antagonist perampanel affords protection against ischemic stroke through caludin-5 mediated regulation of BBB permeability. PMID:26306919

  3. Synthesis of 1,1'-dinitro-3,3'-azo-1,2,4-triazole

    SciTech Connect

    Lee, K.Y.

    1985-04-01

    A novel compound has been prepared and is a candidate for high-energy propellant applications. The 1,1'-dinitro-3,3'-azo-1,2,4-triazole (N-DNAT) has a density of 1.77 g/cm/sup 3/ and can be prepared from inexpensive starting materials.

  4. The Effects of Glucagon-like Peptide-2 on the Tight Junction and Barrier Function in IPEC-J2 Cells through Phosphatidylinositol 3-kinase–Protein Kinase B–Mammalian Target of Rapamycin Signaling Pathway

    PubMed Central

    Yu, Changsong; Jia, Gang; Deng, Qiuhong; Zhao, Hua; Chen, Xiaoling; Liu, Guangmang; Wang, Kangning

    2016-01-01

    Glucagon-like peptide-2 (GLP-2) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER) in lipopolysaccharide (LPS) stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that 100 μg/mL LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1) mRNA, proteins expressions (p<0.01) respectively. GLP-2 (100 nmol/L) promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01) respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01) following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01). In conclusion, these results indicated that GLP-2 can promote TJ’s expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway. PMID:26954146

  5. FL V1.3

    Energy Science and Technology Software Center (ESTSC)

    2009-08-03

    A library of utility classes for computer vision. Contains implementations of various well-known image processing techniques, such as interest point operators and region descriptors. Includes interfaces to various libraries for image and video I/O, as well as an interface to LAPACK/BLAS. FL was developed at the University of Illinois, Urbana-Champaign (UIUC) and released under an open source license. Version 1.2 was a maintenance release provided by SNL under the LGPL license. Version 1.3 is amore » maintenance release, containing the following changes: - Improved image format handling. Now handles strided and planar memory layouts and a wider range of pixel formats. - Improved image file I/O, including better support for metadata, a wider range of stored pixel types, and a couple of new file formats. - Improvements to DOG and SIFT, and efficiency improvements in low-level convolution. - Improvements to networking, including a generic TCP listener. - Various improvements to numerical processing. The HISTORY file included in the distribution contains a more detailed description of the changes.« less

  6. FL V1.3

    SciTech Connect

    Rothganger, Frederick

    2009-08-03

    A library of utility classes for computer vision. Contains implementations of various well-known image processing techniques, such as interest point operators and region descriptors. Includes interfaces to various libraries for image and video I/O, as well as an interface to LAPACK/BLAS. FL was developed at the University of Illinois, Urbana-Champaign (UIUC) and released under an open source license. Version 1.2 was a maintenance release provided by SNL under the LGPL license. Version 1.3 is a maintenance release, containing the following changes: - Improved image format handling. Now handles strided and planar memory layouts and a wider range of pixel formats. - Improved image file I/O, including better support for metadata, a wider range of stored pixel types, and a couple of new file formats. - Improvements to DOG and SIFT, and efficiency improvements in low-level convolution. - Improvements to networking, including a generic TCP listener. - Various improvements to numerical processing. The HISTORY file included in the distribution contains a more detailed description of the changes.

  7. 40 CFR 721.10435 - Phenol, 2-(1-methyl-1-phenylethyl)-4-(1,1,3,3-tetramethylbutyl)-.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Phenol, 2-(1-methyl-1-phenylethyl)-4... Significant New Uses for Specific Chemical Substances § 721.10435 Phenol, 2-(1-methyl-1-phenylethyl)-4-(1,1,3... chemical substance identified as phenol, 2-(1-methyl-1-phenylethyl)-4-(1,1,3,3-tetramethylbutyl)- (PMN...

  8. 40 CFR 721.10435 - Phenol, 2-(1-methyl-1-phenylethyl)-4-(1,1,3,3-tetramethylbutyl)-.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Phenol, 2-(1-methyl-1-phenylethyl)-4... Significant New Uses for Specific Chemical Substances § 721.10435 Phenol, 2-(1-methyl-1-phenylethyl)-4-(1,1,3... chemical substance identified as phenol, 2-(1-methyl-1-phenylethyl)-4-(1,1,3,3-tetramethylbutyl)- (PMN...

  9. 26 CFR 1.861-3 - Dividends.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... or accrued before January 1, 1967, see 26 CFR 1.861-3 (Revised as of January 1, 1972). Paragraph (a... Register citations affecting § 1.861-3, see the List of CFR Sections Affected, which appears in the Finding... 26 Internal Revenue 9 2012-04-01 2012-04-01 false Dividends. 1.861-3 Section 1.861-3...

  10. Trilinos 3.1 tutorial.

    SciTech Connect

    Sala, Marzio; Heroux, Michael Allen

    2004-01-01

    This document introduces the use of Trilinos, version 3.1. Trilinos has been written to support, in a rigorous manner, the solver needs of the engineering and scientific applications at Sandia National Laboratories. Aim of this manuscript is to present the basic features of some of the Trilinos packages. The presented material includes the definition of distributed matrices and vectors with Epetra, the iterative solution of linear system with AztecOO, incomplete factorizations with IFPACK, multilevel methods with ML, direct solution of linear system with Amesos, and iterative solution of nonlinear systems with NOX. With the help of several examples, some of the most important classes and methods are detailed to the inexperienced user. For the most majority, each example is largely commented throughout the text. Other comments can be found in the source of each example. This document is a companion to the Trilinos User's Guide and Trilinos Development Guides. Also, the documentation included in each of the Trilinos' packages is of fundamental importance.

  11. Crystalline 1H-1,2,3-triazol-5-ylidenes

    DOEpatents

    Bertrand, Guy; Gulsado-Barrios, Gregorio; Bouffard, Jean; Donnadieu, Bruno

    2016-08-02

    The present invention provides novel and stable crystalline 1H-1,2,3 triazolium carbenes and metal complexes of 1H-1,2,3 triazolium carbenes. The present invention also provides methods of making 1H-1,2,3 triazolium carbenes and metal complexes of 1H-1,2,3 triazolium carbenes. The present invention also provides methods of using 1H-1,2,3 triazolium carbenes and metal complexes of 1H-1,2,3 triazolium carbenes in catalytic reactions.

  12. 75 FR 28188 - Airworthiness Directives; General Electric Company CF34-1A, -3A, -3A1, -3A2, -3B, and -3B1...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-20

    ... 7, 2010 (75 FR 910), we published a final rule AD, FR Doc, E9-30471, in the Federal Register. That... (GE) CF34-1A, -3A, -3A1, -3A2, -3B, and -3B1 turbofan engines. The GE alert service bulletin...

  13. 18 CFR 3a.1 - Purpose.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Purpose. 3a.1 Section 3a.1 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION General § 3a.1 Purpose. This part 3a describes...

  14. Preparation of 1,1'-dinitro-3,3'-azo-1,2,4-triazole

    DOEpatents

    Lee, Kien-Yin

    1986-01-01

    A new high density composition of matter, 1,1'-dinitro-3,3'-azo-1,2,4-triazole, has been synthesized using inexpensive, commonly available compounds. This compound has been found to be an explosive, and its use as a propellant is anticipated.

  15. 14 CFR 3.1 - Applicability.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Applicability. 3.1 Section 3.1 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION DEFINITIONS GENERAL REQUIREMENTS § 3.1 Applicability. (a) This part applies to any person who makes a record regarding: (1) A...

  16. 26 CFR 1.43-3 - Certification

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 1 2014-04-01 2013-04-01 true Certification 1.43-3 Section 1.43-3 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY INCOME TAX INCOME TAXES Credits Against Tax § 1.43-3 Certification (a) Petroleum engineer's certification of a project—(1) In general. A petroleum engineer must certify, under penalties...

  17. 10 CFR 960.3-1-3 - Regionality.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Regionality. 960.3-1-3 Section 960.3-1-3 Energy DEPARTMENT OF ENERGY GENERAL GUIDELINES FOR THE PRELIMINARY SCREENING OF POTENTIAL SITES FOR A NUCLEAR WASTE... repositories. Such consideration shall take into account the proximity of sites to locations at which waste...

  18. 10 CFR 960.3-1-3 - Regionality.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 4 2011-01-01 2011-01-01 false Regionality. 960.3-1-3 Section 960.3-1-3 Energy DEPARTMENT OF ENERGY GENERAL GUIDELINES FOR THE PRELIMINARY SCREENING OF POTENTIAL SITES FOR A NUCLEAR WASTE... repositories. Such consideration shall take into account the proximity of sites to locations at which waste...

  19. 26 CFR 1.194-3 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 3 2010-04-01 2010-04-01 false Definitions. 1.194-3 Section 1.194-3 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Itemized Deductions for Individuals and Corporations (continued) § 1.194-3 Definitions....

  20. Synthesis of anthracene derivatives of 1,3-diazabicyclo[3.1.0]hex-3-ene

    NASA Astrophysics Data System (ADS)

    Mahmoodi, Nosrat O.; Mirkhaef, Safoura; Ghavidast, Atefeh

    2015-02-01

    Novel mono- and bis-photochromic compounds of 1,3-diazabicyclo[3.1.0]hex-3-enes based on anthracene moiety were synthesized efficiently. Photochromic compounds were synthesized through the reaction of 10-(hydroxymethyl)anthracene-9-carbaldehyde and anthracene-9-carbaldehyde or 9,10-anthracenedicarbaldehyde as bis-aldehydes with ketoaziridines in dry DMF at room temperature. Photochromic compounds exhibited photochromic behavior both in solution and in solid state by irradiation under UV light at 254 nm. Compounds bearing 4-NO2 on aziridine moiety showed intensive color change. Compounds were characterized by IR, 1H NMR, 13C NMR, and UV-Vis.

  1. 48 CFR 3.502-1 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Definitions. 3.502-1 Section 3.502-1 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION GENERAL IMPROPER BUSINESS PRACTICES AND PERSONAL CONFLICTS OF INTEREST Other Improper Business Practices 3.502-1...

  2. 34 CFR 3.1 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Definitions. 3.1 Section 3.1 Education Office of the Secretary, Department of Education OFFICIAL SEAL § 3.1 Definitions. For the purposes of this part: (a) ED means all organizational units of the Department of Education. (b) Embossing Seal means a display of...

  3. 34 CFR 3.1 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Definitions. 3.1 Section 3.1 Education Office of the Secretary, Department of Education OFFICIAL SEAL § 3.1 Definitions. For the purposes of this part: (a) ED means all organizational units of the Department of Education. (b) Embossing Seal means a display of...

  4. 7 CFR 1b.3 - Categorical exclusions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Categorical exclusions. 1b.3 Section 1b.3 Agriculture Office of the Secretary of Agriculture NATIONAL ENVIRONMENTAL POLICY ACT § 1b.3 Categorical exclusions. (a) The following are categories of activities which have been determined not to have a significant individual or cumulative effect on the...

  5. 5 CFR 1.3 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 5 Administrative Personnel 1 2014-01-01 2014-01-01 false Definitions. 1.3 Section 1.3 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE RULES COVERAGE AND DEFINITIONS (RULE I) § 1.3 Definitions. As used in the rules in this subchapter: (a) Competitive service shall have the...

  6. 21 CFR 3.1 - Purpose.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 1 2012-04-01 2012-04-01 false Purpose. 3.1 Section 3.1 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PRODUCT JURISDICTION Assignment of Agency Component for Review of Premarket Applications § 3.1 Purpose. This regulation relates to...

  7. 50 CFR 1.3 - Service.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 1 2011-10-01 2011-10-01 false Service. 1.3 Section 1.3 Wildlife and Fisheries UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF THE INTERIOR GENERAL PROVISIONS DEFINITIONS § 1.3 Service. Service means the United States Fish and Wildlife Service, Department of the Interior....

  8. 50 CFR 1.3 - Service.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 50 Wildlife and Fisheries 1 2012-10-01 2012-10-01 false Service. 1.3 Section 1.3 Wildlife and Fisheries UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF THE INTERIOR GENERAL PROVISIONS DEFINITIONS § 1.3 Service. Service means the United States Fish and Wildlife Service, Department of the Interior....

  9. 50 CFR 1.3 - Service.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 1 2013-10-01 2013-10-01 false Service. 1.3 Section 1.3 Wildlife and Fisheries UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF THE INTERIOR GENERAL PROVISIONS DEFINITIONS § 1.3 Service. Service means the United States Fish and Wildlife Service, Department of the Interior....

  10. 50 CFR 1.3 - Service.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 1 2014-10-01 2014-10-01 false Service. 1.3 Section 1.3 Wildlife and Fisheries UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF THE INTERIOR GENERAL PROVISIONS DEFINITIONS § 1.3 Service. Service means the United States Fish and Wildlife Service, Department of the Interior....