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Sample records for adhesion protein e-cadherin

  1. Restoring E-cadherin-mediated cell-cell adhesion increases PTEN protein level and stability in human breast carcinoma cells

    SciTech Connect

    Li Zengxia; Wang Liying; Zhang Wen; Fu Yi; Zhao Hongbo; Hu Yali; Prins, Bram Peter; Zha Xiliang

    2007-11-09

    The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a well-characterized tumor suppressor that negatively regulates cell growth and survival. Despite the critical role of PTEN in cell signaling, the mechanisms of its regulation are still under investigation. We reported here that PTEN expression could be controlled by overexpression or knock-down of E-cadherin in several mammary carcinoma cell lines. Furthermore, we showed that the accumulation of PTEN protein in E-cadherin overexpressing cells was due to increased PTEN protein stability rather than the regulation of its transcription. The proteasome-dependent PTEN degradation pathway was impaired after restoring E-cadherin expression. Moreover, maintenance of E-cadherin mediated cell-cell adhesion was necessary for its regulating PTEN. Altogether, our results suggested that E-cadherin mediated cell-cell adhesion was essential for preventing the proteasome degradation of PTEN, which might explain how breast carcinoma cells which lost cell-cell contact proliferate rapidly and are prone to metastasis.

  2. Important factors mediates the adhesion of aspergillus fumigatus to alveolar epithelial cells with E-cadherin.

    PubMed

    Xu, Xiao-Yong; Chen, Fei; Sun, He; Chen, Chen; Zhao, Bei-Lei

    2016-01-01

    Aspergillus is widely distributed in the Earth's biosphere. It has strong adaptive capacity, and lives as saprophytic or parasitic life. This study aims to investigate the role of E-cadherin for adhesion of Aspergillus fumigatus blastospores in a human epithelial cell line (A549) and search the correlated molecule in aspergillus. A. fumigatus blastospores were incubated with the total protein of A549 to investigate the binding of E-cadherin and blastospores followed by an affinity purification procedure. After establishing the adhesion model, the adhesion of A. fumigatus blastospores by A549 cells was evaluated by down-regulating E-cadherin of A549 cells with small interfering RNA (siRNA). FVB mice constructed with E-cadherin down-regulation were infected with aspergillus fumigatus. Preliminary exploration of E-cadherin interacting protein on the surface of aspergillus fumigates by immunoprecipitation and mass spectrometry analysis. E-cadherin was adhered to the surface of A. fumigatus blastospore. Adhesion of the blastospores was reduced by blocking or down-regulating E-cadherin in A549 cells. E-cadherin showed limited significance in the process of mice against aspergillus fumigates. Mass spectrometry (MS) analysis indicated the following proteins AFUA_8G07080, AfA24A6.130c, XP_747789 can bind to E-cadherin. In conclusion, E-cadherin is a receptor for adhesion of A. fumigatus blastospores in epithelial cells. This may open a new approach to treat this fungal infection. PMID:27347350

  3. The Metalloprotease Meprinβ Processes E-Cadherin and Weakens Intercellular Adhesion

    PubMed Central

    Huguenin, Maya; Müller, Eliane J.; Trachsel-Rösmann, Sandra; Oneda, Beatrice; Ambort, Daniel; Sterchi, Erwin E.; Lottaz, Daniel

    2008-01-01

    Background Meprin (EC 3.4.24.18), an astacin-like metalloprotease, is expressed in the epithelium of the intestine and kidney tubules and has been related to cancer, but the mechanistic links are unknown. Methodology/Principal Findings We used MDCK and Caco-2 cells stably transfected with meprinα and or meprinβ to establish models of renal and intestinal epithelial cells expressing this protease at physiological levels. In both models E-cadherin was cleaved, producing a cell-associated 97-kDa E-cadherin fragment, which was enhanced upon activation of the meprin zymogen and reduced in the presence of a meprin inhibitor. The cleavage site was localized in the extracellular domain adjacent to the plasma membrane. In vitro assays with purified components showed that the 97-kDa fragment was specifically generated by meprinβ, but not by ADAM-10 or MMP-7. Concomitantly with E-cadherin cleavage and degradation of the E-cadherin cytoplasmic tail, the plaque proteins β-catenin and plakoglobin were processed by an intracellular protease, whereas α-catenin, which does not bind directly to E-cadherin, remained intact. Using confocal microscopy, we observed a partial colocalization of meprinβ and E-cadherin at lateral membranes of incompletely polarized cells at preconfluent or early confluent stages. Meprinβ-expressing cells displayed a reduced strength of cell-cell contacts and a significantly lower tendency to form multicellular aggregates. Conclusions/Significance By identifying E-cadherin as a substrate for meprinβ in a cellular context, this study reveals a novel biological role of this protease in epithelial cells. Our results suggest a crucial role for meprinβ in the control of adhesiveness via cleavage of E-cadherin with potential implications in a wide range of biological processes including epithelial barrier function and cancer progression. PMID:18478055

  4. Protein zero is necessary for E-cadherin-mediated adherens junction formation in Schwann cells.

    PubMed

    Menichella, D M; Arroyo, E J; Awatramani, R; Xu, T; Baron, P; Vallat, J M; Balsamo, J; Lilien, J; Scarlato, G; Kamholz, J; Scherer, S S; Shy, M E

    2001-12-01

    Protein Zero (P0), the major structural protein in the peripheral nervous system (PNS) myelin, acts as a homotypic adhesion molecule and is thought to mediate compaction of adjacent wraps of myelin membrane. E-Cadherin, a calcium-dependent adhesion molecule, is also expressed in myelinating Schwann cells in the PNS and is involved in forming adherens junctions between adjacent loops of membrane at the paranode. To determine the relationship, if any, between P0-mediated and cadherin-mediated adhesion during myelination, we investigated the expression of E-cadherin and its binding partner, beta-catenin, in sciatic nerve of mice lacking P0 (P0(-/-)). We find that in P0(-/-) peripheral myelin neither E-cadherin nor beta-catenin are localized to paranodes, but are instead found in small puncta throughout the Schwann cell. In addition, only occasional, often rudimentary, adherens junctions are formed. Analysis of E-cadherin and beta-catenin expression during nerve development demonstrates that E-cadherin and beta-catenin are localized to the paranodal region after the onset of myelin compaction. Interestingly, axoglial junction formation is normal in P0(-/-) nerve. Taken together, these data demonstrate that P0 is necessary for the formation of adherens junctions but not axoglial junctions in myelinating Schwann cells. PMID:11749037

  5. α-Catenin and Vinculin Cooperate to Promote High E-cadherin-based Adhesion Strength*

    PubMed Central

    Thomas, William A.; Boscher, Cécile; Chu, Yeh-Shiu; Cuvelier, Damien; Martinez-Rico, Clara; Seddiki, Rima; Heysch, Julie; Ladoux, Benoit; Thiery, Jean Paul; Mege, René-Marc; Dufour, Sylvie

    2013-01-01

    Maintaining cell cohesiveness within tissues requires that intercellular adhesions develop sufficient strength to support traction forces applied by myosin motors and by neighboring cells. Cadherins are transmembrane receptors that mediate intercellular adhesion. The cadherin cytoplasmic domain recruits several partners, including catenins and vinculin, at sites of cell-cell adhesion. Our study used force measurements to address the role of αE-catenin and vinculin in the regulation of the strength of E-cadherin-based adhesion. αE-catenin-deficient cells display only weak aggregation and fail to strengthen intercellular adhesion over time, a process rescued by the expression of αE-catenin or chimeric E-cadherin·αE-catenins, including a chimera lacking the αE-catenin dimerization domain. Interestingly, an αE-catenin mutant lacking the modulation and actin-binding domains restores cadherin-dependent cell-cell contacts but cannot strengthen intercellular adhesion. The expression of αE-catenin mutated in its vinculin-binding site is defective in its ability to rescue cadherin-based adhesion strength in cells lacking αE-catenin. Vinculin depletion or the overexpression of the αE-catenin modulation domain strongly decreases E-cadherin-mediated adhesion strength. This supports the notion that both molecules are required for intercellular contact maturation. Furthermore, stretching of cell doublets increases vinculin recruitment and α18 anti-αE-catenin conformational epitope immunostaining at cell-cell contacts. Taken together, our results indicate that αE-catenin and vinculin cooperatively support intercellular adhesion strengthening, probably via a mechanoresponsive link between the E-cadherin·β-catenin complexes and the underlying actin cytoskeleton. PMID:23266828

  6. Microtubules Inhibit E-Cadherin Adhesive Activity by Maintaining Phosphorylated p120-Catenin in a Colon Carcinoma Cell Model

    PubMed Central

    Maiden, Stephanie L.; Petrova, Yuliya I.; Gumbiner, Barry M.

    2016-01-01

    Tight regulation of cadherin-mediated intercellular adhesions is critical to both tissue morphogenesis during development and tissue homeostasis in adults. Cell surface expression of the cadherin-catenin complex is often directly correlated with the level of adhesion, however, examples exist where cadherin appears to be inactive and cells are completely non-adhesive. The state of p120-catenin phosphorylation has been implicated in regulating the adhesive activity of E-cadherin but the mechanism is currently unclear. We have found that destabilization of the microtubule cytoskeleton, independent of microtubule plus-end dynamics, dephosphorylates p120-catenin and activates E-cadherin adhesion in Colo 205 cells. Through chemical screening, we have also identified several kinases as potential regulators of E-cadherin adhesive activity. Analysis of several p120-catenin phosphomutants suggests that gross dephosphorylation of p120-catenin rather than that of specific amino acids may trigger E-cadherin adhesion. Uncoupling p120-catenin binding to E-cadherin at the membrane causes constitutive adhesion in Colo 205 cells, further supporting an inhibitory role of phosphorylated p120-catenin on E-cadherin activity. PMID:26845024

  7. The Integrated Role of Wnt/β-Catenin, N-Glycosylation, and E-Cadherin-Mediated Adhesion in Network Dynamics

    PubMed Central

    Vargas, Diego A.; Sun, Meng; Sadykov, Khikmet; Kukuruzinska, Maria A.; Zaman, Muhammad H.

    2016-01-01

    The cellular network composed of the evolutionarily conserved metabolic pathways of protein N-glycosylation, Wnt/β-catenin signaling pathway, and E-cadherin-mediated cell-cell adhesion plays pivotal roles in determining the balance between cell proliferation and intercellular adhesion during development and in maintaining homeostasis in differentiated tissues. These pathways share a highly conserved regulatory molecule, β-catenin, which functions as both a structural component of E-cadherin junctions and as a co-transcriptional activator of the Wnt/β-catenin signaling pathway, whose target is the N-glycosylation-regulating gene, DPAGT1. Whereas these pathways have been studied independently, little is known about the dynamics of their interaction. Here we present the first numerical model of this network in MDCK cells. Since the network comprises a large number of molecules with varying cell context and time-dependent levels of expression, it can give rise to a wide range of plausible cellular states that are difficult to track. Using known kinetic parameters for individual reactions in the component pathways, we have developed a theoretical framework and gained new insights into cellular regulation of the network. Specifically, we developed a mathematical model to quantify the fold-change in concentration of any molecule included in the mathematical representation of the network in response to a simulated activation of the Wnt/ β-catenin pathway with Wnt3a under different conditions. We quantified the importance of protein N-glycosylation and synthesis of the DPAGT1 encoded enzyme, GPT, in determining the abundance of cytoplasmic β-catenin. We confirmed the role of axin in β-catenin degradation. Finally, our data suggest that cell-cell adhesion is insensitive to E-cadherin recycling in the cell. We validate the model by inhibiting β-catenin-mediated activation of DPAGT1 expression and predicting changes in cytoplasmic β-catenin concentration and stability

  8. The Integrated Role of Wnt/β-Catenin, N-Glycosylation, and E-Cadherin-Mediated Adhesion in Network Dynamics.

    PubMed

    Vargas, Diego A; Sun, Meng; Sadykov, Khikmet; Kukuruzinska, Maria A; Zaman, Muhammad H

    2016-07-01

    The cellular network composed of the evolutionarily conserved metabolic pathways of protein N-glycosylation, Wnt/β-catenin signaling pathway, and E-cadherin-mediated cell-cell adhesion plays pivotal roles in determining the balance between cell proliferation and intercellular adhesion during development and in maintaining homeostasis in differentiated tissues. These pathways share a highly conserved regulatory molecule, β-catenin, which functions as both a structural component of E-cadherin junctions and as a co-transcriptional activator of the Wnt/β-catenin signaling pathway, whose target is the N-glycosylation-regulating gene, DPAGT1. Whereas these pathways have been studied independently, little is known about the dynamics of their interaction. Here we present the first numerical model of this network in MDCK cells. Since the network comprises a large number of molecules with varying cell context and time-dependent levels of expression, it can give rise to a wide range of plausible cellular states that are difficult to track. Using known kinetic parameters for individual reactions in the component pathways, we have developed a theoretical framework and gained new insights into cellular regulation of the network. Specifically, we developed a mathematical model to quantify the fold-change in concentration of any molecule included in the mathematical representation of the network in response to a simulated activation of the Wnt/ β-catenin pathway with Wnt3a under different conditions. We quantified the importance of protein N-glycosylation and synthesis of the DPAGT1 encoded enzyme, GPT, in determining the abundance of cytoplasmic β-catenin. We confirmed the role of axin in β-catenin degradation. Finally, our data suggest that cell-cell adhesion is insensitive to E-cadherin recycling in the cell. We validate the model by inhibiting β-catenin-mediated activation of DPAGT1 expression and predicting changes in cytoplasmic β-catenin concentration and stability

  9. Expression of cell adhesion molecule E-cadherin in Xenopus embryos begins at gastrulation and predominates in the ectoderm

    PubMed Central

    1989-01-01

    The expression of the Ca2+-dependent epithelial cell adhesion molecule E-cadherin (also known as uvomorulin and L-CAM) in the early stages of embryonic development of Xenopus laevis was examined. E-Cadherin was identified in the Xenopus A6 epithelial cell line by antibody cross- reactivity and several biochemical characteristics. Four independent mAbs were generated against purified Xenopus E-cadherin. All four mAbs recognized the same polypeptides in A6 cells, adult epithelial tissues, and embryos. These mAbs inhibited the formation of cell contacts between A6 cells and stained the basolateral plasma membranes of A6 cells, hepatocytes, and alveolar epithelial cells. The time of E- cadherin expression in early Xenopus embryos was determined by immunoblotting. Unlike its expression in early mouse embryos, E- cadherin was not present in the eggs or early blastula of Xenopus laevis. These findings indicate that a different Ca2+-dependent cell adhesion molecule, perhaps another member of the cadherin gene family, is responsible for the Ca2+-dependent adhesion between cleavage stage Xenopus blastomeres. Detectable accumulation of E-cadherin started just before gastrulation at stage 9 1/2 and increased rapidly up to the end of gastrulation at stage 15. In stage 15 embryos, specific immunofluorescence staining of E-cadherin was discernible only in ectoderm, but not in mesoderm and endoderm. The ectoderm at this stage consists of two cell layers. The outer cell layer of ectoderm was stained intensely, and staining was localized to the basolateral plasma membrane of these cells. Lower levels of staining were observed in the inner cell layer of ectoderm. The coincidence of E-cadherin expression with the process of gastrulation and its restriction to the ectoderm indicate that it may play a role in the morphogenetic movements of gastrulation and resulting segregation of embryonic germ layers. PMID:2472408

  10. Molecular mechanisms involved in TFF3 peptide-mediated modulation of the E-cadherin/catenin cell adhesion complex.

    PubMed

    Meyer zum Büschenfelde, Dirk; Hoschützky, Heinz; Tauber, Rudolf; Huber, Otmar

    2004-05-01

    TFF3 is a member of the TFF-domain peptide family which is constitutively expressed in mucous epithelial tissues where it acts as a motogenic factor and plays an important role during epithelial restitution after wounding and during inflammation. In contrast to these beneficial functions, TFFs were also reported to be involved in cell scattering and tumor invasion. These changes in epithelial cell morphology and motility are associated with a modulation of cell contacts. In this respect, we here investigated the E-cadherin/catenin cell adhesion complex in FLAG-hTFF3-transfected HT29/B6 and MDCK cells. In hTFF3-transfected cells the amount of E-cadherin is reduced with a concomitant reduction of alpha- and beta-catenin levels. On one hand, E-cadherin expression is lowered at the transcriptional level as shown by multiplex RT-PCR analysis. This decrease does not depend on differences in the promoter methylation status as shown by methylation-specific PCR. On the other hand, pulse-chase experiments showed a reduction in the E-cadherin half-life in hTFF3-transfected cells reflecting increased E-cadherin degradation. In summary, hTFF3 induces transcriptional and posttranslational processes resulting in a modulation of E-cadherin-mediated cell-cell contacts that may play an important role in the paradoxical benefical and pathogenic function of TFF peptides.

  11. Role of E-cadherin, alpha-, beta-, and gamma-catenins, and p120 (cell adhesion molecules) in prolactinoma behavior.

    PubMed

    Qian, Zhi Rong; Li, Chiun Chei; Yamasaki, Hiroyuki; Mizusawa, Noriko; Yoshimoto, Katsuhiko; Yamada, Shozo; Tashiro, Takashi; Horiguchi, Hidehisa; Wakatsuki, Shingo; Hirokawa, Mitsuyoshi; Sano, Toshiaki

    2002-12-01

    E-cadherin/catenin complex regulates cellular adhesion and motility and is believed to function as an invasion suppressor system. In a number of cancers, abnormal and reduced expression of E-cadherin/catenin complex is associated with tumor invasion and metastasis. Prolactinomas show frequent invasion on the surrounding structures, despite their histologically benign nature. Furthermore, gender-based differences in endocrine and surgical findings are found in patients with prolactinoma. To understand biological factors governing prolactinoma behavior, this study analyzed the expression of E-cadherin; alpha-, beta-, and gamma-catenins; p120; and cell proliferation marker MIB-1 labeling index in 13 invasive tumors (9 in men, 4 in women), 26 noninvasive tumors (4 in men, 22 in women), and 8 normal anterior pituitaries by immunohistochemistry. Immunostaining of E-cadherin; alpha-, beta-, and gamma-catenins; and p120 showed a membranous pattern of reactivity and generally stronger in normal pituitaries than in prolactinomas. Expression of E-cadherin and beta-catenin was significantly lower in invasive than in noninvasive prolactinomas (P <.002 and P <.005, respectively), and reduced expression of E-cadherin and beta-catenin was more frequent in invasive than in noninvasive prolactinomas (P <.001 and P <.05, respectively); in contrast, gamma-catenin expression showed higher in invasive than in noninvasive prolactinomas (P <.05). Expression of E-cadherin was significantly lower in macroprolactinomas than in microprolactinomas (P <.01), and decreased expression of E-cadherin and beta-catenin predicted high MIB-1 expression (P <.05). Moreover, the expression of E-cadherin and beta-catenin was significantly lower in macroprolactinomas in men than in those in women (P <.01 and P <.02, respectively). No statistical correlations were observed between expression of alpha-catenin, p120, and clinicopathologic features. In conclusion, the reduction of E-cadherin and beta

  12. Expression and potential correlation among Forkhead box protein M1, Caveolin-1 and E-cadherin in colorectal cancer

    PubMed Central

    Zhang, Jing; Zhang, Kundong; Zhou, Lisheng; Wu, Weidong; Jiang, Tao; Cao, Jun; Huang, Kejian; Qiu, Zhengjun; Huang, Chen

    2016-01-01

    The aim of the present study was to investigate the expression and functions of Forkhead box protein M1 (FoxM1), Caveolin-1 (Cav-1) and E-cadherin in colorectal cancer (CRC), and to determine the correlations among these proteins in CRC development and progression. The protein expression of FoxM1, Cav-1 and E-cadherin was identified using a human CRC and normal tissue microarray. A standard immunohistochemistry assay was performed employing anti-FoxM1, anti-Cav-1 and anti-E-cadherin antibodies. The clinicopathological significance of FoxM1, Cav-1 and E-cadherin in CRC was determined, and correlations were investigated between FoxM1 and Cav-1, FoxM1 and E-cadherin, Cav-1 and E-cadherin, respectively. The level of FoxM1, Cav-1 and E-Cadherin protein expression in CRC was found to be associated with pathological grade, tumor clinical stages and the presence of metastasis, respectively. Elevated expression of FoxM1 and Cav-1 was observed in the CRC tissues, and a significant correlation was found between the two proteins in CRC. However, it was also observed that FoxM1 was overexpressed while E-cadherin expression was low, indicating that there was a negative correlation between FoxM1 expression and E-cadherin expression. Moreover, there was also a negative correlation between Cav-1 and E-cadherin expression. Overall, the elevated expression of FoxM1 and Cav-1 in a human CRC microarray provided novel clinical evidence to elucidate the fact that they may play a critical role in the development and progression of CRC by negatively regulating E-cadherin expression. Furthermore, the positive correlation between FoxM1 and Cav-1 suggested that the proteins may constitute a novel signaling pathway in human CRC.

  13. CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer

    PubMed Central

    Caldeira, José Roberto F; Prando, Érika C; Quevedo, Francisco C; Neto, Francisco A Moraes; Rainho, Cláudia A; Rogatto, Silvia R

    2006-01-01

    Background The E-cadherin gene (CDH1) maps, at chromosome 16q22.1, a region often associated with loss of heterozygosity (LOH) in human breast cancer. LOH at this site is thought to lead to loss of function of this tumor suppressor gene and was correlated with decreased disease-free survival, poor prognosis, and metastasis. Differential CpG island methylation in the promoter region of the CDH1 gene might be an alternative way for the loss of expression and function of E-cadherin, leading to loss of tissue integrity, an essential step in tumor progression. Methods The aim of our study was to assess, by Methylation-Specific Polymerase Chain Reaction (MSP), the methylation pattern of the CDH1 gene and its possible correlation with the expression of E-cadherin and other standard immunohistochemical parameters (Her-2, ER, PgR, p53, and K-67) in a series of 79 primary breast cancers (71 infiltrating ductal, 5 infiltrating lobular, 1 metaplastic, 1 apocrine, and 1 papillary carcinoma). Results CDH1 hypermethylation was observed in 72% of the cases including 52/71 ductal, 4/5 lobular carcinomas and 1 apocrine carcinoma. Reduced levels of E-cadherin protein were observed in 85% of our samples. Although not statistically significant, the levels of E-cadherin expression tended to diminish with the CDH1 promoter region methylation. In the group of 71 ductal cancinomas, most of the cases of showing CDH1 hypermethylation also presented reduced levels of expression of ER and PgR proteins, and a possible association was observed between CDH1 methylation and ER expression (p = 0.0301, Fisher's exact test). However, this finding was not considered significant after Bonferroni correction of p-value. Conclusion Our preliminary findings suggested that abnormal CDH1 methylation occurs in high frequencies in infiltrating breast cancers associated with a decrease in E-cadherin expression in a subgroup of cases characterized by loss of expression of other important genes to the mammary

  14. Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface.

    PubMed Central

    Iino, R; Koyama, I; Kusumi, A

    2001-01-01

    Single green fluorescent protein (GFP) molecules were successfully imaged for the first time in living cells. GFP linked to the cytoplasmic carboxyl terminus of E-cadherin (E-cad-GFP) was expressed in mouse fibroblast L cells, and observed using an objective-type total internal reflection fluorescence microscope. Based on the fluorescence intensity of individual fluorescent spots, the majority of E-cad-GFP molecules on the free cell surface were found to be oligomers of various sizes, many of them greater than dimers, suggesting that oligomerization of E-cadherin takes place before its assembly at cell-cell adhesion sites. The translational diffusion coefficient of E-cad-GFP is reduced by a factor of 10 to 40 upon oligomerization. Because such large decreases in translational mobility cannot be explained solely by increases in radius upon oligomerization, an oligomerization-induced trapping model is proposed in which, when oligomers are formed, they are trapped in place due to greatly enhanced tethering and corralling effects of the membrane skeleton on oligomers (compared with monomers). The presence of many oligomers greater than dimers on the free surface suggests that these greater oligomers are the basic building blocks for the two-dimensional cell adhesion structures (adherens junctions). PMID:11371443

  15. Slit2-Robo1 signaling promotes the adhesion, invasion and migration of tongue carcinoma cells via upregulating matrix metalloproteinases 2 and 9, and downregulating E-cadherin

    PubMed Central

    Zhao, Yuan; Zhou, Feng-Li; Li, Wei-Ping; Wang, Jing; Wang, Li-Jing

    2016-01-01

    Whether Slit homologue 2 (Slit2) inhibits or promotes tumor cell migration remains controversial, and the role of Slit2-Roundabout 1 (Robo1) signaling in oral cancer remains to be fully elucidated. The aim of the present study was to investigate the role of Slit2-Robo1 signaling in the adhesion, invasion and migration of tongue carcinoma cells, and the mechanism by which Slit2-Robo1 signaling inhibits or promotes tumor cell migration. Tca8113 tongue carcinoma cells were treated with the monoclonal anti-human Robo1 antibody, R5, to inhibit the Slit2-Robo1 signaling pathway, with immunoglobulin (Ig)G2b treatment as a negative control. The expression levels of Slit2 and Robo1 were determined using flow cytometry. The effects of R5 on the adhesion, invasion and migration of Tca8113 tongue carcinoma cells were investigated. Gelatin zymography was used to investigate the activity of matrix metalloproteinase 2 (MMP2) and MMP9. Western blot analysis was used to evaluate the expression levels of E-cadherin in Tca8113 cells treated with 10 µg/ml of either R5 or IgG2b. Slit2 and Robo1 proteins were found to be expressed in the Tca8113 cells. R5 significantly inhibited the adhesion, invasion and migration of Tca8113 cells in vitro. R5 also inhibited the activities of MMP2 and MMP9, and increased the expression of E-cadherin in the Tca8113 cells. These results suggested that Slit2-Robo1 signaling promoted the adhesion, invasion and migration of tongue carcinoma cells by upregulating the expression levels of MMP2 and MMP9 and, downregulating the expression of E-cadherin. PMID:27431199

  16. Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion

    PubMed Central

    Erasmus, Jennifer C.; Welsh, Natalie J.; Braga, Vania M.M.

    2015-01-01

    The precise mechanisms via which Rac1 is activated by cadherin junctions are not fully known. In keratinocytes Rac1 activation by cadherin junctions requires EGFR signalling, but how EGFR does so is unclear. To address which activator could mediate E-cadherin signalling to Rac1, we investigated EGFR and two Rac1 GEFs, SOS1 and DOCK180. EGFR RNAi prevented junction-induced Rac1 activation and led to fragmented localization of E-cadherin at cadherin contacts. In contrast, depletion of another EGFR family member, ErbB3, did not interfere with either process. DOCK180 RNAi, but not SOS1, prevented E-cadherin-induced Rac1 activation. However, in a strong divergence from EGFR RNAi phenotype, DOCK180 depletion did not perturb actin recruitment or cadherin localisation at junctions. Rather, reduced DOCK180 levels impaired the resistance to mechanical stress of pre-formed cell aggregates. Thus, within the same cell type, EGFR and DOCK180 regulate Rac1 activation by newly-formed contacts, but control separate cellular events that cooperate to stabilise junctions. PMID:25957131

  17. Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion.

    PubMed

    Erasmus, Jennifer C; Welsh, Natalie J; Braga, Vania M M

    2015-09-01

    The precise mechanisms via which Rac1 is activated by cadherin junctions are not fully known. In keratinocytes Rac1 activation by cadherin junctions requires EGFR signalling, but how EGFR does so is unclear. To address which activator could mediate E-cadherin signalling to Rac1, we investigated EGFR and two Rac1 GEFs, SOS1 and DOCK180. EGFR RNAi prevented junction-induced Rac1 activation and led to fragmented localization of E-cadherin at cadherin contacts. In contrast, depletion of another EGFR family member, ErbB3, did not interfere with either process. DOCK180 RNAi, but not SOS1, prevented E-cadherin-induced Rac1 activation. However, in a strong divergence from EGFR RNAi phenotype, DOCK180 depletion did not perturb actin recruitment or cadherin localisation at junctions. Rather, reduced DOCK180 levels impaired the resistance to mechanical stress of pre-formed cell aggregates. Thus, within the same cell type, EGFR and DOCK180 regulate Rac1 activation by newly-formed contacts, but control separate cellular events that cooperate to stabilise junctions. PMID:25957131

  18. Dynamic and Static Interactions between p120 Catenin and E-Cadherin Regulate the Stability of Cell-Cell Adhesion

    SciTech Connect

    Ishiyama, Noboru; Lee, Seung-Hye; Liu, Shuang; Li, Guang-Yao; Smith, Matthew J.; Reichardt, Louis F.; Ikura, Mitsuhiko

    2010-04-26

    The association of p120 catenin (p120) with the juxtamembrane domain (JMD) of the cadherin cytoplasmic tail is critical for the surface stability of cadherin-catenin cell-cell adhesion complexes. Here, we present the crystal structure of p120 isoform 4A in complex with the JMD core region (JMD{sub core}) of E-cadherin. The p120 armadillo repeat domain contains modular binding pockets that are complementary to electrostatic and hydrophobic properties of the JMD{sub core}. Single-residue mutations within the JMD{sub core}-binding site of p120 abolished its interaction with E- and N-cadherins in vitro and in cultured cells. These mutations of p120 enabled us to clearly differentiate between N-cadherin-dependent and -independent steps of neuronal dendritic spine morphogenesis crucial for synapse development. NMR studies revealed that p120 regulates the stability of cadherin-mediated cell-cell adhesion by associating with the majority of the JMD, including residues implicated in clathrin-mediated endocytosis and Hakai-dependent ubiquitination of E-cadherin, through its discrete dynamic and static binding sites.

  19. Expression of FoxM1 and the EMT-associated protein E-cadherin in gastric cancer and its clinical significance

    PubMed Central

    Zhang, Jing; Chen, Xiao-Yu; Huang, Ke-Jian; Wu, Wei-Dong; Jiang, Tao; Cao, Jun; Zhou, Li-Sheng; Qiu, Zheng-Jun; Huang, Chen

    2016-01-01

    The aim of the present study was to investigate the expression of forkhead box M1 (FoxM1) and E-cadherin in tissues of gastric cancer in order to reveal any correlation between FoxM1, E-cadherin and clinicopathological parameters. The association between FoxM1 and E-cadherin in the development and progression of gastric cancer was also investigated. The expression of FoxM1 and E-cadherin in gastric cancer and adjacent normal tissue on tissue microarray was detected using immunohistochemistry. The clinicopathological significance of FoxM1 and E-cadherin in gastric cancer was explored, and the association between FoxM1 and E-cadherin was further examined using statistical techniques. In gastric cancer tissues, the expression of FoxM1 and E-cadherin was strongly positive, but it was weak in normal gastric mucosa. Overexpression of FoxM1 was evident in gastric cancer, and was associated with poor tumor differentiation (P<0.05), advanced tumor state (P<0.05) and lymph node (or distant) metastasis (P<0.05), whereas E-cadherin had the opposite effects. Furthermore, the correlation between FoxM1 and E-cadherin expression in gastric cancer tissue was negative. In conclusion, the high FoxM1 expression and low E-cadherin expression in gastric cancer tissue suggests that these proteins play a critical role in the development and progression of gastric cancer.

  20. E-Cadherin-Dependent Stimulation of Traction Force at Focal Adhesions via the Src and PI3K Signaling Pathways

    PubMed Central

    Jasaitis, Audrius; Estevez, Maruxa; Heysch, Julie; Ladoux, Benoit; Dufour, Sylvie

    2012-01-01

    The interplay between cadherin- and integrin-dependent signals controls cell behavior, but the precise mechanisms that regulate the strength of adhesion to the extracellular matrix remains poorly understood. We deposited cells expressing a defined repertoire of cadherins and integrins on fibronectin (FN)-coated polyacrylamide gels (FN-PAG) and on FN-coated pillars used as a micro-force sensor array (μFSA), and analyzed the functional relationship between these adhesion receptors to determine how it regulates cell traction force. We found that cadherin-mediated adhesion stimulated cell spreading on FN-PAG, and this was modulated by the substrate stiffness. We compared S180 cells with cells stably expressing different cadherins on μFSA and found that traction forces were stronger in cells expressing cadherins than in parental cells. E-cadherin-mediated contact and mechanical coupling between cells are required for this increase in cell-FN traction force, which was not observed in isolated cells, and required Src and PI3K activities. Traction forces were stronger in cells expressing type I cadherins than in cells expressing type II cadherins, which correlates with our previous observation of a higher intercellular adhesion strength developed by type I compared with type II cadherins. Our results reveal one of the mechanisms whereby molecular cross talk between cadherins and integrins upregulates traction forces at cell-FN adhesion sites, and thus provide additional insight into the molecular control of cell behavior. PMID:22853894

  1. Zeb1 Regulates E-cadherin and Epcam (Epithelial Cell Adhesion Molecule) Expression to Control Cell Behavior in Early Zebrafish Development*

    PubMed Central

    Vannier, Corinne; Mock, Kerstin; Brabletz, Thomas; Driever, Wolfgang

    2013-01-01

    The ZEB1 transcription factor is best known as an inducer of epithelial-mesenchymal transitions (EMT) in cancer metastasis, acting through transcriptional repression of CDH1 (encoding E-cadherin) and the EMT-suppressing microRNA-200s (miR-200s). Here we analyze roles of the ZEB1 zebrafish orthologs, Zeb1a and Zeb1b, and of miR-200s in control of cell adhesion and morphogenesis during gastrulation and segmentation stages. Loss and gain of function analyses revealed that Zeb1 represses cdh1 expression to fine-tune adhesiveness of migrating deep blastodermal cells. Furthermore, Zeb1 acts as a repressor of epcam in the deep cells of the blastoderm and may contribute to control of epithelial integrity of enveloping layer cells, the outermost cells of the blastoderm. We found a similar ZEB1-dependent repression of EPCAM expression in human pancreatic and breast cancer cell lines, mediated through direct binding of ZEB1 to the EPCAM promoter. Thus, Zeb1 proteins employ several evolutionary conserved mechanisms to regulate cell-cell adhesion during development and cancer. PMID:23667256

  2. Proteomics analysis of E-cadherin knockdown in epithelial breast cancer cells.

    PubMed

    Vergara, Daniele; Simeone, Pasquale; Latorre, Dominga; Cascione, Francesca; Leporatti, Stefano; Trerotola, Marco; Giudetti, Anna Maria; Capobianco, Loredana; Lunetti, Paola; Rizzello, Antonia; Rinaldi, Rosaria; Alberti, Saverio; Maffia, Michele

    2015-05-20

    E-cadherin is the core protein of the epithelial adherens junction. Through its cytoplasmic domain, E-cadherin interacts with several signaling proteins; among them, α- and β-catenins mediate the link of E-cadherin to the actin cytoskeleton. Loss of E-cadherin expression is a crucial step of epithelial-mesenchymal transition (EMT) and is involved in cancer invasion and metastatization. In human tumors, down-regulation of E-cadherin is frequently associated with poor prognosis. Despite the critical role of E-cadherin in cancer progression, little is known about proteome alterations linked with its down-regulation. To address this point, we investigated proteomics, biophysical and functional changes of epithelial breast cancer cell lines upon shRNA-mediated stable knockdown of E-cadherin expression (shEcad). shEcad cells showed a distinct proteomic signature including altered expression of enzymes and proteins involved in cytoskeletal dynamic and migration. Moreover, these results suggest that, besides their role in mechanical adhesion, loss of E-cadherin expression may contribute to cancer progression by modifying a complex network of pathways that tightly regulate fundamental processes as oxidative stress, immune evasion and cell metabolism. Altogether, these results extend our knowledge on the cellular modifications associated with E-cadherin down-regulation in breast cancer cells.

  3. The effects of adriamycin on E-cadherin mediated cell-cell adhesion and apoptosis during early kidney development.

    PubMed

    Yay, A; Ozdamar, S; Balcioglu, E; Baran, M; Akkus, D; Sonmez, M F

    2015-07-01

    Adriamycin (ADR) is strongly teratogenic. We investigated the effects of ADR on apoptosis and the intensity of E-cadherin expression in developing kidneys. An experimental group of rats was given 2 mg/kg/day ADR on days 6-9 of gestation and a control group was given saline on the same schedule. Embryos were decapitated on days 13, 15, 17 and 19 of gestation, and processed and embedded in paraffin for routine light microscopy. Kidney specimens were stained with hematoxylin and eosin or periodic acid-Schiff, or immunostained for E-cadherin. Apoptosis was assessed using the TUNEL method. Weight loss and developmental deficiency were determined in embryos of the experimental group. ADR damaged or destroyed tubule epithelial cells, which caused apparent dilatation of the tubule lumen. Also, the brush borders of proximal tubules were damaged and glomerular spaces were dilated. ADR caused apoptosis of kidney tissue by days 15, 17 and 19 of development and E-cadherin expression was up-regulated during kidney development compared to controls. We found that ADR can cause apoptosis and increased E-cadherin expression in the developing rat kidney. E-cadherin expression and apoptosis may contribute to the development of ADR nephrotoxicity.

  4. Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics.

    PubMed

    Le, T L; Yap, A S; Stow, J L

    1999-07-12

    E-Cadherin plays critical roles in many aspects of cell adhesion, epithelial development, and the establishment and maintenance of epithelial polarity. The fate of E-cadherin once it is delivered to the basolateral cell surface, and the mechanisms which govern its participation in adherens junctions, are not well understood. Using surface biotinylation and recycling assays, we observed that some of the cell surface E-cadherin is actively internalized and is then recycled back to the plasma membrane. The pool of E-cadherin undergoing endocytosis and recycling was markedly increased in cells without stable cell-cell contacts, i.e., in preconfluent cells and after cell contacts were disrupted by depletion of extracellular Ca2+, suggesting that endocytic trafficking of E-cadherin is regulated by cell-cell contact. The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment. The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway. We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.

  5. Cdc42-Interacting Protein 4 Represses E-Cadherin Expression by Promoting β-Catenin Translocation to the Nucleus in Murine Renal Tubular Epithelial Cells.

    PubMed

    Xu, Chuou; Zhou, Qiaodan; Liu, Lili; Liu, Ping; Pei, Guangchang; Zeng, Rui; Han, Min; Xu, Gang

    2015-08-14

    Renal fibrosis is an inevitable outcome of end-stage chronic kidney disease. During this process, epithelial cells lose E-cadherin expression. β-Catenin may act as a mediator by accumulation and translocation to the nucleus. Studies have suggested that CIP4, a Cdc42 effector protein, is associated with β-catenin. However, whether CIP4 contributes to E-cadherin loss in epithelial cells by regulating β-catenin translocation is unclear. In this study, we investigated the involvement of CIP4 in β-catenin translocation. Expression of CIP4 was upregulated in renal tissues of 5/6 nephrectomized rats and mainly distributed in renal tubular epithelia. In TGF-β1-treated NRK-52E cells, upregulation of CIP4 expression was accompanied by reduced expression of E-cadherin. CIP4 overexpression promoted the translocation of β-catenin to the nucleus, which was accompanied by reduced expression of E-cadherin even without TGF-β1 stimulation. In contrast, CIP4 depletion by using siRNA inhibited the translocation of β-catenin to the nucleus and reversed the decrease in expression of E-cadherin. The interaction between CIP4 and β-catenin was detected. We also show that β-catenin depletion could restore the expression of E-cadherin that was suppressed by CIP4 overexpression. In conclusion, these results suggest that CIP4 overexpression represses E-cadherin expression by promoting β-catenin translocation to the nucleus.

  6. Vangl2 Regulates E-Cadherin in Epithelial Cells

    PubMed Central

    Nagaoka, Tadahiro; Inutsuka, Ayumu; Begum, Khadiza; hafiz, Khandakar musabbir bin; Kishi, Masashi

    2014-01-01

    E-cadherin belongs to the classic cadherin subfamily of calcium-dependent cell adhesion molecules and is crucial for the formation and function of epithelial adherens junctions. In this study, we demonstrate that Vangl2, a vertebrate regulator of planar cell polarity (PCP), controls E-cadherin in epithelial cells. E-cadherin co-immunoprecipitates with Vangl2 from embryonic kidney extracts, and this association is also observed in transfected fibroblasts. Vangl2 enhances the internalization of E-cadherin when overexpressed. Conversely, the quantitative ratio of E-cadherin exposed to the cell surface is increased in cultured renal epithelial cells derived from Vangl2Lpt/+ mutant mice. Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis. Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity. This function may be involved in the control of intercellular PCP signalling or in the PCP-related rearrangement of cell adhesions. PMID:25373475

  7. Pancreatic Cancer Cell Glycosylation Regulates Cell Adhesion and Invasion through the Modulation of α2β1 Integrin and E-Cadherin Function

    PubMed Central

    Bassagañas, Sònia; Carvalho, Sandra; Dias, Ana M.; Pérez-Garay, Marta; Ortiz, M. Rosa; Figueras, Joan; Reis, Celso A.; Pinho, Salomé S.; Peracaula, Rosa

    2014-01-01

    In our previous studies we have described that ST3Gal III transfected pancreatic adenocarcinoma Capan-1 and MDAPanc-28 cells show increased membrane expression levels of sialyl-Lewis x (SLex) along with a concomitant decrease in α2,6-sialic acid compared to control cells. Here we have addressed the role of this glycosylation pattern in the functional properties of two glycoproteins involved in the processes of cancer cell invasion and migration, α2β1 integrin, the main receptor for type 1 collagen, and E-cadherin, responsible for cell-cell contacts and whose deregulation determines cell invasive capabilities. Our results demonstrate that ST3Gal III transfectants showed reduced cell-cell aggregation and increased invasive capacities. ST3Gal III transfected Capan-1 cells exhibited higher SLex and lower α2,6-sialic acid content on the glycans of their α2β1 integrin molecules. As a consequence, higher phosphorylation of focal adhesion kinase tyrosine 397, which is recognized as one of the first steps of integrin-derived signaling pathways, was observed in these cells upon adhesion to type 1 collagen. This molecular mechanism underlies the increased migration through collagen of these cells. In addition, the pancreatic adenocarcinoma cell lines as well as human pancreatic tumor tissues showed colocalization of SLex and E-cadherin, which was higher in the ST3Gal III transfectants. In conclusion, changes in the sialylation pattern of α2β1 integrin and E-cadherin appear to influence the functional role of these two glycoproteins supporting the role of these glycans as an underlying mechanism regulating pancreatic cancer cell adhesion and invasion. PMID:24878505

  8. α-catenin, vinculin, and F-actin in strengthening E-cadherin cell–cell adhesions and mechanosensing

    PubMed Central

    Dufour, Sylvie; Mège, René-Marc; Thiery, Jean Paul

    2013-01-01

    Classical cadherins play a crucial role in establishing intercellular adhesion, regulating cortical tension, and maintaining mechanical coupling between cells. The mechanosensitive regulation of intercellular adhesion strengthening depends on the recruitment of adhesion complexes at adhesion sites and their anchoring to the actin cytoskeleton. Thus, the molecular mechanisms coupling cadherin-associated complexes to the actin cytoskeleton are actively being studied, with a particular focus on α-catenin and vinculin. We have recently addressed the role of these proteins by analyzing the consequences of their depletion and the expression of α-catenin mutants in the formation and strengthening of cadherin-mediated adhesions. We have used the dual pipette assay to measure the forces required to separate cell doublets formed in suspension. In this commentary, we briefly summarize the current knowledge on the role of α-catenin and vinculin in cadherin-actin cytoskeletal interactions. These data shed light on the tension-dependent contribution of α-catenin and vinculin in a mechanoresponsive complex that promotes the connection between cadherin and the actin cytoskeleton and their requirement in the development of adhesion strengthening. PMID:23739176

  9. Distinct roles of secreted HtrA proteases from gram-negative pathogens in cleaving the junctional protein and tumor suppressor E-cadherin.

    PubMed

    Hoy, Benjamin; Geppert, Tim; Boehm, Manja; Reisen, Felix; Plattner, Patrick; Gadermaier, Gabriele; Sewald, Norbert; Ferreira, Fatima; Briza, Peter; Schneider, Gisbert; Backert, Steffen; Wessler, Silja

    2012-03-23

    The periplasmic chaperone and serine protease HtrA is important for bacterial stress responses and protein quality control. Recently, we discovered that HtrA from Helicobacter pylori is secreted and cleaves E-cadherin to disrupt the epithelial barrier, but it remained unknown whether this maybe a general virulence mechanism. Here, we show that important other pathogens including enteropathogenic Escherichia coli, Shigella flexneri, and Campylobacter jejuni, but not Neisseria gonorrhoeae, cleaved E-cadherin on host cells. HtrA deletion in C. jejuni led to severe defects in E-cadherin cleavage, loss of cell adherence, paracellular transmigration, and basolateral invasion. Computational modeling of HtrAs revealed a conserved pocket in the active center exhibiting pronounced proteolytic activity. Differential E-cadherin cleavage was determined by an alanine-to-glutamine exchange in the active center of neisserial HtrA. These data suggest that HtrA-mediated E-cadherin cleavage is a prevalent pathogenic mechanism of multiple gram-negative bacteria representing an attractive novel target for therapeutic intervention to combat bacterial infections. PMID:22337879

  10. E-cadherin and alpha-, beta-, and gamma-catenin protein expression is up-regulated in ovarian carcinoma cells in serous effusions.

    PubMed

    Davidson, B; Berner, A; Nesland, J M; Risberg, B; Berner, H S; Tropè, C G; Kristensen, G B; Bryne, M; Ann Florenes, V

    2000-12-01

    The aims of this study were firstly, to investigate the expression of E-cadherin complex proteins in ovarian carcinoma cells in serous effusions and in primary and metastatic lesions; and secondly to study the value of these four proteins and calretinin, a mesothelial marker, in the differential diagnosis of ovarian carcinoma cells from reactive mesothelial cells in effusions. Sixty-seven malignant effusions and 97 corresponding primary (n=36) and metastatic (n=61) lesions were immunohistochemically stained for E-cadherin and alpha-, beta-, and gamma-catenin. Staining extent and intensity were scored. Effusion specimens were additionally analysed for calretinin immunoreactivity. Membrane immunoreactivity for E-cadherin and alpha-, beta-, and gamma-catenin was detected on carcinoma cells in the majority of the effusions, but rarely on reactive mesothelial cells (p<0.001 for all markers). Calretinin immunoreactivity was confined to mesothelial cells (p<0.001). An association was seen between E-cadherin and alpha-catenin expression, in both effusions and solid tumours, and for beta-catenin in solid tumours (range p<0. 001 to p=0.014). Up-regulation of all four cadherin complex proteins was seen in carcinoma cells in effusions, when compared with corresponding primary tumours (range p<0.001 to p=0.028). As with effusions, metastatic lesions showed up-regulation of alpha-, beta-, and gamma-catenin when compared with primary carcinomas (p=0.002-0. 015). Carcinoma cells in effusions showed in addition elevated levels of E-cadherin when compared with metastatic lesions (p<0.001). Staining results in effusions showed no association with effusion site, tumour type or histological grade. Immunoblotting on 29 malignant effusions confirmed the presence of all four proteins in the majority of samples and co-precipitation of E-cadherin and beta-catenin was seen in ten specimens examined. E-cadherin complex proteins are widely expressed in ovarian carcinoma cells. Together with

  11. E-cadherin interactome complexity and robustness resolved by quantitative proteomics

    PubMed Central

    Guo, Zhenhuan; Neilson, Lisa J; Zhong, Hang; Murray, Paul S; Rao, Megha Vaman; Zanivan, Sara; Zaidel-Bar, Ronen

    2016-01-01

    E-cadherin-mediated cell-cell adhesion and signaling plays an essential role in development and maintenance of healthy epithelial tissues. Adhesiveness is conferred by cadherin extracellular domains, and is regulated by an assembly of adaptors and enzymes associated with the cytoplasmic tail. Here, we employed proximity biotinylation and quantitative proteomics to isolate and identify 612 proteins in the vicinity of E-cadherin’s cytoplasmic tail. We used a structure-informed database of protein-protein interactions to construct the most comprehensive E-cadherin interactome to date, containing 89 known E-cadhesome components and 346 novel proteins. Moreover, through cloning and expression of GFP-tagged fusion proteins we localized 26 of the novel proteins to adherens junctions. Finally, employing calcium depletion and myosin inhibition we show the E-cadherin interactome to be remarkably robust to perturbation and essentially independent of cell-cell junctions or actomyosin contractility. PMID:25468996

  12. E-cadherin expression is commonly downregulated by CpG island hypermethylation in esophageal carcinoma cells.

    PubMed

    Si, H X; Tsao, S W; Lam, K Y; Srivastava, G; Liu, Y; Wong, Y C; Shen, Z Y; Cheung, A L

    2001-11-01

    E-cadherin, a cell adhesion molecule, is regarded as an invasion-suppressor molecule and a prognostic marker in many types of human cancers. Downregulation of E-cadherin is common in esophageal carcinoma and is associated with an increase in invasive and metastatic potential. To study the mechanisms responsible for inactivation of this gene in esophageal squamous cell carcinoma (ESCC), we investigated the methylation status around the 5' promoter region of E-cadherin gene of six ESCC cell lines by methylation-specific polymerase chain reaction, and compared it with E-cadherin protein and mRNA expression. We also studied the methylation status of 20 ESCC clinical specimens. Methylation was noted in four of the six cell lines (one fully methylated and three partially methylated). The completely methylated cell line lacked E-cadherin protein expression and mRNA transcription. E-cadherin expression and transcription were reduced in a partially methylated cell line but preserved in the other partially methylated cell lines. Treatment of E-cadherin-negative carcinoma cells with the demethylating agent, 5-aza-2'-deoxycytidine, induced re-expression of the gene. A high frequency of methylation (16/20, 80%) was also noted in the 20 ESCC clinical samples. Our results indicate that 5' CpG island methylation is common in esophageal carcinoma and may play an important role in downregulation of E-cadherin.

  13. Characterization of E-cadherin-dependent and -independent events in a new model of c-Fos-mediated epithelial-mesenchymal transition

    SciTech Connect

    Mejlvang, Jakob; Kriajevska, Marina; Berditchevski, Fedor; Bronstein, Igor; Lukanidin, Eugene M.; Pringle, J. Howard; Mellon, J. Kilian; Tulchinsky, Eugene M. . E-mail: et32@le.ac.uk

    2007-01-15

    Fos proteins have been implicated in control of tumorigenesis-related genetic programs including invasion, angiogenesis, cell proliferation and apoptosis. In this study, we demonstrate that c-Fos is able to induce mesenchymal transition in murine tumorigenic epithelial cell lines. Expression of c-Fos in MT1TC1 cells led to prominent alterations in cell morphology, increased expression of mesenchymal markers, vimentin and S100A4, DNA methylation-dependent down-regulation of E-cadherin and abrogation of cell-cell adhesion. In addition, c-Fos induced a strong {beta}-catenin-independent proliferative response in MT1TC1 cells and stimulated cell motility, invasion and adhesion to different extracellular matrix proteins. To explore whether loss of E-cadherin plays a role in c-Fos-mediated mesenchymal transition, we expressed wild-type E-cadherin and two different E-cadherin mutants in MT1TC1/c-fos cells. Expression of wild-type E-cadherin restored epithelioid morphology and enhanced cellular levels of catenins. However, exogenous E-cadherin did not influence expression of c-Fos-dependent genes, only partly suppressed growth of MT1TC1/c-fos cells and produced no effect on c-Fos-stimulated cell motility and invasion in matrigel. On the other hand, re-expression of E-cadherin specifically negated c-Fos-induced adhesion to collagen type I, but not to laminin or fibronectin. Of interest, mutant E-cadherin which lacks the ability to form functional adhesive complexes had an opposite, potentiating effect on cell adhesion to collagen I. These data suggest that cell adhesion to collagen I is regulated by the functional state of E-cadherin. Overall, our data demonstrate that, with the exception of adhesion to collagen I, c-Fos is dominant over E-cadherin in relation to the aspects of mesenchymal transition assayed in this study.

  14. Direct regulation of E-cadherin by targeted histone methylation of TALE-SET fusion protein in cancer cells.

    PubMed

    Cho, Hyun-Soo; Kang, Jeong Gu; Lee, Jae-Hye; Lee, Jeong-Ju; Jeon, Seong Kook; Ko, Jeong-Heon; Kim, Dae-Soo; Park, Kun-Hyang; Kim, Yong-Sam; Kim, Nam-Soon

    2015-09-15

    TALE-nuclease chimeras (TALENs) can bind to and cleave specific genomic loci and, are used to engineer gene knockouts and additions. Recently, instead of using the FokI domain, epigenetically active domains, such as TET1 and LSD1, have been combined with TAL effector domains to regulate targeted gene expression via DNA and histone demethylation. However, studies of histone methylation in the TALE system have not been performed. Therefore, in this study, we established a novel targeted regulation system with a TAL effector domain and a histone methylation domain. To construct a TALE-methylation fusion protein, we combined a TAL effector domain containing an E-Box region to act as a Snail binding site and the SET domain of EHMT 2 to allow for histone methylation. The constructed TALE-SET module (TSET) repressed the expression of E-cadherin via by increasing H3K9 dimethylation. Moreover, the cells that overexpressed TSET showed increased cell migration and invasion. This is the first phenotype-based study of targeted histone methylation by the TALE module, and this new system can be applied in new cancer therapies to reduce side effects.

  15. The E-cadherin complex contains the src substrate p120.

    PubMed

    Aghib, D F; McCrea, P D

    1995-05-01

    Using normal MDCK cells, and MDCK cells stably transfected with a temperature-sensitive viral src allele (pp60 ts-v-src), we have examined the composition and tyrosine phosphorylation of the E-cadherin complex. E-cadherin is a transmembrane calcium-dependent cell-cell adhesion molecule that is complexed with cytoplasmic proteins including alpha-catenin, beta-catenin, plakoglobin (gamma-catenin), and actin. We have identified two heterodimeric complexes which demonstrate that alpha-catenin interacts directly with beta-catenin, or with plakoglobin, in the absence of E-cadherin. beta-Catenin has previously been shown to bind directly to E-cadherin. We propose that E-cadherin associates with alpha-catenin, and thereby the actin cytoskeleton, via either beta-catenin or plakoglobin. We have further identified three new but related protein components of the E-cadherin complex, which are each cross-reactive by Western blot analysis to antibodies directed against p120, a phosphotyrosine substrate of src, and a phosphotyrosine, phosphoserine, and phosphothreonine substrate of growth factor-stimulated signaling pathways. Greater quantities of the p120-related proteins were found present in the E-cadherin immunoprecipitates of ts-src MDCK cells compared to normal MDCK cells, while two of the p120 cross-reactive species were significantly tyrosine phosphorylated in both normal and ts-src MDCK cells. The association of p120-related species with the E-cadherin complex adds them to our consideration of possible modulators of cadherin function. Likewise, the newly identified alpha-catenin-beta-catenin and alpha-catenin-plakoglobin dimers may have interesting biological properties, conceivably including the titration of catenins between cadherin and APC complexes.

  16. Aberrant expression of E-cadherin and β-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer.

    PubMed

    Kohan-Ghadr, H R; Smith, L C; Arnold, D R; Murphy, B D; Lefebvre, R C

    2012-01-01

    Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and β-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n = 6) and control pregnancies (n = 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or β-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total β-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-β-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/β-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and β-catenin proteins, along with defective β-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

  17. Fabrication of a Dual Substrate Display to Test Roles of Cell Adhesion Proteins in Vesicle Targeting to Plasma Membrane Domains

    PubMed Central

    Hunt, Stephen J.; Nelson, W. James

    2009-01-01

    While much is known of the molecular machinery involved in protein sorting during exocytosis, less is known about the spatial regulation of exocytosis at the plasma membrane (PM). This study outlines a novel method, Dual Substrate Display, used to formally test the hypothesis that E-cadherin-mediated adhesion directs basolateral vesicle exocytosis to specific sites at the PM. We show that vesicles containing the basolateral marker protein VSV-G preferentially target to sites of adhesion to E-cadherin rather than collagen VI or a control peptide. These results support the hypothesis that E-cadherin adhesion initiates signaling at the PM resulting in targeted sites for exocytosis. PMID:17803993

  18. Melanocyte migration is influenced by E-cadherin-dependent adhesion of keratinocytes in both two- and three-dimensional in vitro wound models.

    PubMed

    Keswell, Dheshnie; Kidson, Susan H; Davids, Lester M

    2015-02-01

    During wound healing, melanocytes are required to migrate into the wounded area that is still in the process of re-construction. The role and behaviour of melanocytes during this process is poorly understood, that is, whether melanocyte migration into the wound is keratinocyte-dependent or not. This paper attempts, through the use of both two- and three-dimensional in vitro models, to understand the role and behaviour of melanocytes during the process of wound healing. In addition, it sheds light on whether keratinocytes influence/contribute toward melanocyte migration and ultimately wound healing. Scratch assays were performed to analyse migration and Western blot analyses measured cellular E-cadherin expression. Immunohistochemistry was used to analyse the in vivo 3D wound healing effect. Scratch assays performed on co-cultures of melanocytes and keratinocytes demonstrated that melanocytes actively migrated, with the use of their dendrites, into the scratch ahead of the proliferating keratinocyte sheet. Migration of the melanocyte into the wound bed was accompanied by loss of attachment to keratinocytes at the wound front with concomitant downregulation of E-cadherin expression as observed through immunocytochemistry. This result suggests that, in vitro, melanocyte migration occurs independently of keratinocytes but that the migration is influenced by keratinocyte E-cadherin expression. We now demonstrate that melanocyte migration during re-pigmentation is an active process, and suggest that targeting of mechanisms involved in active melanocyte migration (e.g. the melanocyte dendrite) may enhance the re-pigmentation process.

  19. The Role of E-Cadherin in Maintaining the Barrier Function of Corneal Epithelium after Treatment with Cultured Autologous Oral Mucosa Epithelial Cell Sheet Grafts for Limbal Stem Deficiency

    PubMed Central

    Hoft, Richard H.; Wood, Andrew; Oliva, Joan; Niihara, Hope; Makalinao, Andrew; Thropay, Jacquelyn; Pan, Derek; Tiger, Kumar; Garcia, Julio; Laporte, Amanda; French, Samuel W.; Niihara, Yutaka

    2016-01-01

    The role of E-cadherin in epithelial barrier function of cultured autologous oral mucosa epithelial cell sheet (CAOMECS) grafts was examined. CAOMECS were cultured on a temperature-responsive surface and grafted onto rabbit corneas with Limbal Stem Cell Deficiency (LSCD). E-cadherin levels were significantly higher in CAOMECS compared to normal and LSCD epithelium. Beta-catenin colocalized with E-cadherin in CAOMECS cell membranes while phosphorylated beta-catenin was significantly increased. ZO-1, occludin, and Cnx43 were also strongly expressed in CAOMECS. E-cadherin and beta-catenin localization at the cell membrane was reduced in LSCD corneas, while CAOMECS-grafted corneas showed a restoration of E-cadherin and beta-catenin expression. LSCD corneas did not show continuous staining for ZO-1 or for Cnx43, while CAOMECS-grafted corneas showed a positive expression of ZO-1 and Cnx43. Cascade Blue® hydrazide did not pass through CAOMECS. Because E-cadherin interactions are calcium-dependent, EGTA was used to chelate calcium and disrupt cell adhesion. EGTA-treated CAOMECS completely detached from cell culture surface, and E-cadherin levels were significantly decreased. In conclusion, E cadherin high expression contributed to CAOMECS tight and gap junction protein recruitment at the cell membrane, thus promoting cellular adhesion and a functional barrier to protect the ocular surface. PMID:27777792

  20. Polycystin-1 and Gα12 regulate the cleavage of E-cadherin in kidney epithelial cells

    PubMed Central

    Xu, Jen X.; Lu, Tzong-Shi; Li, Suyan; Wu, Yong; Ding, Lai; Denker, Bradley M.; Bonventre, Joseph V.

    2014-01-01

    Interaction of polycystin-1 (PC1) and Gα12 is important for development of kidney cysts in autosomal dominant polycystic kidney disease (ADPKD). The integrity of cell polarity and cell-cell adhesions (mainly E-cadherin-mediated adherens junction) is altered in the renal epithelial cells of ADPKD. However, the key signaling pathway for this alteration is not fully understood. Madin-Darby canine kidney (MDCK) cells maintain the normal integrity of epithelial cell polarity and adherens junctions. Here, we found that deletion of Pkd1 increased activation of Gα12, which then promoted the cystogenesis of MDCK cells. The morphology of these cells was altered after the activation of Gα12. By using liquid chromatography-mass spectrometry, we found several proteins that could be related this change in the extracellular milieu. E-cadherin was one of the most abundant peptides after active Gα12 was induced. Gα12 activation or Pkd1 deletion increased the shedding of E-cadherin, which was mediated via increased ADAM10 activity. The increased shedding of E-cadherin was blocked by knockdown of ADAM10 or specific ADAM10 inhibitor GI254023X. Pkd1 deletion or Gα12 activation also changed the distribution of E-cadherin in kidney epithelial cells and caused β-catenin to shift from cell membrane to nucleus. Finally, ADAM10 inhibitor, GI254023X, blocked the cystogenesis induced by PC1 knockdown or Gα12 activation in renal epithelial cells. Our results demonstrate that the E-cadherin/β-catenin signaling pathway is regulated by PC1 and Gα12 via ADAM10. Specific inhibition of this pathway, especially ADAM10 activity, could be a novel therapeutic regimen for ADPKD. PMID:25492927

  1. E-cadherin expression in transitional cell carcinomas.

    PubMed

    Székely, Eszter; Török, Virág; Székely, Tamás; Riesz, Péter; Romics, Imre

    2006-01-01

    The authors analyzed the expression of E-cadherin, one of the most important cell adhesion molecules, on histological slides of tumors of bladder cancer patients. The aim of the study was to see whether there is any association between E-cadherin expression and tumor grade, stage, age and gender of the patients, number of recurrences, or overall survival. The samples were examined in 51 primary bladder transitional cell carcinomas (TCC) of 50 patients, resected by transurethral resection (TUR) between January 1, 1996 and January 1, 1997. Immunoreactions were performed with monoclonal anti-human E-cadherin antibody. Forty of the fifty patients could be clinically followed. The analysis of the results on these forty patients was performed by contingency analysis and significance was assessed by chi2 test. No significant association between E-cadherin expression and tumor grade, stage, age or gender of the patients, the number of recurrences, or overall survival could be seen.

  2. Diffuse Type Gastric and Lobular Breast Carcinoma in a Familial Gastric Cancer Patient with an E-Cadherin Germline Mutation

    PubMed Central

    Keller, Gisela; Vogelsang, Holger; Becker, Ingrid; Hutter, Jörg; Ott, Katja; Candidus, Sonja; Grundei, Tobias; Becker, Karl-Friedrich; Mueller, James; Siewert, Jörg R.; Höfler, Heinz

    1999-01-01

    E-Cadherin alterations have been reported frequently in sporadic diffuse type gastric and lobular breast carcinomas. Germline mutations of this gene have been identified recently in several gastric cancer families. We analyzed seven patients with a family history of the disease who had diffuse type gastric cancer diagnosed before the age of 45 for germline mutations in CDH1, the gene encoding the E-cadherin protein. We identified a frameshift mutation in exon 3 in one patient with a strong family history of gastric cancer. The same germline mutation was found in the patient’s mother, who had metachronous development of lobular breast and diffuse type gastric carcinomas. Immunohistochemistry for E-cadherin protein expression revealed an abnormal staining pattern in both of these tumors, suggesting complete inactivation of the cell adhesion molecule. Thus, our finding suggests that besides diffuse type gastric cancer, lobular breast carcinomas may be associated with germline CDH1 mutations. PMID:10433926

  3. Significance of the hedgehog pathway-associated proteins Gli-1 and Gli-2 and the epithelial-mesenchymal transition-associated proteins Twist and E-cadherin in hepatocellular carcinoma

    PubMed Central

    Chun, Hyung Wook; Hong, Ran

    2016-01-01

    It has been found that abnormal activation of the hedgehog (Hh) signaling pathway is involved in the occurrence, invasion and metastasis of malignant tumors. In addition, epithelial-mesenchymal transition (EMT) also performs an important function in the invasion and metastasis of malignant tumors. However, the significance of the Hh signaling pathway and EMT in hepatocellular carcinoma (HCC) remains unknown. In the present study, the expression of Gli family zinc finger 1 (Gli-1) and Gli family zinc finger 2 (Gli-2), which are key transcriptional factors in the Hh signaling pathway, and Twist and E-cadherin, which are two factors involved in EMT, was examined in 42 patients with HCC and 20 cases of non-tumorous liver (NTL) tissue by immunohistochemistry. Clinicopathological information was collected in order to analyze the correlation of the Hh signaling pathway with EMT. The present study aimed to examine the difference in the expression of Gli-1, Gli-2, E-cadherin and Twist in HCC and NTL to assess the diagnostic value of these factors in HCC. Additionally, the present study aimed to elucidate the correlation between those proteins and other clinicopathological parameters. Whether abnormal activation of the Hh signaling pathway is closely associated with EMT was also evaluated. Gli-1 and Twist expression was found to be significantly increased and E-cadherin expression was found to be decreased in HCC in contrast to NTL (Gli-1, P=0.019; Twist, P=0.003; E-cadherin, P<0.001). Increased Twist expression was associated with the tumor size (P=0.043), and loss of or decreased E-cadherin expression was associated with the histological type of HCC (P=0.021). There was an inverse association between the expression of Twist and E-cadherin (P=0.006). These results showed that Twist overexpression by induction of EMT changes is involved in the occurrence and progression of HCC. However, the role of Hh signaling pathway-associated proteins in HCC may require elucidation by

  4. Significance of the hedgehog pathway-associated proteins Gli-1 and Gli-2 and the epithelial-mesenchymal transition-associated proteins Twist and E-cadherin in hepatocellular carcinoma

    PubMed Central

    Chun, Hyung Wook; Hong, Ran

    2016-01-01

    It has been found that abnormal activation of the hedgehog (Hh) signaling pathway is involved in the occurrence, invasion and metastasis of malignant tumors. In addition, epithelial-mesenchymal transition (EMT) also performs an important function in the invasion and metastasis of malignant tumors. However, the significance of the Hh signaling pathway and EMT in hepatocellular carcinoma (HCC) remains unknown. In the present study, the expression of Gli family zinc finger 1 (Gli-1) and Gli family zinc finger 2 (Gli-2), which are key transcriptional factors in the Hh signaling pathway, and Twist and E-cadherin, which are two factors involved in EMT, was examined in 42 patients with HCC and 20 cases of non-tumorous liver (NTL) tissue by immunohistochemistry. Clinicopathological information was collected in order to analyze the correlation of the Hh signaling pathway with EMT. The present study aimed to examine the difference in the expression of Gli-1, Gli-2, E-cadherin and Twist in HCC and NTL to assess the diagnostic value of these factors in HCC. Additionally, the present study aimed to elucidate the correlation between those proteins and other clinicopathological parameters. Whether abnormal activation of the Hh signaling pathway is closely associated with EMT was also evaluated. Gli-1 and Twist expression was found to be significantly increased and E-cadherin expression was found to be decreased in HCC in contrast to NTL (Gli-1, P=0.019; Twist, P=0.003; E-cadherin, P<0.001). Increased Twist expression was associated with the tumor size (P=0.043), and loss of or decreased E-cadherin expression was associated with the histological type of HCC (P=0.021). There was an inverse association between the expression of Twist and E-cadherin (P=0.006). These results showed that Twist overexpression by induction of EMT changes is involved in the occurrence and progression of HCC. However, the role of Hh signaling pathway-associated proteins in HCC may require elucidation by

  5. Acute and chronic cadmium exposure promotes E-cadherin degradation in MCF7 breast cancer cells.

    PubMed

    Ponce, Esmeralda; Louie, Maggie C; Sevigny, Mary B

    2015-10-01

    Cadmium is an environmental carcinogen that usually enters the body at minute concentrations through diet or cigarette smoke and bioaccumulates in soft tissues. In past studies, cadmium has been shown to contribute to the development of more aggressive cancer phenotypes including increased cell migration and invasion. This study aims to determine if cadmium exposure-both acute and chronic-contributes to breast cancer progression by interfering with the normal functional relationship between E-cadherin and β-catenin. An MCF7 breast cancer cell line (MCF7-Cd) chronically exposed to 10(-7)  M CdCl2 was previously developed and used as a model system to study chronic exposures, whereas parental MCF7 cells exposed to 10(-6)  M CdCl2 for short periods of time were used to study acute exposures. Cadmium exposure of MCF7 cells led to the degradation of the E-cadherin protein via the ubiquitination pathway. This resulted in fewer E-cadherin/β-catenin complexes and the relocation of active β-catenin to the nucleus, where it interacted with transcription factor TCF-4 to modulate gene expression. Interestingly, only cells chronically exposed to cadmium showed a significant decrease in the localization of β-catenin to the plasma membrane and an increased distance between cells. Our data suggest that cadmium exposure promotes breast cancer progression by (1) down-regulating E-cadherin, thus decreasing the number of E-cadherin/β-catenin adhesion complexes, and (2) enhancing the nuclear translocation of β-catenin to increase expression of cancer-promoting proteins (i.e., c-Jun and cyclin D1).

  6. E-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components

    PubMed Central

    Kim, Nam-Gyun; Koh, Eunjin; Chen, Xiao; Gumbiner, Barry M.

    2011-01-01

    Contact inhibition of cell growth is essential for embryonic development and maintenance of tissue architecture in adult organisms, and the growth of tumors is characterized by a loss of contact inhibition of proliferation. The recently identified Hippo signaling pathway has been implicated in contact inhibition of proliferation as well as organ size control. The modulation of the phosphorylation and nuclear localization of Yes-associated protein (YAP) by the highly conserved kinase cascade of the Hippo signaling pathway has been intensively studied. However, cell-surface receptors regulating the Hippo signaling pathway in mammals are not well understood. In this study, we show that Hippo signaling pathway components are required for E-cadherin–dependent contact inhibition of proliferation. Knockdown of the Hippo signaling components or overexpression of YAP inhibits the decrease in cell proliferation caused by E-cadherin homophilic binding at the cell surface, independent of other cell–cell interactions. We also demonstrate that the E-cadherin/catenin complex functions as an upstream regulator of the Hippo signaling pathway in mammalian cells. Expression of E-cadherin in MDA-MB-231 cells restores the density-dependent regulation of YAP nuclear exclusion. Knockdown of β-catenin in densely cultured MCF10A cells, which mainly depletes E-cadherin–bound β-catenin, induces a decrease in the phosphorylation of S127 residue of YAP and its nuclear accumulation. Moreover, E-cadherin homophilic binding independent of other cell interactions is sufficient to control the subcellular localization of YAP. Therefore, Our results indicate that, in addition to its role in cell–cell adhesion, E-cadherin-mediated cell–cell contact directly regulates the Hippo signaling pathway to control cell proliferation. PMID:21730131

  7. Expression of e-cadherin, alpha-catenins and Beta-catenins in human gastric carcinomas - correlation with histology and tumor progression.

    PubMed

    Yasui, W; Kuniyasu, H; Akama, Y; Kitahara, K; Nagafuchi, A; Ishihara, S; Tsukita, S; Tahara, E

    1995-01-01

    The expression of cell-cell adhesion molecule, E-cadherin and its associated proteins, alpha- and beta-catenins in human gastric carcinomas was examined by Western blotting. All the seven gastric carcinoma cell lines expressed E-cadherin except KATOIII, which was derived from pleural effusion of a scirrhous type stomach cancer or Borrmann's type-4 carcinoma. The expression of alpha-catenin was not detected in HSC43 derived from scirrhous carcinoma, while HSC39 expressed abnormal beta-catenin caused by genetic alteration. In gastric carcinoma cases, the levels of E-cadherin and alpha-catenin were significantly lower in poorly differentiated adenocarcinomas and scirrhous carcinomas when compared to other types of gastric carcinomas. Deeply invasive carcinomas expressed E-cadherin and alpha-catenin at lower levels. However, the expression level of alpha-catenin was not necessarily consistent with that of E-cadherin. One of 10 gastric carcinomas examined showed complete deletion of alpha-catenin gene in Southern blotting. beta-catenin was expressed at lower level in poorly differentiated adenocarcinomas than in well-differentiated adenocarcinomas. These findings suggest that reduction in the expression of E-cadherin and its associated molecules, catenins, is involved in the development and infiltrative growth of scirrhous type gastric carcinomas. PMID:21597700

  8. Reggies/flotillins regulate E-cadherin-mediated cell contact formation by affecting EGFR trafficking.

    PubMed

    Solis, Gonzalo P; Schrock, Yvonne; Hülsbusch, Nikola; Wiechers, Marianne; Plattner, Helmut; Stuermer, Claudia A O

    2012-05-01

    The reggie/flotillin proteins are implicated in membrane trafficking and, together with the cellular prion protein (PrP), in the recruitment of E-cadherin to cell contact sites. Here, we demonstrate that reggies, as well as PrP down-regulation, in epithelial A431 cells cause overlapping processes and abnormal formation of adherens junctions (AJs). This defect in cell adhesion results from reggie effects on Src tyrosine kinases and epidermal growth factor receptor (EGFR): loss of reggies reduces Src activation and EGFR phosphorylation at residues targeted by Src and c-cbl and leads to increased surface exposure of EGFR by blocking its internalization. The prolonged EGFR signaling at the plasma membrane enhances cell motility and macropinocytosis, by which junction-associated E-cadherin is internalized and recycled back to AJs. Accordingly, blockage of EGFR signaling or macropinocytosis in reggie-deficient cells restores normal AJ formation. Thus, by promoting EGFR internalization, reggies restrict the EGFR signaling involved in E-cadherin macropinocytosis and recycling and regulate AJ formation and dynamics and thereby cell adhesion.

  9. Enhanced G2/M Arrest, Caspase Related Apoptosis and Reduced E-Cadherin Dependent Intercellular Adhesion by Trabectedin in Prostate Cancer Stem Cells

    PubMed Central

    Uslu, Ruchan; Kara, Mikail; Soner, Burak Cem; Oktem, Gulperi

    2015-01-01

    Trabectedin (Yondelis, ET-743) is a marine-derived tetrahydroisoquinoline alkaloid. It is originally derived from the Caribbean marine tunicate Ecteinascidia turbinata and currently produced synthetically. Trabectedin is active against a variety of tumor cell lines growing in culture. The present study focused on the effect of trabectedin in cell proliferation, cell cycle progression, apoptosis and spheroid formation in prostate cancer stem cells (CSCs). Cluster of differentiation (CD) 133+high/CD44+high prostate CSCs were isolated from the DU145 and PC-3 human prostate cancer cell line through flow cytometry. We studied the growth-inhibitory effects of trabectedin and its molecular mechanisms on human prostate CSCs and non-CSCs. DU-145 and PC-3 CSCs were treated with 0.1, 1, 10 and 100 nM trabectedin for 24, 48 and 72 h and the growth inhibition rates were examined using the sphere-forming assay. Annexin-V assay and immunofluorescence analyses were performed for the detection of the cell death. Concentration-dependent effects of trabectedin on the cell cycle were also evaluated. The cells were exposed to the different doses of trabectedin for 24, 48 and 72 h to evaluate the effect of trabectedin on the number and diameter of spheroids. According to the results, trabectedin induced cytotoxicity and apoptosis at the IC50 dose, resulting in a significant increase expression of caspase-3, caspase-8, caspase-9, p53 and decrease expression of bcl-2 in dose-dependent manner. Cell cycle analyses revealed that trabectedin induces dose-dependent G2/M-phase cell cycle arrest, particularly at high-dose treatments. Three-dimensional culture studies showed that trabectedin reduced the number and diameter of spheroids of DU145 and PC3 CSCs. Furthermore, we have found that trabectedin disrupted cell-cell interactions via E-cadherin in prostasphere of DU-145 and PC-3 CSCs. Our results showed that trabectedin inhibits cellular proliferation and accelerates apoptotic events in

  10. Enhanced G2/M Arrest, Caspase Related Apoptosis and Reduced E-Cadherin Dependent Intercellular Adhesion by Trabectedin in Prostate Cancer Stem Cells.

    PubMed

    Acikgoz, Eda; Guven, Ummu; Duzagac, Fahriye; Uslu, Ruchan; Kara, Mikail; Soner, Burak Cem; Oktem, Gulperi

    2015-01-01

    Trabectedin (Yondelis, ET-743) is a marine-derived tetrahydroisoquinoline alkaloid. It is originally derived from the Caribbean marine tunicate Ecteinascidia turbinata and currently produced synthetically. Trabectedin is active against a variety of tumor cell lines growing in culture. The present study focused on the effect of trabectedin in cell proliferation, cell cycle progression, apoptosis and spheroid formation in prostate cancer stem cells (CSCs). Cluster of differentiation (CD) 133+high/CD44+high prostate CSCs were isolated from the DU145 and PC-3 human prostate cancer cell line through flow cytometry. We studied the growth-inhibitory effects of trabectedin and its molecular mechanisms on human prostate CSCs and non-CSCs. DU-145 and PC-3 CSCs were treated with 0.1, 1, 10 and 100 nM trabectedin for 24, 48 and 72 h and the growth inhibition rates were examined using the sphere-forming assay. Annexin-V assay and immunofluorescence analyses were performed for the detection of the cell death. Concentration-dependent effects of trabectedin on the cell cycle were also evaluated. The cells were exposed to the different doses of trabectedin for 24, 48 and 72 h to evaluate the effect of trabectedin on the number and diameter of spheroids. According to the results, trabectedin induced cytotoxicity and apoptosis at the IC50 dose, resulting in a significant increase expression of caspase-3, caspase-8, caspase-9, p53 and decrease expression of bcl-2 in dose-dependent manner. Cell cycle analyses revealed that trabectedin induces dose-dependent G2/M-phase cell cycle arrest, particularly at high-dose treatments. Three-dimensional culture studies showed that trabectedin reduced the number and diameter of spheroids of DU145 and PC3 CSCs. Furthermore, we have found that trabectedin disrupted cell-cell interactions via E-cadherin in prostasphere of DU-145 and PC-3 CSCs. Our results showed that trabectedin inhibits cellular proliferation and accelerates apoptotic events in

  11. Letter to the Editor: Human Pluripotent Stem Cells Release Oncogenic Soluble E-Cadherin.

    PubMed

    Rosner, Margit; Hengstschläger, Markus

    2016-09-01

    Since their discovery, human pluripotent stem cells (hPSCs) including embryonic and induced pluripotent stem cells hold great promise in disease modeling and regenerative medicine. Despite intensive research and remarkable progress, it is becoming increasingly acknowledged that their yet incomplete, biological characterisation represents one of the major drawbacks to their successful translation into the clinics. The expression of the transmembrane protein E-cadherin in hPSCs is well defined to be pivotal to the maintenance of the pluripotent state by mediating intercellular adhesion and intracellular signaling. Next to these canonical functions, were here report for the first time that hPSCs are subject to matrix metalloproteinase-dependent E-cadherin ectodomain shedding. This generates a ∼80-kD, soluble E-cadherin fragment which is released into the extracellular space, and which is well described to exert paracrine signaling activity and classified as being oncogenic. Collectively, this finding does not only improve our knowledge on the signaling crosstalk between hPSCs and their cellular environment and the type and nature of the paracrine signals produced by these cells, but also has clear implications for the development of efficient and safe stem cell-based therapies. Stem Cells 2016;34:2443-2446. PMID:27399873

  12. Dehydropeptidase 1 promotes metastasis through regulation of E-cadherin expression in colon cancer

    PubMed Central

    Park, Sang Yoon; Lee, Seon-Jin; Cho, Hee Jun; Kim, Tae Woo; Kim, Jong-Tae; Kim, Jae Wha; Lee, Chul-Ho; Kim, Bo-Yeon; Yeom, Young Il; Lim, Jong-Seok; Lee, Younghee; Lee, Hee Gu

    2016-01-01

    Dehydropeptidase 1 (DPEP1) is a zinc-dependent metalloproteinase that is expressed aberrantly in several cancers. The role of DPEP1 in cancer remain controversial. In this study, we demonstrate that DPEP1 functions as a positive regulator for colon cancer cell metastasis. The expression of DPEP1 mRNA and proteins were upregulated in colon cancer tissues compared to normal mucosa. Gain-of-function and loss-of-function approaches were used to examine the malignant phenotype of DPEP1-expressing or DPEP1-depleted cells. DPEP1 expression caused a significant increase in colon cancer cell adhesion and invasion in vitro, and metastasis in vivo. In contrast, DPEP1 depletion induced opposite effects. Furthermore, cilastatin, a DPEP1 inhibitor, suppressed the invasion and metastasis of DPEP1-expressing cells. DPEP1 inhibited the leukotriene D4 signaling pathway and increased the expression of E-cadherin. We also show that DPEP1 mediates TGF-β-induced EMT. TGF-β transcriptionally repressed DPEP1 expression. TGF-β treatment decreased E-cadherin expression and promoted cell invasion in DPEP1-expressing colon cancer cell lines, whereas it did not affect these parameters in DPEP1-depleted cell lines. These results suggest that DPEP1 promotes cancer metastasis by regulating E-cadherin plasticity and that it might be a potential therapeutic target for preventing the progression of colon cancer. PMID:26824987

  13. Three mechanisms control E-cadherin localization to the zonula adherens.

    PubMed

    Woichansky, Innokenty; Beretta, Carlo Antonio; Berns, Nicola; Riechmann, Veit

    2016-01-01

    E-cadherin localization to the zonula adherens is fundamental for epithelial differentiation but the mechanisms controlling localization are unclear. Using the Drosophila follicular epithelium we genetically dissect E-cadherin transport in an in vivo model. We distinguish three mechanisms mediating E-cadherin accumulation at the zonula adherens. Two membrane trafficking pathways deliver newly synthesized E-cadherin to the plasma membrane. One is Rab11 dependent and targets E-cadherin directly to the zonula adherens, while the other transports E-cadherin to the lateral membrane. Lateral E-cadherin reaches the zonula adherens by endocytosis and targeted recycling. We show that this pathway is dependent on RabX1, which provides a functional link between early and recycling endosomes. Moreover, we show that lateral E-cadherin is transported to the zonula adherens by an apically directed flow within the plasma membrane. Differential activation of these pathways could facilitate cell shape changes during morphogenesis, while their misregulation compromises cell adhesion and tissue architecture in differentiated epithelia. PMID:26960923

  14. 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.

  15. Wheat proteins enhance stability and function of adhesion molecules in cryopreserved hepatocytes.

    PubMed

    Grondin, Mélanie; Hamel, Francine; Averill-Bates, Diana A; Sarhan, Fathey

    2009-01-01

    Cryopreserved hepatocytes with good hepatospecific functions upon thawing are important for clinical transplantation and for in vitro drug toxicity testing. However, cryopreservation reduces viability and certain hepatospecific functions, but the most pronounced change is diminished attachment efficiency of hepatocytes. Adhesion of cells to the extracellular matrix and cell-cell contacts are crucial for many aspects of cellular function. These processes are partly mediated and controlled by cellular adhesion molecules. The mechanisms responsible for reduced attachment efficiency of cryopreserved hepatocytes are not well understood. To address this question, we investigated the effect of a new cryopreservation procedure, using wheat proteins (WPs) or mixtures of recombinant forms of wheat freezing tolerance-associated proteins, on the stability of three important adhesion molecules (beta1-integrin, E-cadherin, and beta-catenin). Immunoblot analyses revealed that the levels of beta1-integrin, E-cadherin, and beta-catenin were much lower in cryopreserved rat hepatocytes, when compared to fresh cells. Protein expression of the adhesion molecules was generally lower in cells cryopreserved with DMSO, compared to WPs. Moreover, the stability of the adhesion molecules was not affected by cryopreservation to the same degree, with more pronounced decreases occurring for beta1-integrin (62-74%) > beta-catenin (51-58%) > E-cadherin (21-37%). However, when hepatocytes were cryopreserved with partially purified WPs (SulWPE, AcWPE) or with mixtures of recombinant wheat proteins, there was a clear protective effect against the loss of protein expression of beta1-integrin, E-cadherin, and beta-catenin. Protein expression was only 10-20% lower than that observed in fresh hepatocytes. These findings clearly demonstrate that WPs, and more particularly, partially purified WPs and recombinant wheat proteins, were more efficient for cryopreservation of rat hepatocytes by maintaining good

  16. Sustained α-catenin Activation at E-cadherin Junctions in the Absence of Mechanical Force.

    PubMed

    Biswas, Kabir H; Hartman, Kevin L; Zaidel-Bar, Ronen; Groves, Jay T

    2016-09-01

    Mechanotransduction at E-cadherin junctions has been postulated to be mediated in part by a force-dependent conformational activation of α-catenin. Activation of α-catenin allows it to interact with vinculin in addition to F-actin, resulting in a strengthening of junctions. Here, using E-cadherin adhesions reconstituted on synthetic, nanopatterned membranes, we show that activation of α-catenin is dependent on E-cadherin clustering, and is sustained in the absence of mechanical force or association with F-actin or vinculin. Adhesions were formed by filopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters, which could be distinguished as either peripheral or central assemblies depending on their relative location at the cell-bilayer adhesion. Whereas F-actin, vinculin, and phosphorylated myosin light chain associated only with the peripheral assemblies, activated α-catenin was present in both peripheral and central assemblies, and persisted in the central assemblies in the absence of actomyosin tension. Impeding filopodia-mediated nucleation and micron-scale assembly of E-cadherin adhesion complexes by confining the movement of bilayer-bound E-cadherin on nanopatterned substrates reduced the levels of activated α-catenin. Taken together, these results indicate that although the initial activation of α-catenin requires micron-scale clustering that may allow the development of mechanical forces, sustained force is not required for maintaining α-catenin in the active state. PMID:27602732

  17. Slug-upregulated miR-221 promotes breast cancer progression through suppressing E-cadherin expression

    PubMed Central

    Pan, Yi; Li, Jing; Zhang, Yaqin; Wang, Nan; Liang, Hongwei; Liu, Yuan; Zhang, Chen-Yu; Zen, Ke; Gu, Hongwei

    2016-01-01

    It is generally regarded that E-cadherin is downregulated during tumorigenesis via Snail/Slug-mediated E-cadherin transcriptional reduction. However, this transcriptional suppressive mechanism cannot explain the failure of producing E-cadherin protein in metastatic breast cancer cells after overexpressing E-cadherin mRNA. Here we reveal a novel mechanism that E-cadherin is post-transcriptionally regulated by Slug-promoted miR-221, which serves as an additional blocker for E-cadherin expression in metastatic tumor cells. Profiling the predicted E-cadherin-targeting miRNAs in breast cancer tissues and cells showed that miR-221 was abundantly expressed in breast tumor and metastatic MDA-MB-231 cells and its level was significantly higher in breast tumor or MDA-MB-231 cells than in distal non-tumor tissue and low-metastatic MCF-7 cells, respectively. MiR-221, which level inversely correlated with E-cadherin level in breast cancer cells, targeted E-cadherin mRNA open reading frame (ORF) and suppressed E-cadherin protein expression. Depleting or increasing miR-221 level in breast cancer cells induced or decreased E-cadherin protein level, leading to suppressing or promoting tumor cell progression, respectively. Moreover, miR-221 was specifically upregulated by Slug but not Snail. TGF-β treatment enhanced Slug activity and thus increased miR-221 level in MCF-7 cells. In summary, our results provide the first evidence that Slug-upregulated miR-221 promotes breast cancer progression via reducing E-cadherin expression. PMID:27174021

  18. Regulation of E-cadherin: does hypoxia initiate the metastatic cascade?

    PubMed Central

    Beavon, I R

    1999-01-01

    The ability of tumours to metastasis is regarded as one of the hallmarks of malignancy. The process through which tumours evolve to achieve this has been termed the metastatic cascade. This cascade has been the subject of much investigation over many years. One of the vital events identified by these investigations is the reduction of adhesion between tumour cells facilitating invasion of the surrounding tissues and vascular channels, ultimately leading to the development of a distant metastasis. E-cadherin and its associated catenin complex have been identified as key molecules in cell adhesion. This review looks at the structure and interaction of the E-cadherin-catenin complex and the factors that appear to regulate E-cadherin expression and thus cell adhesion. From the data gathered, it has become possible to propose the hypothesis that the development of tumour hypoxia is the initiating factor that sets the tumour on the road to metastasis. PMID:10694937

  19. Epithelial cell-derived periostin functions as a tumor suppressor in gastric cancer through stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14ARF/Mdm2 signaling pathway.

    PubMed

    Lv, Hongjun; Liu, Rui; Fu, Jiao; Yang, Qi; Shi, Jing; Chen, Pu; Ji, Meiju; Shi, Bingyin; Hou, Peng

    2014-01-01

    Periostin is usually considered as an oncogene in diverse human cancers, including breast, prostate, colon, esophagus, and pancreas cancers, whereas it acts as a tumor suppressor in bladder cancer. In gastric cancer, it has been demonstrated that periglandular periostin expression is decreased whereas stromal periostin expression is significantly increased as compared with normal gastric tissues. Moreover, periostin produced by stromal myofibroblasts markedly promotes gastric cancer cell growth. These observations suggest that periostin derived from different types of cells may play distinct biological roles in gastric tumorigenesis. The aim of this study was to explore the biological functions and related molecular mechanisms of epithelial cell-derived periostin in gastric cancer. Our data showed that periglandular periostin was significantly down-regulated in gastric cancer tissues as compared with matched normal gastric mucosa. In addition, its expression in metastatic lymph nodes was significantly lower than that in their primary cancer tissues. Our data also demonstrated that periglandular periostin expression was negatively associated with tumor stage. More importantly, restoration of periostin expression in gastric cancer cells dramatically suppressed cell growth and invasiveness. Elucidation of the mechanisms involved revealed that periostin restoration enhanced Rb phosphorylation and sequentially activated the transcription of E2F1 target gene p14(ARF), leading to Mdm2 inactivation and the stabilization of p53 and E-cadherin proteins. Strikingly, these effects of periostin were abolished upon Rb deletion. Collectively, we have for the first time demonstrated that epithelial cell-derived periostin exerts tumor-suppressor activities in gastric cancer through stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14(ARF)/Mdm2 signaling pathway. PMID:25486483

  20. E-cadherin is required for cranial neural crest migration in Xenopus laevis.

    PubMed

    Huang, Chaolie; Kratzer, Marie-Claire; Wedlich, Doris; Kashef, Jubin

    2016-03-15

    The cranial neural crest (CNC) is a highly motile and multipotent embryonic cell population, which migrates directionally on defined routes throughout the embryo, contributing to facial structures including cartilage, bone and ganglia. Cadherin-mediated cell-cell adhesion is known to play a crucial role in the directional migration of CNC cells. However, migrating CNC co-express different cadherin subtypes, and their individual roles have yet to be fully explored. In previous studies, the expression of individual cadherin subtypes has been analysed using different methods with varying sensitivities, preventing the direct comparison of expression levels. Here, we provide the first comprehensive and comparative analysis of the expression of six cadherin superfamily members during different phases of CNC cell migration in Xenopus. By applying a quantitative RT-qPCR approach, we can determine the copy number and abundance of each expressed cadherin through different phases of CNC migration. Using this approach, we show for the first time expression of E-cadherin and XB/C-cadherin in CNC cells, adding them as two new members of cadherins co-expressed during CNC migration. Cadherin co-expression during CNC migration in Xenopus, in particular the constant expression of E-cadherin, contradicts the classical epithelial-mesenchymal transition (EMT) model postulating a switch in cadherin expression. Loss-of-function experiments further show that E-cadherin is required for proper CNC cell migration in vivo and also for cell protrusion formation in vitro. Knockdown of E-cadherin is not rescued by co-injection of other classical cadherins, pointing to a specific function of E-cadherin in mediating CNC cell migration. Finally, through reconstitution experiments with different E-cadherin deletion mutants in E-cadherin morphant embryos, we demonstrate that the extracellular domain, but not the cytoplasmic domain, of E-cadherin is sufficient to rescue CNC cell migration in vivo.

  1. E-cadherin is required for cranial neural crest migration in Xenopus laevis.

    PubMed

    Huang, Chaolie; Kratzer, Marie-Claire; Wedlich, Doris; Kashef, Jubin

    2016-03-15

    The cranial neural crest (CNC) is a highly motile and multipotent embryonic cell population, which migrates directionally on defined routes throughout the embryo, contributing to facial structures including cartilage, bone and ganglia. Cadherin-mediated cell-cell adhesion is known to play a crucial role in the directional migration of CNC cells. However, migrating CNC co-express different cadherin subtypes, and their individual roles have yet to be fully explored. In previous studies, the expression of individual cadherin subtypes has been analysed using different methods with varying sensitivities, preventing the direct comparison of expression levels. Here, we provide the first comprehensive and comparative analysis of the expression of six cadherin superfamily members during different phases of CNC cell migration in Xenopus. By applying a quantitative RT-qPCR approach, we can determine the copy number and abundance of each expressed cadherin through different phases of CNC migration. Using this approach, we show for the first time expression of E-cadherin and XB/C-cadherin in CNC cells, adding them as two new members of cadherins co-expressed during CNC migration. Cadherin co-expression during CNC migration in Xenopus, in particular the constant expression of E-cadherin, contradicts the classical epithelial-mesenchymal transition (EMT) model postulating a switch in cadherin expression. Loss-of-function experiments further show that E-cadherin is required for proper CNC cell migration in vivo and also for cell protrusion formation in vitro. Knockdown of E-cadherin is not rescued by co-injection of other classical cadherins, pointing to a specific function of E-cadherin in mediating CNC cell migration. Finally, through reconstitution experiments with different E-cadherin deletion mutants in E-cadherin morphant embryos, we demonstrate that the extracellular domain, but not the cytoplasmic domain, of E-cadherin is sufficient to rescue CNC cell migration in vivo

  2. A protein interaction map for cell-cell adhesion regulators identifies DUSP23 as a novel phosphatase for β-catenin

    PubMed Central

    Gallegos, Lisa Leon; Ng, Mei Rosa; Sowa, Mathew E.; Selfors, Laura M.; White, Anne; Zervantonakis, Ioannis K.; Singh, Pragya; Dhakal, Sabin; Harper, J. Wade; Brugge, Joan S.

    2016-01-01

    Cell-cell adhesion is central to morphogenesis and maintenance of epithelial cell state. We previously identified 27 candidate cell-cell adhesion regulatory proteins (CCARPs) whose down-regulation disrupts epithelial cell-cell adhesion during collective migration. Using a protein interaction mapping strategy, we found that 18 CCARPs link to core components of adherens junctions or desmosomes. We further mapped linkages between the CCARPs and other known cell-cell adhesion proteins, including hits from recent screens uncovering novel components of E-cadherin adhesions. Mechanistic studies of one novel CCARP which links to multiple cell-cell adhesion proteins, the phosphatase DUSP23, revealed that it promotes dephosphorylation of β-catenin at Tyr 142 and enhances the interaction between α- and β-catenin. DUSP23 knockdown specifically diminished adhesion to E-cadherin without altering adhesion to fibronectin matrix proteins. Furthermore, DUSP23 knockdown produced “zipper-like” cell-cell adhesions, caused defects in transmission of polarization cues, and reduced coordination during collective migration. Thus, this study identifies multiple novel connections between proteins that regulate cell-cell interactions and provides evidence for a previously unrecognized role for DUSP23 in regulating E-cadherin adherens junctions through promoting the dephosphorylation of β-catenin. PMID:27255161

  3. Intestinal trefoil factor controls the expression of the adenomatous polyposis coli-catenin and the E-cadherin-catenin complexes in human colon carcinoma cells.

    PubMed

    Efstathiou, J A; Noda, M; Rowan, A; Dixon, C; Chinery, R; Jawhari, A; Hattori, T; Wright, N A; Bodmer, W F; Pignatelli, M

    1998-03-17

    Intestinal trefoil factor 3 (TFF3) is a member of the trefoil family of peptides, small molecules constitutively expressed in epithelial tissues, including the gastrointestinal tract. TFF3 has been shown to promote migration of intestinal epithelial cells in vitro and to enhance mucosal healing and epithelial restitution in vivo. In this study, we evaluated the effect of recombinant TFF3 (rTFF3) stimulation on the expression and cellular localization of the epithelial (E)-cadherin-catenin complex, a prime mediator of Ca2+ dependent cell-cell adhesion, and the adenomatous polyposis coli (APC)-catenin complex in HT29, HCT116, and SW480 colorectal carcinoma cell lines. Stimulation by rTFF3 (10(-9) M and 10(-8) M) for 20-24 hr led to cell detachment and to a reduction in intercellular adhesion in HT29 and HCT116 cells. In both cell lines, E-cadherin expression was down-regulated. The expression of APC, alpha-catenin and beta-catenin also was decreased in HT29 cells, with a translocation of APC into the nucleus. No change in either cell adhesion or in the expression of E-cadherin, the catenins, and APC was detected in SW480 cells. In addition, TFF3 induced DNA fragmentation and morphological changes characteristic of apoptosis in HT29. Tyrphostin, a competitive inhibitor of protein tyrosine kinases, inhibited the effects of TFF3. Our results indicate that by perturbing the complexes between E-cadherin, beta-catenin, and associated proteins, TFF3 may modulate epithelial cell adhesion, migration, and survival.

  4. Octamer-binding protein 4 affects the cell biology and phenotypic transition of lung cancer cells involving β-catenin/E-cadherin complex degradation.

    PubMed

    Chen, Zhong-Shu; Ling, Dong-Jin; Zhang, Yang-De; Feng, Jian-Xiong; Zhang, Xue-Yu; Shi, Tian-Sheng

    2015-03-01

    Clinical studies have reported evidence for the involvement of octamer‑binding protein 4 (Oct4) in the tumorigenicity and progression of lung cancer; however, the role of Oct4 in lung cancer cell biology in vitro and its mechanism of action remain to be elucidated. Mortality among lung cancer patients is more frequently due to metastasis rather than their primary tumors. Epithelial‑mesenchymal transition (EMT) is a prominent biological event for the induction of epithelial cancer metastasis. The aim of the present study was to investigate whether Oct4 had the capacity to induce lung cancer cell metastasis via the promoting the EMT in vitro. Moreover, the effect of Oct4 on the β‑catenin/E‑cadherin complex, associated with EMT, was examined using immunofluorescence and immunoprecipitation assays as well as western blot analysis. The results demonstrated that Oct4 enhanced cell invasion and adhesion accompanied by the downregulation of epithelial marker cytokeratin, and upregulation of the mesenchymal markers vimentin and N‑cadherin. Furthermore, Oct4 induced EMT of lung cancer cells by promoting β‑catenin/E‑cadherin complex degradation and regulating nuclear localization of β‑catenin. In conclusion, the present study indicated that Oct4 affected the cell biology of lung cancer cells in vitro through promoting lung cancer cell metastasis via EMT; in addition, the results suggested that the association and degradation of the β‑catenin/E‑cadherin complex was regulated by Oct4 during the process of EMT.

  5. CUGBP1 and HuR regulate E-cadherin translation by altering recruitment of E-cadherin mRNA to processing bodies and modulate epithelial barrier function.

    PubMed

    Yu, Ting-Xi; Gu, Bei-Lin; Yan, Jun-Kai; Zhu, Jie; Yan, Wei-Hui; Chen, Jie; Qian, Lin-Xi; Cai, Wei

    2016-01-01

    The effectiveness and stability of epithelial barrier depend on apical junctional complexes, which consist of tight junctions (TJs) and adherens junctions (AJs). E-cadherin is the primary component of AJs, and it is essential for maintenance of cell-to-cell interactions and regulates the epithelial barrier. However, the exact mechanism underlying E-cadherin expression, particularly at the posttranscriptional level, remains largely unknown. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HU antigen R (HuR) are highly expressed in the intestinal epithelial tissues and modulate the stability and translation of target mRNAs. Here, we present evidence that CUGBP1 and HuR interact directly with the 3'-untranslated region of E-cadherin mRNA and regulate E-cadherin translation. CUGBP1 overexpression in Caco-2 cells inhibited E-cadherin translation by increasing the recruitment of E-cadherin mRNA to processing bodies (PBs), thus resulting in an increase in paracellular permeability. Overexpression of HuR exhibited an opposite effect on E-cadherin expression by preventing the translocation of E-cadherin mRNA to PBs and therefore prevented CUGBP1-induced repression of E-cadherin expression. Elevation of HuR also abolished the CUGBP1-induced epithelial barrier dysfunction. These findings indicate that CUGBP1 and HuR negate each other's effects in regulating E-cadherin translation by altering the recruitment of E-cadherin mRNA to PBs and play an important role in the regulation of intestinal barrier integrity under various pathophysiological conditions.

  6. Drosophila E-cadherin is required for the maintenance of ring canals anchoring to mechanically withstand tissue growth

    PubMed Central

    Loyer, Nicolas; Kolotuev, Irina; Pinot, Mathieu; Le Borgne, Roland

    2015-01-01

    Intercellular bridges called “ring canals” (RCs) resulting from incomplete cytokinesis play an essential role in intercellular communication in somatic and germinal tissues. During Drosophila oogenesis, RCs connect the maturing oocyte to nurse cells supporting its growth. Despite numerous genetic screens aimed at identifying genes involved in RC biogenesis and maturation, how RCs anchor to the plasma membrane (PM) throughout development remains unexplained. In this study, we report that the clathrin adaptor protein 1 (AP-1) complex, although dispensable for the biogenesis of RCs, is required for the maintenance of the anchorage of RCs to the PM to withstand the increased membrane tension associated with the exponential tissue growth at the onset of vitellogenesis. Here we unravel the mechanisms by which AP-1 enables the maintenance of RCs’ anchoring to the PM during size expansion. We show that AP-1 regulates the localization of the intercellular adhesion molecule E-cadherin and that loss of AP-1 causes the disappearance of the E-cadherin–containing adhesive clusters surrounding the RCs. E-cadherin itself is shown to be required for the maintenance of the RCs’ anchorage, a function previously unrecognized because of functional compensation by N-cadherin. Scanning block-face EM combined with transmission EM analyses reveals the presence of interdigitated, actin- and Moesin-positive, microvilli-like structures wrapping the RCs. Thus, by modulating E-cadherin trafficking, we show that the sustained E-cadherin–dependent adhesion organizes the microvilli meshwork and ensures the proper attachment of RCs to the PM, thereby counteracting the increasing membrane tension induced by exponential tissue growth. PMID:26424451

  7. TLE1 promotes EMT in A549 lung cancer cells through suppression of E-cadherin

    PubMed Central

    Yao, Xin; Ireland, Shubha Kale; Pham, Tri; Temple, Brandi; Chen, Renwei; Raj, Madhwa HG; Biliran, Hector

    2014-01-01

    The Groucho transcriptional corepressor TLE1 protein has recently been shown to be a putative lung specific oncogene, but its underlying oncogenic activity in lung cancer has not been fully elucidated. In this report, we investigated whether TLE1 regulates lung cancer aggressiveness using the human lung adenocarcinoma cell line A549 as a model system. Through a combination of genetic approaches, we found that TLE1 potentiates Epithelial-to-Mesenchymal Transition (EMT) in A549 cells in part through suppression of the tumor suppressor gene E-cadherin. Exogenous expression of TLE1 in A549 cells resulted in heightened EMT phenotypes (enhanced fibroblastoid morphology and increased cell migratory potential) and in molecular alterations characteristic of EMT (downregulation of the epithelial marker E-cadherin and upregulation of the mesenchymal marker Vimentin). Conversely, downregulation of endogenous TLE1 expression in these cells resulted in reversal of basal EMT characterized by a cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Mechanistic studies showed that TLE1 suppresses E-cadherin expression at the transcriptional level in part by recruiting Histone Deacetylase (HDAC) activity to the E-cadherin promoter. Consistently, the HDAC inhibitor TSA partially reversed the TLE1-induced E-cadherin downregulation and cell migration, suggesting a role for HDACs in TLE1-mediated transcriptional repression of E-cadherin and EMT function. These findings uncover a novel role of TLE1 in regulating EMT in A549 cells through its repressive effect on E-cadherin and provide a mechanism for TLE1 oncogenic activity in lung cancer. PMID:25446087

  8. PI3K/AKT pathway regulates E-cadherin and Desmoglein 2 in aggressive prostate cancer

    PubMed Central

    Barber, Alison G; Castillo-Martin, Mireia; Bonal, Dennis M; Jia, Angela J; Rybicki, Benjamin A; Christiano, Angela M; Cordon-Cardo, Carlos

    2015-01-01

    Reduced expression of both classical and desmosomal cadherins has been associated with different types of carcinomas, including prostate cancer. This study aims to provide a comprehensive view of the role and regulation of cell–cell adhesion in prostate cancer aggressiveness by examining the functional implications of both E-cadherin and Desmoglein 2 (DSG2). E-cadherin expression was first examined using immunofluorescence in 50 normal prostate tissues and in a cohort of 414 prostate cancer patients. Correlation and survival analyses were performed to assess its clinical significance. In primary prostate cancer patients, reduced expression of both E-cadherin and DSG2 is significantly associated with an earlier biochemical recurrence. Transgenic DU145 E-cadherin knockdown and constitutively active AKT overexpression lines were generated. Functional implications of such genetic alterations were analyzed in vitro and in vivo, the latter by using tumorigenesis as well as extravasation and metastatic tumor formation assays. We observed that loss of E-cadherin leads to impaired primary and metastatic tumor formation in vivo, suggesting a tumor promoter role for E-cadherin in addition to its known role as a tumor suppressor. Activation of AKT leads to a significant reduction in E-cadherin expression and nuclear localization of Snail, suggesting a role for the PI3K/AKT signaling pathway in the transient repression of E-cadherin. This reduced expression may be regulated by separate mechanisms as neither the loss of E-cadherin nor activation of AKT significantly affected DSG2 expression. In conclusion, these findings illustrate the critical role of cell–cell adhesion in the progression to aggressive prostate cancer, through regulation by the PI3K pathway. PMID:26033689

  9. Suppression of E-cadherin Mediates Gallotannin Induced Apoptosis in Hep G2 Hepatocelluar Carcinoma Cells

    PubMed Central

    Han, Hee Jeong; Kwon, Hee Young; Sohn, Eun Jung; Ko, Hyunsuk; Kim, Bogeun; Jung, Kwon; Lew, Jae Hwan; Kim, Sung-Hoon

    2014-01-01

    Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin β1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells. PMID:24795530

  10. E-cadherin determines Caveolin-1 tumor suppression or metastasis enhancing function in melanoma cells.

    PubMed

    Lobos-González, Lorena; Aguilar, Lorena; Diaz, Jorge; Diaz, Natalia; Urra, Hery; Torres, Vicente A; Silva, Veronica; Fitzpatrick, Christopher; Lladser, Alvaro; Hoek, Keith S; Leyton, Lisette; Quest, Andrew F G

    2013-07-01

    The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10 (E-cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10 (cav-1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity, and cell migration observed with B16F10 (cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells.

  11. E-cadherin junction formation involves an active kinetic nucleation process

    SciTech Connect

    Biswas, Kabir H.; Hartman, Kevin L.; Yu, Cheng -han; Harrison, Oliver J.; Song, Hang; Smith, Adam W.; Huang, William Y. C.; Lin, Wan -Chen; Guo, Zhenhuan; Padmanabhan, Anup; Troyanovsky, Sergey M.; Dustin, Michael L.; Shapiro, Lawrence; Honig, Barry; Zaidel-Bar, Ronen; Groves, Jay T.

    2015-08-19

    Epithelial (E)-cadherin-mediated cell–cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.

  12. E-cadherin determines Caveolin-1 tumor suppression or metastasis enhancing function in melanoma cells

    PubMed Central

    Lobos-González, L; Aguilar, L; Diaz, J; Diaz, N; Urra, H; Torres, V; Silva, V; Fitzpatrick, C; Lladser, A; Hoek, K.S.; Leyton, L; Quest, AFG

    2013-01-01

    SUMMARY The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis, and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10(cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10(E-cad) cells reduces subcutaneous tumor formation, and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10(cav1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity and cell migration observed with B16F10(cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells. PMID:23470013

  13. E-cadherin junction formation involves an active kinetic nucleation process

    PubMed Central

    Biswas, Kabir H.; Hartman, Kevin L.; Yu, Cheng-han; Harrison, Oliver J.; Song, Hang; Smith, Adam W.; Huang, William Y. C.; Lin, Wan-Chen; Guo, Zhenhuan; Padmanabhan, Anup; Troyanovsky, Sergey M.; Dustin, Michael L.; Shapiro, Lawrence; Honig, Barry; Zaidel-Bar, Ronen; Groves, Jay T.

    2015-01-01

    Epithelial (E)-cadherin-mediated cell−cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role. PMID:26290581

  14. E-cadherin junction formation involves an active kinetic nucleation process

    DOE PAGES

    Biswas, Kabir H.; Hartman, Kevin L.; Yu, Cheng -han; Harrison, Oliver J.; Song, Hang; Smith, Adam W.; Huang, William Y. C.; Lin, Wan -Chen; Guo, Zhenhuan; Padmanabhan, Anup; et al

    2015-08-19

    Epithelial (E)-cadherin-mediated cell–cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest thatmore » the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.« less

  15. E-cadherin determines Caveolin-1 tumor suppression or metastasis enhancing function in melanoma cells.

    PubMed

    Lobos-González, Lorena; Aguilar, Lorena; Diaz, Jorge; Diaz, Natalia; Urra, Hery; Torres, Vicente A; Silva, Veronica; Fitzpatrick, Christopher; Lladser, Alvaro; Hoek, Keith S; Leyton, Lisette; Quest, Andrew F G

    2013-07-01

    The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10 (E-cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10 (cav-1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity, and cell migration observed with B16F10 (cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells. PMID:23470013

  16. Cloning and characterization of the human invasion suppressor gene E-cadherin (CDH1)

    SciTech Connect

    Berx, G.; Staes, K.; Hengel, J. van

    1995-03-20

    E-cadherin is a Ca{sup 2+}-dependent epithelial cell-cell adhesion molecule. Downregulation of E-cadherin expression often correlates with strong invasive potential and poor prognosis of human carcinomas. By using recombinant {lambda} phage, cosmid, and P1 phage clones, we isolated the full-length human E-cadherin gene (CDH1). The gene spans a region of approximately 100 kb, and its location on chromosome 16q22.1 was confirmed by FISH analysis. Detailed restriction mapping and partial sequence analysis of the gene allowed us to identify 16 exons and a 65-kb-long intron 2. The intron-exon boundaries are highly conserved in comparison with other {open_quotes}classical cadherins.{close_quotes} In intron 1 we identified a high-density CpG island that may be implicated in transcription regulation during embryogenesis and malignancy. 52 refs., 2 figs., 2 tabs.

  17. Preventing E-cadherin aberrant N-glycosylation at Asn-554 improves its critical function in gastric cancer.

    PubMed

    Carvalho, S; Catarino, T A; Dias, A M; Kato, M; Almeida, A; Hessling, B; Figueiredo, J; Gärtner, F; Sanches, J M; Ruppert, T; Miyoshi, E; Pierce, M; Carneiro, F; Kolarich, D; Seruca, R; Yamaguchi, Y; Taniguchi, N; Reis, C A; Pinho, S S

    2016-03-31

    E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with β1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell-cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression.

  18. Preventing E-cadherin aberrant N-glycosylation at Asn-554 improves its critical function in gastric cancer.

    PubMed

    Carvalho, S; Catarino, T A; Dias, A M; Kato, M; Almeida, A; Hessling, B; Figueiredo, J; Gärtner, F; Sanches, J M; Ruppert, T; Miyoshi, E; Pierce, M; Carneiro, F; Kolarich, D; Seruca, R; Yamaguchi, Y; Taniguchi, N; Reis, C A; Pinho, S S

    2016-03-31

    E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with β1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell-cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression. PMID:26189796

  19. Intravital FRAP Imaging using an E-cadherin-GFP Mouse Reveals Disease- and Drug-Dependent Dynamic Regulation of Cell-Cell Junctions in Live Tissue

    PubMed Central

    Erami, Zahra; Herrmann, David; Warren, Sean C.; Nobis, Max; McGhee, Ewan J.; Lucas, Morghan C.; Leung, Wilfred; Reischmann, Nadine; Mrowinska, Agata; Schwarz, Juliane P.; Kadir, Shereen; Conway, James R.W.; Vennin, Claire; Karim, Saadia A.; Campbell, Andrew D.; Gallego-Ortega, David; Magenau, Astrid; Murphy, Kendelle J.; Ridgway, Rachel A.; Law, Andrew M.; Walters, Stacey N.; Grey, Shane T.; Croucher, David R.; Zhang, Lei; Herzog, Herbert; Hardeman, Edna C.; Gunning, Peter W.; Ormandy, Christopher J.; Evans, T.R. Jeffry; Strathdee, Douglas; Sansom, Owen J.; Morton, Jennifer P.; Anderson, Kurt I.; Timpson, Paul

    2015-01-01

    Summary E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments. PMID:26725115

  20. Clinicopathologic Correlations of E-cadherin and Prrx-1 Expression Loss in Hepatocellular Carcinoma

    PubMed Central

    Yi, Kijong; Kim, Hyunsung; Chung, Yumin; Ahn, Hyein; Sim, Jongmin; Wi, Young Chan; Pyo, Ju Yeon; Song, Young-Soo; Paik, Seung Sam; Oh, Young-Ha

    2016-01-01

    Background Developing predictive markers for hepatocellular carcinoma (HCC) is important, because many patients experience recurrence and metastasis. Epithelial to mesenchymal transition (EMT) is a developmental process that plays an important role during embryogenesis and also during cancer metastasis. Paired-related homeobox protein 1 (Prrx-1) is an EMT inducer that has recently been introduced, and its prognostic significance in HCC is largely unknown. Methods Tissue microarray was constructed using surgically resected primary HCCs from 244 cases. Immunohistochemical staining of E-cadherin and Prrx-1 was performed. The correlation between E-cadherin loss and Prrx-1 expression, as well as other clinicopathologic factors, was evaluated. Results E-cadherin expression was decreased in 96 cases (39.4%). Loss of E-cadherin correlated with a higher recurrence rate (p < .001) but was not correlated with patient’s survival. Thirty-two cases (13.3%) showed at least focal nuclear Prrx-1 immunoreactivity while all non-neoplastic livers (n = 22) were negative. Prrx-1 expression was not associated with E-cadherin loss, survival or recurrence rates, pathologic factors, or the Ki-67 labeling index. Twenty tumors that were positive for E-cadherin and Prrx-1 had significantly higher nuclear grades than the rest of the cohort (p = .037). In Cox proportional hazard models, E-cadherin loss and large vessel invasion were independent prognostic factors for shorter disease-free survival. Cirrhosis and high Ki-67 index (> 40%) were independent prognostic factors for shorter overall survival. Conclusions Prrx-1 was expressed in small portions of HCCs but not in normal livers. Additional studies with a large number of Prrx-1-positive cases are required to confirm the results of this study. PMID:27580127

  1. Regulation of Embryonic Cell Adhesion by the Prion Protein

    PubMed Central

    Schrock, Yvonne; Geiss, Corinna; Luncz, Lydia; Thomanetz, Venus; Stuermer, Claudia A. O

    2009-01-01

    Prion proteins (PrPs) are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1) mediates Ca+2-independent homophilic cell adhesion and signaling; and (2) modulates Ca+2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin–based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development. PMID:19278297

  2. CDH1/E-cadherin and solid tumors. An updated gene-disease association analysis using bioinformatics tools.

    PubMed

    Abascal, María Florencia; Besso, María José; Rosso, Marina; Mencucci, María Victoria; Aparicio, Evangelina; Szapiro, Gala; Furlong, Laura Inés; Vazquez-Levin, Mónica Hebe

    2016-02-01

    Cancer is a group of diseases that causes millions of deaths worldwide. Among cancers, Solid Tumors (ST) stand-out due to their high incidence and mortality rates. Disruption of cell-cell adhesion is highly relevant during tumor progression. Epithelial-cadherin (protein: E-cadherin, gene: CDH1) is a key molecule in cell-cell adhesion and an abnormal expression or/and function(s) contributes to tumor progression and is altered in ST. A systematic study was carried out to gather and summarize current knowledge on CDH1/E-cadherin and ST using bioinformatics resources. The DisGeNET database was exploited to survey CDH1-associated diseases. Reported mutations in specific ST were obtained by interrogating COSMIC and IntOGen tools. CDH1 Single Nucleotide Polymorphisms (SNP) were retrieved from the dbSNP database. DisGeNET analysis identified 609 genes annotated to ST, among which CDH1 was listed. Using CDH1 as query term, 26 disease concepts were found, 21 of which were neoplasms-related terms. Using DisGeNET ALL Databases, 172 disease concepts were identified. Of those, 80 ST disease-related terms were subjected to manual curation and 75/80 (93.75%) associations were validated. On selected ST, 489 CDH1 somatic mutations were listed in COSMIC and IntOGen databases. Breast neoplasms had the highest CDH1-mutation rate. CDH1 was positioned among the 20 genes with highest mutation frequency and was confirmed as driver gene in breast cancer. Over 14,000 SNP for CDH1 were found in the dbSNP database. This report used DisGeNET to gather/compile current knowledge on gene-disease association for CDH1/E-cadherin and ST; data curation expanded the number of terms that relate them. An updated list of CDH1 somatic mutations was obtained with COSMIC and IntOGen databases and of SNP from dbSNP. This information can be used to further understand the role of CDH1/E-cadherin in health and disease. PMID:26674224

  3. CDH1/E-cadherin and solid tumors. An updated gene-disease association analysis using bioinformatics tools.

    PubMed

    Abascal, María Florencia; Besso, María José; Rosso, Marina; Mencucci, María Victoria; Aparicio, Evangelina; Szapiro, Gala; Furlong, Laura Inés; Vazquez-Levin, Mónica Hebe

    2016-02-01

    Cancer is a group of diseases that causes millions of deaths worldwide. Among cancers, Solid Tumors (ST) stand-out due to their high incidence and mortality rates. Disruption of cell-cell adhesion is highly relevant during tumor progression. Epithelial-cadherin (protein: E-cadherin, gene: CDH1) is a key molecule in cell-cell adhesion and an abnormal expression or/and function(s) contributes to tumor progression and is altered in ST. A systematic study was carried out to gather and summarize current knowledge on CDH1/E-cadherin and ST using bioinformatics resources. The DisGeNET database was exploited to survey CDH1-associated diseases. Reported mutations in specific ST were obtained by interrogating COSMIC and IntOGen tools. CDH1 Single Nucleotide Polymorphisms (SNP) were retrieved from the dbSNP database. DisGeNET analysis identified 609 genes annotated to ST, among which CDH1 was listed. Using CDH1 as query term, 26 disease concepts were found, 21 of which were neoplasms-related terms. Using DisGeNET ALL Databases, 172 disease concepts were identified. Of those, 80 ST disease-related terms were subjected to manual curation and 75/80 (93.75%) associations were validated. On selected ST, 489 CDH1 somatic mutations were listed in COSMIC and IntOGen databases. Breast neoplasms had the highest CDH1-mutation rate. CDH1 was positioned among the 20 genes with highest mutation frequency and was confirmed as driver gene in breast cancer. Over 14,000 SNP for CDH1 were found in the dbSNP database. This report used DisGeNET to gather/compile current knowledge on gene-disease association for CDH1/E-cadherin and ST; data curation expanded the number of terms that relate them. An updated list of CDH1 somatic mutations was obtained with COSMIC and IntOGen databases and of SNP from dbSNP. This information can be used to further understand the role of CDH1/E-cadherin in health and disease.

  4. p120 Catenin-Mediated Stabilization of E-Cadherin Is Essential for Primitive Endoderm Specification.

    PubMed

    Pieters, Tim; Goossens, Steven; Haenebalcke, Lieven; Andries, Vanessa; Stryjewska, Agata; De Rycke, Riet; Lemeire, Kelly; Hochepied, Tino; Huylebroeck, Danny; Berx, Geert; Stemmler, Marc P; Wirth, Dagmar; Haigh, Jody J; van Hengel, Jolanda; van Roy, Frans

    2016-08-01

    E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their in vitro differentiation was incomplete. In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment. PMID:27556156

  5. p120 Catenin-Mediated Stabilization of E-Cadherin Is Essential for Primitive Endoderm Specification

    PubMed Central

    Haenebalcke, Lieven; Stryjewska, Agata; De Rycke, Riet; Lemeire, Kelly; Huylebroeck, Danny; Stemmler, Marc P.; Wirth, Dagmar; Haigh, Jody J.; van Hengel, Jolanda; van Roy, Frans

    2016-01-01

    E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their in vitro differentiation was incomplete. In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment. PMID:27556156

  6. ADAM12-directed ectodomain shedding of E-cadherin potentiates trophoblast fusion.

    PubMed

    Aghababaei, M; Hogg, K; Perdu, S; Robinson, W P; Beristain, A G

    2015-12-01

    Trophoblasts, placental cells of epithelial lineage, undergo extensive differentiation to form the cellular components of the placenta. Trophoblast progenitor cell differentiation into the multinucleated syncytiotrophoblast is a key developmental process required for placental function, where defects in syncytiotrophoblast formation and turnover associate with placental pathologies and link to poor pregnancy outcomes. The cellular and molecular processes governing syncytiotrophoblast formation are poorly understood, but require the activation of pathways that direct cell fusion. The protease, A Disintegrin and Metalloproteinase 12 (ADAM12), controls cell fusion in myoblasts and is highly expressed in the placenta localizing to multiple trophoblast populations. However, the importance of ADAM12 in regulating trophoblast fusion is unknown. Here, we describe a function for ADAM12 in regulating trophoblast fusion. Using two distinct trophoblast models of cell fusion, we show that ADAM12 is dynamically upregulated and is under the transcriptional control of protein kinase A. siRNA-directed loss of ADAM12 impedes spontaneous fusion of primary cytotrophoblasts, whereas overexpression of the secreted variant, ADAM12S, potentiates cell fusion in the Bewo trophoblast cell line. Mechanistically, both ectopic and endogenous levels of ADAM12 were shown to control trophoblast fusion through E-cadherin ectodomain shedding and remodeling of intercellular boundaries. This study describes a novel role for ADAM12 in placental development, specifically highlighting its importance in controlling the differentiation of villous cytotrophoblasts into multinucleated cellular structures. Moreover, this work identifies E-cadherin as a novel ADAM12 substrate, and highlights the significance that cell adhesion molecule ectodomain shedding has in normal development.

  7. CAR regulates epithelial cell junction stability through control of E-cadherin trafficking

    PubMed Central

    Morton, Penny E.; Hicks, Alexander; Nastos, Theodoros; Santis, George; Parsons, Maddy

    2013-01-01

    CAR (Coxsackie and Adenovirus Receptor) is the primary docking receptor for typeB coxsackie viruses and subgroup C adenoviruses. CAR is a member of the JAM family of adhesion receptors and is located to both tight and adherens junctions between epithelial cells where it can assemble adhesive contacts through homodimerisation in trans. However, the role of CAR in controlling epithelial junction dynamics remains poorly understood. Here we demonstrate that levels of CAR in human epithelial cells play a key role in determining epithelial cell adhesion through control of E-cadherin stability at cell-cell junctions. Mechanistically, we show that CAR is phosphorylated within the C-terminus by PKCδ and that this in turn controls Src-dependent endocytosis of E-cadherin at cell junctions. This data demonstrates a novel role for CAR in regulating epithelial homeostasis. PMID:24096322

  8. XPC inhibits NSCLC cell proliferation and migration by enhancing E-Cadherin expression

    PubMed Central

    Cui, Tiantian; Srivastava, Amit Kumar; Han, Chunhua; Yang, Linlin; Zhao, Ran; Zou, Ning; Qu, Meihua; Duan, Wenrui; Zhang, Xiaoli; Wang, Qi-En

    2015-01-01

    Xeroderma pigmentosum complementation group C (XPC) protein is an important DNA damage recognition factor in nucleotide excision repair. Deletion of XPC is associated with early stages of human lung carcinogenesis, and reduced XPC mRNA levels predict poor patient outcome for non-small cell lung cancer (NSCLC). However, the mechanisms linking loss of XPC expression and poor prognosis in lung cancer are still unclear. Here, we report evidence that XPC silencing drives proliferation and migration of NSCLC cells by down-regulating E-Cadherin. XPC knockdown enhanced proliferation and migration while decreasing E-Cadherin expression in NSCLC cells with an epithelial phenotype. Restoration of E-Cadherin in these cells suppressed XPC knockdown-induced cell growth both in vitro and in vivo. Mechanistic studies showed that the loss of XPC repressed E-Cadherin expression by activating the ERK pathway and upregulating Snail expression. Our findings indicate that XPC silencing-induced reduction of E-Cadherin expression contributes, at least in part, to the poor outcome of NSCLC patients with low XPC expression. PMID:25871391

  9. Involvement of the MEK/ERK pathway in EGF-induced E-cadherin down-regulation.

    PubMed

    Tashiro, Etsu; Henmi, Shizuka; Odake, Hiroyuki; Ino, Seitaro; Imoto, Masaya

    2016-09-01

    E-cadherin is a major component of the epithelial adherens junction. However, the regulatory mechanism of E-cadherin expression is still poorly understood. In this study, we found that EGF decreased E-cadherin expression at both mRNA and protein levels in colorectal carcinoma LoVo cells. Since E-cadherin down-regulation is a well-known hallmark of the EMT (Epithelial-Mesenchymal Transition), we investigated whether EGF induced E-cadherin down-regulation during the EMT. EGF was unable to affect the expression of mesenchymal markers (such as N-cadherin, vimentin or fibronectin) or EMT-regulating transcription factors (such as SNAIL, SLUG, ZEB1, ZEB2 or TWIST), suggesting that EGF induced E-cadherin down-regulation via an EMT-independent mechanism. On the other hand, the MEK inhibitor U0126 was found to suppress EGF-induced E-cadherin down-regulation at the transcriptional level, suggesting that the MEK/ERK pathway is involved in EGF-induced E-cadherin down-regulation. Moreover, we also found that EGF disrupted cell-cell contact, stimulated cells to form an elongated shape with filamentous protrusions, and induced cell migration in LoVo cells. These effects were suppressed by U0126. Therefore, EGF is suggested to induce E-cadherin down-regulation at the transcriptional level through the MEK/ERK pathway, which might result in, at least in part, the induction of cellular morphological changes and cell migration in LoVo cells.

  10. SLIT2 attenuation during lung cancer progression deregulates beta-catenin and E-cadherin and associates with poor prognosis.

    PubMed

    Tseng, Ruo-Chia; Lee, Shih-Hua; Hsu, Han-Shui; Chen, Ben-Han; Tsai, Wan-Ching; Tzao, Ching; Wang, Yi-Ching

    2010-01-15

    Chromosome 4p15.3 is frequently deleted in late-stage lung cancer. We investigated the significance of the SLIT2 gene located in this region to lung cancer progression. SLIT2 encodes an extracellular glycoprotein that can suppress breast cancer by regulating beta-catenin. In this study, we examined alterations in the structure or expression of SLIT2, its receptor ROBO1, and beta-catenin, along with the AKT/glycogen synthase kinase 3beta (GSK3beta)/beta-transducin repeat-containing protein (betaTrCP) pathway in lung cancer cell lines and patients. Low SLIT2 expression correlated with an upward trend of pathological stage and poorer survival in lung cancer patients. Importantly, SLIT2, betaTrCP, and beta-catenin expression levels predicted postoperative recurrence of lung cancer in patients. Stimulating SLIT2 expression by various methods increased the level of E-cadherin caused by attenuation of its transcriptional repressor SNAI1. Conversely, knocking down SLIT2 expression increased cell migration and reduced cell adhesion through coordinated deregulation of beta-catenin and E-cadherin/SNAI1 in the AKT/GSK3beta/betaTrCP pathway. Our findings indicate that SLIT2 suppresses lung cancer progression, defining it as a novel "theranostic" factor with potential as a therapeutic target and prognostic predictor in lung cancer. Cancer Res; 70(2); 543-51.

  11. Mir-373 affects human lung cancer cells' growth and its E-cadherin expression.

    PubMed

    Wu, Weihua; He, Xiaoyan; Kong, Jing; Ye, Bin

    2012-01-01

    The aims of this study was to elucidate whether the expression of E-cadherin can be affected by the recombinant has-mir-373 eukaryotic expression plasmid vector through tests in vitro, and to analyze the relationship between the expression of E-cadherin and tumor growth. According to the has-mir-373 sequence in miRBase database, two template DNA sequences were designed. The has-mir-373 sequence and a control sequence were synthesized and cloned into pGenesil-1 eukaryotic expression plasmid vector. The recombinant plasmids were transfected into human lung cancer A549 cells by liposome-mediated method. The mir-373 expression in A549 cells was detected by using real-time quantitative polymerase chain reaction (real-time PCR). MTT (methyl thiazolyl tetrazolium) was used to analyze the growth of cancer cell cycle. RT-PCR and Western blotting were used to evaluate the levels of E-cadherin mRNA and protein expression, respectively. The expression of E-cadherin in cells was determined by immunocytochemistry. The mobility capability of transfected cells were evaluated by using wound healing assay in vitro. The fluorescent light was observed under fluorescent microscope. RT-PCR indicated that the mRNA of E-cadherin increased, and the Western blotting results also displayed that mir-373 promoted the expression of the E-cadherin protein. Compared with the control groups, MTT method and wound healing assay demonstrated that both the growth rate and migration of A549 cells transfected with the recombinant has-mir-373 eukaryotic expression plasmid was also decreased significantly (p < 0.001). The differences between the other two control groups were not significant (p > 0.05). The immunocytochemistry demonstrated a significant increase of E-cadherin protein levels in the cells transfected with mir-373, but not in the cells of the control group. Mir-373 could increase the expression levels of the E-cadherin and decrease the migration ability of human lung cancer A549 cells in

  12. Hedgehog signaling regulates E-cadherin expression for the maintenance of the actin cytoskeleton and tight junctions.

    PubMed

    Xiao, Chang; Ogle, Sally A; Schumacher, Michael A; Schilling, Neal; Tokhunts, Robert A; Orr-Asman, Melissa A; Miller, Marian L; Robbins, David J; Hollande, Frederic; Zavros, Yana

    2010-12-01

    In the stomach, strictly regulated cell adherens junctions are crucial in determining epithelial cell differentiation. Sonic Hedgehog (Shh) regulates epithelial cell differentiation in the adult stomach. We sought to identify whether Shh plays a role in regulating adherens junction protein E-cadherin as a mechanism for epithelial cell differentiation. Mouse nontumorigenic gastric epithelial (IMGE-5) cells treated with Hedgehog signaling inhibitor cyclopamine and anti-Shh 5E1 antibody or transduced with short hairpin RNA against Skinny Hedgehog (IMGE-5(Ski)) were cultured. A mouse model expressing a parietal cell-specific deletion of Shh (HKCre/Shh(KO)) was used to identify further changes in adherens and tight junctions. Inhibition of Hedgehog signaling in IMGE-5 cells caused loss of E-cadherin expression accompanied by disruption of F-actin cortical expression and relocalization of zonula occludens-1 (ZO-1). Loss of E-cadherin was also associated with increased proliferation in IMGE-5(Ski) cells and increased expression of the mucous neck cell lineage marker MUC6. Compared with membrane-expressed E-cadherin and ZO-1 protein in controls, dissociation of E-cadherin/β-catenin and ZO-1/occludin protein complexes was observed in HKCre/Shh(KO) mice. In conclusion, we demonstrate that Hedgehog signaling regulates E-cadherin expression that is required for the maintenance of F-actin cortical expression and stability of tight junction protein ZO-1.

  13. Rb depletion results in deregulation of E-cadherin and induction of cellular phenotypic changes that are characteristic of the epithelial-to-mesenchymal transition.

    PubMed

    Arima, Yoshimi; Inoue, Yasumichi; Shibata, Tatsuhiro; Hayashi, Hidemi; Nagano, Osamu; Saya, Hideyuki; Taya, Yoichi

    2008-07-01

    The retinoblastoma tumor suppressor protein (Rb) is mutated or expressed at very low levels in several tumor types, including retinoblastoma and osteosarcoma, as well as small cell lung, colon, prostate, bladder, and breast carcinomas. Loss or reduction of Rb expression is seen most commonly in high-grade breast adenocarcinomas, suggesting that a relationship may exist between loss of Rb function and a less-differentiated state, increased proliferation, and high metastatic potential. In this study, we found that knockdown of Rb by small interfering RNA in MCF7 breast cancer cells disrupts cell-cell adhesion and induces a mesenchymal-like phenotype. The epithelial-to-mesenchymal transition (EMT), a key event in embryonic morphogenesis, is implicated in the metastasis of primary tumors. Additionally, Rb is decreased during growth factor- and cytokine-induced EMT and overexpression of Rb inhibits the EMT in MCF10A human mammary epithelial cells. Ectopic expression and knockdown of Rb resulted in increased or reduced expression of E-cadherin, which is specifically involved in epithelial cell-cell adhesion. Other EMT-related transcriptional factors, including Slug and Zeb-1, are also induced by Rb depletion. Furthermore, we confirmed that Rb binds to an E-cadherin promoter sequence in association with the transcription factor activator protein-2alpha. Finally, in breast cancer specimens, we observed a concurrent down-regulation of Rb and E-cadherin expression in mesenchymal-like invasive cancers. These findings suggest that Rb inactivation contributes to tumor progression due to not only loss of cell proliferation control but also conversion to an invasive phenotype and that the inhibition of EMT is a novel tumor suppressor function of Rb.

  14. Effects of Cd{sup 2+} on cis-dimer structure of E-cadherin in living cells

    SciTech Connect

    Takeda, Hiroshi

    2014-02-21

    Highlights: • The effects of Cd on the dimer of cadherin in living cells was analyzed. • Cd induced cadherin dimer formation was not detected in living cell with low Ca. • Ca mediated structural cooperativity and allostery in the native cadherin. • Ca concentration-dependent competitive displacement of Cd from cadherin is proposed. - Abstract: E-cadherin, a calcium (Ca{sup 2+})-dependent cell–cell adhesion molecule, plays a key role in the maintenance of tissue integrity. We have previously demonstrated that E-cadherin functions in vivo as a cis-dimer through chemical cross-linking reagents. Ca{sup 2+} plays an important role in the cis-dimer formation of cadherin. However, the molecular mechanisms by which Ca{sup 2+} interacts with the binding sites that regulate cis-dimer structures have not been completely elucidated. As expected for a Ca{sup 2+} antagonist, cadmium (Cd{sup 2+}) disrupts cadherin function by displacing Ca{sup 2+} from its binding sites on the cadherin molecules. We used Cd{sup 2+} as a probe for investigating the role of Ca{sup 2+} in the dynamics of the E-cadherin extracellular region that involve cis-dimer formation and adhesion. While cell–cell adhesion assembly was completely disrupted in the presence of Cd{sup 2+}, the amount of cis-dimers of E-cadherin that formed at the cell surface was not affected. In our “Cd{sup 2+}-switch” experiments, we did not find that Cd{sup 2+}-induced E-cadherin cis-dimer formation in EL cells when they were incubated in low-Ca{sup 2+} medium. In the present study, we demonstrated for the first time the effects of Cd{sup 2+} on the cis-dimer structure of E-cadherin in living cells using a chemical cross-link analysis.

  15. UV-induction of keratinocyte endothelin-1 downregulates E-cadherin in melanocytes and melanoma cells.

    PubMed

    Jamal, Sumayah; Schneider, Robert J

    2002-08-01

    Endothelin-1 (ET-1), a peptide that is secreted by keratinocytes in the skin in response to ultraviolet irradiation, is a ligand for the endothelin-B (ET(B)) receptor. Blockade of this receptor inhibits melanoma cell growth and induces cell death in vivo and in vitro. Additionally, ET(B) is a melanoma progression marker. These findings suggest that the ET-1/ET(B) receptor pathway contributes to melanoma development or progression. Here, we demonstrate that activation of the ET-1/ET(B) pathway downregulates E-cadherin and associated catenin proteins in human melanocytes and melanoma cells. E-cadherin is an established suppressor of melanoma cell invasion in vitro and in vivo. Downregulation of E-cadherin by ET-1/ET(B) involves the downstream activation of caspase-8 but not of distal, executioner caspases, and does not lead to apoptosis. ET-1 also induces a transient association between caspase-8 and E-cadherin:beta-catenin complexes. Hence, activation of the ET-1/ET(B) pathway promotes molecular events known to promote melanoma invasion.

  16. Collagen type I may influence the expression of E-cadherin and beta-catenin in carcinoma ex-pleomorphic adenoma.

    PubMed

    Araújo, Vera C; Demasi, Ana Paula Dias; Furuse, Cristiane; Altemani, Albina; Alves, Venâncio A; Freitas, Leandro L; Araújo, Ney S

    2009-07-01

    Carcinoma ex-pleomorphic adenoma (CXPA) is an aggressive salivary gland malignancy, usually derived from a long-standing or a recurrent benign tumor, the pleomorphic adenoma (PA). In the context of dynamic reciprocity, changes in the composition and structure of extracellular matrix proteins and cell surface receptors have been frequently associated with dysfunctional adhesion and invasive behavior of tumor cells. It is not fully understood if these changes are involved in the conversion of PA to CXPA. In this study, different progression stages of CXPA were investigated regarding the expression of the major extracellular matrix proteins, collagen type I, and of E-cadherin and beta-catenin, the components of adherens junctions. By immunohistochemical analysis, we have demonstrated that direct contact of tumor cells with fibrillar type I collagen, particularly near the invasive front and in invasive areas prevailing small nests of CXPA cells, could be associated with reduced expression of the E-cadherin and beta-catenin adhesion molecules and with invasive behavior of epithelial, but not of CXPA with myoepithelial component. Our results also suggested that this association could depend on the organization of collagen molecules, being prevented by high-order polymeric structures. These findings could implicate the local microenvironment in the transition from the premalignant PA to invasive CXPA.

  17. Force via integrins but not E-cadherin decreases Oct3/4 expression in embryonic stem cells

    SciTech Connect

    Uda, Yuhei; Poh, Yeh-Chuin; Chowdhury, Farhan; Wu, Douglas C.; Tanaka, Tetsuya S.; Sato, Masaaki; Wang, Ning

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Force via integrins or cadherins induces similar cell stiffening responses. Black-Right-Pointing-Pointer Force via integrins but not cadherins induces cell spreading. Black-Right-Pointing-Pointer Force via integrins but not cadherins induces differentiation of embryonic stem cells. -- Abstract: Increasing evidence suggests that mechanical factors play a critical role in fate decisions of stem cells. Recently we have demonstrated that a local force applied via Arg-Gly-Asp (RGD) peptides coated magnetic beads to mouse embryonic stem (ES) cells increases cell spreading and cell stiffness and decreases Oct3/4 (Pou5f1) gene expression. However, it is not clear whether the effects of the applied stress on these functions of ES cells can be extended to natural extracellular matrix proteins or cell-cell adhesion molecules. Here we show that a local cyclic shear force applied via fibronectin or laminin to integrin receptors increased cell spreading and stiffness, downregulated Oct3/4 gene expression, and decreased cell proliferation rate. In contrast, the same cyclic force applied via cell-cell adhesion molecule E-cadherin (Cdh1) had no effects on cell spreading, Oct3/4 gene expression, and the self-renewal of mouse ES cells, but induced significant cell stiffening. Our findings demonstrate that biological responses of ES cells to force applied via integrins are different from those to force via E-cadherin, suggesting that mechanical forces might play different roles in different force transduction pathways to shape early embryogenesis.

  18. Clinico-pathological correlation of E-cadherin expression at the invasive tumor front of Indian oral squamous cell carcinomas: An immunohistochemical study

    PubMed Central

    Mehendiratta, Monica; Solomon, Monica Charlotte; Boaz, Karen; Guddattu, Vasudeva; Mohindra, Aashima

    2014-01-01

    Background: Recent studies have indicated that although malignant cells at the invasive tumor front, bare morphological resemblance to the cells at central portion of the tumor, their molecular character differs significantly. E-cadherin is a cell-cell adhesion molecule that connects epithelial cells. This study attempts to correlate the E-cadherin expression at the invasive tumor front with tumor differentiation along with its clinico-pathological parameters. Materials and Methods: Immunohistochemical staining with E-cadherin was carried out on archival cases of primary oral squamous cell carcinomas (n = 30). The E-cadherin expression at the invasive tumor front was analyzed and was linked to clinico-pathological parameters including patient prognosis. Results: The downregulation of E-cadherin expression at the invasive tumor edge when compared with patient's prognosis yielded a significant correlation (P = 0.041) but its correlation with the degree of differentiation determined was not significant (P = 0.27). Also, its association with tumor size and lymph node status was negative. Conclusions: Loss of E-cadherin expression at the invasive tumor front is an important event in the progression of oral squamous cell carcinomas. Tumors with a loss of expression of E-cadherin are those which had a poor prognosis PMID:25328302

  19. Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature-sensitive v-SRC gene

    PubMed Central

    1993-01-01

    Loss of histotypic organization of epithelial cells is a common feature in normal development as well as in the invasion of carcinomas. Here we show that the v-src oncogene is a potent effector of epithelial differentiation and invasiveness. MDCK epithelial cells transformed with a temperature-sensitive mutant of v-src exhibit a strictly epithelial phenotype at the nonpermissive temperature for pp60v-src activity (40.5 degrees C) but rapidly loose cell-to-cell contacts and acquire a fibroblast-like morphology after culture at the permissive temperature (35 degrees C). Furthermore, the invasiveness of the cells into collagen gels or into chick heart fragments was increased at the permissive temperature. The profound effects of v-src on intercellular adhesion were not linked to changes in the levels of expression of the epithelial cell adhesion molecule E-cadherin. Rather, we observed an increase in tyrosine phosphorylation of E-cadherin and, in particular, of the associated protein beta-catenin. These results suggest a mechanism by which v-src counteracts junctional assembly and thereby promotes invasiveness and dedifferentiation of epithelial cells through phosphorylation of the E-cadherin/catenin complex. PMID:8425900

  20. Smoking induces epithelial-to-mesenchymal transition in non-small cell lung cancer through HDAC-mediated downregulation of E-cadherin.

    PubMed

    Nagathihalli, Nagaraj S; Massion, Pierre P; Gonzalez, Adriana L; Lu, Pengcheng; Datta, Pran K

    2012-11-01

    Epidemiological studies have shown that most cases of lung cancers (85%-90%) are directly attributable to tobacco smoking. Although association between cigarette smoking and lung cancer is well documented, surprisingly little is known about the molecular mechanisms of how smoking is involved in epithelial-to-mesenchymal transition (EMT) through epigenetic changes. Here, we show that lung cancer patients with a smoking history have low E-cadherin levels and loss of E-cadherin is a poor prognostic factor in smokers. Moreover, the downregulation of E-cadherin correlates with the number of pack years. In an attempt to determine the role of long-term cigarette smoking on EMT, we observed that treatment of lung cell lines with cigarette smoke condensate (CSC) induces EMT through downregulation of epithelial markers, including E-cadherin and upregulation of mesenchymal markers. CSC decreases E-cadherin expression at the transcriptional level through upregulation of LEF1 and Slug, and knockdown of these two proteins increases E-cadherin expression. Importantly, chromatin immunoprecipitation assays suggest that LEF-1 and Slug binding to E-cadherin promoter is important for CSC-mediated downregulation of E-cadherin. The histone deacetylase (HDAC) inhibitor MS-275 reverses CSC-induced EMT, migration, and invasion through the restoration of E-cadherin expression. These results suggest that recruitment of HDACs by transcriptional repressors LEF-1 and Slug is responsible for E-cadherin suppression and EMT in cigarette smokers and provide a potential drug target toward the treatment of lung cancer.

  1. Self-assembly of DNA and cell-adhesive proteins onto pH-sensitive inorganic crystals for precise and efficient transgene delivery.

    PubMed

    Chowdhury, E H

    2008-01-01

    Intracellular delivery of a functional gene or a gene-silencing DNA or RNA sequence is expected to be a powerful tool for treating critical human diseases very precisely and effectively. One of the major hurdles to the successful delivery of a nucleic acid with nanoparticles is the transport across the plasma membrane. The existence of various and numerous cell surface receptors with potential capability of being internalized by cells upon ligand binding unveils the ways of overcoming the barrier by targeting the nanoparticles to specific receptor. This review will reveal the current progress on utilizing the cell adhesion molecules as targeting receptors for transgene delivery, with a special focus on the design of bio-functionalized inorganic nanocrystals using both naturally occurring and genetically engineered cell adhesive proteins for high efficiency transfection of embryonic stem cells. Self-assembly of both DNA and cell-adhesive proteins, such as fibronectin and E-cadherin-Fc into the growing nanocrystals of carbonate apatite leads to their high affinity interactions with fibronectin-specific integrins and E-cadherin in embryonic stem cell surface and accelerates transgene delivery for subsequent expression. While only apatite nano-particles were very inefficient in transfecting embryonic stem cells, fibronectin-anchored particles and to a more significant extent, fibronectin and E-cadherin-Fc-associated particles dramatically enhanced transgene delivery with a value notably higher than that of commercially available lipofection system. Activation of protein kinase C (PKC) dramatically enhances transgene expression probably by up-regulating both integrin and E-cadherin. Thus, the new establishment of a bio-functional hybrid gene-carrier would promote and facilitate development of stem cell-based therapy in regenerative medicine.

  2. Interaction of E-cadherin and PTEN regulates morphogenesis and growth arrest in human mammary epithelial cells

    SciTech Connect

    Fournier, Marcia V.; Fata, Jimmie E.; Martin, Katherine J.; Yaswen, Paul; Bissell, Mina J.

    2009-06-03

    PTEN is a dual function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two non-malignant human mammary epithelial cell lines (HMECs) that form polarized, growth-arrested structures (acini) when cultured in 3-dimensional laminin-rich extracellular matrix gels (3D lrECM). As acini begin to form, PTEN accumulates in both the cytoplasm, and at cell-cell contacts where it colocalizes with E-cadherin/{beta}-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function-blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in SKBR3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in 3D lrECM indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus appears to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells.

  3. Adhesives from modified soy protein

    DOEpatents

    Sun, Susan; Wang, Donghai; Zhong, Zhikai; Yang, Guang

    2008-08-26

    The, present invention provides useful adhesive compositions having similar adhesive properties to conventional UF and PPF resins. The compositions generally include a protein portion and modifying ingredient portion selected from the group consisting of carboxyl-containing compounds, aldehyde-containing compounds, epoxy group-containing compounds, and mixtures thereof. The composition is preferably prepared at a pH level at or near the isoelectric point of the protein. In other preferred forms, the adhesive composition includes a protein portion and a carboxyl-containing group portion.

  4. Loss of functional E-cadherin renders cells more resistant to the apoptotic agent taxol in vitro

    SciTech Connect

    Ferreira, Paulo; Oliveira, Maria Jose; Beraldi, Eliana; Mateus, Ana Rita; Nakajima, Takashi; Gleave, Martin; Yokota, Jun; Carneiro, Fatima; Huntsman, David; Seruca, Raquel; Suriano, Gianpaolo . E-mail: gsuriano@ipatimup.pt

    2005-10-15

    Experimental evidence supports a role for E-cadherin in suppressing invasion, metastasis, and proliferation. Germline mutations of the E-cadherin represent the genetic cause of hereditary diffuse gastric cancer (HDGC). In this type of tumor, isolated cancer cells permeate the basal membrane and paradoxically survive in the gastric wall in the absence of contact with neighbor epithelial cells or with the extracellular matrix. This suggests that upon E-cadherin deregulation, cells acquired resistance to apoptosis. To test this hypothesis, CHO cells stably expressing either wild-type E-cadherin or the HDGC-related germline mutations T340A and V832M were seeded either on a thin layer of collagen type I or on plastic and then subjected to the apoptotic agent taxol. We found that in vitro functional E-cadherin renders cells more sensitive to the effect of taxol. Our results also indicate that this effect is associated to decreased level of the anti-apoptotic bcl-2 protein.

  5. Induction of E-cadherin+ human amniotic fluid cell differentiation into oocyte-like cells via culture in medium supplemented with follicular fluid.

    PubMed

    Liu, Te; Huang, Yongyi; Bu, Yanzhen; Zhao, Yanhui; Zou, Gang; Liu, Zhixue

    2014-07-01

    Pluripotent human amniotic fluid cells (HuAFCs) can differentiate into various types of somatic cell in vitro. However, their differentiation into oocyte-like cells has never been described to the best of our knowledge. In the present study, differentiation of E-cadherin+ and E-cadherin- HuAFC sub-populations into oocyte-like cells was induced via culture in medium containing bovine follicular fluid and β-mercaptoethanol. The E-cadherin+ HuAFCs expressed DAZL highly. Post-induction, cells with an oocyte-like phenotype were found among the E-cadherin+ HuAFCs, expressing markers specific to germ cells and oocytes (VASA, ZP3 and GDF9) and meiosis (DMC1 and SCP3). When specific small interfering RNA (siRNA) was used to suppress E-cadherin in the E-cadherin+ HuAFCs, the levels of DAZL expression were reduced. Post-induction, the morphology of the siRNA‑E‑cadherin HuAFCs was poorer and the expression levels of germ cell-specific markers were lower compared with those of the siRNA-mock HuAFCs. Therefore, E-cadherin+ HuAFCs could be more easily induced to differentiate into oocyte-like cells by bovine follicular fluid and β-mercaptoethanol. In addition, the E-cadherin+ HuAFCs exhibited potential characteristics of DAZL protein expression, and thus it was conjectured that bovine follicular fluid acts on DAZL protein and promotes E-cadherin+ HuAFC differentiation into oocyte-like cells.

  6. Induction of E-cadherin+ human amniotic fluid cell differentiation into oocyte-like cells via culture in medium supplemented with follicular fluid.

    PubMed

    Liu, Te; Huang, Yongyi; Bu, Yanzhen; Zhao, Yanhui; Zou, Gang; Liu, Zhixue

    2014-07-01

    Pluripotent human amniotic fluid cells (HuAFCs) can differentiate into various types of somatic cell in vitro. However, their differentiation into oocyte-like cells has never been described to the best of our knowledge. In the present study, differentiation of E-cadherin+ and E-cadherin- HuAFC sub-populations into oocyte-like cells was induced via culture in medium containing bovine follicular fluid and β-mercaptoethanol. The E-cadherin+ HuAFCs expressed DAZL highly. Post-induction, cells with an oocyte-like phenotype were found among the E-cadherin+ HuAFCs, expressing markers specific to germ cells and oocytes (VASA, ZP3 and GDF9) and meiosis (DMC1 and SCP3). When specific small interfering RNA (siRNA) was used to suppress E-cadherin in the E-cadherin+ HuAFCs, the levels of DAZL expression were reduced. Post-induction, the morphology of the siRNA‑E‑cadherin HuAFCs was poorer and the expression levels of germ cell-specific markers were lower compared with those of the siRNA-mock HuAFCs. Therefore, E-cadherin+ HuAFCs could be more easily induced to differentiate into oocyte-like cells by bovine follicular fluid and β-mercaptoethanol. In addition, the E-cadherin+ HuAFCs exhibited potential characteristics of DAZL protein expression, and thus it was conjectured that bovine follicular fluid acts on DAZL protein and promotes E-cadherin+ HuAFC differentiation into oocyte-like cells. PMID:24788191

  7. Type Iγ phosphatidylinositol phosphate kinase modulates adherens junction and E-cadherin trafficking via a direct interaction with μ1B adaptin

    PubMed Central

    Ling, Kun; Bairstow, Shawn F.; Carbonara, Chateen; Turbin, Dmitry A.; Huntsman, David G.; Anderson, Richard A.

    2007-01-01

    Assembly of E-cadherin–based adherens junctions (AJ) is obligatory for establishment of polarized epithelia and plays a key role in repressing the invasiveness of many carcinomas. Here we show that type Iγ phosphatidylinositol phosphate kinase (PIPKIγ) directly binds to E-cadherin and modulates E-cadherin trafficking. PIPKIγ also interacts with the μ subunits of clathrin adaptor protein (AP) complexes and acts as a signalling scaffold that links AP complexes to E-cadherin. Depletion of PIPKIγ or disruption of PIPKIγ binding to either E-cadherin or AP complexes results in defects in E-cadherin transport and blocks AJ assembly. An E-cadherin germline mutation that loses PIPKIγ binding and shows disrupted basolateral membrane targeting no longer forms AJs and leads to hereditary gastric cancers. These combined results reveal a novel mechanism where PIPKIγ serves as both a scaffold, which links E-cadherin to AP complexes and the trafficking machinery, and a regulator of trafficking events via the spatial generation of phosphatidylinositol-4,5-bisphosphate. PMID:17261850

  8. Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition.

    PubMed

    Li, Qisheng; Sodroski, Catherine; Lowey, Brianna; Schweitzer, Cameron J; Cha, Helen; Zhang, Fang; Liang, T Jake

    2016-07-01

    Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry.

  9. Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition.

    PubMed

    Li, Qisheng; Sodroski, Catherine; Lowey, Brianna; Schweitzer, Cameron J; Cha, Helen; Zhang, Fang; Liang, T Jake

    2016-07-01

    Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry. PMID:27298373

  10. Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells.

    PubMed

    Efstathiou, J A; Liu, D; Wheeler, J M; Kim, H C; Beck, N E; Ilyas, M; Karayiannakis, A J; Mortensen, N J; Kmiot, W; Playford, R J; Pignatelli, M; Bodmer, W F

    1999-03-01

    Epithelial (E)-cadherin and its associated cytoplasmic proteins (alpha-, beta-, and gamma-catenins) are important mediators of epithelial cell-cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin-catenin-mediated adhesion/signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2.

  11. E-cadherin downregulation at the infiltrating tumour front is associated with histological grade and stage in colorectal carcinoma of Malaysians.

    PubMed

    Dass, Serena Diane; Cheah, Phaik-Leng; Ong, Diana Bee-Lan; Teoh, Kean-Hooi; Looi, Lai-Meng

    2015-04-01

    Loss of E-cadherin, a 120 kDA transmembrane glycoprotein responsible for cell-cell adhesion, is one of the hallmarks of epithelial-mesenchymal-transition (EMT). E-cadherin expression was immunohistochemically studied in 94 histopathologically re-confirmed colorectal carcinomas (CRC) using a monoclonal antibody to E-cadherin (Dako: Clone NCH-38) on a Ventana Benchmark XT automated system. Each case was assessed for E-cadherin immunopositivity at two separate locations viz the tumour centre (TC) as well as the infiltrating front (IF). Expression was semiquantitated for proportion of immunopositive malignant cells as 0 (negative), 1 (1-25% staining), 2 (26-50% staining), 3 (51-75% staining) and 4 (>75% staining) and staining intensity: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The final histoscore of E-cadherin immunopositivity was arbitrarily computed as proportion of immunopositivity multiplied by staining intensity of the malignant cells. E-cadherin histoscores were significantly lower at the IF (4.5±2.5) compared with TC (10.7±2.4). Furthermore, the histoscores were significantly reduced at the IF of 49 TNM III+IV tumours (3.6±2.5) compared with 45 II+III CRC (5.4±2.2). Reduction of E-cadherin expression was also noted in the 23 high grade (TC=8.6±3.2; IF=2.6±2.3) compared with 71 low grade tumours (TC=11.4±1.5; IF=5.1±2.3). E-cadherin is downregulated at the infiltrating front of CRC, possibly marking for EMT at this location. The downregulation is further enhanced amongst late stage and high grade tumours compared with earlier stage and low grade tumours; findings which are similar to that noted in CRC of other populations.

  12. Armc8 regulates the invasive ability of hepatocellular carcinoma through E-cadherin/catenin complex.

    PubMed

    Zhao, Yang; Peng, Songlin; Jia, Changjun; Xu, Feng; Xu, Yongqing; Dai, Chaoliu

    2016-08-01

    Armc8 (armadillo-repeat-containing protein 8) was proved to promote disruption of E-cadherin complex through regulating α-catenin degradation. In this study, we investigated Armc8 expression in hepatocellular carcinoma using immunohistochemistry (IHC). The positive rate of Armc8 expression in hepatocellular carcinoma was 53.9 % and higher than that in normal hepatic tissues (9.2 %) (p < 0.05). Clinicopathological analysis shows that Armc8 expression in hepatocellular carcinoma was significantly associated with larger tumor size (≥5 cm), multiple tumor numbers, higher pathological grade (media and poor), advanced TNM stages (II/III), and advanced BCLC stages (B/C). Western blot study also detected higher Armc8 expression in hepatocellular carcinoma cells including HepG2, HCC97L, and SMMC-7721 than in human hepatic cell Bel-7402. We further use specific small interfering RNAs (siRNAs) to knock down Armc8 expression in HepG2 cells and found that knockdown of Armc8 expression significantly inhibited the invasive ability of HepG2 cells. Downregulation of Armc8 expression significantly upregulated α-catenin, β-catenin, and E-cadherin expression in HepG2 cells. Immunofluorescent study shows that knockdown of Armc8 expression restored E-cadherin expression in membrane of HepG2 cells. These results indicate that Armc8 may be a potential cancer marker in hepatocellular carcinoma and may regulate cancer invasion through E-cadherin/catenin complex. PMID:26944057

  13. Pituitary tumor transforming gene PTTG2 induces psoriasis by regulating vimentin and E-cadherin expression.

    PubMed

    Liu, Xiao-Bing; Li, Feng; Li, Ya-Qin; Yang, Fan

    2015-01-01

    Psoriasis is a common and intractable skin disease affecting the physical and mental health of patients. This study focused on the roles of pituitary tumor transforming gene 2 (PTTG2) in psoriasis. Using real-time quantitative PCR and western blot, the expression patterns of PTTG2 were compared in psoriatic epidermis cells and normal cells, from both mRNA levels and protein levels. Knockdown of PTTG2 by siRNA was conducted in HaCaT cells to investigate the changes in cell viability and migration in vitro. Expression changes of vimentin and E-cadherin were also detected in the transfected cells. Results showed PTTG2 was significantly overexpressed in the psoriatic epidermis cells (P < 0.05). The cell viability and migration were inhibited by the knockdown of PTTG2. Besides, knockdown of PTTG2 resulted in down-regulation of vimentin and up-regulation of E-cadherin, with significant differences compared to the siRNA control group (P < 0.05). This study indicated the involvement of PTTG2 in mediating epidermis cell viability and migration and in pathogenesis of psoriasis. PTTG2 might be a potential therapeutic target for psoriasis through inducing epithelial-to-mesenchymal transition (EMT) via regulating the expression of vimentin and E-cadherin. PMID:26617803

  14. Immunohistochemical expression of E-cadherin and β-catenin in feline mammary tumours.

    PubMed

    Zappulli, V; De Cecco, S; Trez, D; Caliari, D; Aresu, L; Castagnaro, M

    2012-01-01

    E-cadherin and β-catenin have been studied in carcinogenesis and tumour progression and reduced membrane expression of these molecules in canine mammary tumours has been associated with a poor prognosis. The present study investigated immunohistochemically the expression of E-cadherin and β-catenin in 53 mammary tumours and 48 hyperplastic or dysplastic lesions from 57 queens. E-cadherin and β-catenin expression was membranous in all samples and there was a significant decrease in expression in malignant tumours and metastases. Cytoplasmic expression of both markers was inversely correlated to the membrane localization. β-catenin nuclear labelling was detected in one lymph node metastasis (60% positive cells) and in the basal/myoepithelial cells of 6/7 ductal tumours. No correlation with survival was found for either marker. These results confirm the role of these proteins in maintaining tissue architecture and in inhibiting cell invasiveness and potentially indicate the oncogenic potential of the Wnt/β-catenin transduction pathway in feline mammary tumours. In addition, specific independent expression of β-catenin in the nuclei of basal/myoepithelial cells might suggest that this molecule is involved in regulation of the mammary stem/pluripotent cell component. Further studies should include more cases of benign mammary neoplasia and further investigate β-catenin nuclear expression in ductal tumours. PMID:22520821

  15. Cleavage of E-cadherin and β-catenin by calpain affects Wnt signaling and spheroid formation in suspension cultures of human pluripotent stem cells.

    PubMed

    Konze, Sarah A; van Diepen, Laura; Schröder, Anke; Olmer, Ruth; Möller, Hanna; Pich, Andreas; Weißmann, Robert; Kuss, Andreas W; Zweigerdt, Robert; Buettner, Falk F R

    2014-04-01

    The envisioned clinical and industrial use of human pluripotent stem cells and their derivatives has given major momentum to the establishment of suspension culture protocols that enable the mass production of cells. Understanding molecular changes accompanying the transfer from adherent to suspension culture is of utmost importance because this information can have a direct effect on the development of optimized culture conditions. In this study we assessed the gene expression of human embryonic stem cells and induced pluripotent stem cells grown in surface-adherent culture (two-dimensional) versus free-floating suspension culture spheroids (three-dimensional). We combined a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture with deep-sequencing-based transcriptomics. Cells in three-dimensional culture showed reduced expression of proteins forming structural components of cell-cell and cell-extracellular matrix junctions. However, fully unexpected, we found up-regulation of secreted inhibitors of the canonical Wnt signaling pathway and, concomitantly, a reduction in the level of active β-catenin and in the expression of Wnt target genes. In Western blot analyses the cysteine protease calpain was shown to cleave E-cadherin and β-catenin under three-dimensional culture conditions. Our data allowed the development of a model in which calpain cleavage of E-cadherin induces the disintegration of focal cell contacts and generates a 100-kDa E-cadherin fragment required for the formation of three-dimensional cell-cell contacts in spheroids. The parallel release of β-catenin and its potential activation by calpain cleavage are counterbalanced by the overexpression of soluble Wnt pathway inhibitors. According to this model, calpain has a key function in the interplay between E-cadherin and β-catenin-mediated intercellular adhesion and the canonical Wnt signaling pathway. Supporting this model, we show that pharmacological

  16. Cleavage of E-Cadherin and β-Catenin by Calpain Affects Wnt Signaling and Spheroid Formation in Suspension Cultures of Human Pluripotent Stem Cells*

    PubMed Central

    Konze, Sarah A.; van Diepen, Laura; Schröder, Anke; Olmer, Ruth; Möller, Hanna; Pich, Andreas; Weißmann, Robert; Kuss, Andreas W.; Zweigerdt, Robert; Buettner, Falk F. R.

    2014-01-01

    The envisioned clinical and industrial use of human pluripotent stem cells and their derivatives has given major momentum to the establishment of suspension culture protocols that enable the mass production of cells. Understanding molecular changes accompanying the transfer from adherent to suspension culture is of utmost importance because this information can have a direct effect on the development of optimized culture conditions. In this study we assessed the gene expression of human embryonic stem cells and induced pluripotent stem cells grown in surface-adherent culture (two-dimensional) versus free-floating suspension culture spheroids (three-dimensional). We combined a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture with deep-sequencing-based transcriptomics. Cells in three-dimensional culture showed reduced expression of proteins forming structural components of cell–cell and cell–extracellular matrix junctions. However, fully unexpected, we found up-regulation of secreted inhibitors of the canonical Wnt signaling pathway and, concomitantly, a reduction in the level of active β-catenin and in the expression of Wnt target genes. In Western blot analyses the cysteine protease calpain was shown to cleave E-cadherin and β-catenin under three-dimensional culture conditions. Our data allowed the development of a model in which calpain cleavage of E-cadherin induces the disintegration of focal cell contacts and generates a 100-kDa E-cadherin fragment required for the formation of three-dimensional cell–cell contacts in spheroids. The parallel release of β-catenin and its potential activation by calpain cleavage are counterbalanced by the overexpression of soluble Wnt pathway inhibitors. According to this model, calpain has a key function in the interplay between E-cadherin and β-catenin-mediated intercellular adhesion and the canonical Wnt signaling pathway. Supporting this model, we show that

  17. Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II–dependent manner

    PubMed Central

    le Duc, Quint; Shi, Quanming; Blonk, Iris; Sonnenberg, Arnoud

    2010-01-01

    Cell surface receptors integrate chemical and mechanical cues to regulate a wide range of biological processes. Integrin complexes are the mechanotransducers between the extracellular matrix and the actomyosin cytoskeleton. By analogy, cadherin complexes may function as mechanosensors at cell–cell junctions, but this capacity of cadherins has not been directly demonstrated. Furthermore, the molecular composition of the link between E-cadherin and actin, which is needed to sustain such a function, is unresolved. In this study, we describe nanomechanical measurements demonstrating that E-cadherin complexes are functional mechanosensors that transmit force between F-actin and E-cadherin. Imaging experiments reveal that intercellular forces coincide with vinculin accumulation at actin-anchored cadherin adhesions, and nanomechanical measurements show that vinculin potentiates the E-cadherin mechanosensory response. These investigations directly demonstrate the mechanosensory capacity of the E-cadherin complex and identify a novel function for vinculin at cell–cell junctions. These findings have implications for barrier function, morphogenesis, cell migration, and invasion and may extend to all soft tissues in which classical cadherins regulate cell–cell adhesion. PMID:20584916

  18. E-cadherin enhances neuregulin signaling and promotes Schwann cell myelination.

    PubMed

    Basak, Sayantani; Desai, Darshan J; Rho, Esther H; Ramos, Roselle; Maurel, Patrice; Kim, Haesun A

    2015-09-01

    In myelinating Schwann cells, E-cadherin is a component of the adherens junctions that stabilize the architecture of the noncompact myelin region. In other cell types, E-cadherin has been considered as a signaling receptor that modulates intracellular signal transduction and cellular responses. To determine whether E-cadherin plays a regulatory role during Schwann cell myelination, we investigated the effects of E-cadherin deletion and over-expression in Schwann cells. In vivo, Schwann cell-specific E-cadherin ablation results in an early myelination delay. In Schwann cell-dorsal root ganglia neuron co-cultures, E-cadherin deletion attenuates myelin formation and shortens the myelin segment length. When over-expressed in Schwann cells, E-cadherin improves myelination on Nrg1 type III(+/-) neurons and induces myelination on normally non-myelinated axons of sympathetic neurons. The pro-myelinating effect of E-cadherin is associated with an enhanced Nrg1-erbB receptor signaling, including activation of the downstream Akt and Rac. Accordingly, in the absence of E-cadherin, Nrg1-signaling is diminished in Schwann cells. Our data also show that E-cadherin expression in Schwann cell is induced by axonal Nrg1 type III, indicating a reciprocal interaction between E-cadherin and the Nrg1 signaling. Altogether, our data suggest a regulatory function of E-cadherin that modulates Nrg1 signaling and promotes Schwann cell myelin formation.

  19. Analysis of cytoskeleton dynamics and cell migration in drosophila ovaries using GFP-actin and E-cadherin-GFP fusion molecules

    NASA Astrophysics Data System (ADS)

    Verkhusha, Vladyslav V.; Tsukita, Shoichiro; Oda, Hiroki

    1999-06-01

    Coordination of cell migration and adhesion is essential for movement of tissues during morphogenesis. During Drosophila oogenesis so called border cells (BCs) break from an anterior epithelium of egg chamber, acquire a mesenchymal-like morphology, and migrate posteriorly between nurse cells to oocyte. The confocal microscopic observation of BCs has revealed well-developed forepart lamellipodium stained with Drosophila E-cadherin (DE-cadherin), PS2 integrin, cytoplasmic myosin and F-actin. To investigate mechanism of BC migration in vivo we have constructed a DE-cadherin-GFP and a GFP-actin fusion proteins and induced their expression BCs utilizing the UAS/GAL4 system. The DE-cadherin-GFP signal as well as immunostaining of PS2 integrin visualized a track of migrating BCs providing an evidence that adhesive molecules are pulled out and left behind on the surface of nurse cells. Our data suggest that two distinct adhesive systems, DE-cadherins and PS2 integrins simultaneously mediate the migration of BCs. Release of adhesive contacts in the tail region is a rate- limited event in BC migration. The spatial-temporal sequence of actin-based events visualized by the GFP-actin suggest a treadmilling model for actin behavior in BC lamellipodium. BC migration can be considered as simultaneous reiterating processes of lamellipodium extension and adhesive attachment, cytoskeletal contraction, and rear detachment.

  20. Aberrant Methylation of the E-Cadherin Gene Promoter Region in the Endometrium of Women With Uterine Fibroids.

    PubMed

    Li, Yan; Ran, Ran; Guan, Yingxia; Zhu, Xiaoxiong; Kang, Shan

    2016-08-01

    A uterine fibroid is a leiomyoma that originates from the smooth muscle layer of the uterus. A variety of endometrial abnormalities are associated with uterine fibroids. This study aims to investigate the methylation status of the E-cadherin gene (CDH1) promoter region in the endometrium of patients with uterine fibroids. The methylation of CDH1 was studied using methylation-specific polymerase chain reaction in the endometrial tissue of 102 patients with uterine fibroids and 50 control patients. The E-cadherin expression was examined by flow cytometry. The methylation rate of CDH1 promoter region was 33.3% in the endometrium of patients with uterine fibroids and 8% in the endometrium of women without fibroids. The frequency of CDH1 promoter methylation in the endometrium of patients with fibroids was significantly higher than that in the endometrium of women without fibroids (P = .001). Furthermore, the E-cadherin expression level in methylation-positive tissues was significantly lower than that in methylation-negative tissues (P = .017). These results suggest that epigenetic aberration of CDH1 may occur in the endometrium of patients with fibroids, which may be associated with E-cadherin protein expression in endometrial tissue. PMID:26880767

  1. Aberrant Methylation of the E-Cadherin Gene Promoter Region in the Endometrium of Women With Uterine Fibroids.

    PubMed

    Li, Yan; Ran, Ran; Guan, Yingxia; Zhu, Xiaoxiong; Kang, Shan

    2016-08-01

    A uterine fibroid is a leiomyoma that originates from the smooth muscle layer of the uterus. A variety of endometrial abnormalities are associated with uterine fibroids. This study aims to investigate the methylation status of the E-cadherin gene (CDH1) promoter region in the endometrium of patients with uterine fibroids. The methylation of CDH1 was studied using methylation-specific polymerase chain reaction in the endometrial tissue of 102 patients with uterine fibroids and 50 control patients. The E-cadherin expression was examined by flow cytometry. The methylation rate of CDH1 promoter region was 33.3% in the endometrium of patients with uterine fibroids and 8% in the endometrium of women without fibroids. The frequency of CDH1 promoter methylation in the endometrium of patients with fibroids was significantly higher than that in the endometrium of women without fibroids (P = .001). Furthermore, the E-cadherin expression level in methylation-positive tissues was significantly lower than that in methylation-negative tissues (P = .017). These results suggest that epigenetic aberration of CDH1 may occur in the endometrium of patients with fibroids, which may be associated with E-cadherin protein expression in endometrial tissue.

  2. Curcumin Suppresses Metastasis via Sp-1, FAK Inhibition, and E-Cadherin Upregulation in Colorectal Cancer.

    PubMed

    Chen, Chun-Chieh; Sureshbabul, Munisamy; Chen, Huei-Wen; Lin, Yu-Shuang; Lee, Jen-Yi; Hong, Qi-Sheng; Yang, Ya-Chien; Yu, Sung-Liang

    2013-01-01

    Colorectal cancer (CRC) is a serious public health problem that results due to changes of diet and various environmental stress factors in the world. Curcumin is a traditional medicine used for treatment of a wide variety of tumors. However, antimetastasis mechanism of curcumin on CRC has not yet been completely investigated. Here, we explored the underlying molecular mechanisms of curcumin on metastasis of CRC cells in vitro and in vivo. Curcumin significantly inhibits cell migration, invasion, and colony formation in vitro and reduces tumor growth and liver metastasis in vivo. We found that curcumin suppresses Sp-1 transcriptional activity and Sp-1 regulated genes including ADEM10, calmodulin, EPHB2, HDAC4, and SEPP1 in CRC cells. Curcumin inhibits focal adhesion kinase (FAK) phosphorylation and enhances the expressions of several extracellular matrix components which play a critical role in invasion and metastasis. Curcumin reduces CD24 expression in a dose-dependent manner in CRC cells. Moreover, E-cadherin expression is upregulated by curcumin and serves as an inhibitor of EMT. These results suggest that curcumin executes its antimetastasis function through downregulation of Sp-1, FAK, and CD24 and by promoting E-cadherin expression in CRC cells. PMID:23970932

  3. MiR-429 reverses epithelial-mesenchymal transition by restoring E-cadherin expression in bladder cancer

    PubMed Central

    Wu, Chia-Lun; Ho, Jar-Yi; Chou, Sheng-Chieh; Yu, Dah-Shyong

    2016-01-01

    Epithelial-mesenchymal transition (EMT) accompanying loss of E-cadherin is important for invasiveness and metastasis of bladder cancer. MicroRNAs (miRs) had been associated with cancer progression and differentiation in several cancers. Our goal is to find out the specific miR which modulates EMT in bladder cancer. Real-time quantitative polymerase chain reaction was used to measure the miRs expression in urothelial cell carcinoma (UCC) cell lines. MiR or siRNA mimics was used to regulate miR and mRNA level respectively. Migration and scratch assays were used to determine the migratory ability. Zymography assay was used to confirm the metalloproteinase activity. Western blotting was used to elucidate the mechanism which regulated by specific miR. MiR-429 was highly expressed in low grade UCC cell lines. Exogenous mimic of miR-429 treatment dramatically inhibited the migratory ability of T24 cells. MiR-429 downstream target ZEB1 was decreased, E-cadherin was restored, and β-catenin was contrarily decreased by exogenous mimic of miR-429 treatment in T24 cells. Cell invasive ability was also inhibited by exogenous mimic of miR-429 treatment through inactivating the MMP-2 activity in T24 cells. E-cadherin protein expression level was inhibited by E-cadherin siRNA accompanied with increasing cell migratory ability when compared with control group in low grade TSGH8301 cells. MiR-429 decreased the cell migratory and invasive abilities through reducing ZEB1 and β-catenin, restoring the E-cadherin expression and inactivation of MMP-2 of UCC cells. MiR-429 might be used as a progression marker of bladder cancer. PMID:27058893

  4. The expressions of NEDD9 and E-cadherin correlate with metastasis and poor prognosis in triple-negative breast cancer patients

    PubMed Central

    Li, Peng; Sun, Tingting; Yuan, Qingzhong; Pan, Guozheng; Zhang, Jian; Sun, Diwen

    2016-01-01

    Background Neural precursor cell expressed, developmentally downregulated 9 (NEDD9), a member of Crk-associated substrate family, is involved in cancer cell adhesion, migration, invasion, and epithelial–mesenchymal transition. E-cadherin is a key event in the cellular invasion during the epithelial–mesenchymal transition mechanism. The aim of this study was to evaluate the association among NEDD9 expression, E-cadherin expression, and survival in triple-negative breast cancer (TNBC) patients. Methods NEDD9 and E-cadherin expressions were analyzed by immunohistochemistry in 106 TNBC patients and 120 non-TNBC patients. And the association of clinicopathological factors with survival was analyzed using Kaplan–Meier analysis and Cox regression in TNBC patients. Results The results revealed that the rate of increased expression of NEDD9 and reduced expression of E-cadherin was significantly higher in TNBC group than that in non-TNBC group (P<0.001, both). Comparison of features between TNBC and non-TNBC groups showed that histological type (P=0.026) and lymph node metastasis (P=0.001) were significantly different. Correlation analysis showed that positive NEDD9 expression and negative E-cadherin expression were significantly correlated with lymph node metastasis and tumor-node-metastasis stage (P<0.05). In addition, the enhanced NEDD9 expression was significantly associated with a reduced 5-year survival for TNBC patients (overall survival [OS]: P=0.013; disease-free survival [DFS]: P=0.021). Negative E-cadherin expression showed a significantly worse 5-year OS and DFS (OS: P=0.011; DFS: P=0.012). Multivariate analysis showed that lymph node metastasis (OS: P=0.006; DFS: P=0.004), tumor-node-metastasis stage (OS: P=0.012; DFS: P=0.001), NEDD9 (OS: P=0.046; DFS: P=0.022), and E-cadherin (OS: P=0.022; DFS: P=0.025) independently predicted a poor prognosis of OS and DFS. Moreover, patients with NEDD9-positive/E-cadherin-negative expression had a significantly worse

  5. Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion

    PubMed Central

    1996-01-01

    Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta- catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549- 557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E- cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion. PMID:8991100

  6. Helicobacter spp. infection induces changes in epithelial proliferation and E-cadherin expression in the gastric mucosa of pigs.

    PubMed

    Bracarense, A P F R L; Yamasaki, L; Silva, E O; Oliveira, R L; Alfieri, A A

    2013-11-01

    Gastric disease is common in finishing pigs. Helicobacter spp. infection has been associated with gastritis, gastric ulcers and gastric neoplasia in man and animals. The aim of this study was to determine the effects of Helicobacter spp. infection on gastric morphology in pigs, with emphasis on glandular cell proliferation and E-cadherin expression. Samples of fundus and antrum from 67 finishing pigs were examined microscopically and by immunohistochemistry. The presence of Helicobacter spp. was confirmed by polymerase chain reaction (PCR). Mucosal changes were evaluated and epithelial proliferation was determined by evaluation of the morphometry of nucleolar organizer regions and counting proliferating cell nuclear antigen-positive cells and mitotic figures. Intercellular adhesion was evaluated by E-cadherin expression. In 47 (70%) pigs, Helicobacter spp. infection was confirmed by PCR. Histological findings associated with the infection included mononuclear cell infiltration of the lamina propria and glandular degeneration. There was a significant association between infection and epithelial proliferation in both regions as well as a decrease in the expression of E-cadherin in the antrum.

  7. E-cadherin inhibits nuclear accumulation of Nrf2: implications for chemoresistance of cancer cells.

    PubMed

    Kim, Won Dong; Kim, Young Woo; Cho, Il Je; Lee, Chang Ho; Kim, Sang Geon

    2012-03-01

    Nrf2 has an anti-carcinogenic effect. However, an increase in Nrf2 activity is also implicated in cancer chemoresistance. A switch from E-cadherin to N-cadherin affects the transdifferentiation and metastasis of cancer cells. In view of the key role of this switch in cancer malignancy, we investigated the regulatory effect of E-cadherin on Nrf2. In HEK293 cells, overexpression of E-cadherin inhibited the nuclear accumulation of Nrf2, and prevented Nrf2-dependent gene induction. GST pull-down and immunocytochemical assays verified the interaction between E-cadherin and Nrf2: E-cadherin bound the C-terminus of Nrf2, but not its N-terminus, which comprises the Neh2 domain responsible for phosphorylation of Ser40. Our finding that the mutation of Ser40 to alanine in Nrf2 did not affect the ability of E-cadherin to bind Nrf2 and repress target gene transactivation suggests that E-cadherin might not disturb the phosphorylation. Studies using mutant constructs of E-cadherin suggested that the β-catenin-binding domain contributes to the inhibitory effect of E-cadherin on Nrf2. Consistently, knockdown of β-catenin attenuated not only the effect of E-cadherin binding to Nrf2, but also Keap1-dependent ubiquitylation of Nrf2, and thereby increased Nrf2 activity, supporting the involvement of β-catenin in the interactions. Collectively, E-cadherin recruits Nrf2 through β-catenin, and assists the function of Keap1 for the inhibition of nuclear localization and transcriptional activity of Nrf2. In HepG2 cells, the loss of E-cadherin by either siRNA knockdown or treatment with TGFβ1 enhanced the constitutive or inducible activity of Nrf2, implying that chemoresistance of cancer cells upon the loss of E-cadherin might be associated with Nrf2. PMID:22302998

  8. Dissociation of E-cadherin/β-catenin complex by MG132 and bortezomib enhances CDDP induced cell death in oral cancer SCC-25 cells.

    PubMed

    Lü, Lanhai; Liu, Xiqiang; Wang, Cheng; Hu, Fengchun; Wang, Jianning; Huang, Hongzhang

    2015-12-01

    E-cadherin/β-catenin complex plays an important role in maintaining the homeostasis of tissues and regulating cell proliferation, survival and apoptosis. To address the relationships between the change of E-cadherin/β-catenin complex and cell apoptosis, human oral squamous carcinoma SCC-25 cells were used to investigate whether the dissociation of the E-cadherin/β-catenin complex was the main reason of MG132- or bortezomib-induced apoptosis. We found that MG132 or bortezomib alone induced remarkable loss of cell integrity and contact, inhibited cell growth, survival, migration and caused cell cycle arrest, intracellular ROS production. Further experiments showed that colony formations were significantly decreased by MG132 and bortezomib alone or plus cis-diaminedichloroplatinum (CDDP). Immunofluorescence staining showed that SCC-25 cells exhibited remarkable accumulations of β-catenin in cytoplasm and few E-cadherin in cell membranes after MG132 or bortezomib treatment. Western blot results showed that MG132 or bortezomib induced high accumulation of ubiquitinated proteins and activation of apoptosis related protein caspase-3. Meanwhile, the combinational use of MG132 or bortezomib with CDDP led to synergistic effects on SCC-25 cells. However, knockdown of β-catenin could decrease MG132 or bortezomib induced cell death. Taken together, our data suggest that the regulation of E-cadherin/β-catenin complex could be a promising therapeutic target to overcome the multidrug resistance of oral cancer.

  9. Quantification of mutant E-cadherin using bioimaging analysis of in situ fluorescence microscopy. A new approach to CDH1 missense variants

    PubMed Central

    Sanches, João Miguel; Figueiredo, Joana; Fonseca, Martina; Durães, Cecília; Melo, Soraia; Esménio, Sofia; Seruca, Raquel

    2015-01-01

    Missense mutations result in full-length proteins containing an amino acid substitution that can be neutral or deleterious, interfering with the normal conformation, localization, and function of a protein. A striking example is the presence of CDH1 (E-cadherin gene) germline missense variants in hereditary diffuse gastric cancer (HDGC), which represent a clinical burden for genetic counseling and surveillance of mutation carriers and their families. CDH1 missense variants can compromise not only the function of E-cadherin but also its expression pattern. Here, we propose a novel method to characterize E-cadherin signature in order to identify cases with E-cadherin deregulation and functional impairment. The strategy includes a bioimaging pipeline to quantify the expression level and characterize the distribution of the protein from in situ immunofluorescence images. The algorithm computes 1D (dimension intensity) radial and internuclear fluorescence profiles to generate expression outlines and 2D virtual cells representing a typical cell within the populations analyzed. Using this new approach, we verify that cells expressing mutant forms of E-cadherin display fluorescence profiles distinct from those of the wild-type cells. Mutant proteins showed a significantly decrease of fluorescence intensity at the membrane and often abnormal expression peaks in the cytoplasm, reflecting the underlying molecular mechanism of trafficking deregulation. Our results suggest employing this methodology as a complementary approach to evaluate the pathogenicity of E-cadherin missense variants. Moreover, it can be applied to a wide range of proteins and, more importantly, to diseases characterized by aberrant protein expression or trafficking deregulation. PMID:25388006

  10. Altered E-Cadherin Levels and Distribution in Melanocytes Precede Clinical Manifestations of Vitiligo.

    PubMed

    Wagner, Roselyne Y; Luciani, Flavie; Cario-André, Muriel; Rubod, Alain; Petit, Valérie; Benzekri, Laila; Ezzedine, Khaled; Lepreux, Sébastien; Steingrimsson, Eirikur; Taieb, A; Gauthier, Yvon; Larue, Lionel; Delmas, Véronique

    2015-07-01

    Vitiligo is the most common depigmenting disorder resulting from the loss of melanocytes from the basal epidermal layer. The pathogenesis of the disease is likely multifactorial and involves autoimmune causes, as well as oxidative and mechanical stress. It is important to identify early events in vitiligo to clarify pathogenesis, improve diagnosis, and inform therapy. Here, we show that E-cadherin (Ecad), which mediates the adhesion between melanocytes and keratinocytes in the epidermis, is absent from or discontinuously distributed across melanocyte membranes of vitiligo patients long before clinical lesions appear. This abnormality is associated with the detachment of the melanocytes from the basal to the suprabasal layers in the epidermis. Using human epidermal reconstructed skin and mouse models with normal or defective Ecad expression in melanocytes, we demonstrated that Ecad is required for melanocyte adhesiveness to the basal layer under oxidative and mechanical stress, establishing a link between silent/preclinical, cell-autonomous defects in vitiligo melanocytes and known environmental stressors accelerating disease expression. Our results implicate a primary predisposing skin defect affecting melanocyte adhesiveness that, under stress conditions, leads to disappearance of melanocytes and clinical vitiligo. Melanocyte adhesiveness is thus a potential target for therapy aiming at disease stabilization.

  11. The Anoikis Effector Bit1 Inhibits EMT through Attenuation of TLE1-Mediated Repression of E-Cadherin in Lung Cancer Cells

    PubMed Central

    Yao, Xin; Pham, Tri; Temple, Brandi; Gray, Selena; Cannon, Cornita; Chen, Renwei; Abdel-Mageed, Asim B.; Biliran, Hector

    2016-01-01

    The mitochondrial Bcl-2 inhibitor of transcription 1 (Bit1) protein is part of an anoikis-regulating pathway that is selectively dependent on integrins. We previously demonstrated that the caspase-independent apoptotic effector Bit1 exerts tumor suppressive function in lung cancer in part by inhibiting anoikis resistance and anchorage-independent growth in vitro and tumorigenicity in vivo. Herein we show a novel function of Bit1 as an inhibitor cell migration and epithelial–mesenchymal transition (EMT) in the human lung adenocarcinoma A549 cell line. Suppression of endogenous Bit1 expression via siRNA and shRNA strategies promoted mesenchymal phenotypes, including enhanced fibroblastoid morphology and cell migratory potential with concomitant downregulation of the epithelial marker E-cadherin expression. Conversely, ectopic Bit1 expression in A549 cells promoted epithelial transition characterized by cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Specific downregulation of E-cadherin in Bit1-transfected cells was sufficient to block Bit1-mediated inhibition of cell motility while forced expression of E-cadherin alone attenuated the enhanced migration of Bit1 knockdown cells, indicating that E-cadherin is a downstream target of Bit1 in regulating cell motility. Furthermore, quantitative real-time PCR and reporter analyses revealed that Bit1 upregulates E-cadherin expression at the transcriptional level through the transcriptional regulator Amino-terminal Enhancer of Split (AES) protein. Importantly, the Bit1/AES pathway induction of E-cadherin expression involves inhibition of the TLE1-mediated repression of E-cadherin, by decreasing TLE1 corepressor occupancy at the E-cadherin promoter as revealed by chromatin immunoprecipitation assays. Consistent with its EMT inhibitory function, exogenous Bit1 expression significantly suppressed the formation of lung metastases of A549 cells in an in vivo experimental

  12. E-cadherin germline mutation carriers: clinical management and genetic implications.

    PubMed

    Corso, Giovanni; Figueiredo, Joana; Biffi, Roberto; Trentin, Chiara; Bonanni, Bernardo; Feroce, Irene; Serrano, Davide; Cassano, Enrico; Annibale, Bruno; Melo, Soraia; Seruca, Raquel; De Lorenzi, Francesca; Ferrara, Francesco; Piagnerelli, Riccardo; Roviello, Franco; Galimberti, Viviana

    2014-12-01

    Hereditary diffuse gastric cancer is an autosomic dominant syndrome associated with E-cadherin protein (CDH1) gene germline mutations. Clinical criteria for genetic screening were revised in 2010 by the International Gastric Cancer Linkage Consortium at the Cambridge meeting. About 40 % of families fulfilling clinical criteria for this inherited disease present deleterious CDH1 germline mutations. Lobular breast cancer is a neoplastic condition associated with hereditary diffuse gastric cancer syndrome. E-cadherin constitutional mutations have been described in both settings, in gastric and breast cancers. The management of CDH1 asymptomatic mutation carriers requires a multidisciplinary approach; the only life-saving procedure is the prophylactic total gastrectomy after thorough genetic counselling. Several prophylactic gastrectomies have been performed to date; conversely, no prophylactic mastectomies have been described in CDH1 mutant carriers. However, the recent discovery of novel germline alterations in pedigree clustering only for lobular breast cancer opens up a new debate in the management of these individuals. In this critical review, we describe the clinical management of CDH1 germline mutant carriers providing specific recommendations for genetic counselling, clinical criteria, surveillance and/ or prophylactic surgery.

  13. Copper modulates zinc metalloproteinase-dependent ectodomain shedding of key signaling and adhesion proteins and promotes the invasion of prostate cancer epithelial cells.

    PubMed

    Parr-Sturgess, Catherine A; Tinker, Claire L; Hart, Claire A; Brown, Michael D; Clarke, Noel W; Parkin, Edward T

    2012-10-01

    A disintegrin and metalloproteinases (ADAMs) and matrix metalloproteinases (MMPs) are zinc metalloproteinases (ZMPs) that catalyze the "ectodomain shedding" of a range of cell surface proteins including signaling and adhesion molecules. These "sheddases" are associated with the invasion and metastasis of a range of cancers. Increased serum and tumor tissue levels of copper are also observed in several cancers, although little is known about how the metal might promote disease progression at the molecular level. In the current study, we investigated whether copper might regulate the ectodomain shedding of two key cell surface proteins implicated in the invasion and metastasis of prostate cancer, the Notch ligand Jagged1 and the adhesion molecule E-cadherin, and whether the metal was able to influence the invasion of the prostate cancer epithelial cell line PC3. Physiological copper concentrations stimulated the ZMP-mediated proteolysis of Jagged1 and E-cadherin in cell culture models, whereas other divalent metals had no effect. Copper-mediated Jagged1 proteolysis was also observed following the pretreatment of cells with cycloheximide and in a cell-free membrane system, indicating a posttranslational mechanism of sheddase activation. Finally, the concentrations of copper that stimulated ZMP-mediated protein shedding also enhanced PC3 invasion; an effect that could be negated using a sheddase inhibitor or copper chelators. Collectively, these data implicate copper as an important factor in promoting prostate cancer cell invasion and indicate that the selective posttranslational activation of ZMP-mediated protein shedding might play a role in this process.

  14. E-cadherin dis-engagement activates the Rap1 GTPase

    PubMed Central

    Asuri, Sirisha; Yan, Jingliang; Paranavitana, Nivanka C.; Quilliam, Lawrence A.

    2008-01-01

    E-cadherin based adherens junctions are finely regulated by multiple cellular signaling events. Here we show that the Ras-related Rap1 GTPase is enriched in regions of nascent cell-cell contacts and strengthens E-cadherin junctions: constitutively active Rap1 expressing MDCK cells exhibit increased junctional contact and resisted calcium depletion-induced cell-cell junction disruption. E-cadherin disengagement activated Rap1 and this correlated with E-cadherin association with the Rap GEFs, C3G and PDZ-GEF I. PDZ-GEF I associated with E-cadherin and β-catenin whereas C3G interaction with E-cadherin did not involve β-catenin. Knockdown of PDZ-GEF I in MDCK cells decreased Rap1 activity following E-cadherin junction disruption. We hereby show that Rap1 plays a role in the maintenance and repair of E-cadherin junctions and is activated via an “outside-in” signaling pathway initiated by E-cadherin and mediated at least in part by PDZ-GEF I. PMID:18767072

  15. Inhibition of p300 histone acetyltransferase activity in palate mesenchyme cells attenuates Wnt signaling via aberrant E-cadherin expression.

    PubMed

    Warner, Dennis R; Smith, Scott C; Smolenkova, Irina A; Pisano, M Michele; Greene, Robert M

    2016-03-01

    p300 is a multifunctional transcriptional coactivator that interacts with numerous transcription factors and exhibits protein/histone acetyltransferase activity. Loss of p300 function in humans and in mice leads to craniofacial defects. In this study, we demonstrated that inhibition of p300 histone acetyltransferase activity with the compound, C646, altered the expression of several genes, including Cdh1 (E-cadherin) in mouse maxillary mesenchyme cells, which are the cells that give rise to the secondary palate. The increased expression of plasma membrane-bound E-cadherin was associated with reduced cytosolic β-catenin, that led to attenuated signaling through the canonical Wnt pathway. Furthermore, C646 reduced both cell proliferation and the migratory ability of these cells. These results suggest that p300 histone acetyltransferase activity is critical for Wnt-dependent palate mesenchymal cell proliferation and migration, both processes that play a significant role in morphogenesis of the palate.

  16. Regulatory Variants and Disease: The E-Cadherin −160C/A SNP as an Example

    PubMed Central

    Li, Gongcheng; Pan, Tiejun; Guo, Dan

    2014-01-01

    Single nucleotide polymorphisms (SNPs) occurring in noncoding sequences have largely been ignored in genome-wide association studies (GWAS). Yet, amounting evidence suggests that many noncoding SNPs especially those that are in the vicinity of protein coding genes play important roles in shaping chromatin structure and regulate gene expression and, as such, are implicated in a wide variety of diseases. One of such regulatory SNPs (rSNPs) is the E-cadherin (CDH1) promoter −160C/A SNP (rs16260) which is known to affect E-cadherin promoter transcription by displacing transcription factor binding and has been extensively scrutinized for its association with several diseases especially malignancies. Findings from studying this SNP highlight important clinical relevance of rSNPs and justify their inclusion in future GWAS to identify novel disease causing SNPs. PMID:25276428

  17. A Pathway for the Control of Anoikis Sensitivity by E-Cadherin and Epithelial-to-Mesenchymal Transition▿‡

    PubMed Central

    Kumar, Sanjeev; Park, Sun Hee; Cieply, Benjamin; Schupp, Jane; Killiam, Elizabeth; Zhang, Fan; Rimm, David L.; Frisch, Steven M.

    2011-01-01

    Detachment of epithelial cells from matrix or attachment to an inappropriate matrix engages an apoptotic response known as anoikis, which prevents metastasis. Cellular sensitivity to anoikis is compromised during the oncogenic epithelial-to-mesenchymal transition (EMT), through unknown mechanisms. We report here a pathway through which EMT confers anoikis resistance. NRAGE (neurotrophin receptor-interacting melanoma antigen) interacted with a component of the E-cadherin complex, ankyrin-G, maintaining NRAGE in the cytoplasm. Oncogenic EMT downregulated ankyrin-G, enhancing the nuclear localization of NRAGE. The oncogenic transcriptional repressor protein TBX2 interacted with NRAGE, repressing the tumor suppressor gene p14ARF. P14ARF sensitized cells to anoikis; conversely, the TBX2/NRAGE complex protected cells against anoikis by downregulating this gene. This represents a novel pathway for the regulation of anoikis by EMT and E-cadherin. PMID:21746881

  18. Expression of E-cadherin and vimentin in oral squamous cell carcinoma

    PubMed Central

    Zhou, Jingping; Tao, Detao; Xu, Qing; Gao, Zhenlin; Tang, Daofang

    2015-01-01

    The aim of the study is to determine the levels of E-cadherin, vimentin expression in tumor tissues from patients with oral squamous cell carcinoma (OSCC), and the relationship between the expression of E-cadherin, vimentin and epithelial-mesenchymal transition, in order to explore its values for predicting the invasion and metastasis of oral squamous cell carcinoma, short survival of patients in many types of cancer. E-cadherin and vimentin expression of 10 benign and 42 OSCC tumor tissues was examined by immunohistochemical staining. E-cadherin is positively expressed in normal oral mucosa epithelium, but vimentin expression is not found in normal oral mucosa epithelia; the E-cadherin and vimentin were expressed in 26 of 42 (61.9%) and 16 of 42 (38.1%), respectively. No statistically difference was found for E-cadherin and vimentin expression in patients with different age, gender and tumor location, E-cadherin and vimentin expression was significantly associated with lymph node metastasis and tissue location (P < 0.05); E-cadherin expression was also significantly associated with tumor stage (P < 0.05); there are significantly difference between infiltrative margin and central area in patients with oral squamous cell carcinoma for E-cadherin and vimentin positive expression (P < 0.05). E-cadherin and vimentin positive expression was associated with tumor metastasis of oral squamous cell carcinoma. Our study preliminarily confirmed that EMT phenomenon is existed during the development of oral squamous cell carcinoma. Co-evaluation of E-cadherin and vimentin might be a valuable tool for predicting OSCC patient outcome. PMID:26045832

  19. Defining the E-cadherin repressor interactome in epithelial-mesenchymal transition: the PMC42 model as a case study.

    PubMed

    Hugo, Honor J; Kokkinos, Maria I; Blick, Tony; Ackland, M Leigh; Thompson, Erik W; Newgreen, Donald F

    2011-01-01

    Epithelial-mesenchymal transition (EMT) is a feature of migratory cellular processes in all stages of life, including embryonic development and wound healing. Importantly, EMT features cluster with disease states such as chronic fibrosis and cancer. The dissolution of the E-cadherin-mediated adherens junction (AJ) is a key preliminary step in EMT and may occur early or late in the growing epithelial tumour. This is a first step for tumour cells towards stromal invasion, intravasation, extravasation and distant metastasis. The AJ may be inactivated in EMT by directed E-cadherin cleavage; however, it is increasingly evident that the majority of AJ changes are transcriptional and mediated by an expanding group of transcription factors acting directly or indirectly to repress E-cadherin expression. A review of the current literature has revealed that these factors may regulate each other in a hierarchical pattern where Snail1 (formerly Snail) and Snail2 (formerly Slug) are initially induced, leading to the activation of Zeb family members, TCF3, TCF4, Twist, Goosecoid and FOXC2. Within this general pathway, many inter-regulatory relationships have been defined which may be important in maintaining the EMT phenotype. This may be important given the short half-life of Snail1 protein. We have investigated these inter-regulatory relationships in the mesenchymal breast carcinoma cell line PMC42 (also known as PMC42ET) and its epithelial derivative, PMC42LA. This review also discusses several newly described regulators of E-cadherin repressors including oestrogen receptor-α and new discoveries in hypoxia- and growth factor-induced EMT. Finally, we evaluated how these findings may influence approaches to current cancer treatment.

  20. Enhanced Biological Functions of Human Mesenchymal Stem-Cell Aggregates Incorporating E-Cadherin-Modified PLGA Microparticles.

    PubMed

    Zhang, Yan; Mao, Hongli; Gao, Chao; Li, Suhua; Shuai, Qizhi; Xu, Jianbin; Xu, Ke; Cao, Lei; Lang, Ren; Gu, Zhongwei; Akaike, Toshihiro; Yang, Jun

    2016-08-01

    Mesenchymal stem cells (MSCs) have emerged as a promising source of multipotent cells for various cell-based therapies due to their unique properties, and formation of 3D MSC aggregates has been explored as a potential strategy to enhance therapeutic efficacy. In this study, poly(lactic-co-glycolic acid) (PLGA) microparticles modified with human E-cadherin fusion protein (hE-cad-PLGA microparticles) have been fabricated and integrated with human MSCs to form 3D cell aggregates. The results show that, compared with the plain PLGA, the hE-cad-PLGA microparticles distribute within the aggregates more evenly and further result in a more significant improvement of cellular proliferation and secretion of a series of bioactive factors due to the synergistic effects from the bioactive E-cadherin fragments and the PLGA microparticles. Meanwhile, the hE-cad-PLGA microparticles incorporated in the aggregates upregulate the phosphorylation of epidermal growth factor receptors and activate the AKT and ERK1/2 signaling pathways in the MSCs. Additionally, the E-cadherin/β-catenin cellular membrane complex in the MSCs is markedly stimulated by the hE-cad-PLGA microparticles. Therefore, engineering 3D cell aggregates with hE-cad-PLGA microparticles can be a promising method for ex vivo multipotent stem-cell expansion with enhanced biological functions and may offer a novel route to expand multipotent stem-cell-based clinical applications. PMID:27245478

  1. Protein adhesion force dynamics and single adhesion events.

    PubMed Central

    Sagvolden, G

    1999-01-01

    Using the manipulation force microscope, a novel atomic force microscope, the adhesion forces of bovine serum albumin, myoglobin, ferritin, and lysozyme proteins to glass and polystyrene substrates were characterized by following the force necessary to displace an adsorbed protein-covered microsphere over several orders of magnitude in time. This force was consistent with a power law with exponent a = 0.37 +/- 0.03 on polystyrene, indicating that there is no typical time scale for adhesion on this substrate. On glass, the rate of adhesion depended strongly on protein charge. Forces corresponding to single protein adhesion events were identified. The typical rupture force of a single lysozyme, ferritin, bovine serum albumin, and myoglobin protein adhering to glass was estimated to be 90 +/- 10 pN, 115 +/- 13 pN, 277 +/- 44 pN, and 277 +/- 44 pN, respectively, using a model of the experimental system. These forces, as well as the force amplitudes on hydrophobic polystyrene, correlate with protein stiffness. PMID:10388777

  2. Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

    SciTech Connect

    Kouchi, Zen; Fujiwara, Yuki; Yamaguchi, Hideki; Nakamura, Yoshikazu; Fukami, Kiyoko

    2011-05-20

    Highlights: {yields} We analyzed Phosphatidylinositol 5-phosphate kinase II{beta} (PIPKII{beta}) function in cancer. {yields} PIPKII{beta} is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. {yields} PIPKII{beta} suppresses cellular motility through E-cadherin induction in SW480 cells. {yields} Nuclear PIP{sub 2} but not plasma membrane-localized PIP{sub 2} mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1{alpha},25-dihydroxyvitamin D{sub 3} (1{alpha},25(OH){sub 2}D{sub 3}) has anti-cancer activity in several colon cancers. 1{alpha},25(OH){sub 2}D{sub 3} induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKII{beta}) but not PIPKII{alpha} is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLC{delta}1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P{sub 2}) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLC{delta}1 PHD inhibited 1{alpha},25(OH){sub 2}D{sub 3}-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P{sub 2} production mediates E-cadherin expression through PIPKII{beta} in a VDR-dependent manner. PIPKII{beta} is also involved in the suppression of the cell motility induced by 1{alpha},25(OH){sub 2}D{sub 3}. These results indicate that PIPKII{beta}-mediated PI(4,5)P{sub 2} signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.

  3. Estrogen-mediated down-regulation of E-cadherin in breast cancer cells.

    PubMed

    Oesterreich, Steffi; Deng, Wanleng; Jiang, Shiming; Cui, Xiaojiang; Ivanova, Margarita; Schiff, Rachel; Kang, Kaiyan; Hadsell, Darryl L; Behrens, Jürgen; Lee, Adrian V

    2003-09-01

    E-cadherin is an important mediator of cell-cell interactions, and has been shown to play a crucial role in breast tumor suppression. Its inactivation occurs through instability at its chromosomal locus and mutations, but also through epigenetic mechanisms such as promoter hypermethylation and transcriptional silencing. We show here that the potent mitogen estrogen causes down-regulation of E-cadherin levels in both normal and tumorigenic breast epithelial cells, and that this down-regulation is reversed by antiestrogens. The reduction in E-cadherin levels is via a decrease in promoter activity and subsequent mRNA levels. Chromatin immunoprecipitation assays revealed that estrogen receptor and corepressors were bound to the E-cadherin promoter, and that overexpression of corepressors such as scaffold attachment factor B resulted in enhanced repression of E-cadherin. We propose that estrogen-mediated down-regulation of E-cadherin is a novel way of reducing E-cadherin levels in estrogen receptor-positive breast cancer.

  4. Epigenetic repression of E-cadherin expression by hepatitis B virus x antigen in liver cancer.

    PubMed

    Arzumanyan, A; Friedman, T; Kotei, E; Ng, I O L; Lian, Z; Feitelson, M A

    2012-02-01

    Loss of E-cadherin is associated with acquisition of metastatic capacity. Numerous studies suggest that histone deacetylation and/or hypermethylation of CpG islands in E-cadherin gene (CDH1) are major mechanisms responsible for E-cadherin silencing in different tumors and cancer cell lines. The hepatitis B virus (HBV)-encoded X antigen, HBx, contributes importantly to the development of hepatocellular carcinoma using multiple mechanisms. Experiments were designed to test if in addition to CDH1 hypermethylation HBx promotes epigenetic modulation of E-cadherin transcriptional activity through histone deacetylation and miR-373. The relationships between HBx, E-cadherin, mSin3A, Snail-1 and miR-373 were evaluated in HBx expressing (HepG2X) and control (HepG2CAT) cells by western blotting, immunoprecipitation (IP), chromatin IP as well as by immunohistochemical staining of liver and tumor tissue sections from HBV-infected patients. In HepG2X cells, decreased levels of E-cadherin and elevated levels of mSin3A and Snail-1 were detected. Reciprocal IP with anti-HBx and anti-mSin3A demonstrated mutual binding. Furthermore, HBx-mSin3A colocalization was detected by immunofluorescent staining. HBx downregulated E-cadherin expression by the recruitment of the mSin3A/histone deacetylase complex to the Snail-binding sites in human CDH1. Histone deacetylation inhibition by Trichostatin-A treatment restored E-cadherin expression. Mir-373, a positive regulator of E-cadherin expression, was downregulated by HBx in HepG2X cells and tissue sections from HBV-infected patients. Thus, histone deacetylation of CDH1 and downregulation of miR-373, together with the previously demonstrated hypermethylation of CDH1 by HBx, may be important for the understanding of HBV-related carcinogenesis.

  5. CD133 expression correlates with membrane beta-catenin and E-cadherin loss from human hair follicle placodes during morphogenesis.

    PubMed

    Gay, Denise L; Yang, Chao-Chun; Plikus, Maksim V; Ito, Mayumi; Rivera, Charlotte; Treffeisen, Elsa; Doherty, Laura; Spata, Michelle; Millar, Sarah E; Cotsarelis, George

    2015-01-01

    Genetic studies suggest that the major events of human hair follicle development are similar to those in mice, but detailed analyses of this process are lacking. In mice, hair follicle placode "budding" is initiated by invagination of Wnt-induced epithelium into the underlying mesenchyme. Modification of adherens junctions (AJs) is clearly required for budding. Snail-mediated downregulation of AJ component E-cadherin is important for placode budding in mice. Beta-catenin, another AJ component, has been more difficult to study owing to its essential functions in Wnt signaling, a prerequisite for hair follicle placode induction. Here, we show that a subset of human invaginating hair placode cells expresses the stem cell marker CD133 during early morphogenesis. CD133 associates with membrane beta-catenin in early placodes, and its continued expression correlates with loss of beta-catenin and E-cadherin from the cell membrane at a time when E-cadherin transcriptional repressors Snail and Slug are not implicated. Stabilization of CD133 via anti-CD133 antibody treatment of human fetal scalp explants depresses beta-catenin and E-cadherin membrane localization. We discuss this unique correlation and suggest a hypothetical model whereby CD133 promotes morphogenesis in early hair follicle placodes through the localized removal of membrane beta-catenin proteins and subsequent AJ dissolution.

  6. Dog as model for down-expression of E-cadherin and beta-catenin in tubular epithelial cells in renal fibrosis.

    PubMed

    Aresu, Luca; Rastaldi, Maria Pia; Pregel, Paola; Valenza, Federico; Radaelli, Enrico; Scanziani, Eugenio; Castagnaro, Massimo

    2008-12-01

    Mechanism of renal fibrosis leading to end stage kidney remains still a challenge of interest in humans. The pathogenesis of chronic kidney disease is characterized by progressive loss of kidney function and fibrosis. The mechanism of epithelial-mesenchymal transition (EMT) has been predominantly studied in in vitro studies, and we previously demonstrated the EMT of tubular epithelial cells in dogs. In this study, we examined and quantified the modifications of cadherin-catenin complex by immunohistochemistry of E-cadherin and beta-catenin and the mesenchymal marker vimentin in 25 dogs with three different spontaneous inflammatory renal diseases. Results showed a significant down-expression of levels of E-cadherin and beta-catenin directly correlated with the tubular-interstitial damage (TID). In TID grades 2 and 3, E-cadherin expression was significantly reduced (p < 0.001). beta-catenin expression was overall similar to E-cadherin. The mesenchymal-associated protein, vimentin, was de novo identified in tubules within areas of inflammation. In this work, we identified the loss of cadherin or catenin expression as a progressive mechanism in tubulo-interstitial fibrosis, which allows dissociation of structural integrity of renal epithelia and loss of epithelial polarity. The dog might result more significant as model for new therapies.

  7. CD133 expression correlates with membrane beta-catenin and e-cadherin loss from human hair follicle placodes during morphogenesis

    PubMed Central

    Gay, Denise; Yang, Chao-Chun; Plikus, Maksim; Ito, Mayumi; Rivera, Charlotte; Treffeisen, Elsa; Doherty, Laura; Spata, Michelle; Millar, Sarah E.; Cotsarelis, George

    2014-01-01

    Genetic studies suggest that the major events of human hair follicle development are similar to those in mice, but detailed analyses of this process are lacking. In mice, hair follicle placode ‘budding’ is initiated by invagination of Wnt-induced epithelium into the underlying mesenchyme. Modification of adherens junctions is clearly required for budding. Snail-mediated downregulation of adherens junction component E-cadherin is important for placode budding in mice. Beta-catenin, another adherens junction component, has been more difficult to study due to its essential functions in Wnt signaling, a prerequisite for hair follicle placode induction. Here, we show that a subset of human invaginating hair placode cells expresses the stem cell marker CD133 during early morphogenesis. CD133 associates with membrane beta-catenin in early placodes and its continued expression correlates with loss of beta-catenin and E-cadherin from the cell membrane at a time when E-cadherin transcriptional repressors Snail and Slug are not implicated. Stabilization of CD133 via anti-CD133 antibody treatment of human fetal scalp explants depresses beta-catenin and E-cadherin membrane localization. We discuss this unique correlation and suggest a hypothetical model whereby CD133 promotes morphogenesis in early hair follicle placodes through the localized removal of membrane beta-catenin proteins and subsequent adherens junction dissolution. PMID:25010141

  8. An E-cadherin-mediated hitchhiking mechanism for C. elegans germ cell internalization during gastrulation

    PubMed Central

    Chihara, Daisuke; Nance, Jeremy

    2012-01-01

    Gastrulation movements place endodermal precursors, mesodermal precursors and primordial germ cells (PGCs) into the interior of the embryo. Somatic cell gastrulation movements are regulated by transcription factors that also control cell fate, coupling cell identity and position. By contrast, PGCs in many species are transcriptionally quiescent, suggesting that they might use alternative gastrulation strategies. Here, we show that C. elegans PGCs internalize by attaching to internal endodermal cells, which undergo morphogenetic movements that pull the PGCs into the embryo. We show that PGCs enrich HMR-1/E-cadherin at their surfaces to stick to endoderm. HMR-1 expression in PGCs is necessary and sufficient to ensure internalization, suggesting that HMR-1 can promote PGC-endoderm adhesion through a mechanism other than homotypic trans interactions between the two cell groups. Finally, we demonstrate that the hmr-1 3′ untranslated region promotes increased HMR-1 translation in PGCs. Our findings reveal that quiescent PGCs employ a post-transcriptionally regulated hitchhiking mechanism to internalize during gastrulation, and demonstrate a morphogenetic role for the conserved association of PGCs with the endoderm. PMID:22675206

  9. Neural Cell Adhesion Protein CNTN1 Promotes the Metastatic Progression of Prostate Cancer.

    PubMed

    Yan, Judy; Ojo, Diane; Kapoor, Anil; Lin, Xiaozeng; Pinthus, Jehonathan H; Aziz, Tariq; Bismar, Tarek A; Wei, Fengxiang; Wong, Nicholas; De Melo, Jason; Cutz, Jean-Claude; Major, Pierre; Wood, Geoffrey; Peng, Hao; Tang, Damu

    2016-03-15

    Prostate cancer metastasis is the main cause of disease-related mortality. Elucidating the mechanisms underlying prostate cancer metastasis is critical for effective therapeutic intervention. In this study, we performed gene-expression profiling of prostate cancer stem-like cells (PCSC) derived from DU145 human prostate cancer cells to identify factors involved in metastatic progression. Our studies revealed contactin 1 (CNTN1), a neural cell adhesion protein, to be a prostate cancer-promoting factor. CNTN1 knockdown reduced PCSC-mediated tumor initiation, whereas CNTN1 overexpression enhanced prostate cancer cell invasion in vitro and promoted xenograft tumor formation and lung metastasis in vivo. In addition, CNTN1 overexpression in DU145 cells and corresponding xenograft tumors resulted in elevated AKT activation and reduced E-cadherin (CDH1) expression. CNTN1 expression was not readily detected in normal prostate glands, but was clearly evident on prostate cancer cells in primary tumors and lymph node and bone metastases. Tumors from 637 patients expressing CNTN1 were associated with prostate cancer progression and worse biochemical recurrence-free survival following radical prostatectomy (P < 0.05). Collectively, our findings demonstrate that CNTN1 promotes prostate cancer progression and metastasis, prompting further investigation into the mechanisms that enable neural proteins to become aberrantly expressed in non-neural malignancies.

  10. Immunolocalization of E-cadherin and β-catenin in the cyclic and early pregnant canine endometrium.

    PubMed

    Payan-Carreira, R; Pires, M A; Santos, C; Holst, B Ström; Colaço, J; Rodriguez-Martinez, H

    2016-09-01

    Putative changes in E-cadherin and β-catenin during implantation in dogs are of interest to study, as they are relevant proteins for epithelial integrity. E-cadherin and β-catenin were immunolocalized in the canine endometrium during the estrous cycle and early pregnancy, using monoclonal antibodies. Both proteins were detected in all types of endometrial epithelia (surface epithelium [SE], superficial glandular, and deep glandular epithelia) at all stages of the estrous cycle and in early placental structures. E-cadherin depicted a gradient of intensity apparently being lowest in the SE to progressively increase toward the deepness of the endometrial glands, regardless of the stage of estrous cycle. The overall immunostaining was, however, weaker at diestrus. In pregnant samples, the trophoblast was conspicuously immunolabeled compared with the endometrial surface lining epithelium. In the latter, the cytoplasmic pattern predominated over the membrane-bound, as was also seen in the decidual cells of the placental labyrinth. In the early placenta, only trophoblast cells and lacunae retained membrane signals. β-Catenin membrane labeling appeared relatively constant throughout the cycle, although a tendency toward a decrease in intensity was detected at the secretory stages. In addition, a dislocation of the immunoreaction from membrane to the cytoplasm was observed in both the SE and the glandular epithelia at particular stages of the cycle. In early pregnancy, a loss of the membranous pattern was observed in the SE and labyrinth, but neither on trophoblast nor in lacunae. The results show the existence of a softening of the adherens junctional complex in progestagen-dominated stages favoring embryo-maternal interactions and endometrial invasion during canine implantation. PMID:27155731

  11. Loss of heterozygosity at E-cadherin and other loci on chromosome 16 in ovarian cancer

    SciTech Connect

    Han, H.; Chen, S.S.; Yang-Feng, T.L.

    1994-09-01

    Our study of genome-wide screening and mapping of genetic aberrations by comparative genomic hybridization (CGH) revealed that the copy number of DNA sequences on chromosome 16q was reduced in a significant number of ovarian tumors. Loss of heterozygosity (LOH) on 16q has only been reported in ovarian tumors from the Japanese population; this arm has not been thoroughly studied in most LOH analyses of ovarian cancers. We have investigated LOH status at four chromosome 16q loci in 74 common epithelial ovarian tumors (benign and borderline 16, grade I 5, GII 6 and GIII 47). LOH frequencies of .27 (9/33), .19 (10/54), .23 (8/35) and .24 (10/42) were found at HP, D16S408, D16S421 and E-cadherin (E-cad) loci, respectively. Considering overall LOH on chromosome 16q, 26 out of 62 cases (41.9%) heterozygous for one or more loci showed LOH at a minimum of one locus. Our results are consistent with the observation made by CGH and also imply that most LOH in 16q is associated with physical deletion of at least a portion of 16q. E-cad is an intercellular adhesion molecule of epithelial tissues, and reduced E-cad expression is associated with invasiveness of several cancers (breast, bladder, lung, etc.). Thus E-cad may function as a tumor suppressor gene. In addition, the cell matrix adhesion regulator gene (CMAR), which is located distal to E-cad, may be a second candidate. This possibility is currently being investigated by additional LOH analysis. Southern analysis of DNA from tumors with LOH using a full-length E-cad cDNA probe detected only Pvull and MspI polymorphisms but no gross alterations in the remaining alleles. Further studies by SSCP, which explore the possible inactivating mutations in the remaining E-cad alleles, are in progress.

  12. Hypoxia induced E-cadherin involving regulators of Hippo pathway due to HIF-1α stabilization/nuclear translocation in bone metastasis from breast carcinoma

    SciTech Connect

    Maroni, Paola; Matteucci, Emanuela; Drago, Lorenzo; Banfi, Giuseppe; Bendinelli, Paola; Desiderio, Maria Alfonsina

    2015-01-15

    The present study deals with the molecular mechanisms involved in the regulation of E-cadherin expression under hypoxia, because the adjustment of the amount of E-cadherin due to physical stimuli of the microenvironment might influence the colonization of metastasis to skeleton. We analyzed the effect of 1% oxygen tension, that is similar to that encountered in the bone marrow by metastatic cells spreading from breast carcinoma. The purpose was to evaluate the hypoxia-orchestrated control of E-cadherin transactivation via hypoxia inducible factor-1 (HIF-1) and peroxisome proliferator activated receptor-γ (PPARγ), and the involvement of Hippo pathway members, as regulators of transcription factors. To give a translational significance to the study, we took into consideration human pair-matched ductal breast carcinoma and bone metastasis: E-cadherin and Wwox were expressed in bone metastasis but not in breast carcinoma, while HIF-1α and TAZ seemed localized principally in nuclei of metastasis and were found in all cell compartments of breast carcinoma. A close examination of the regulatory mechanisms underlying E-cadherin expression in bone metastasis was done in 1833 clone derived from MDA-MB231 cells. Hypoxia induced E-cadherin only in 1833 clone, but not in parental cells, through HIF-1 and PPARγ activities, while Wwox decreased. Since Wwox was highly expressed in bone metastasis, the effect of ectopic Wwox was evaluated, and we showed E-cadherin transactivation and enhanced invasiveness in WWOX transfected 1833 cells. Also, hypoxia was additive with ectopic Wwox remarkably enhancing HIF-1α nuclear shuttle and accumulation due to the lengthening of the half-life of HIF-1α protein; under this experimental condition HIF-1α appeared as a slower migrated band compared with control, in agreement with the phosphorylation state. The in vitro data strongly supported the almost exclusive presence of HIF-1α in nuclei of human-bone metastasis. Thus, we identified

  13. Dragon (repulsive guidance molecule RGMb) inhibits E-cadherin expression and induces apoptosis in renal tubular epithelial cells.

    PubMed

    Liu, Wenjing; Li, Xiaoling; Zhao, Yueshui; Meng, Xiao-Ming; Wan, Chao; Yang, Baoxue; Lan, Hui-Yao; Lin, Herbert Y; Xia, Yin

    2013-11-01

    Dragon is one of the three members of the repulsive guidance molecule (RGM) family, i.e. RGMa, RGMb (Dragon), and RGMc (hemojuvelin). We previously identified the RGM members as bone morphogenetic protein (BMP) co-receptors that enhance BMP signaling. Our previous studies found that Dragon is highly expressed in the tubular epithelial cells of mouse kidneys. However, the roles of Dragon in renal epithelial cells are yet to be defined. We now show that overexpression of Dragon increased cell death induced by hypoxia in association with increased cleaved poly(ADP-ribose) polymerase and cleaved caspase-3 levels in mouse inner medullary collecting duct (IMCD3) cells. Dragon also inhibited E-cadherin expression but did not affect epithelial-to-mesenchymal transition induced by TGF-β in IMCD3 cells. Previous studies suggest that the three RGM members can function as ligands for the receptor neogenin. Interestingly, our present study demonstrates that the Dragon actions on apoptosis and E-cadherin expression in IMCD3 cells were mediated by the neogenin receptor but not through the BMP pathway. Dragon expression in the kidney was up-regulated by unilateral ureteral obstruction in mice. Compared with wild-type mice, heterozygous Dragon knock-out mice exhibited 45-66% reduction in Dragon mRNA expression, decreased epithelial apoptosis, and increased tubular E-cadherin expression and had attenuated tubular injury after unilateral ureteral obstruction. Our results suggest that Dragon may impair tubular epithelial integrity and induce epithelial apoptosis both in vitro and in vivo.

  14. Molecular mechanics of mussel adhesion proteins

    NASA Astrophysics Data System (ADS)

    Qin, Zhao; Buehler, Markus J.

    2014-01-01

    Mussel foot protein (mfp), a natural glue produced by marine mussel, is an intriguing material because of its superior ability for adhesion in various environments. For example, a very small amount of this material is sufficient to affix a mussel to a substrate in water, providing structural support under extreme forces caused by the dynamic effects of waves. Towards a more complete understanding of its strength and underwater workability, it is necessary to understand the microscropic mechanisms by which the protein structure interacts with various substrates. However, none of the mussel proteins' structure is known, preventing us from directly using atomistic modeling to probe their structural and mechanical properties. Here we use an advanced molecular sampling technique to identify the molecular structures of two mussel foot proteins (mfp-3 and mfp-5) and use those structures to study their mechanics of adhesion, which is then incorporated into a continuum model. We calculate the adhesion energy of the mussel foot protein on a silica substrate, compute the adhesion strength based on results obtained from molecular modeling, and compare with experimental data. Our results show good agreement with experimental measurements, which validates the multiscale model. We find that the molecular structure of the folded mussel foot protein (ultimately defined by its genetic sequence) favors strong adhesion to substrates, where L-3,4-dihydroxyphenylalanine (or DOPA) protein subunits work in a cooperative manner to enhance adhesion. Our experimental data suggests a peak attachment force of 0.4±0.1 N, which compares favorably with the prediction from the multiscale model of Fc=0.21-0.33 N. The principles learnt from those results could guide the fabrication of new interfacial materials (e.g. composites) to integrate organic with inorganic surfaces in an effective manner.

  15. Effects of Arg-Gly-Asp-modified elastin-like polypeptide on pseudoislet formation via up-regulation of cell adhesion molecules and extracellular matrix proteins.

    PubMed

    Lee, Kyeong-Min; Jung, Gwon-Soo; Park, Jin-Kyu; Choi, Seong-Kyoon; Jeon, Won Bae

    2013-03-01

    Extracellular matrix (ECM) plays an important role in controlling the β-cell morphology, survival and insulin secretary functions. An RGD-modified elastin-like polypeptide (RGD-ELP), TGPG[VGRGD(VGVPG)(6)](20)WPC, has been reported previously as a bioactive matrix. In this study, to investigate whether RGD-ELP affects β-cell growth characteristics and insulin secretion, β-TC6 cells were cultured on the RGD-ELP coatings prepared via thermally induced phase transition. On RGD-ELP, β-TC6 cells clustered into an islet-like architecture with high cell viability. Throughout 7days' culture, the proliferation rate of the cells within a pseudoislet was similar to that of monolayer culture. Under high glucose (25mM), β-TC6 pseudoislets showed up-regulated insulin gene expression and exhibited glucose-stimulated insulin secretion. Importantly, the mRNA and protein abundances of cell adhesion molecules (CAM) E-cadherin and connexin-36 were much higher in pseudoislets than in monolayer cells. The siRNA-mediated inhibition of E-cadherin or connexin-36 expression severely limited pseudoislet formation. In addition, the mRNA levels of collagen types I and IV, fibronectin and laminin were significantly elevated in pseudoislets. The results suggest that RGD-ELP promotes pseudoislet formation via up-regulation of the CAM and ECM components. The functional roles of RGD-ELP are discussed in respect of its molecular composition.

  16. Isoform-specific function of calpains in cell adhesion disruption: studies in postlactational mammary gland and breast cancer.

    PubMed

    Rodríguez-Fernández, Lucía; Ferrer-Vicens, Iván; García, Concha; Oltra, Sara S; Zaragozá, Rosa; Viña, Juan R; García-Trevijano, Elena R

    2016-09-15

    Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in pathological states such as breast cancer. The intracellular Ca(2+)-dependent proteases, calpains (CAPNs), are known to cleave adhesion proteins. The isoform-specific function of CAPN1 and CAPN2 was explored and compared in two models of cell adhesion disruption: mice mammary gland during weaning-induced involution and breast cancer cell lines according to tumor subtype classification. In both models, E-cadherin, β-catenin, p-120, and talin-1 were cleaved as assessed by western blot analysis. Both CAPNs were able to cleave adhesion proteins from lactating mammary gland in vitro Nevertheless, CAPN2 was the only isoform found to co-localize with E-cadherin in cell junctions at the peak of lactation. CAPN2/E-cadherin in vivo interaction, analyzed by proximity ligation assay, was dramatically increased during involution. Calpain inhibitor administration prevented the cytosolic accumulation of truncated E-cadherin cleaved by CAPN2. Conversely, in breast cancer cells, CAPN2 was restricted to the nuclear compartment. The isoform-specific expression of CAPNs and CAPN activity was dependent on the breast cancer subtype. However, CAPN1 and CAPN2 knockdown cells showed that cleavage of adhesion proteins and cell migration was mediated by CAPN1, independently of the breast cancer cell line used. Data presented here suggest that the subcellular distribution of CAPN1 and CAPN2 is a major issue in target-substrate recognition; therefore, it determines the isoform-specific role of CAPNs during disruption of cell adhesion in either a physiological or a pathological context.

  17. Isoform-specific function of calpains in cell adhesion disruption: studies in postlactational mammary gland and breast cancer.

    PubMed

    Rodríguez-Fernández, Lucía; Ferrer-Vicens, Iván; García, Concha; Oltra, Sara S; Zaragozá, Rosa; Viña, Juan R; García-Trevijano, Elena R

    2016-09-15

    Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in pathological states such as breast cancer. The intracellular Ca(2+)-dependent proteases, calpains (CAPNs), are known to cleave adhesion proteins. The isoform-specific function of CAPN1 and CAPN2 was explored and compared in two models of cell adhesion disruption: mice mammary gland during weaning-induced involution and breast cancer cell lines according to tumor subtype classification. In both models, E-cadherin, β-catenin, p-120, and talin-1 were cleaved as assessed by western blot analysis. Both CAPNs were able to cleave adhesion proteins from lactating mammary gland in vitro Nevertheless, CAPN2 was the only isoform found to co-localize with E-cadherin in cell junctions at the peak of lactation. CAPN2/E-cadherin in vivo interaction, analyzed by proximity ligation assay, was dramatically increased during involution. Calpain inhibitor administration prevented the cytosolic accumulation of truncated E-cadherin cleaved by CAPN2. Conversely, in breast cancer cells, CAPN2 was restricted to the nuclear compartment. The isoform-specific expression of CAPNs and CAPN activity was dependent on the breast cancer subtype. However, CAPN1 and CAPN2 knockdown cells showed that cleavage of adhesion proteins and cell migration was mediated by CAPN1, independently of the breast cancer cell line used. Data presented here suggest that the subcellular distribution of CAPN1 and CAPN2 is a major issue in target-substrate recognition; therefore, it determines the isoform-specific role of CAPNs during disruption of cell adhesion in either a physiological or a pathological context. PMID:27402795

  18. E-cadherin Controls Bronchiolar Progenitor Cells and Onset of Preneoplastic Lesions in Mice12

    PubMed Central

    Ceteci, Fatih; Ceteci, Semra; Zanucco, Emanuele; Thakur, Chitra; Becker, Matthias; El-Nikhely, Nefertiti; Fink, Ludger; Seeger, Werner; Savai, Rajkumar; Rapp, Ulf R

    2012-01-01

    Although progenitor cells of the conducting airway have been spatially localized and some insights have been gained regarding their molecular phenotype, relatively little is known about the mechanisms regulating their maintenance, activation, and differentiation. This study investigates the potential roles of E-cadherin in mouse Clara cells, as these cells were shown to represent the progenitor/stem cells of the conducting airways and have been implicated as the cell of origin of human non-small cell lung cancer. Postnatal inactivation of E-cadherin affected Clara cell differentiation and compromised airway regeneration under injury conditions. In steady-state adult lung, overexpression of the dominant negative E-cadherin led to an expansion of the bronchiolar stem cells and decreased differentiation concomitant with canonical Wnt signaling activation. Expansion of the bronchiolar stem cell pool was associated with an incessant proliferation of neuroepithelial body.associated Clara cells that ultimately gave rise to bronchiolar hyperplasia. Despite progressive hyperplasia, only a minority of the mice developed pulmonary solid tumors, suggesting that the loss of E-cadherin function leads to tumor formation when additional mutations are sustained. The present study reveals that E-cadherin plays a critical role in the regulation of proliferation and homeostasis of the epithelial cells lining the conducting airways. PMID:23308049

  19. Application of APTES-Anti-E-cadherin film for early cancer monitoring.

    PubMed

    Ben Ismail, Manel; Carreiras, Franck; Agniel, Rémy; Mili, Donia; Sboui, Dejla; Zanina, Nahla; Othmane, Ali

    2016-10-01

    Cancer staging is a way to classify cancer according to the extent of the disease in the body. The stage is usually determined by several factors such as the location of the primary tumor, the tumor size, the degree of spread in the surrounding tissues, etc. The study of E-cadherin (EC) expression on cancerous cells of patients has revealed variations in the molecular expression patterns of primary tumors and metastatic tumors. The detection of these cells requires a long procedure involving conventional techniques, thus, the requirement for development of new rapid devices that permit direct and highly sensitive detection stimulates the sensing field progress. Here, we explore if E-cadherin could be used as a biomarker to bind and detect epithelial cancer cells. Hence, the sensitive and specific detection of E-cadherin expressed on epithelial cells is approached by immobilizing anti-E-cadherin antibody (AEC) onto aminosilanized indium-tin oxide (ITO) surface. The immunosensing surfaces have been characterized by electrochemical measurements, wettability and confocal microscopy and their performance has been assessed in the presence of cancer cell lines. Under optimal conditions, the resulting immunosensor displayed a selective detection of E-cadherin expressing cells, which could be detected either by fluorescence or electrochemical techniques. The developed immunosensing surface could provide a simple tool that can be applied to cancer staging. PMID:27423102

  20. Reduced expression of E-cadherin and p120-catenin and elevated expression of PLC-γ1 and PIKE are associated with aggressiveness of oral squamous cell carcinoma.

    PubMed

    Jiang, Yi; Liao, Liyan; Shrestha, Chandrama; Ji, Shangli; Chen, Ying; Peng, Jian; Wang, Larry; Liao, Eryuan; Xie, Zhongjian

    2015-01-01

    Oral squamous cell carcinoma (OSCC) is one of the most lethal malignant tumors. The cadherin/catenin cell-cell adhesion complex plays a major role in cancer development and progression. p120-catenin (p120) is a cytoplasmic molecule closely associated with E-cadherin which activates phospholipase C-γ1 (PLC-γ1). Our previous studies indicate that activation of PLC-γ1 plays a critical role in epidermal growth factor (EGF)-induced migration and proliferation of squamous cell carcinoma (SCC) cells and phosphatidylinositol 3-kinase enhancer (PIKE) is highly expressed in SCC cells and mediates EGFR-dependent SCC cell proliferation. Our current study was to determine whether the expression of E-cadherin, p120, PLC-γ1, and PIKE, is associated with OSCC. To address this issue, we assessed levels and localization of E-cadherin, p120, PLC-γ1, and PIKE in specimen of 92 patients with OSCC by immunohistochemistry. The results showed that the expression of E-cadherin, and p120 negatively correlated with the tumor differentiation and the expression of PLC-γ1 and PIKE positively correlated with the tumor differentiation. The expression of PLC-γ1 and PIKE in OSCC stage T3 + T4 or in OSCC with lymph node metastasis was significantly higher than that in OSCC stage T1 + T2 or in OSCC without lymph node metastasis. The expression of p120 positively correlated with levels of E-cadherin but negatively correlated with levels of PLC-γ1 and PIKE in OSCC. These data indicate that increased expression of PLC-γ1 and PIKE and decreased expression of E-cadherin and p120 are associated with the aggressiveness of OSCC.

  1. Carcinoma cells induce lumen filling and EMT in epithelial cells through soluble E-cadherin-mediated activation of EGFR.

    PubMed

    Patil, Pratima U; D'Ambrosio, Julia; Inge, Landon J; Mason, Robert W; Rajasekaran, Ayyappan K

    2015-12-01

    In epithelial cancers, carcinoma cells coexist with normal cells. Although it is known that the tumor microenvironment (TME) plays a pivotal role in cancer progression, it is not completely understood how the tumor influences adjacent normal epithelial cells. In this study, a three-dimensional co-culture system comprising non-transformed epithelial cells (MDCK) and transformed carcinoma cells (MSV-MDCK) was used to demonstrate that carcinoma cells sequentially induce preneoplastic lumen filling and epithelial-mesenchymal transition (EMT) in epithelial cysts. MMP-9 secreted by carcinoma cells cleaves cellular E-cadherin (encoded by CDH1) from epithelial cells to generate soluble E-cadherin (sE-cad), a pro-oncogenic protein. We show that sE-cad induces EGFR activation, resulting in lumen filling in MDCK cysts. Long-term sE-cad treatment induced EMT. sE-cad caused lumen filling by induction of the ERK signaling pathway and triggered EMT through the sustained activation of the AKT pathway. Although it is known that sE-cad induces MMP-9 release and consequent EGFR activation in tumor cells, our results, for the first time, demonstrate that carcinoma cells can induce sE-cad shedding in adjacent epithelial cells, which leads to EGFR activation and the eventual transdifferentiation of the normal epithelial cells. PMID:26483386

  2. From cell membrane to the nucleus: an emerging role of E-cadherin in gene transcriptional regulation

    PubMed Central

    Du, Wenjun; Liu, Xi; Fan, Guiling; Zhao, Xingsheng; Sun, Yanying; Wang, Tianzhen; Zhao, Ran; Wang, Guangyu; Zhao, Ci; Zhu, Yuanyuan; Ye, Fei; Jin, Xiaoming; Zhang, Fengmin; Zhong, Zhaohua; Li, Xiaobo

    2014-01-01

    E-cadherin is a well-known mediator of cell–cell adherens junctions. However, many other functions of E-cadherin have been reported. Collectively, the available data suggest that E-cadherin may also act as a gene transcriptional regulator. Here, evidence supporting this claim is reviewed, and possible mechanisms of action are discussed. E-cadherin has been shown to modulate the activity of several notable cell signalling pathways, and given that most of these pathways in turn regulate gene expression, we proposed that E-cadherin may regulate gene transcription by affecting these pathways. Additionally, E-cadherin has been shown to accumulate in the nucleus where documentation of an E-cadherin fragment bound to DNA suggests that E-cadherin may directly regulate gene transcription. In summary, from the cell membrane to the nucleus, a role for E-cadherin in gene transcription may be emerging. Studies specifically focused on this potential role would allow for a more thorough understanding of this transmembrane glycoprotein in mediating intra- and intercellular activities. PMID:25164084

  3. An adhesive protein capsule of Escherichia coli.

    PubMed Central

    Orskov, I; Birch-Andersen, A; Duguid, J P; Stenderup, J; Orskov, F

    1985-01-01

    The nature of the adhesive capacity of three hemagglutinating Escherichia coli strains that had earlier been described as nonfimbriated was studied. The strains that were isolated from human disease adhered to human buccal and urinary tract epithelial cells, an adhesion that was not inhibited by D-mannose. By crossed immunoelectrophoresis it was shown that the three strains produced a common antigen, Z1, developed after growth at 37 degrees C but not 18 degrees C. One of the strains produced an additional antigen, Z2, of almost the same electrophoretic mobility in crossed immunoelectrophoresis. A mutant of this strain deficient of its polysaccharide K antigen had maintained the adhesive capacity, indicating that the K antigen was not responsible for adhesion. A further mutant of the acapsular mutant produced a strongly reduced amount of the Z antigens and had lost the ability to adhere. The Z1 (and Z2?) antigens were therefore deemed to be responsible for adhesion. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of cells of the three strains, a heavy Coomassie-blue stained line was seen, indicating the presence of a protein subunit of molecular weight slightly above 14,400. By immunoblotting with absorbed antiserum, it was shown that this protein was the same as that detected by crossed immunoelectrophoresis. Protease from Streptomyces griseus, but not trypsin, digested the protein. Heating to 100 degrees C did not affect it. By immunoelectron microscopy of embedded and sectioned bacteria that had first been treated with specific antisera and ferritin-labeled antirabbit immunoglobulin, the protein adhesin-antibody complex was found to surround the bacteria as a heavy capsule. After negative staining with uranylacetate (pH approximately 4), the capsule appeared as a mesh of very fine filaments. The possible role of this capsule in the pathogenesis of disease is discussed. Images PMID:2856913

  4. Soy and cottonseed protein blends as wood adhesives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As an environmentally friendlier alternative to adhesives from petroleum feedstock, soy proteins are currently being formulated as wood adhesives. Cottonseed proteins have also been found to provide good adhesive properties. In at least some cases, cottonseed proteins appear to form greater shear ...

  5. Brain Metastases from Lung Cancer Show Increased Expression of DVL1, DVL3 and Beta-Catenin and Down-Regulation of E-Cadherin

    PubMed Central

    Kafka, Anja; Tomas, Davor; Beroš, Vili; Pećina, Hrvoje Ivan; Zeljko, Martina; Pećina-Šlaus, Nives

    2014-01-01

    The susceptibility of brain to secondary formation from lung cancer primaries is a well-known phenomenon. In contrast, the molecular basis for invasion and metastasis to the brain is largely unknown. In the present study, 31 brain metastases that originated from primary lung carcinomas were analyzed regarding over expression of Dishevelled-1 (DVL1), Dishevelled-3 (DVL3), E-cadherin (CDH1) and beta-catenin (CTNNB1). Protein expressions and localizations were analyzed by immunohistochemistry. Genetic alterations of E-cadherin were tested by polymerase chain reaction (PCR)/loss of heterozygosity (LOH). Heteroduplex was used to investigate mutations in beta-catenin. DVL1 and DVL3 showed over expression in brain metastasis in 87.1% and 90.3% of samples respectively. Nuclear staining was observed in 54.8% of cases for DVL1 and 53.3% for DVL3. The main effector of the Wnt signaling, beta-catenin, was up-regulated in 56%, and transferred to the nucleus in 36% of metastases. When DVL1 and DVL3 were up-regulated the number of cases with nuclear beta-catenin significantly increased (p = 0.0001). Down-regulation of E-cadherin was observed in 80% of samples. Genetic analysis showed 36% of samples with LOH of the CDH1. In comparison to other lung cancer pathologies, the diagnoses adenocarcinoma and small cell lung cancer (SCLC) were significantly associated to CDH1 LOH (p = 0.001). Microsatellite instability was detected in one metastasis from adenocarcinoma. Exon 3 of beta-catenin was not targeted. Altered expression of Dishevelled-1, Dishevelled-3, E-cadherin and beta-catenin were present in brain metastases which indicates that Wnt signaling is important and may contribute to better understanding of genetic profile conditioning lung cancer metastasis to the brain. PMID:24933634

  6. Brain metastases from lung cancer show increased expression of DVL1, DVL3 and beta-catenin and down-regulation of E-cadherin.

    PubMed

    Kafka, Anja; Tomas, Davor; Beroš, Vili; Pećina, Hrvoje Ivan; Zeljko, Martina; Pećina-Šlaus, Nives

    2014-06-13

    The susceptibility of brain to secondary formation from lung cancer primaries is a well-known phenomenon. In contrast, the molecular basis for invasion and metastasis to the brain is largely unknown. In the present study, 31 brain metastases that originated from primary lung carcinomas were analyzed regarding over expression of Dishevelled-1 (DVL1), Dishevelled-3 (DVL3), E-cadherin (CDH1) and beta-catenin (CTNNB1). Protein expressions and localizations were analyzed by immunohistochemistry. Genetic alterations of E-cadherin were tested by polymerase chain reaction (PCR)/loss of heterozygosity (LOH). Heteroduplex was used to investigate mutations in beta-catenin. DVL1 and DVL3 showed over expression in brain metastasis in 87.1% and 90.3% of samples respectively. Nuclear staining was observed in 54.8% of cases for DVL1 and 53.3% for DVL3. The main effector of the Wnt signaling, beta-catenin, was up-regulated in 56%, and transferred to the nucleus in 36% of metastases. When DVL1 and DVL3 were up-regulated the number of cases with nuclear beta-catenin significantly increased (p=0.0001). Down-regulation of E-cadherin was observed in 80% of samples. Genetic analysis showed 36% of samples with LOH of the CDH1. In comparison to other lung cancer pathologies, the diagnoses adenocarcinoma and small cell lung cancer (SCLC) were significantly associated to CDH1 LOH (p=0.001). Microsatellite instability was detected in one metastasis from adenocarcinoma. Exon 3 of beta-catenin was not targeted. Altered expression of Dishevelled-1, Dishevelled-3, E-cadherin and beta-catenin were present in brain metastases which indicates that Wnt signaling is important and may contribute to better understanding of genetic profile conditioning lung cancer metastasis to the brain.

  7. Polarized E-cadherin endocytosis directs actomyosin remodeling during embryonic wound repair.

    PubMed

    Hunter, Miranda V; Lee, Donghoon M; Harris, Tony J C; Fernandez-Gonzalez, Rodrigo

    2015-08-31

    Embryonic epithelia have a remarkable ability to rapidly repair wounds. A supracellular actomyosin cable around the wound coordinates cellular movements and promotes wound closure. Actomyosin cable formation is accompanied by junctional rearrangements at the wound margin. We used in vivo time-lapse quantitative microscopy to show that clathrin, dynamin, and the ADP-ribosylation factor 6, three components of the endocytic machinery, accumulate around wounds in Drosophila melanogaster embryos in a process that requires calcium signaling and actomyosin contractility. Blocking endocytosis with pharmacological or genetic approaches disrupted wound repair. The defect in wound closure was accompanied by impaired removal of E-cadherin from the wound edge and defective actomyosin cable assembly. E-cadherin overexpression also resulted in reduced actin accumulation around wounds and slower wound closure. Reducing E-cadherin levels in embryos in which endocytosis was blocked rescued actin localization to the wound margin. Our results demonstrate a central role for endocytosis in wound healing and indicate that polarized E-cadherin endocytosis is necessary for actomyosin remodeling during embryonic wound repair.

  8. Polarized E-cadherin endocytosis directs actomyosin remodeling during embryonic wound repair

    PubMed Central

    Hunter, Miranda V.; Lee, Donghoon M.; Harris, Tony J.C.

    2015-01-01

    Embryonic epithelia have a remarkable ability to rapidly repair wounds. A supracellular actomyosin cable around the wound coordinates cellular movements and promotes wound closure. Actomyosin cable formation is accompanied by junctional rearrangements at the wound margin. We used in vivo time-lapse quantitative microscopy to show that clathrin, dynamin, and the ADP-ribosylation factor 6, three components of the endocytic machinery, accumulate around wounds in Drosophila melanogaster embryos in a process that requires calcium signaling and actomyosin contractility. Blocking endocytosis with pharmacological or genetic approaches disrupted wound repair. The defect in wound closure was accompanied by impaired removal of E-cadherin from the wound edge and defective actomyosin cable assembly. E-cadherin overexpression also resulted in reduced actin accumulation around wounds and slower wound closure. Reducing E-cadherin levels in embryos in which endocytosis was blocked rescued actin localization to the wound margin. Our results demonstrate a central role for endocytosis in wound healing and indicate that polarized E-cadherin endocytosis is necessary for actomyosin remodeling during embryonic wound repair. PMID:26304727

  9. Synthetic (glyco-)peptides of the homophilic recognition domain of E-cadherin lead to increased E-cadherin mRNA synthesis and are inductors of cell differentiation in primary lung cancer cell lines.

    PubMed

    Zeckey, Christian; Dahm, Manfred; Wallrath, Anja; Herr, Monika; Walter Kunz, Horst; Vahl, Christian-Friedrich; Horn, Sigrid

    2010-07-15

    E-cadherin is one of the critical molecules involved in the metastatic process in many types of cancer. Once combined, E-cadherin exceeds the amount of membranous E-cadherin on the cellular surface by activation of intracellular signaling cascades. Studies on transformed keratinocytes of the HaCat cell line showed induction of differentiation by synthetical partial structures of the homophilic binding region of E-cadherin. The knowledge of effects in lung cancer cells is sparse. Therefore, the effects in primary lung cancer cell lines were investigated. Four primary lung cancer cell lines were incubated for 3, 6, 12, 15, 18, and 24h with synthetic partial structures (peptide and glycopeptide). The control substance was sodium butyrate. mRNA was isolated, and relative quantification of E-cadherin was performed using the Real-Time PCR. During the stimulation period, morphologic pictures were taken, and immunohistochemical staining of membranous E-cadherin was performed. Life/dead assays were used to display cell vitality. The intracellular E-cadherin mRNA amount was increased after incubation with the synthetic partial structures. Life/dead assays showed improved survival and integrated cell/cell bindings after stimulation with the partial structures. Increased cell mortality was revealed after sodium butyrate incubation. An effect mediated via E-cadherin on the cellular surface is proposed. The two synthetic partial structures of the homophilic binding region of E-cadherin increased the intracellular E-cadherin mRNA amount, cell-cell bindings, and survival of the tumor cells. Extracellular binding by synthetic partial structures to the binding region may have a beneficial influence on tumor progression in the metastatic process. PMID:20403671

  10. Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation

    NASA Astrophysics Data System (ADS)

    Meng, Qing-Yuan; Akaike, Toshihiro

    2013-03-01

    Induced embryonic stem (ES) cells are expected to be promising cell resources for the observation of the cell behaviors in developmental biology as well as the implantation in cell treatments in human diseases. A recombinant E-cadherin substratum was developed as a cell recognizable substratum to maintain the ES cells' self-renewal and pluripotency at single cell level. Furthermore, the generation of various cell lineages in different germ layers, including hepatic or neural cells, was achieved on the chimeric protein layer precisely and effectively. The induction and isolation of specific cell population was carried out with the enhancing effect of other artificial extracellular matrices (ECMs) in enzyme-free process. The murine ES cell-derived cells showed highly morphological similarities and functional expressions to matured hepatocytes or neural progenitor cells.

  11. The Frizzled-dependent planar polarity pathway locally promotes E-cadherin turnover via recruitment of RhoGEF2

    PubMed Central

    Warrington, Samantha J.; Strutt, Helen; Strutt, David

    2013-01-01

    Polarised tissue elongation during morphogenesis involves cells within epithelial sheets or tubes making and breaking intercellular contacts in an oriented manner. Growing evidence suggests that cell adhesion can be modulated by endocytic trafficking of E-cadherin (E-cad), but how this process can be polarised within individual cells is poorly understood. The Frizzled (Fz)-dependent core planar polarity pathway is a major regulator of polarised cell rearrangements in processes such as gastrulation, and has also been implicated in regulation of cell adhesion through trafficking of E-cad; however, it is not known how these functions are integrated. We report a novel role for the core planar polarity pathway in promoting cell intercalation during tracheal tube morphogenesis in Drosophila embryogenesis, and present evidence that this is due to regulation of turnover and levels of junctional E-cad by the guanine exchange factor RhoGEF2. Furthermore, we show that core pathway activity leads to planar-polarised recruitment of RhoGEF2 and E-cad turnover in the epidermis of both the embryonic germband and the pupal wing. We thus reveal a general mechanism by which the core planar polarity pathway can promote polarised cell rearrangements. PMID:23364328

  12. Heme-oxygenase-1 implications in cell morphology and the adhesive behavior of prostate cancer cells

    PubMed Central

    Gueron, Geraldine; Giudice, Jimena; Valacco, Pia; Paez, Alejandra; Elguero, Belen; Toscani, Martin; Jaworski, Felipe; Leskow, Federico Coluccio; Cotignola, Javier; Marti, Marcelo; Binaghi, Maria; Navone, Nora; Vazquez, Elba

    2014-01-01

    Prostate cancer (PCa) is the second leading cause of cancer death in men. Although previous studies in PCa have focused on cell adherens junctions (AJs), key players in metastasis, they have left the molecular mechanisms unexplored. Inflammation and the involvement of reactive oxygen species (ROS) are critical in the regulation of cell adhesion and the integrity of the epithelium. Heme oxygenase-1 (HO-1) counteracts oxidative and inflammatory damage. Here, we investigated whether HO-1 is implicated in the adhesive and morphological properties of tumor cells. Genes differentially regulated by HO-1 were enriched for cell motility and adhesion biological processes. HO-1 induction, increased E-cadherin and β-catenin levels. Immunofluorescence analyses showed a striking remodeling of E-cadherin/β-catenin based AJs under HO-1 modulation. Interestingly, the enhanced levels of E-cadherin and β-catenin coincided with a markedly change in cell morphology. To further our analysis we sought to identify HO-1 binding proteins that might participate in the regulation of cell morphology. A proteomics approach identified Muskelin, as a novel HO-1 partner, strongly implicated in cell morphology regulation. These results define a novel role for HO-1 in modulating the architecture of cell-cell interactions, favoring a less aggressive phenotype and further supporting its anti-tumoral function in PCa. PMID:24961479

  13. Bromodichloromethane induces cell proliferation in different tissues of male F344 rats by suppression of E-cadherin expression via hypermethylation or transcriptional activation of c-myc and cyclin D1.

    PubMed

    Liao, Jing; Li, Xiao-Feng; Zhou, Shun-Chang; Luo, Yan; Liu, Ai-Lin; Lu, Wen-Qing

    2013-11-25

    The aim of this study was to investigate the mechanism of bromodichloromethane (BDCM) - induced cell proliferation in different tissues of male F344 rats. Rats were administered at doses of 0 and 100mg/kg/day BDCM dissolved in corn oil by gavage for 5 days/week for 1, 4, 8 and 12 weeks. Then the colon, kidney and liver were collected. No histologic lesions were observed in the colon of rats exposed to BDCM, while there were mild nephrotoxicity and marginal hepatotoxicity related to BDCM treatment. Moreover, BDCM enhanced cell proliferation in the colon and kidney but not in the liver. In colons, hypermethylation in E-cadherin promoter might be associated with inhibition of mRNA and protein expression after 12 weeks of BDCM exposure. In kidneys, BDCM decreased E-cadherin mRNA expression, accompanying with transcriptional activation of c-myc and cyclin D1. However, suppression of E-cadherin mRNA and protein expression occurred in the absence of significant changes in DNA methylation. Therefore, suppression of E-cadherin expression via hypermethylation or transcriptional activation of c-myc and cyclin D1 may be involved in BDCM-induced cell proliferation in different tissues of male F344 rats.

  14. Halogenated DOPA in a Marine Adhesive Protein

    PubMed Central

    Sun, Cheng Jun; Srivastava, Aasheesh; Reifert, Jack R.; Waite, J. Herbert

    2009-01-01

    The sandcastle worm Phragmatopoma californica, a marine polychaete, constructs a tube-like shelter by cementing together sand grains using a glue secreted from the building organ in its thorax. The glue is a mixture of post-translationally modified proteins, notably the cement proteins Pc-1 and Pc-2 with the amino acid, 3,4-dihydroxyphenyl-L-alanine (DOPA). Significant amounts of a halogenated derivative of DOPA were isolated from the worm cement following partial acid hydrolysis and capture of catecholic amino acids by phenylboronate affinity chromatography. Analysis by tandem mass spectrometry and 1H NMR indicates the DOPA derivative to be 2-chloro-4, 5-dihydroxyphenyl-L-alanine. The potential roles of 2-chloro-DOPA in chemical defense and underwater adhesion are considered. PMID:20126508

  15. Halogenated DOPA in a Marine Adhesive Protein.

    PubMed

    Sun, Cheng Jun; Srivastava, Aasheesh; Reifert, Jack R; Waite, J Herbert

    2009-02-01

    The sandcastle worm Phragmatopoma californica, a marine polychaete, constructs a tube-like shelter by cementing together sand grains using a glue secreted from the building organ in its thorax. The glue is a mixture of post-translationally modified proteins, notably the cement proteins Pc-1 and Pc-2 with the amino acid, 3,4-dihydroxyphenyl-L-alanine (DOPA). Significant amounts of a halogenated derivative of DOPA were isolated from the worm cement following partial acid hydrolysis and capture of catecholic amino acids by phenylboronate affinity chromatography. Analysis by tandem mass spectrometry and (1)H NMR indicates the DOPA derivative to be 2-chloro-4, 5-dihydroxyphenyl-L-alanine. The potential roles of 2-chloro-DOPA in chemical defense and underwater adhesion are considered.

  16. Level of expression of E-cadherin mRNA in colorectal cancer correlates with clinical outcome.

    PubMed Central

    Dorudi, S.; Hanby, A. M.; Poulsom, R.; Northover, J.; Hart, I. R.

    1995-01-01

    A series of colorectal carcinomas (n = 49) resected from patients with known clinical outcomes were analysed for E-cadherin expression using in situ hybridisation to measure mRNA. Patients surviving 5 years or longer (n = 31) exhibited significantly higher levels of E-cadherin mRNA than those surviving less than 5 years (n = 18, P = 0.003). These preliminary results from this small sample suggest that E-cadherin expression may be a useful prognostic marker in colorectal cancer patients. Images Figure 1 PMID:7880746

  17. The Characteristics and Prognostic Effect of E-Cadherin Expression in Colorectal Signet Ring Cell Carcinoma

    PubMed Central

    Wang, Renjie; Ma, Xiaoji; Li, Yaqi; He, Yiping; Huang, Dan; Cai, Sanjun; Peng, Junjie

    2016-01-01

    Purpose Signet ring cell carcinoma (SRCC) is rare. The aim of this study is to understand the clinicopathological features and identify the possible prognostic factors in colorectal SRCC. Methods Patients with SRCC who underwent primary lesion resection at Fudan University Shanghai Cancer Center from September 2008 to July 2014 were retrospectively analyzed. Patient’s gender, age, tumor location, depth of invasion, lymph node metastasis, synchronous distant metastasis, perineural invasion, lymphovascular invasion, and E-cadherin expression were studied with prognosis, and the correlation between E-cadherin expression and clinicopathological features were analyzed. All clinicopathological and molecular factors were put into multivariate analysis using Cox proportional hazards model for detecting independent prognostic factors. Results 59 patients accounting for 0.89% of total colorectal cancer patients met the criteria and were enrolled in the study. The median survival time is 28.9 months, and the 3-year survival rate is 62.7%. SRCC were seen more common in young male patients. Advanced stage was more common in SRCC, 58 (98.3%) patients had T3/T4 lesions, 52 (88.1%) patients had lymph node metastasis, and 14 (23.7%) patients had distant metastasis. Distant metastases were seen more common in peritoneal cavity. Distant metastasis (HR = 4.194, 95% CI: 1.297–13.567), lymphovascular invasion (HR = 2.888, 95% CI: 1.115–7.483), and E-cadherin expression (HR = 0.272, 95% CI: 0.096–0.768) were independent predictors for survival. Conclusions SRCC is a rare subtype of colorectal cancer with poor prognosis. Distant metastasis, lymphovascular invasion, and E-cadherin expression can predict prognosis of colorectal SRCCs independently. More precise therapy and more close surveillance are needed for these patients. PMID:27509205

  18. Quantification of topological features in cell meshes to explore E-cadherin dysfunction

    PubMed Central

    Mestre, Tânia; Figueiredo, Joana; Ribeiro, Ana Sofia; Paredes, Joana; Seruca, Raquel; Sanches, João Miguel

    2016-01-01

    In cancer, defective E-cadherin leads to cell detachment, migration and metastization. Further, alterations mediated by E-cadherin dysfunction affect cell topology and tissue organization. Herein, we propose a novel quantitative approach, based on microscopy images, to analyse abnormal cellular distribution patterns. We generated undirected graphs composed by sets of triangles which accurately reproduce cell positioning and structural organization within each image. Network analysis was developed by exploring triangle geometric features, namely area, edges length and formed angles, as well as their variance, when compared with the respective equilateral triangles. We generated synthetic networks, mimicking the diversity of cell-cell interaction patterns, and evaluated the applicability of the selected metrics to study topological features. Cells expressing wild-type E-cadherin and cancer-related mutants were used to validate our strategy. Specifically, A634V, R749W and P799R cancer-causing mutants present more disorganized spatial distribution when compared with wild-type cells. Moreover, P799R exhibited higher length and angle distortions and abnormal cytoskeletal organization, suggesting the formation of very dynamic and plastic cellular interactions. Hence, topological analysis of cell network diagrams is an effective tool to quantify changes in cell-cell interactions and, importantly, it can be applied to a myriad of processes, namely tissue morphogenesis and cancer. PMID:27151223

  19. Persisting and Increasing Neutrophil Infiltration Associates with Gastric Carcinogenesis and E-cadherin Downregulation

    PubMed Central

    Fu, Hualin; Ma, Yue; Yang, Meng; Zhang, Chunlei; Huang, Hai; Xia, Ying; Lu, Lungen; Jin, Weilin; Cui, Daxiang

    2016-01-01

    H. pylori-induced chronic inflammation is considered the most important cause of gastric cancer. The actual process how chronic inflammation triggers gastric carcinogenesis is still not clear. In this study, neutrophils and relative markers in gastric cancer development were examined with immunohistochemistry and fluorescence RNA in situ hybridization methods. On average, 24 times more neutrophils were found in gastric cancer tissues and about 9 times more neutrophils were found in gastric intestinal metaplasia tissues comparing to normal gastric tissue controls. CagA+ H. pylori infection in cancer adjacent tissues or EBV infection in cancer tissues did not increase neutrophil infiltration into gastric cancer tissues significantly. Neutrophil density was positively correlated with cell proliferation while negatively correlated with E-cadherin intensity. E-cadherin is also transcriptionally downregulated in gastric cancer tissues comparing to adjacent tissue controls. The increased neutrophils in the gastric cancer tissues appear to be related to increased chemoattractant IL-8 levels. In gastric cancers, neutrophil numbers were higher comparing to cancer adjacent tissues and not associated with patient ages, tumor invasion depth, tumor staging, metastasis or cancer types. The conclusion is that persisting and increasing neutrophil infiltration is associated with E-cadherin downregulation, cell proliferation and gastric carcinogenesis. PMID:27412620

  20. Benign, malignant salivary gland tumors: comparison of immunohistochemical expression of e-cadherin.

    PubMed

    Prabhu, Sudeendra; Kaveri, H; Rekha, K

    2009-07-01

    The aim of the present study was to assess any variation in the immunohistochemical expression of E-cadherin in benign and malignant salivary gland tumors. A total of 60 cases of benign and malignant salivary gland tumors were evaluated immunohistochemically for E-cadherin expression. These included 10 cases of pleomorphic adenoma (PA), 2 cases of canalicular adenoma (CA), 2 cases of myoepithelioma (MY), 24 cases of adenoid cystic carcinoma (ACC), 12 cases of mucoepidermoid carcinoma (MEC), 9 cases of adenocarcinoma (AC) and 1 case of carcinoma ex pleomorphic adenoma (Ca Ex PA). 48 cases (80%) showed positive expression, in which benign tumors exhibited relatively increased reactivity (85.7%) as compared to the malignant tumors (78.3%). 10 PA, 2 MY, 20 ACC, 9 MEC, 6 AC and 1 Ca Ex PA expressed E-cadherin. Negative expression was evident in CA, ACC, MEC and AC. Statistically significant reduction in reactivity was evident in mucoepidermoid carcinoma and adenocarcinoma, when compared to pleomorphic adenoma.

  1. Gene expression of WNTs, β-catenin and E-cadherin during the periimplantation period of pregnancy in pigs--involvement of steroid hormones.

    PubMed

    Kiewisz, Jolanta; Kaczmarek, Monika M; Andronowska, Aneta; Blitek, Agnieszka; Ziecik, Adam J

    2011-09-01

    WNTs (wingless-type MMTV integration site family, member) are morphogenes considered as important factors taking part in uterus developmental processes and implantation. β-catenin is a downstream effector of WNTs action within the cell as well as, through E-cadherin, affecting epithelial organization and function. This study was conducted to investigate WNT4, WNT5A, WNT7A, β-catenin (CTNNB1) and E-cadherin (CDH1) gene expression and protein localization in the endometrium during the periimplantation period. Furthermore, the effect of 17β-estradiol (E(2)) and progesterone (P(4)) on WNTs, CTNNB1 and CDH1 gene expression in the porcine endometrium in vitro was examined. WNT4 protein was localized in the luminal and glandular epithelium as well as in the basal lamina of the uterine mucosa. WNT5A protein was detected only in the luminal epithelium. WNT7A, β-catenin and E-cadherin protein were identified both in the luminal and glandular epithelial cells, however, WNT7A protein immunoreactivity varied during respective days of estrous cycle and/or pregnancy. Despite unchanged expression of WNT4 mRNA in the endometrium of cyclic and early pregnant pigs, the negative influence of E(2) on WNT4 gene during in vitro experiment was observed. WNT4 and CDH1 gene expression was negatively correlated with blood plasma E(2) and P(4) level in uterine luminal flushings (ULFs) on Day 12 of pregnancy. Expression of WNT5A gene was up-regulated in the endometrium on Day 9 of pregnancy when compared to the respective day of the estrous cycle. A significant decrease of WNT7A gene expression and increase of CDH1 mRNA amount was detected on Day 12 of pregnancy. Overall, the results show the spatial localization of WNT4, WNT5A, WNT7A, β-catenin and E-cadherin proteins in porcine endometrium during periimplantation period of pregnancy and indicate significant changes of WNT5A, WNT7A and CDH1 gene expression before implantation in the pig.

  2. A role for E-cadherin in ensuring cohesive migration of a heterogeneous population of non-epithelial cells

    PubMed Central

    Campbell, Kyra; Casanova, Jordi

    2015-01-01

    Collective cell migration is a key process underlying the morphogenesis of many organs as well as tumour invasion, which very often involves heterogeneous cell populations. Here we investigated how such populations can migrate cohesively in the Drosophila posterior midgut, comprised of epithelial and mesenchymal cells and show a novel role for the epithelial adhesion molecule E-cadherin (E-Cad) in mesenchymal cells. Despite a lack of junctions at the ultrastructure level, reducing E-Cad levels causes mesenchymal cells to detach from one another and from neighbouring epithelial cells; as a result, coordination between the two populations is lost. Moreover, Bazooka and recycling mechanisms are also required for E-Cad accumulation in mesenchymal cells. These results indicate an active role for E-Cad in mediating cohesive and ordered migration of non-epithelial cells, and discount the notion of E-Cad as just an epithelial feature that has to be switched off to enable migration of mesenchymal cells. PMID:26272476

  3. Exon 11 skipping of E-cadherin RNA downregulates its expression in Head and Neck cancer cells

    PubMed Central

    Sharma, Sanjai; Liao, Wei; Zhou, Xiaofeng; Wong, David T.W.; Lichtenstein, Alan

    2011-01-01

    E-cadherin is an important tumor suppressor gene whose expression is lost when cells acquire a metastatic phenotype. We analyzed the role of E-cadherin mis-splicing as a mechanism of its downregulation by analyzing a mis-spliced E-cadherin transcript that lacks exon 11 of this gene. This results in a frame shift and a premature termination codon which targets this transcript for degradation. Tumor tissues including breast (20%, n=9)), prostate (30%, n=9) and Head and Neck (H&N) (75%, n=8) cancer, express the exon 11 skipped transcripts (versus non-malignant controls) and its levels inversely correlate with E-cadherin expression. This is a novel mechanism of E-cadherin downregulation by mis-splicing in tumor cells which is observed in highly prevalent human tumors. In the H&N cancer model, non-tumorigenic keratinocytes express exon 11 skipped splice product 2–6 fold lower than the H&N tumor cell lines. Mechanistic studies reveal that SFRS2 (SC35), a splicing factor as one of the regulators that increases mis-splicing and downregulates E-cadherin expression. Furthermore, this splicing factor was found to be over expressed in five out of seven H&N cell lines and primary H&N tumors. Also, methylation of E-cadherin gene acts as a regulator of this aberrant splicing process. In two H&N cell lines, wild type transcript expression increased 16–25 folds while the percentage of exon 11 skipped transcripts in both the cell lines decreased 5–30 folds when cells were treated with a hypomethylating agent, azacytidine. Our findings reveal that promoter methylation and an upregulated splicing factor (SFRS2) are involved in the E-cadherin mis-splicing in tumors. PMID:21764905

  4. Transcriptional regulation of E-cadherin and oncoprotein E7 by valproic acid in HPV positive cell lines

    PubMed Central

    Faghihloo, Ebrahim; Akbari, Abolfazl; Adjaminezhad-Fard, Fatemeh; Mokhtari-Azad, Talat

    2016-01-01

    Objective(s): Valproic acid (VPA) has proven to be as one of the most promising useful drug with anticancer properties. In this study, we investigate the VPA effects on E-cadherin expression in HeLa, TC1, MKN45, and HCT116 cell lines. This study assesses the effects of VPA on human papillomavirus E7 expression in HPV positive cell lines. Materials and Methods: Cell lines were treated by 2 mmol/l VPA and expression of E-cadherin and E7 was analyzed by quantitative real-time PCR. Student’s t test and ANOVA were used to determine changes in expression levels. Results: The results revealed that mean of E-cadherin expression is increased by VPA 1.8 times in HCT116 and MKN45 cell lines, also the mean of E-cadherin mRNA levels is up-regulated 2.9 times in HeLa and TC1 cell lines. So, E-cadherin augmentation induced by VPA in HeLa and TC-1, HPV positive cell lines, is higher than HPV negative cell lines MKN45 and HCT116. The mean of HPV E7 expression is decreased by VPA, 4.6 times in in HeLa and TC-1 cell lines. Conclusion: This study demonstrates that re-expression of E-cadherin by VPA in HPV positive cell lines is more than HPV negative cell lines. Whereas, HPV E7 reduces the expression of E-cadherin, reduction of HPV E7 expression by VPA is related to more augmentation of E-cadherin in HPV positive cell lines. So, this study demonstrates that VPA has more anticancer properties in HPV positive cell lines, and could potentially be a promising candidate for cervical cancer treatment. PMID:27482340

  5. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

    PubMed Central

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N.; Patil, Navinkumar J.; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-01-01

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives. PMID:26508080

  6. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

    NASA Astrophysics Data System (ADS)

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N.; Patil, Navinkumar J.; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-10-01

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives.

  7. Betacellulin induces Slug-mediated down-regulation of E-cadherin and cell migration in ovarian cancer cells

    PubMed Central

    Zhao, Jianfang; Klausen, Christian; Qiu, Xin; Cheng, Jung-Chien; Chang, Hsun-Ming; Leung, Peter C.K.

    2016-01-01

    Epithelial ovarian cancer is the leading cause of death among gynaecological cancers. Previous studies have demonstrated that epidermal growth factor receptor (EGFR) ligands can induce ovarian cancer cell invasion by down-regulating E-cadherin. Betacellulin is a unique member of the EGF family. It is overexpressed in a variety of cancers and is associated with reduced survival. However, the biological functions and clinical significance of betacellulin in ovarian cancer remain unknown. In the current study, we tested the hypothesis that betacellulin induces ovarian cancer cell migration by suppressing E-cadherin expression. Treatment of SKOV3 and OVCAR5 ovarian cancer cell lines with betacellulin down-regulated E-cadherin, but not N-cadherin. In addition, betacellulin treatment increased the expression of Snail and Slug, and these effects were completely blocked by pre-treatment with EGFR inhibitor AG1478. Interestingly, only knockdown of Slug reversed the down-regulation of E-cadherin by betacellulin. Betacellulin treatment induced the activation of both the MEK-ERK and PI3K-Akt signaling pathways, and it also significantly increased ovarian cancer cell migration. Importantly, the effects of betacellulin on E-cadherin, Slug and cell migration were attenuated by pre-treatment with either U0126 or LY294002. Our results suggest that betacellulin induces ovarian cancer migration and Slug-dependent E-cadherin down-regulation via EGFR-mediated MEK-ERK and PI3K-Akt signaling. PMID:27129169

  8. Investigation of modified cottonseed protein adhesives for wood composites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several modified cottonseed protein isolates were studied and compared to corresponding soy protein isolates for their adhesive properties when bonded to wood composites. Modifications included treatments with alkali, guanidine hydrochloride, sodium dodecyl sulfate (SDS), and urea. Wood composites...

  9. PKCζ Promotes Breast Cancer Invasion by Regulating Expression of E-cadherin and Zonula Occludens-1 (ZO-1) via NFκB-p65

    PubMed Central

    Paul, Arindam; Danley, Marsha; Saha, Biswarup; Tawfik, Ossama; Paul, Soumen

    2015-01-01

    Atypical Protein Kinase C zeta (PKCζ) forms Partitioning-defective (PAR) polarity complex for apico-basal distribution of membrane proteins essential to maintain normal cellular junctional complexes and tissue homeostasis. Consistently, tumor suppressive role of PKCζ has been established for multiple human cancers. However, recent studies also indicate pro-oncogenic function of PKCζ without firm understanding of detailed molecular mechanism. Here we report a possible mechanism of oncogenic PKCζ signaling in the context of breast cancer. We observed that depletion of PKCζ promotes epithelial morphology in mesenchymal-like MDA-MB-231 cells. The induction of epithelial morphology is associated with significant upregulation of adherens junction (AJ) protein E-cadherin and tight junction (TJ) protein Zonula Occludens-1 (ZO-1). Functionally, depletion of PKCζ significantly inhibits invasion and metastatic progression. Consistently, we observed higher expression and activation of PKCζ signaling in invasive and metastatic breast cancers compared to non-invasive diseases. Mechanistically, an oncogenic PKCζ– NFκB-p65 signaling node might be involved to suppress E-cadherin and ZO-1 expression and ectopic expression of a constitutively active form of NFκB-p65 (S536E-NFκB-p65) significantly rescues invasive potential of PKCζ-depleted breast cancer cells. Thus, our study discovered a PKCζ - NFκB-p65 signaling pathway might be involved to alter cellular junctional dynamics for breast cancer invasive progression. PMID:26218882

  10. The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an adaptive state?

    PubMed

    Murphy-Ullrich, J E

    2001-04-01

    The process of cellular de-adhesion is potentially important for the ability of a cell to participate in morphogenesis and to respond to injurious stimuli. Cellular de-adhesion is induced by the highly regulated matricellular proteins TSP1 and 2, tenascin-C, and SPARC. These proteins induce a rapid transition to an intermediate state of adhesiveness characterized by loss of actin-containing stress fibers and restructuring of the focal adhesion plaque that includes loss of vinculin and alpha-actinin, but not of talin or integrin. This process involves intracellular signaling mediators, which are engaged in response to matrix protein-receptor interactions. Each of these proteins employs different receptors and signaling pathways to achieve this common morphologic endpoint. What is the function of this intermediate adhesive state and what is the physiologic significance of this action of the matricellular proteins? Given that matricellular proteins are expressed in response to injury and during development, one can speculate that the intermediate adhesive state is an adaptive condition that facilitates expression of specific genes that are involved in repair and adaptation. Since cell shape is maintained in weakly adherent cells, this state might induce survival signals to prevent apoptosis due to loss of strong cell adhesion, but yet allow for cell locomotion. The three matricellular proteins considered here might each preferentially facilitate one or more aspects of this adaptive response rather than all of these equally. Currently, we have only preliminary data to support the specific ideas proposed in this article. It will be interesting in the next several years to continue to elucidate the biological roles of the intermediate adhesive state induced by these matricellular proteins. and focal adhesions in a cell that nevertheless maintains a spread, extended morphology and integrin clustering. TSP1, tenascin-C, and SPARC induce the intermediate adhesive state, as

  11. Characterization of Ascites-Derived Ovarian Tumor Cells from Spontaneously Occurring Ovarian Tumors of the Chicken: Evidence for E-Cadherin Upregulation

    PubMed Central

    Tiwari, Anupama; Hadley, Jill A.; Hendricks, Gilbert L.; Elkin, Robert G.; Cooper, Timothy; Ramachandran, Ramesh

    2013-01-01

    Ovarian cancer, a highly metastatic disease, is the fifth leading cause of cancer-related deaths in women. Chickens are widely used as a model for human ovarian cancer as they spontaneously develop epithelial ovarian tumors similar to humans. The cellular and molecular biology of chicken ovarian cancer (COVCAR) cells, however, have not been studied. Our objectives were to culture COVCAR cells and to characterize their invasiveness and expression of genes and proteins associated with ovarian cancer. COVCAR cell lines (n = 13) were successfully maintained in culture for up to19 passages, cryopreserved and found to be viable upon thawing and replating. E-cadherin, cytokeratin and α-smooth muscle actin were localized in COVCAR cells by immunostaining. COVCAR cells were found to be invasive in extracellular matrix and exhibited anchorage-independent growth forming colonies, acini and tube-like structures in soft agar. Using RT-PCR, COVCAR cells were found to express E-cadherin, N-cadherin, cytokeratin, vimentin, mesothelin, EpCAM, steroidogenic enzymes/proteins, inhibin subunits-α, βA, βB, anti-müllerian hormone, estrogen receptor [ER]-α, ER-β, progesterone receptor, androgen receptor, and activin receptors. Quantitative PCR analysis revealed greater N-cadherin, vimentin, and VEGF mRNA levels and lesser cytokeratin mRNA levels in COVCAR cells as compared with normal ovarian surface epithelial (NOSE) cells, which was suggestive of epithelial-mesenchymal transformation. Western blotting analyses revealed significantly greater E-cadherin levels in COVCAR cell lines compared with NOSE cells. Furthermore, cancerous ovaries and COVCAR cell lines expressed higher levels of an E-cadherin cleavage product when compared to normal ovaries and NOSE cells, respectively. Cancerous ovaries were found to express significantly higher ovalbumin levels whereas COVCAR cell lines did not express ovalbumin thus suggesting that the latter did not originate from oviduct. Taken

  12. Characterization of canine platelet adhesion to extracellular matrix proteins.

    PubMed

    Pelagalli, Alessandra; Pero, Maria Elena; Mastellone, Vincenzo; Cestaro, Anna; Signoriello, Simona; Lombardi, Pietro; Avallone, Luigi

    2011-07-01

    Canine platelets have been extensively studied but little is known about specific aspects such as adhesion. Platelet adhesion is a critical step during haemostasis and thrombosis as well as during inflammatory and immunopathogenic responses. The aim of this study was to evaluate the adhesive properties of canine platelets using fibrinogen and collagen as substrates immobilized on plates. Adhesion was monitored for 120 min and the effect of adenosine 5'-diphosphate (ADP) was assayed. The results showed that canine platelets displayed good adhesion activity that was significantly time-dependent. Moreover, ADP was able to enhance platelet adhesion in a dose-dependent manner. The findings aid knowledge of the adhesion process and suggest a specific role of surface platelet receptors in mediating the interaction with extracellular matrix proteins.

  13. Syndecan-2 enhances E-cadherin shedding and fibroblast-like morphological changes by inducing MMP-7 expression in colon cancer cells.

    PubMed

    Jang, Bohee; Jung, Hyejung; Chung, Heesung; Moon, Byung-In; Oh, Eok-Soo

    2016-08-12

    E-cadherin plays a mechanical role in mediating cell-cell interactions and maintaining epithelial tissue integrity, and the loss of E-cadherin function has been implicated in cancer progression and metastasis. Syndecan-2, a cell-surface heparan sulfate proteoglycan, is upregulated during the development of colon cancer. Here, we assessed the functional relationship between E-cadherin and syndecan-2. We found that stable overexpression of syndecan-2 in a human colorectal adenocarcinoma cell line (HT29) enhanced the proteolytic shedding of E-cadherin to conditioned-media. Either knockdown of matrix metalloproteinase 7 (MMP-7) or inhibition of MMP-7 activity using GM6001 significantly reduced the extracellular shedding of E-cadherin, suggesting that syndecan-2 mediates E-cadherin shedding via MMP-7. Consistent with this notion, enhancement of MMP-7 expression by interleukin-1α treatment increased the shedding of E-cadherin. Conversely, the specific reduction of either syndecan-2 or MMP-7 reduced the shedding of E-cadherin. HT29 cells overexpressing syndecan-2 showed significantly lower cell-surface expression of E-cadherin, decreased cell-cell contact, a more fibroblastic cell morphology, and increased expression levels of ZEB-1. Taken together, these data suggest that syndecan-2 induces extracellular shedding of E-cadherin and supports the acquisition of a fibroblast-like morphology by regulating MMP-7 expression in a colon cancer cell line.

  14. Cell-cell contacts mediated by E-cadherin (uvomorulin) restrict invasive behavior of L-cells

    PubMed Central

    1991-01-01

    L-cells were cotransfected with plasmids coding for mouse E-cadherin (uvomorulin) and the neophosphotransferase gene, and stable transfectants expressing E-cadherin at the cell surface were selected and cloned. Control transfection was done with the neophosphotransferase gene alone. The invasive migration of transfected and untransfected L-cells into three-dimensional collagen gels was then analyzed. L-cells not expressing E-cadherin migrated efficiently into the gels, whereas invasion of the E-cadherin-expressing L-cells was restricted in a cell density dependent manner. At sparse density, when the cells exhibited little cell-cell contacts, no difference was observed between the level of invasion of the cadherin-expressing cells and the control cells. However, with increasing cell density, decreasing amounts of the cadherin-expressing cells but increasing amounts of the control cells migrated into the gels. At confluent density hardly any cadherin-expressing cells were able to migrate into the gels. The inhibition of the invasion of the cadherin-expressing cells could be reverted if confluent cells were cultured in the presence of monoclonal antibodies against E-cadherin. Since the expression of E-cadherin did not influence the invasive mobility of single cells, these results indicate that E-cadherin-mediated cell-cell contacts inhibited invasive cellular migration. Time-lapse videoscopy and studies of cell migration from a monolayer into a cell-free area demonstrated that the restricted invasion could be explained by contact inhibition of cell movement of the cadherin-expressing cells. PMID:1649199

  15. Nuclear factor I-C (NFIC) regulates dentin sialophosphoprotein (DSPP) and E-cadherin via control of Krüppel-like factor 4 (KLF4) during dentinogenesis.

    PubMed

    Lee, Hye-Kyung; Lee, Dong-Seol; Park, Su-Jin; Cho, Kwang-Hee; Bae, Hyun-Sook; Park, Joo-Cheol

    2014-10-10

    Odontoblasts are a type of terminally differentiated matrix-secreting cells. A number of molecular mechanisms are involved in the differentiation of odontoblasts. Several studies demonstrated that Krüppel-like factor 4 (KLF4) promotes odontoblast differentiation via control of dentin sialophosphoprotein (DSPP). Because nuclear factor I-C (NFIC) is also known to control DSPP, we investigated the relationship between NFIC and KLF4 during odontoblast differentiation. Klf4 mRNA expression was significantly decreased in Nfic(-/-) pulp cells compared with wild type cells. In immunohistochemistry assays, dentin matrix protein 1 (Dmp1), and DSP protein expression was barely observed in Nfic(-/-) odontoblasts and dentin matrix. Nfic bound directly to the Klf4 promoter and stimulated Klf4 transcriptional activity, thereby regulating Dmp1 and DSPP expression during odontoblast differentiation. Nfic or Klf4 overexpression promoted mineralized nodule formation in MDPC-23 cells. In addition, Nfic overexpression also decreased Slug luciferase activity but augmented E-cadherin promoter activity via up-regulation of Klf4 in odontoblasts. Our study reveals important signaling pathways during dentinogenesis: the Nfic-Klf4-Dmp1-Dspp and the Nfic-Klf4-E-cadherin pathways in odontoblasts. Our results indicate the important role of NFIC in regulating KLF4 during dentinogenesis.

  16. Thrombomodulin reduces tumorigenic and metastatic potential of lung cancer cells by up-regulation of E-cadherin and down-regulation of N-cadherin expression.

    PubMed

    Zheng, Nana; Huo, Zihe; Zhang, Bin; Meng, Mei; Cao, Zhifei; Wang, Zhiwei; Zhou, Quansheng

    2016-08-01

    Thrombomodulin (TM) is an endothelial cell membrane protein and plays critical roles in anti-thrombosis, anti-inflammation, vascular endothelial protection, and is traditionally regarded as a "vascular protection god". In recent years, although TM has been reported to be down-regulated in a variety of malignant tumors including lung cancer, the role and mechanism of TM in lung cancer are enigmatic. In this study, we found that induction of TM overexpression by cholesterol-reducing drug atorvastatin significantly diminished the tumorigenic capability of the lung cancer cells. Moreover, we demonstrated that TM overexpression caused G0/G1 phase arrest and markedly reduced the colony forming capability of the cells. Furthermore, overexpression of TM inhibited cell migration and invasion. Consistently, depletion of TM promoted cell growth, reduced the cell population at the G0/G1 phase, and enhanced cell migratory ability. Mechanistic study revealed that TM up-regulated E-cadherin but down-regulated N-cadherin expression, resulting in reversal of epithelial-mesenchymal transition (EMT) in the lung cancer cells. Moreover, silencing TM expression led to decreased E-cadherin and increased N-cadherin. Taken together, our study suggests that TM functions as a tumor suppressive protein, providing a conceptual framework for inducing TM overexpression as a sensible strategy and approach for novel anti-lung cancer drug discovery. PMID:27223053

  17. Soy protein isolate molecular level contributions to bulk adhesive properties

    NASA Astrophysics Data System (ADS)

    Shera, Jeanne Norton

    Increasing environmental awareness and the recognized health hazards of formaldehyde-based resins has prompted a strong demand for environmentally-responsible adhesives for wood composites. Soy protein-based adhesives have been shown to be commercially viable with 90-day shelf stability and composite physical properties comparable to those of commercial formaldehyde-based particleboards. The main research focus is to isolate and characterize the molecular level features in soy protein isolate responsible for providing mechanical properties, storage stability, and water resistance during adhesive formulation, processing, and wood composite fabrication. Commercial composite board will be reviewed to enhance our understanding of the individual components and processes required for particleboard production. The levels of protein structure will be defined and an overview of current bio-based technology will be presented. In the process, the logic for utilizing soy protein as a sole binder in the adhesive will be reinforced. Variables such as adhesive components, pH, divalent ions, blend aging, protein molecular weight, formulation solids content, and soy protein functionalization will relate the bulk properties of soy protein adhesives to the molecular configuration of the soybean protein. This work has demonstrated that when intermolecular beta-sheet interactions and protein long-range order is disrupted, viscosity and mechanical properties decrease. Storage stability can be maintained through the stabilization of intermolecular beta-sheet interactions. When molecular weight is reduced through enzymatic digestion, long-range order is disrupted and viscosity and mechanical properties decrease accordingly. Processibility and physical properties must be balanced to increase solids while maintaining low viscosity, desirable mechanical properties, and adequate storage stability. The structure of the soybean protein must be related to the particleboard bulk mechanical

  18. Transcytosis of Listeria monocytogenes across the intestinal barrier upon specific targeting of goblet cell accessible E-cadherin

    PubMed Central

    Nikitas, Georgios; Deschamps, Chantal; Disson, Olivier; Niault, Théodora; Cossart, Pascale

    2011-01-01

    Listeria monocytogenes (Lm) is a foodborne pathogen that crosses the intestinal barrier upon interaction between its surface protein InlA and its species-specific host receptor E-cadherin (Ecad). Ecad, the key constituent of adherens junctions, is typically situated below tight junctions and therefore considered inaccessible from the intestinal lumen. In this study, we investigated how Lm specifically targets its receptor on intestinal villi and crosses the intestinal epithelium to disseminate systemically. We demonstrate that Ecad is luminally accessible around mucus-expelling goblet cells (GCs), around extruding enterocytes at the tip and lateral sides of villi, and in villus epithelial folds. We show that upon preferential adherence to accessible Ecad on GCs, Lm is internalized, rapidly transcytosed across the intestinal epithelium, and released in the lamina propria by exocytosis from where it disseminates systemically. Together, these results show that Lm exploits intrinsic tissue heterogeneity to access its receptor and reveal transcytosis as a novel and unanticipated pathway that is hijacked by Lm to breach the intestinal epithelium and cause systemic infection. PMID:21967767

  19. Spatial distribution of proteins in the quagga mussel adhesive apparatus.

    PubMed

    Rees, David J; Hanifi, Arash; Manion, Joseph; Gantayet, Arpita; Sone, Eli D

    2016-01-01

    The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious 'anchor' (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion.

  20. URG11 mediates hypoxia-induced epithelial-to-mesenchymal transition by modulation of E-cadherin and {beta}-catenin

    SciTech Connect

    Du, Rui; Huang, Chen; Bi, Qian; Zhai, Ying; Xia, Lin; Liu, Jie; Sun, Shiren; Fan, Daiming

    2010-01-01

    Upregulated gene 11 (URG11), recently identified as a new HBx-upregulated gene that may activate {beta}-catenin and Wnt signaling, was found to be upregulated in a human tubule cell line under low oxygen. Here, we investigated the potential role of URG11 in hypoxia-induced renal tubular epithelial-to-mesenchymal (EMT). Overexpression of URG11 in a human proximal tubule cell line (HK2) promoted a mesenchymal phenotype accompanied by reduced expression of the epithelial marker E-cadherin and increased expression of the mesenchymal markers vimentin and {alpha}-SMA, while URG11 knockdown by siRNA effectively reversed hypoxia-induced EMT. URG11 promoted the expression of {beta}-catenin and increased its nuclear accumulation under normoxic conditions through transactivation of the {beta}-catenin promoter. This in turn upregulated {beta}-catenin/T-cell factor (TCF) and its downstream effector genes, vimentin, and {alpha}-SMA. In vivo, strong expression of URG11 was observed in the tubular epithelia of 5/6-nephrectomized rats, and a Western blot analysis demonstrated a close correlation between HIF-1{alpha} and URG11 protein levels. Altogether, our results indicate that URG11 mediates hypoxia-induced EMT through the suppression of E-cadherin and the activation of the {beta}-catenin/TCF pathway.

  1. Intrinsic Surface-Drying Properties of Bio-adhesive Proteins

    PubMed Central

    Akdogan, Yasar; Wei, Wei; Huang, Kuo-Ying; Kageyama, Yoshiyuki; Danner, Eric W.; Miller, Dusty R.; Martinez Rodriguez, Nadine R.; Herbert Waite, J.

    2014-01-01

    Sessile marine mussels must “dry” underwater surfaces before adhering to them. Synthetic adhesives have yet to overcome this fundamental challenge. Previous studies of bio-inspired adhesion have largely been performed under applied compressive forces but these are poor predictors of an adhesive’s ability to spontaneously penetrate surface hydration layers. In a force-free approach to measuring molecular-level interaction via the surface water diffusivity, different mussel foot proteins were found to have differential abilities to evict hydration layers from the surfaces—a necessary step for adsorption and adhesion. It was anticipated that Dopa would mediate dehydration given its efficacy forbio-inspired wet adhesion. Instead, hydrophobic side-chains are found to be a critical component in bringing about protein-surface intimacy. This is the first direct measurement of interfacial water dynamics during force-free adsorptive interactions at solid surfaces, and offers guidance for engineering wet adhesives and coatings. PMID:25168789

  2. Expression of p27Kip1 and E-cadherin in Head and Neck Squamous Cell Carcinoma of Indonesian Patients.

    PubMed

    E I, Auerkari; V, Joewono; D R, Handjari; A T, Sarwono; A W, Suhartono; K, Eto; M A, Ikeda

    2014-01-01

    Cancer cells exhibit characteristic damage of DNA and its expression. The expression of the tumor suppressors E-cadherin and p27(Kip1) has been tested on 57 head and neck squamous cell carcinomas (HNSCC) of Indonesian subjects. HNSCC tumor samples including both primary and (unrelated) nodal cases were obtained from the archives of Indonesian hospitals, in accordance with acknowledged ethical requirements. Only modest correlation was found between reduced expression of E-cadherin or p27(Kip1) with increased malignancy of primary and nodal growth. The observed strong correlation regardless of malignancy between the expressed levels of E-cadherin and p27(Kip1) suggests that also in combination these would not help to better predict the outcome of HNSCC.

  3. Polymer adhesion at surfaces: biological adhesive proteins and their synthetic mimics

    NASA Astrophysics Data System (ADS)

    Messersmith, Phillip

    2008-03-01

    Mussels are famous for their ability to permanently adhere to a wide variety of wet surfaces, such as rocks, metal and polymer ship hulls, and wood structures. They accomplish this through specialized proteins collectively referred to as mussel adhesive proteins (MAPs). The biophysical aspects of MAP adhesion is being revealed through the use of single molecule force measurements. The results provide insight into the adhesive roles of key amino acids found in these proteins, including the magnitude of adhesive forces, cooperative effects, and their self-healing properties. This molecular-level information is being incorporated into designs of biomimetic polymer coatings for a variety of applications. Our biomimetic approach to polymer design will be illustrated by a few examples where adhesive constituents found in MAPs are exploited to make wet-adhesive polymer coatings. In addition, small molecule analogs of MAPs can be used to apply thin functional films onto virtually any material surface using a facile approach. These coatings have a variety of potential uses in microelectronics, water treatment, prevention of environmental biofouling, and for control of biointerfacial phenomena at the surfaces of medical/diagnostic devices.

  4. Novel protein-repellent dental adhesive containing 2-methacryloyloxyethyl phosphorylcholine

    PubMed Central

    Zhang, Ning; Melo, Mary Anne S.; Bai, Yuxing; Xu, Hockin H. K.

    2015-01-01

    Objectives Biofilms at tooth-restoration margins can produce acids and cause secondary caries. A protein-repellent adhesive resin can potentially inhibition bacteria attachment and biofilm growth. However, there has been no report on protein-repellent dental resins. The objectives of this study were to develop a protein-repellent bonding agent incorporating 2-methacryloyloxyethyl phosphorylcholine (MPC), and to investigate its resistance to protein adsorption and biofilm growth for the first time. Methods MPC was incorporated into Scotchbond Multi-Purpose (SBMP) at 0%, 3.75%, 7.5%, 11.25%, and 15% by mass. Extracted human teeth were used to measure dentin shear bond strengths. Protein adsorption onto resins was determined by a micro bicinchoninic acid (BCA) method. A dental plaque microcosm biofilm model with human saliva as inoculum was used to measure biofilm metabolic activity and colony-forming unit (CFU) counts. Results Adding 7.5% MPC into primer and adhesive did not decrease the dentin bond strength, compared to control (p > 0.1). Incorporation of 7.5% of MPC achieved the lowest protein adsorption, which was 20-fold less than that of control. Incorporation of 7.5% of MPC greatly reduced bacterial adhesion, yielding biofilm total microorganism, total streptococci, and mutans streptococci CFU that were an order of magnitude less than control. Conclusions A protein-repellent dental adhesive resin was developed for the first time. Incorporation of MPC into primer and adhesive at 7.5% by mass greatly reduced the protein adsorption and bacterial adhesion, without compromising the dentin bond strength. The novel protein-repellent primer and adhesive are promising to inhibit biofilm formation and acid production, to protect the tooth-restoration margins and prevent secondary caries. PMID:25234652

  5. Interaction with Suv39H1 is Critical for Snail-mediated E-cadherin Repression in Breast Cancer

    PubMed Central

    Dong, Chenfang; Wu, Yadi; Wang, Yifan; Wang, Chi; Kang, Tiebang; Rychahou, Piotr G.; Chi, Young-In; Evers, B. Mark; Zhou, Binhua P.

    2013-01-01

    Expression of E-cadherin, a hallmark of epithelial-mesenchymal transition (EMT), is often lost due to promoter DNA methylation in basal-like breast cancer (BLBC), which contributes to the metastatic advantage of this disease; however, the underlying mechanism remains unclear. Here we identified that Snail interacted with Suv39H1, a major methyltransferase responsible for H3K9me3 that intimately links to DNA methylation. We demonstrated that the SNAG domain of Snail and the SET domain of Suv39H1 were required for their mutual interactions. We found that H3K9me3 and DNA methylation on the E-cadherin promoter were higher in BLBC cell lines. We showed that Snail interacted with Suv39H1 and recruited it to the E-cadherin promoter for transcriptional repression. Knockdown of Suv39H1 restored E-cadherin expression by blocking H3K9me3 and DNA methylation and resulted in the inhibition of cell migration, invasion and metastasis of BLBC. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT, but also paves a way for the development of new treatment strategies against this disease. PMID:22562246

  6. E-cadherin expression in macrophages dampens their inflammatory responsiveness in vitro, but does not modulate M2-regulated pathologies in vivo

    PubMed Central

    Van den Bossche, Jan; Laoui, Damya; Naessens, Thomas; Smits, Hermelijn H.; Hokke, Cornelis H.; Stijlemans, Benoît; Grooten, Johan; De Baetselier, Patrick; Van Ginderachter, Jo A.

    2015-01-01

    IL-4/IL-13-induced alternatively activated macrophages (M(IL-4/IL-13), AAMs or M2) are known to express E-cadherin, enabling them to engage in heterotypic cellular interactions and IL-4-driven macrophage fusion in vitro. Here we show that E-cadherin overexpression in Raw 264.7 macrophages inhibits their inflammatory response to LPS stimulation, as demonstrated by a reduced secretion of inflammatory mediators like interleukin (IL)-6, tumor necrosis factor (TNF) and nitric oxide (NO). To study the function of E-cadherin in M(IL-4/IL-13) macrophages in vivo, we generated macrophage-specific E-cadherin-deficient C57BL/6 mice. Using this new tool, we analyzed immunological parameters during two typical AAM-associated Th2-driven diseases and assessed Th2-associated granuloma formation. Although E-cadherin is strongly induced in AAMs during Taenia crassiceps helminth infections and allergic airway inflammation, its deletion in macrophages does not affect the course of both Th2 cytokine-driven diseases. Moreover, macrophage E-cadherin expression is largely redundant for granuloma formation around Schistosoma mansoni ova. Overall, we conclude that E-cadherin is a valuable AAM marker which suppresses the inflammatory response when overexpressed. Yet E-cadherin deletion in macrophages does not affect M(LPS+IFNγ) and M(IL-4) polarization in vitro, nor in vivo macrophage function, at least in the conditions tested. PMID:26226941

  7. Adhesion Proteins - An Impact on Skeletal Myoblast Differentiation

    PubMed Central

    Przewoźniak, Marta; Czaplicka, Iwona; Czerwińska, Areta M.; Markowska-Zagrajek, Agnieszka; Moraczewski, Jerzy; Stremińska, Władysława; Jańczyk-Ilach, Katarzyna; Ciemerych, Maria A.; Brzoska, Edyta

    2013-01-01

    Formation of mammalian skeletal muscle myofibers, that takes place during embryogenesis, muscle growth or regeneration, requires precise regulation of myoblast adhesion and fusion. There are few evidences showing that adhesion proteins play important role in both processes. To follow the function of these molecules in myoblast differentiation we analysed integrin alpha3, integrin beta1, ADAM12, CD9, CD81, M-cadherin, and VCAM-1 during muscle regeneration. We showed that increase in the expression of these proteins accompanies myoblast fusion and myotube formation in vivo. We also showed that during myoblast fusion in vitro integrin alpha3 associates with integrin beta1 and ADAM12, and also CD9 and CD81, but not with M-cadherin or VCAM-1. Moreover, we documented that experimental modification in the expression of integrin alpha3 lead to the modification of myoblast fusion in vitro. Underexpression of integrin alpha3 decreased myoblasts' ability to fuse. This phenomenon was not related to the modifications in the expression of other adhesion proteins, i.e. integrin beta1, CD9, CD81, ADAM12, M-cadherin, or VCAM-1. Apparently, aberrant expression only of one partner of multiprotein adhesion complexes necessary for myoblast fusion, in this case integrin alpha3, prevents its proper function. Summarizing, we demonstrated the importance of analysed adhesion proteins in myoblast fusion both in vivo and in vitro. PMID:23671573

  8. Growth differentiation factor 8 induces SKOV3 ovarian cancer cell migration and E-cadherin down-regulation.

    PubMed

    Zhao, Jianfang; Klausen, Christian; Xiong, Siyuan; Cheng, Jung-Chien; Chang, Hsun-Ming; Leung, Peter C K

    2016-11-01

    Epithelial ovarian cancer is the most lethal gynecological malignancy because most women present with late stage disseminated disease. Epithelial-mesenchymal transition (EMT) is characterized by the down-regulation of E-cadherin and up-regulation of N-cadherin, and is a crucial event in the pathogenesis of ovarian cancer. Transforming growth factor-β (TGF-β) is a major regulator of EMT in many normal and neoplastic cell types. Growth differentiation factor 8 (GDF8), which also activates TGF-β-like SMAD2/3 signaling, is best known for negatively regulating muscle growth. Though recent studies suggest that GDF8 enhances placental trophoblast cell migration, little is known about the role of GDF8 in EMT and cancer metastasis. We hypothesized that GDF8 could enhance ovarian cancer cell migration by inducing EMT. Here we demonstrate for the first time that GDF8 down-regulates E-cadherin but does not alter N-cadherin in SKOV3 ovarian cancer cells. This effect is abolished by the activin receptor-like kinase (ALK)4/5/7 inhibitor SB431542 or siRNA-mediated knockdown of ALK5, whereas knockdown of ALK4 is only partially inhibitory. GDF8 treatment increases the phosphorylation of SMAD2/3 and up-regulates the E-cadherin transcriptional repressors Snail and Slug; and these effects are abolished by pre-treatment with SB431542. Knockdown of common SMAD4 fully reverses the effects of GDF8 on E-cadherin and partially attenuates its effects on Snail and Slug. Importantly, GDF8 treatment increases SKOV3 cell migration and this effect is blocked by SB431542. Our study suggests that GDF8 promotes ovarian cancer cell migration via ALK4/5-SMAD2/3-E-cadherin signaling. PMID:27481097

  9. Correlation Between E-cadherin Immunoexpression and Efficacy of First Line Platinum-Based Chemotherapy in Advanced High Grade Serous Ovarian Cancer.

    PubMed

    Miše, Branka Petrić; Telesmanić, Vesna Dobrić; Tomić, Snježana; Šundov, Dinka; Čapkun, Vesna; Vrdoljak, Eduard

    2015-04-01

    To analyze correlation between immunoexpression of E-cadherin and efficacy of first line platinum-based chemotherapy in patients with advanced-stage high-grade serous ovarian carcinoma. The expression of E-cadherin was analyzed immunohistochemically in formalin-fixed, paraffin-embedded samples from 98 patients with advanced-stage high-grade serous ovarian cancer and related to clinical features (stage according to the International Federation of Gynecology and Obstetrics (FIGO) and residual tumors after initial cytoreductive surgery), response to platinum-based chemotherapy (according to Response Evaluation Criteria in Solid tumors (RECIST 1.1 criteria)), platinum sensitivity (according to platinum free interval (PFI) as platinum-refractory, platinum-resistant and platinum-sensitive) and patients progression free survival (PFS) and overall survival (OS). E-cadherin immunostaining was positive in 74 and negative in 24 serous ovarian carcinomas. E-cadherin immunoreactivity was not associated with FIGO stage, residual tumor after initial cytoreductive surgery and number of chemotherapy cycles. Positive E-cadherin expression predict significantly better response to first line platinum-based chemotherapy (p < 0.001) and platinum sensitivity (p < 0.001). Moreover, positive E-cadherin expression predict significantly longer PFS (p < 0.001) and OS (p < 0.001). The multivariate analysis for OS showed that positive E-cadherin expression is predictor to platinum sensitivity (p < 0.001) and longer OS (p = 0.01). Positive E-cadherin expression seems to be a predictor of better response to first line platinum-based chemotherapy, platinum sensitivity and favorable clinical outcome in patients with advanced-stage serous ovarian cancer. Negative E-cadherin expression was shown to be significant, independent predictor of poorer PFS and OS. E-cadherin as a marker has predictive and prognostic value.

  10. A correlation between altered O-GlcNAcylation, migration and with changes in E-cadherin levels in ovarian cancer cells

    SciTech Connect

    Jin, Feng-zhen; Yu, Chao; Zhao, De-zhang; Wu, Ming-jun; Yang, Zhu

    2013-06-10

    O-GlcNAcylation is a dynamic and reversible posttranslational modification of nuclear and cytoplasmic proteins. In recent years, the roles of O-GlcNAcylation in several human malignant tumors have been investigated, and O-GlcNAcylation was found to be linked to cellular features relevant to metastasis. In this study, we modeled four diverse ovarian cancer cells and investigated the effects of O-GlcNAcylation on ovarian cancer cell migration. We found that total O-GlcNAcylation level was elevated in HO-8910PM cells compared to OVCAR3 cells. Additionally, through altering the total O-GlcNAcylation level by OGT silencing or OGA inhibition, we found that the migration of OVCAR3 cells was dramatically enhanced by PUGNAc and Thiamet G treatment, and the migration ability of HO-8910PM cells was significantly inhibited by OGT silencing. Furthermore, we also found that the expression of E-cadherin, an O-GlcNAcylated protein in ovarian cancer cells, was reduced by OGA inhibition in OVCAR3 cells and elevated by OGT silencing in HO-8910PM cells. These results indicate that O-GlcNAcylation could enhance ovarian cancer cell migration and decrease the expression of E-cadherin. Our studies also suggest that O-GlcNAcylation might become another potential target for the therapy of ovarian cancer. -- Highlights: • We examine the migration potential of diverse ovarian cancer cells. • We examine the total O-GlcNAcylation level of diverse ovarian cancer cells. • Increasing O-GlcNAcylation level will enhance the migration of ovarian cancer cells. • Reducing O-GlcNAcylation level will inhibit the migration of ovarian cancer cells. • The mechanism explains O-GlcNAcylation enhance ovarian cancer cell migration.

  11. Hypermethylation Status of E-Cadherin Gene in Gastric Cancer Patients in a High Incidence Area.

    PubMed

    Rashid, Haroon; Alam, Khursheed; Afroze, Dil; Yousuf, Adfar; Banday, Manzoor; Kawoosa, Fizalah

    2016-01-01

    Gastric cancer (GC) is the fourth most prevalant cancer and the second leading cause of cancer-related mortality worldwide. As in other cancers gastric carcinogenesis is multifactorial involving environmental, genetic and epigenetic components. Epigenetic silencing due to hypermethylation of tumour suppressor genes is one of the key events in gastric carcinogenesis. This study was aimed to analyse the hypermethylation status of the E-Cadherin (CDH1) gene promoter in GCs in the ethnic Kashmiri population. In this study a total of 80 GC patients were recruited. Hypermethylation in tumour tissue was detected by methylation specific PCR (MS-PCR). Hypermethylation of CDH1 promoter was observed in 52 (65%) of gastric carcinoma cases which was significantly much higher than adjacent normal tissue [p≤0.0001]. Further the frequency of CDH1 promoter methylation was significantly different with intestinal and diffuse types of gastric cancer [55.7% vs 82.1%; <0.05]. Moreover females and cases with lymph node invasion had higher frequencies of CDH1 hypermethylation [P≤0.05]. Thus the current data indicate a vital role of epigenetic alteration of CDH1 in the causation and development of gastric cancer, particularly of diffuse type, in our population.

  12. 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.

  13. Prognostic and Clinicopathological Significance of Downregulated E-Cadherin Expression in Patients with Non-Small Cell Lung Cancer (NSCLC): A Meta-Analysis

    PubMed Central

    Xian, Lei

    2014-01-01

    Background Many studies have investigated the prognostic role of E-cadherin in patients with NSCLC; however, the result still remains inconclusive. An up-to data system review and meta-analysis was necessary to give a comprehensive evaluation of prognostic role of E-cadherin in NSCLC. Methods Eligible studies were searched in Pubmed, Embase and Web of Science databases. The inclusion criteria were studies that assessed the relationship between E-cadherin expression detected by immunohistochemistry (IHC) and the prognosis or clinicopathological features in patients with NSCLC. Subgroup analysis according to race, percentage of reduced/negative E-cadherin expression, histological type, and sample size were also conducted. Odds ratio (OR) or hazard ratio (HR) with 95% confidence interval (CI) were calculated to examine the risk or hazard association. Results A total of 29 studies including 4010 patients were qualified for analysis. The analysis suggested that downregulated E-cadherin expression was significant associated with unfavorable overall survival (OS) and disease-free survival/progression-free survival (DFS/PFS) in patients with NSCLC. Subgroup analysis by race, percentage of reduced/negative E-cadherin expression, sample size also found the significant association in OS. When only the stage I NSCLC were considered, downregulated E-cadherin expression still had an unfavorable impact on OS. Additionally, downregulated E-cadherin expression was significantly associated with differentiation grade, lymphnode metastasis, vascular invasion, and TNM stage. Conclusion Downregulated E-cadherin expression detected by IHC seems to correlate with tumour progression and could serve as an important prognostic factor in patients with NSCLC. PMID:24978478

  14. Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/β-catenin signaling via its FadA adhesin

    PubMed Central

    Liu, Wendy; Hao, Yujun; Cai, Guifang; Han, Yiping W.

    2013-01-01

    SUMMARY Fusobacterium nucleatum (Fn) has been associated with colorectal cancer (CRC), but causality and underlying mechanisms remain to be established. We demonstrate that Fn adheres to, invades and induces oncogenic and inflammatory responses to stimulate growth of CRC cells through its unique FadA adhesin. FadA binds to E-cadherin, activates β-catenin signaling, and differentially regulates the inflammatory and oncogenic responses. The FadA-binding site on E-cadherin is mapped to an 11 amino acid region. A synthetic peptide derived from this region of E-cadherin abolishes FadA-induced CRC cell growth, and oncogenic and inflammatory responses. FadA levels in the colon tissue from patients with adenomas and adenocarcinomas is >10–100 times higher compared to normal individuals. The increased FadA expression in CRC correlates with increased expression of oncogenic and inflammatory genes. This study unveils a mechanism by which Fn can drive CRC and identifies FadA as a potential diagnostic and therapeutic target for CRC. PMID:23954158

  15. Alternate RASSF1 Transcripts Control SRC Activity, E-Cadherin Contacts, and YAP-Mediated Invasion

    PubMed Central

    Vlahov, Nikola; Scrace, Simon; Soto, Manuel Sarmiento; Grawenda, Anna M.; Bradley, Leanne; Pankova, Daniela; Papaspyropoulos, Angelos; Yee, Karen S.; Buffa, Francesca; Goding, Colin R.; Timpson, Paul; Sibson, Nicola; O’Neill, Eric

    2015-01-01

    Summary Tumor progression to invasive carcinoma is associated with activation of SRC family kinase (SRC, YES, FYN) activity and loss of cellular cohesion. The hippo pathway-regulated cofactor YAP1 supports the tumorigenicity of RAS mutations but requires both inactivation of hippo signaling and YES-mediated phosphorylation of YAP1 for oncogenic activity. Exactly how SRC kinases are activated and hippo signaling is lost in sporadic human malignancies remains unknown. Here, we provide evidence that hippo-mediated inhibition of YAP1 is lost upon promoter methylation of the RAS effector and hippo kinase scaffold RASSF1A. We find that RASSF1A promoter methylation reduces YAP phospho-S127, which derepresses YAP1, and actively supports YAP1 activation by switching RASSF1 transcription to the independently transcribed RASSF1C isoform that promotes Tyr kinase activity. Using affinity proteomics, proximity ligation, and real-time molecular visualization, we find that RASSF1C targets SRC/YES to epithelial cell-cell junctions and promotes tyrosine phosphorylation of E-cadherin, β-catenin, and YAP1. RASSF1A restricts SRC activity, preventing motility, invasion, and tumorigenesis in vitro and in vivo, with epigenetic inactivation correlating with increased inhibitory pY527-SRC in breast tumors. These data imply that distinct RASSF1 isoforms have opposing functions, which provide a biomarker for YAP1 activation and explain correlations of RASSF1 methylation with advanced invasive disease in humans. The ablation of epithelial integrity together with subsequent YAP1 nuclear localization allows transcriptional activation of β-catenin/TBX-YAP/TEAD target genes, including Myc, and an invasive phenotype. These findings define gene transcript switching as a tumor suppressor mechanism under epigenetic control. PMID:26549256

  16. Distinct E-cadherin-based complexes regulate cell behaviour through miRNA processing or Src and p120 catenin activity

    PubMed Central

    Kourtidis, Antonis; Ngok, Siu P.; Pulimeno, Pamela; Feathers, Ryan W.; Carpio, Lomeli R.; Baker, Tiffany R.; Carr, Jennifer M.; Yan, Irene K.; Borges, Sahra; Perez, Edith A.; Storz, Peter; Copland, John A.; Patel, Tushar; Thompson, E. Aubrey; Citi, Sandra; Anastasiadis, Panos Z.

    2016-01-01

    E-cadherin and p120 catenin (p120) are essential for epithelial homeostasis, but can also exert pro-tumorigenic activities. Here, we resolve this apparent paradox by identifying two spatially and functionally distinct junctional complexes in non-transformed polarized epithelial cells: one growth suppressing at the apical zonula adherens (ZA), defined by the p120 partner PLEKHA7 and a non-nuclear subset of the core microprocessor components DROSHA and DGCR8, and one growth promoting at basolateral areas of cell–cell contact containing tyrosine-phosphorylated p120 and active Src. Recruitment of DROSHA and DGCR8 to the ZA is PLEKHA7 dependent. The PLEKHA7–microprocessor complex co-precipitates with primary microRNAs (pri-miRNAs) and possesses pri-miRNA processing activity. PLEKHA7 regulates the levels of select miRNAs, in particular processing of miR-30b, to suppress expression of cell transforming markers promoted by the basolateral complex, including SNAI1, MYC and CCND1. Our work identifies a mechanism through which adhesion complexes regulate cellular behaviour and reveals their surprising association with the microprocessor. PMID:26302406

  17. Adhesion

    MedlinePlus

    ... as the shoulder Eyes Inside the abdomen or pelvis Adhesions can become larger or tighter over time. ... Other causes of adhesions in the abdomen or pelvis include: Appendicitis , most often when the appendix breaks ...

  18. Transition of Immunohistochemical Expression of E-Cadherin and Vimentin from Premalignant to Malignant Lesions of Oral Cavity and Oropharynx

    PubMed Central

    Akhtar, Kafil; Ara, Anjum; Siddiqui, Shahid A; Sherwani, Rana K

    2016-01-01

    Objectives We sought to study the expression of epithelial-to-mesenchymal transition markers E-cadherin and vimentin in precancerous lesions of the oral cavity and oropharynx and to use the specific pattern of expression to predict invasiveness. Methods This cross-sectional study looked at 87 cases of oral and oropharyngeal lesions obtained between December 2012 and November 2014 in the Department of Pathology, Jawaharlal Nehru Medical College, Aligarh Muslim University, India. Fifty-three biopsies from the buccal mucosa, tongue, and pharynx and 34 resected oral specimens were evaluated for premalignant and malignant lesions using hematoxylin and eosin and immunohistochemical stains. Immunohistochemical expression of epithelial marker E-cadherin and mesenchymal marker vimentin was evaluated wherever possible. Slides were examined for staining pattern (cytoplasmic or membrane), proportion, and intensity of staining of tumor cells. Patients follow-up and therapy related changes were also studied. Results There were 64 premalignant and 23 malignant cases in our study with 65 (74.7%) cases seen in males and 22 (25.3%) cases seen in females. The majority of malignant cases, (n = 15; 64.2%) were seen in the fifth and sixth decades of life while most of the premalignant lesions (n = 36; 56.4%) were seen in the fourth and fifth decade. Amongst the 64 premalignant oral lesions, leukoplakia comprised of 14 cases (21.9%), of which three cases had associated mild to moderate dysplasia. The majority of premalignant lesions showed strong E-cadherin expression and decreased expression of vimentin with negative and weak expression in both dysplasias and carcinoma in situ (p = 0.013). E-cadherin expression was significantly reduced in invasive carcinomas compared to dysplasias and carcinoma in situ and the difference in immunoreactivity was statistically significant (p < 0.050). Vimentin expression increased as the tumor progressed from dysplasias to carcinoma in situ to invasive

  19. Silk Fibroin Aqueous-Based Adhesives Inspired by Mussel Adhesive Proteins.

    PubMed

    Burke, Kelly A; Roberts, Dane C; Kaplan, David L

    2016-01-11

    Silk fibroin from the domesticated silkworm Bombyx mori is a naturally occurring biopolymer with charged hydrophilic terminal regions that end-cap a hydrophobic core consisting of repeating sequences of glycine, alanine, and serine residues. Taking inspiration from mussels that produce proteins rich in L-3,4-dihydroxyphenylalanine (DOPA) to adhere to a variety of organic and inorganic surfaces, the silk fibroin was functionalized with catechol groups. Silk fibroin was selected for its high molecular weight, tunable mechanical and degradation properties, aqueous processability, and wide availability. The synthesis of catechol-functionalized silk fibroin polymers containing varying amounts of hydrophilic polyethylene glycol (PEG, 5000 g/mol) side chains was carried out to balance silk hydrophobicity with PEG hydrophilicity. The efficiency of the catechol functionalization reaction did not vary with PEG conjugation over the range studied, although tuning the amount of PEG conjugated was essential for aqueous solubility. Adhesive bonding and cell compatibility of the resulting materials were investigated, where it was found that incorporating as little as 6 wt % PEG prior to catechol functionalization resulted in complete aqueous solubility of the catechol conjugates and increased adhesive strength compared with silk lacking catechol functionalization. Furthermore, PEG-silk fibroin conjugates maintained their ability to form β-sheet secondary structures, which can be exploited to reduce swelling. Human mesenchymal stem cells (hMSCs) proliferated on the silks, regardless of PEG and catechol conjugation. These materials represent a protein-based approach to catechol-based adhesives, which we envision may find applicability as biodegradable adhesives and sealants. PMID:26674175

  20. Silk Fibroin Aqueous-Based Adhesives Inspired by Mussel Adhesive Proteins.

    PubMed

    Burke, Kelly A; Roberts, Dane C; Kaplan, David L

    2016-01-11

    Silk fibroin from the domesticated silkworm Bombyx mori is a naturally occurring biopolymer with charged hydrophilic terminal regions that end-cap a hydrophobic core consisting of repeating sequences of glycine, alanine, and serine residues. Taking inspiration from mussels that produce proteins rich in L-3,4-dihydroxyphenylalanine (DOPA) to adhere to a variety of organic and inorganic surfaces, the silk fibroin was functionalized with catechol groups. Silk fibroin was selected for its high molecular weight, tunable mechanical and degradation properties, aqueous processability, and wide availability. The synthesis of catechol-functionalized silk fibroin polymers containing varying amounts of hydrophilic polyethylene glycol (PEG, 5000 g/mol) side chains was carried out to balance silk hydrophobicity with PEG hydrophilicity. The efficiency of the catechol functionalization reaction did not vary with PEG conjugation over the range studied, although tuning the amount of PEG conjugated was essential for aqueous solubility. Adhesive bonding and cell compatibility of the resulting materials were investigated, where it was found that incorporating as little as 6 wt % PEG prior to catechol functionalization resulted in complete aqueous solubility of the catechol conjugates and increased adhesive strength compared with silk lacking catechol functionalization. Furthermore, PEG-silk fibroin conjugates maintained their ability to form β-sheet secondary structures, which can be exploited to reduce swelling. Human mesenchymal stem cells (hMSCs) proliferated on the silks, regardless of PEG and catechol conjugation. These materials represent a protein-based approach to catechol-based adhesives, which we envision may find applicability as biodegradable adhesives and sealants.

  1. Expression of Tenascin C, EGFR, E-Cadherin, and TTF-1 in Medullary Thyroid Carcinoma and the Correlation with RET Mutation Status.

    PubMed

    Steiner, Florian; Hauser-Kronberger, Cornelia; Rendl, Gundula; Rodrigues, Margarida; Pirich, Christian

    2016-01-01

    Tenascin C expression correlates with tumor grade and indicates worse prognosis in several tumors. Epidermal growth factor receptor (EGFR) plays an important role in driving proliferation in many tumors. Loss of E-cadherin function is associated with tumor invasion and metastasis. Thyroid transcription factor-1 (TTF-1) is involved in rearranged during transfection (RET) transcription in Hirschsprung's disease. Tenascin C, EGFR, E-cadherin, TTF-1-expression, and their correlations with RET mutation status were investigated in 30 patients with medullary thyroid carcinoma (MTC) (n = 26) or C-cell hyperplasia (n = 4). Tenascin C was found in all, EGFR in 4/26, E-cadherin in 23/26, and TTF-1 in 25/26 MTC. Tenascin C correlated significantly with tumor proliferation (overall, r = 0.61, p < 0.005; RET-mutated, r = 0.81, p < 0.01). E-cadherin showed weak correlation, whereas EGFR and TTF-1 showed no significant correlation with tumor proliferation. EGFR, E-cadherin, and TTF-1 showed weak correlation with proliferation of RET-mutated tumors. Correlation between TTF-1 and tenascin C, E-cadherin, and EGFR was r = -0.10, 0.37, and 0.21, respectively. In conclusion, MTC express tenascin C, E-cadherin, and TTF-1. Tenascin C correlates significantly with tumor proliferation, especially in RET-mutated tumors. EGFR is low, and tumors expressing EGFR do not exhibit higher proliferation. TTF-1 does not correlate with RET mutation status and has a weak correlation with tenascin C, E-cadherin, and EGFR expression. PMID:27409604

  2. Expression of Tenascin C, EGFR, E-Cadherin, and TTF-1 in Medullary Thyroid Carcinoma and the Correlation with RET Mutation Status.

    PubMed

    Steiner, Florian; Hauser-Kronberger, Cornelia; Rendl, Gundula; Rodrigues, Margarida; Pirich, Christian

    2016-07-09

    Tenascin C expression correlates with tumor grade and indicates worse prognosis in several tumors. Epidermal growth factor receptor (EGFR) plays an important role in driving proliferation in many tumors. Loss of E-cadherin function is associated with tumor invasion and metastasis. Thyroid transcription factor-1 (TTF-1) is involved in rearranged during transfection (RET) transcription in Hirschsprung's disease. Tenascin C, EGFR, E-cadherin, TTF-1-expression, and their correlations with RET mutation status were investigated in 30 patients with medullary thyroid carcinoma (MTC) (n = 26) or C-cell hyperplasia (n = 4). Tenascin C was found in all, EGFR in 4/26, E-cadherin in 23/26, and TTF-1 in 25/26 MTC. Tenascin C correlated significantly with tumor proliferation (overall, r = 0.61, p < 0.005; RET-mutated, r = 0.81, p < 0.01). E-cadherin showed weak correlation, whereas EGFR and TTF-1 showed no significant correlation with tumor proliferation. EGFR, E-cadherin, and TTF-1 showed weak correlation with proliferation of RET-mutated tumors. Correlation between TTF-1 and tenascin C, E-cadherin, and EGFR was r = -0.10, 0.37, and 0.21, respectively. In conclusion, MTC express tenascin C, E-cadherin, and TTF-1. Tenascin C correlates significantly with tumor proliferation, especially in RET-mutated tumors. EGFR is low, and tumors expressing EGFR do not exhibit higher proliferation. TTF-1 does not correlate with RET mutation status and has a weak correlation with tenascin C, E-cadherin, and EGFR expression.

  3. LINKIN, a new transmembrane protein necessary for cell adhesion

    PubMed Central

    Kato, Mihoko; Chou, Tsui-Fen; Yu, Collin Z; DeModena, John; Sternberg, Paul W

    2014-01-01

    In epithelial collective migration, leader and follower cells migrate while maintaining cell–cell adhesion and tissue polarity. We have identified a conserved protein and interactors required for maintaining cell adhesion during a simple collective migration in the developing C. elegans male gonad. LINKIN is a previously uncharacterized, transmembrane protein conserved throughout Metazoa. We identified seven atypical FG–GAP domains in the extracellular domain, which potentially folds into a β-propeller structure resembling the α-integrin ligand-binding domain. C. elegans LNKN-1 localizes to the plasma membrane of all gonadal cells, with apical and lateral bias. We identified the LINKIN interactors RUVBL1, RUVBL2, and α-tubulin by using SILAC mass spectrometry on human HEK 293T cells and testing candidates for lnkn-1-like function in C. elegans male gonad. We propose that LINKIN promotes adhesion between neighboring cells through its extracellular domain and regulates microtubule dynamics through RUVBL proteins at its intracellular domain. DOI: http://dx.doi.org/10.7554/eLife.04449.001 PMID:25437307

  4. CCM1 and the second life of proteins in adhesion complexes

    PubMed Central

    van den Berg, Maaike CW; Burgering, Boudewijn MT

    2014-01-01

    It is well recognized that a number of proteins present within adhesion complexes perform discrete signaling functions outside these adhesion complexes, including transcriptional control. In this respect, β-catenin is a well-known example of an adhesion protein present both in cadherin complexes and in the nucleus where it regulates the TCF transcription factor. Here we discuss nuclear functions of adhesion complex proteins with a special focus on the CCM-1/KRIT-1 protein, which may turn out to be yet another adhesion complex protein with a second life. PMID:24714220

  5. ERβ1 inhibits the migration and invasion of breast cancer cells through upregulation of E-cadherin in a Id1-dependent manner

    SciTech Connect

    Zhou, Yan; Ming, Jia; Xu, Yan; Zhang, Yi; Jiang, Jun

    2015-02-06

    Highlights: • Expression of ERβ1 was positively correlated with E-cadherin in breast cancer cell. • ERβ1 upregulates E-cadherin expression in breast cancer cell lines. • ERβ1 upregulates E-cadherin expression in a Id1-dependent manner. - Abstract: ERβ1 is a member of the nuclear receptor superfamily of ligand-regulated transcription factors. It plays an important role in regulating the progression of breast cancer. However, the mechanisms of ERβ1 in tumorigenesis, metastasis and prognosis are still not fully clear. In this study, we showed that the expression of ERβ1 was positively correlated with E-cadherin expression in breast cancer cell lines. In addition, we found that ERβ1 upregulates E-cadherin expression in breast cancer cell lines. Furthermore, we also found that ERβ1 inhibits the migration and invasion of breast cancer cells and upregulated E-cadherin expression in a Id1-dependent manner. Taken together, our study provides further understanding of the molecular mechanism of ERβ1 in tumor metastasis and suggests the feasibility of developing novel therapeutic approaches to target Id1 to inhibit breast cancer metastasis.

  6. Cytoskeletal and cellular adhesion proteins in zebrafish (Danio rerio) myogenesis.

    PubMed

    Costa, M L; Escaleira, R; Manasfi, M; de Souza, L F; Mermelstein, C S

    2003-08-01

    The current myogenesis and myofibrillogenesis model has been based mostly on in vitro cell culture studies, and, to a lesser extent, on in situ studies in avian and mammalian embryos. While the more isolated artificial conditions of cells in culture permitted careful structural analysis, the actual in situ cellular structures have not been described in detail because the embryos are more difficult to section and manipulate. To overcome these difficulties, we used the optically clear and easy to handle embryos of the zebrafish Danio rerio. We monitored the expression of cytoskeletal and cell-adhesion proteins (actin, myosin, desmin, alpha-actinin, troponin, titin, vimentin and vinculin) using immunofluorescence microscopy and video-enhanced, background-subtracted, differential interference contrast of 24- to 48-h zebrafish embryos. In the mature myotome, the mononucleated myoblasts displayed periodic striations for all sarcomeric proteins tested. The changes in desmin distribution from aggregates to perinuclear and striated forms, although following the same sequence, occurred much faster than in other models. All desmin-positive cells were also positive for myofibrillar proteins and striated, in contrast to that which occurs in cell cultures. Vimentin appeared to be striated in mature cells, while it is developmentally down-regulated in vitro. The whole connective tissue septum between the somites was positive for adhesion proteins such as vinculin, instead of the isolated adhesion plaques observed in cell cultures. The differences in the myogenesis of zebrafish in situ and in cell culture in vitro suggest that some of the previously observed structures and protein distributions in cultures could be methodological artifacts.

  7. Dimethoxy Curcumin Induces Apoptosis by Suppressing Survivin and Inhibits Invasion by Enhancing E-Cadherin in Colon Cancer Cells.

    PubMed

    Chen, Dong; Dai, Fang; Chen, Zhehang; Wang, Saisai; Cheng, Xiaobin; Sheng, Qinsong; Lin, Jianjiang; Chen, Wenbin

    2016-01-01

    BACKGROUND Dimethoxy curcumin (DMC) is a kind of lipophilic analog of curcumin with great improvement in chemical and metabolic stability. DMC has been studied in breast and renal cancer, but no research in colon cancer has been found yet. MATERIAL AND METHODS Two colon cancer cells (HT-29 and SW480) and one normal human colon mucosal epithelial cell (NCM460) were used in this study. We studied the effect of DMC on the proliferation in vitro and in vivo. Transwell migration assay was used to estimate the inhibition of DMC on invasion. Moreover, the expressions of PARP, caspase-3, survivin and E-cadherin were detected to uncover the related signaling pathways by western blotting assay both in vitro and in vivo. RESULTS DMC significantly inhibited the growth of colon cancer cells in dose-dependent manner; IC50 for DMC was calculated to be 43.4, 28.2 and 454.8µM on HT-29, SW480 and NCM460. DMC significantly increased the apoptosis in both HT-29 (p=0.0051) and SW480 (p=0.0013) cells in vitro, and significantly suppressed the growth of both cell lines in vivo. Moreover, DMC reduced the number of migrated cells in both HT-29 (p=0.007) and SW480 (p=0.004) cells. By western blotting analysis, the cleavage of pro-caspases-3 and PARP were clearly induced by DMC to their active form, while the expression of survivin was reduced and E-cadherin was enhanced in both cells in vitro and in vivo. CONCLUSIONS DMC may exert an effective anti-tumor effect in colon cancer cells by down-regulating survivin and upregulating E-cadherin. PMID:27614381

  8. Dimethoxy Curcumin Induces Apoptosis by Suppressing Survivin and Inhibits Invasion by Enhancing E-Cadherin in Colon Cancer Cells

    PubMed Central

    Chen, Dong; Dai, Fang; Chen, Zhehang; Wang, Saisai; Cheng, Xiaobin; Sheng, Qinsong; Lin, Jianjiang; Chen, Wenbin

    2016-01-01

    Background Dimethoxy curcumin (DMC) is a kind of lipophilic analog of curcumin with great improvement in chemical and metabolic stability. DMC has been studied in breast and renal cancer, but no research in colon cancer has been found yet. Material/Methods Two colon cancer cells (HT-29 and SW480) and one normal human colon mucosal epithelial cell (NCM460) were used in this study. We studied the effect of DMC on the proliferation in vitro and in vivo. Transwell migration assay was used to estimate the inhibition of DMC on invasion. Moreover, the expressions of PARP, caspase-3, survivin and E-cadherin were detected to uncover the related signaling pathways by western blotting assay both in vitro and in vivo. Results DMC significantly inhibited the growth of colon cancer cells in dose-dependent manner; IC50 for DMC was calculated to be 43.4, 28.2 and 454.8μM on HT-29, SW480 and NCM460. DMC significantly increased the apoptosis in both HT-29 (p=0.0051) and SW480 (p=0.0013) cells in vitro, and significantly suppressed the growth of both cell lines in vivo. Moreover, DMC reduced the number of migrated cells in both HT-29 (p=0.007) and SW480 (p=0.004) cells. By western blotting analysis, the cleavage of pro-caspases-3 and PARP were clearly induced by DMC to their active form, while the expression of survivin was reduced and E-cadherin was enhanced in both cells in vitro and in vivo. Conclusions DMC may exert an effective anti-tumor effect in colon cancer cells by down-regulating survivin and upregulating E-cadherin. PMID:27614381

  9. Celastrol inhibits TGF-β1-induced epithelial–mesenchymal transition by inhibiting Snail and regulating E-cadherin expression

    SciTech Connect

    Kang, Hyereen; Lee, Minjae; Jang, Sung-Wuk

    2013-08-09

    Highlights: •We investigated the effects of celastrol on TGF-β1-induced EMT in epithelial cells. •Celastrol regulates TGF-β1-induced morphological changes and E-cadherin expression. •Celastrol inhibits TGF-β1-induced Snail expression. •Celastrol strongly suppresses TGF-β1-induced invasion in MDCK and A549 cells. -- Abstract: The epithelial–mesenchymal transition (EMT) is a pivotal event in the invasive and metastatic potentials of cancer progression. Celastrol inhibits the proliferation of a variety of tumor cells including leukemia, glioma, prostate, and breast cancer; however, the possible role of celastrol in the EMT is unclear. We investigated the effect of celastrol on the EMT. Transforming growth factor-beta 1 (TGF-β1) induced EMT-like morphologic changes and upregulation of Snail expression. The downregulation of E-cadherin expression and upregulation of Snail in Madin–Darby Canine Kidney (MDCK) and A549 cell lines show that TGF-β1-mediated the EMT in epithelial cells; however, celastrol markedly inhibited TGF-β1-induced morphologic changes, Snail upregulation, and E-cadherin expression. Migration and invasion assays revealed that celastrol completely inhibited TGF-β1-mediated cellular migration in both cell lines. These findings indicate that celastrol downregulates Snail expression, thereby inhibiting TGF-β1-induced EMT in MDCK and A549 cells. Thus, our findings provide new evidence that celastrol suppresses lung cancer invasion and migration by inhibiting TGF-β1-induced EMT.

  10. Iron Overload Causes Alterations of E-Cadherin in the Liver.

    PubMed

    Fujikura, Y; Krijt, J; Povýšil, C; Mělková, Z; Přikryl, P; Vokurka, M; Nečas, E

    2016-01-01

    Iron overload causes tissue damage in the liver, but its initial effects at the molecular and cellular level are not well understood. Epithelial cadherin (E-cad) is a major adhesion protein in adherens junctions and is associated with several signal transduction pathways. Dysfunction of E-cad causes instability of adherens junctions, which leads to cell invasion, cell migration, and carcinogenesis. We found in liver samples from iron-overloaded mice that the apparent molecular mass of E-cad was reduced from 125 to 115 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting, and that the cellular expression of E-cad was decreased in immunohistochemistry. The mRNA level of E-cad, however, did not change significantly, suggesting that the alterations are posttranslational. Interestingly, incubation of control liver extracts with Fe2+ alone also produced the same mobility shift. Neither an oxidant nor an antioxidant influenced this shift in vitro, suggesting that reactive oxygen species, which are generated by iron and known to cause damage to macromolecules, are not involved. Treatment of the 115 kDa E-cad with deferoxamine, an iron chelator, thus removing Fe2+, shifted the molecular mass back to 125 kDa, demonstrating that the shift is reversible. The observation also implies that the alteration that causes the mobility shift is not due to transcriptional control, deglycosylation, and proteolysis. This reversible mobility shift of E-cad has not been previously known. The alteration of E-cad that causes the mobility shift might be an initial step to liver diseases by iron overload. PMID:27516188

  11. Expression of E-Cadherin, Leukemia Inhibitory Factor and Progesterone Receptor in Mouse Blastocysts after Ovarian Stimulation

    PubMed Central

    Movaghar, Bahar; Askarian, Saeedeh

    2012-01-01

    Objective: The appropriate interaction between a blastocyst and the endometrium is essential for successful implantation. Numerous factors, including hormone receptors (progesterone receptor), cytokines [leukemia inhibitory factors (LIF)], and adherence molecules such as E-cadherin are involved in the cross-talk that occurs between the embryo and endometrium. Studies show that a lack of these genes impact endometrial receptivity. In this study, we compare the expression levels of E-cadherin, LIF, and progesterone receptor (PgR) genes in blastocysts that have been obtained from superovulated mice to those obtained from natural cycles. Materials and Methods: In this experimental study, for the experimental group, a total of 17 virgin female NMRI mice (6- 8 weeks old) were injected with 7.5 IU pregnant mare serum gonadotropin (PMSG). Their blastocysts (approximately n= 120) were flushed out after 3.5 days, following administration of human chorionic gonadotropin (hCG). The control group consisted of blastocysts from 62 female mice that were mated with male mice. The natural cycle blastocysts were flushed out from the female mice uteri 3.5 days after mating. The expression levels of E-cadherin, LIF, t PgR genes were examined by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Data were analyzed by the student’s t-test (one sample t-test). Results: Expression levels of all studied genes were significantly lower in the hormone-treated group compared to the natural cycle blastocysts (p<0.05). Conclusion: Although ovarian stimulation is utilized to obtain more oocytes in ART cycles, it seems that this could disadvantageous to implantation because of the decrease in expression levels of certain genes. Because of the important roles of E-cadherin, LIF, and progesterone receptor in the implantation process, we have shown lower expression levels of these genes in mouse blastocysts obtained from ovarian-stimulated mice than those derived from

  12. Cancer Cell Invasion in Three-dimensional Collagen Is Regulated Differentially by Gα13 Protein and Discoidin Domain Receptor 1-Par3 Protein Signaling.

    PubMed

    Chow, Christina R; Ebine, Kazumi; Knab, Lawrence M; Bentrem, David J; Kumar, Krishan; Munshi, Hidayatullah G

    2016-01-22

    Cancer cells can invade in three-dimensional collagen as single cells or as a cohesive group of cells that require coordination of cell-cell junctions and the actin cytoskeleton. To examine the role of Gα13, a G12 family heterotrimeric G protein, in regulating cellular invasion in three-dimensional collagen, we established a novel method to track cell invasion by membrane type 1 matrix metalloproteinase-expressing cancer cells. We show that knockdown of Gα13 decreased membrane type 1 matrix metalloproteinase-driven proteolytic invasion in three-dimensional collagen and enhanced E-cadherin-mediated cell-cell adhesion. E-cadherin knockdown reversed Gα13 siRNA-induced cell-cell adhesion but failed to reverse the effect of Gα13 siRNA on proteolytic invasion. Instead, concurrent knockdown of E-cadherin and Gα13 led to an increased number of single cells rather than groups of cells. Significantly, knockdown of discoidin domain receptor 1 (DDR1), a collagen-binding protein that also co-localizes to cell-cell junctions, reversed the effects of Gα13 knockdown on cell-cell adhesion and proteolytic invasion in three-dimensional collagen. Knockdown of the polarity protein Par3, which can function downstream of DDR1, also reversed the effects of Gα13 knockdown on cell-cell adhesion and proteolytic invasion in three-dimensional collagen. Overall, we show that Gα13 and DDR1-Par3 differentially regulate cell-cell junctions and the actin cytoskeleton to mediate invasion in three-dimensional collagen.

  13. Polymorphism of the E-cadherin gene CDH1 is associated with susceptibility to vitiligo.

    PubMed

    Tarlé, Roberto Gomes; Silva de Castro, Caio Cesar; do Nascimento, Liliane Machado; Mira, Marcelo Távora

    2015-04-01

    Vitiligo is a depigmenting disorder characterized by loss of functional melanocytes from the epidermis. Experimental data suggest that defective melanocyte adhesion may underlie the pathogenesis of the disease. In particular, association between vitiligo and genetic variants of the DDR1 gene involved in melanocyte adhesion has been recently published. A subsequent, independent study revealed lower expression of DDR1 in vitiligo lesions. Here, we expand this investigation by testing for association between vitiligo and polymorphisms of CDH1, IL1B and NOV (formerly CCN3), genes belonging to the DDR1 adhesion pathway, in two population samples of distinct design. Our results reveal that alleles of marker rs10431924 of the CDH1 gene are associated with vitiligo, especially in the presence of autoimmune comorbidities.

  14. Formation of post-confluence structure in human parotid gland acinar cells on PLGA through regulation of E-cadherin.

    PubMed

    Chan, Yen-Hui; Huang, Tsung-Wei; Chou, Ya-Shuan; Hsu, Sheng-Hao; Su, Wei-Fang; Lou, Pei-Jen; Young, Tai-Horng

    2012-01-01

    As a potential solution for patients to retrieve their lost salivary gland functions, tissue engineering of an auto-secretory device is profoundly needed. Under serum-free environment, primary human parotid gland acinar (PGAC) cells can be obtained. After reaching confluence, PGAC cells spontaneously form three-dimension (3D) cell aggregations, termed post-confluence structure (PCS), and change their behaviors. Poly (lactic-co-glycolic acid) (PLGA) has been widely used in the field of biomedical applications because of its biodegradable properties for desired functions. Nonetheless, the role of PLGA in facilitating PGAC cells to form PCS has seldom been explored to recover epithelial characteristics. In this study, PGAC cells were found to have a greater tendency to form PCS on PLGA than on tissue culture polystyrene (TCPS). By tracing cell migration paths and modulating E-cadherin activity with specific inhibitor or antibody, we demonstrated that the static force of homophilic interaction on surfaces of individual cells, but not the dynamics of cell migration, played a more important role in PCS formation. Thus, PLGA was successfully confirmed to support PGAC cells to form more PCS through the effects on enhancing E-cadherin expression, which is associated with FAK/ILK/Snail expression in PGAC cells. This result indicates that selective appropriate biomaterials may be potentially useful in generating 3D PCS on two-dimension (2D) substrate without fabricating a complex 3D scaffold.

  15. Elevated Src family kinase activity stabilizes E-cadherin-based junctions and collective movement of head and neck squamous cell carcinomas

    PubMed Central

    Veracini, Laurence; Grall, Dominique; Schaub, Sébastien; Divonne, Stéphanie Beghelli-de la Forest; Etienne-Grimaldi, Marie-Christine; Milano, Gérard; Bozec, Alexandre; Babin, Emmanuel; Sudaka, Anne; Thariat, Juliette; Van Obberghen-Schilling, Ellen

    2015-01-01

    EGF receptor (EGFR) overexpression is thought to drive head and neck carcinogenesis however clinical responses to EGFR-targeting agents have been modest and alternate targets are actively sought to improve results. Src family kinases (SFKs), reported to act downstream of EGFR are among the alternative targets for which increased expression or activity in epithelial tumors is commonly associated to the dissolution of E-cadherin-based junctions and acquisition of a mesenchymal-like phenotype. Robust expression of total and activated Src was observed in advanced stage head and neck tumors (N=60) and in head and neck squamous cell carcinoma lines. In cultured cancer cells Src co-localized with E-cadherin in cell-cell junctions and its phosphorylation on Y419 was both constitutive and independent of EGFR activation. Selective inhibition of SFKs with SU6656 delocalized E-cadherin and disrupted cellular junctions without affecting E-cadherin expression and this effect was phenocopied by knockdown of Src or Yes. These findings reveal an EGFR-independent role for SFKs in the maintenance of intercellular junctions, which likely contributes to the cohesive invasion E-cadherin-positive cells in advanced tumors. Further, they highlight the need for a deeper comprehension of molecular pathways that drive collective cell invasion, in absence of mesenchymal transition, in order to combat tumor spread. PMID:25779657

  16. EZH2 promotes cell migration and invasion but not alters cell proliferation by suppressing E-cadherin, partly through association with MALAT-1 in pancreatic cancer

    PubMed Central

    Han, Ting; Jiao, Feng; Hu, Hai; Yuan, Cuncun; Wang, Lei; Jin, Zi-Liang; Song, Wei-feng; Wang, Li-Wei

    2016-01-01

    Enhancer of zeste homolog 2 (EZH2) is an essential component of the polycomb repressive complex 2 (PRC2), which is required for epigenetic silencing of target genes, including those affecting cancer progression. Its role in pancreatic cancer remains to be clarified; therefore, we investigated the effects of aberrantly expressed EZH2 on pancreatic cancer. We found that EZH2 expression is up-regulated in pancreatic cancer tissues and positively correlated with lymph node metastasis and advanced clinical stage in pancreatic cancer patients. EZH2 knockdown in pancreatic cancer cell lines inhibited cell migration and invasion, but did not alter cell proliferation. Silencing of EZH2 also increased E-cadherin expression in vitro, and E-cadherin expression was inversely correlated with EZH2 expression in pancreatic cancer tissue samples. Patients with high EZH2 and low E-cadherin expression had the worst prognosis. RIP and ChIP assays suggest that EZH2 is recruited to the E-cadherin promoter by the long non-coding RNA, MALAT-1 (metastasis associated in lung adenocarcinoma transcript 1), where it represses E-cadherin expression. Our results show that EZH2-based therapies may be an option for the treatment of pancreatic cancer. PMID:26848980

  17. EZH2 promotes cell migration and invasion but not alters cell proliferation by suppressing E-cadherin, partly through association with MALAT-1 in pancreatic cancer.

    PubMed

    Han, Ting; Jiao, Feng; Hu, Hai; Yuan, Cuncun; Wang, Lei; Jin, Zi-Liang; Song, Wei-Feng; Wang, Li-Wei

    2016-03-01

    Enhancer of zeste homolog 2 (EZH2) is an essential component of the polycomb repressive complex 2 (PRC2), which is required for epigenetic silencing of target genes, including those affecting cancer progression. Its role in pancreatic cancer remains to be clarified; therefore, we investigated the effects of aberrantly expressed EZH2 on pancreatic cancer. We found that EZH2 expression is up-regulated in pancreatic cancer tissues and positively correlated with lymph node metastasis and advanced clinical stage in pancreatic cancer patients. EZH2 knockdown in pancreatic cancer cell lines inhibited cell migration and invasion, but did not alter cell proliferation. Silencing of EZH2 also increased E-cadherin expression in vitro, and E-cadherin expression was inversely correlated with EZH2 expression in pancreatic cancer tissue samples. Patients with high EZH2 and low E-cadherin expression had the worst prognosis. RIP and ChIP assays suggest that EZH2 is recruited to the E-cadherin promoter by the long non-coding RNA, MALAT-1 (metastasis associated in lung adenocarcinoma transcript 1), where it represses E-cadherin expression. Our results show that EZH2-based therapies may be an option for the treatment of pancreatic cancer.

  18. Clinicopathological significance of SIRT1 and p300/CBP expression in gastroesophageal junction (GEJ) cancer and the correlation with E-cadherin and MLH1.

    PubMed

    Zhang, Li-Hua; Huang, Qin; Fan, Xiang-Shan; Wu, Hong-Yan; Yang, Jun; Feng, An-Ning

    2013-10-01

    SIRT1 and p300/CBP, which are considered to be essential histone deacetylases and acetyltransferases, are also considered to be relative to tumorigenesis because they modulate the expression of several tumor suppressor genes. Therefore, this study investigated the expression of SIRT1 and p300/CBP in gastroesophageal junction (GEJ) cancer and their correlation with E-cadherin and MLH1 in order to explore the clinicopathological significance of SIRT1 and p300/CBP expression and their possible effects involving E-cadherin and MLH1 expression. Tissue microarray technique and immunohistochemical stains were applied to evaluate the SIRT1, p300/CBP, E-cadherin, and MLH1 expression in 176 GEJ cancer tissues and 32 normal GEJ region tissues. The results showed that the over-expression of SIRT1 was associated with a higher number of metastasis lymph nodes, more advanced staging, and shorter mean survival time. SIRT1 and p300/CBP were negatively and positively correlated with the expression of E-cadherin and MLH1, respectively, in the cancer cases. These results indicated a possible effect of SIRT1 and p300/CBP involved in regulating the expression of E-cadherin and MLH1, thus participating in the tumor progression of GEJ cancer.

  19. RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs.

    PubMed

    Kajiho, Hiroaki; Kajiho, Yuko; Frittoli, Emanuela; Confalonieri, Stefano; Bertalot, Giovanni; Viale, Giuseppe; Di Fiore, Pier Paolo; Oldani, Amanda; Garre, Massimiliano; Beznoussenko, Galina V; Palamidessi, Andrea; Vecchi, Manuela; Chavrier, Philippe; Perez, Frank; Scita, Giorgio

    2016-07-01

    The mechanisms of tumor cell dissemination and the contribution of membrane trafficking in this process are poorly understood. Through a functional siRNA screening of human RAB GTPases, we found that RAB2A, a protein essential for ER-to-Golgi transport, is critical in promoting proteolytic activity and 3D invasiveness of breast cancer (BC) cell lines. Remarkably, RAB2A is amplified and elevated in human BC and is a powerful and independent predictor of disease recurrence in BC patients. Mechanistically, RAB2A acts at two independent trafficking steps. Firstly, by interacting with VPS39, a key component of the late endosomal HOPS complex, it controls post-endocytic trafficking of membrane-bound MT1-MMP, an essential metalloprotease for matrix remodeling and invasion. Secondly, it further regulates Golgi transport of E-cadherin, ultimately controlling junctional stability, cell compaction, and tumor invasiveness. Thus, RAB2A is a novel trafficking determinant essential for regulation of a mesenchymal invasive program of BC dissemination. PMID:27255086

  20. The Hedgehog Inhibitor Cyclopamine Reduces β-Catenin-Tcf Transcriptional Activity, Induces E-Cadherin Expression, and Reduces Invasion in Colorectal Cancer Cells

    PubMed Central

    Qualtrough, David; Rees, Phil; Speight, Beverley; Williams, Ann C.; Paraskeva, Christos

    2015-01-01

    Colorectal cancer is a major global health problem resulting in over 600,000 deaths world-wide every year with the majority of these due to metastatic disease. Wnt signalling, and more specifically β-catenin-related transcription, has been shown to drive both tumorigenesis and the metastatic process in colorectal neoplasia, yet its complex interactions with other key signalling pathways, such as hedgehog, remain to be elucidated. We have previously shown that the Hedgehog (HH) signalling pathway is active in cells from colorectal tumours, and that inhibition of the pathway with cyclopamine induces apoptosis. We now show that cyclopamine treatment reduces β-catenin related transcription in colorectal cancer cell lines, and that this effect can be reversed by addition of Sonic Hedgehog protein. We also show that cyclopamine concomitantly induces expression of the tumour suppressor and prognostic indicator E-cadherin. Consistent with a role for HH in regulating the invasive potential we show that cyclopamine reduces the expression of transcription factors (Slug, Snail and Twist) associated with the epithelial-mesenchymal transition and reduces the invasiveness of colorectal cancer cells in vitro. Taken together, these data show that pharmacological inhibition of the hedgehog pathway has therapeutic potential in the treatment of colorectal cancer. PMID:26393651

  1. Adhesion and structure properties of protein nanomaterials containing hydrophobic and charged amino acids.

    PubMed

    Shen, Xinchun; Mo, Xiaoqun; Moore, Robyn; Frazier, Shawnalea J; Iwamoto, Takeo; Tomich, John M; Sun, Xiuzhi Susan

    2006-03-01

    Protein polymers are being used or considered for biobased adhesives and coating materials. Most adhesives derived from macro protein molecules work through receptors or cross-links to bring about adhesion. The adhesion mechanism of protein polymers would lead to better understanding of adhesives and the discovery of new practical properties of protein polymers at both nano- and macro-scales. The objective of this research work was to study adhesion properties of protein polymers at nanoscale (a peptide adhesive with nanometer-scale units that range in size of several nanometers, defined as protein nanomaterial). Seven protein nanomaterial samples with different degrees of adhesive strength were designed and synthesized using solid phase chemistries. All protein nanomaterials contain a common hydrophobic core flanked by charged amino acid sequences. The adhesion properties of the protein nanomaterials were investigated at different pH values and curing temperatures. The protein nanomaterials self aggregate and interact with the wood surface. The protein nanomaterial KKK-FLIVIGSII-KKK identified in this study had high adhesive strength toward wood. It had the highest shear strength at pH 12, with an amino acid sequence that was very hydrophobic and uncharged. This protein nanomaterial underwent structural analyses using circular dichroism, laser-Fourier transform infrared, and laser desorption mass spectrometry. At pH 12 this peptide adopted a pH-induced beta-like conformation. Adhesive strength reflects contributions of both hydrogen bonding and van der Waals interactions. Ionic and covalent bonds do not appear to be significant factors for adhesion in this study.

  2. Use of additives to enhance the properties of cottonseed protein as wood adhesives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soy protein is currently being used commercially as a “green” wood adhesive. Previous work in this laboratory has shown that cottonseed protein isolate, tested on maple wood veneer, produced higher adhesive strength and hot water resistance relative to soy protein. In the present study, cottonseed...

  3. Expression of epithelial adhesion proteins and integrins in chronic inflammation.

    PubMed Central

    Haapasalmi, K.; Mäkelä, M.; Oksala, O.; Heino, J.; Yamada, K. M.; Uitto, V. J.; Larjava, H.

    1995-01-01

    Epithelial cell behavior in chronic inflammation is poorly characterized. During inflammation of tooth-supporting structures (periodontal disease), increased proliferation of epithelial cells into the inflamed connective tissue stroma is commonly seen. In some areas ulceration and degeneration take place. We studied alterations in the expression of adhesion molecules and integrins during chronic periodontal inflammation. In inflamed tissue, laminin-1 and type IV collagen were still present in the basement membrane and surrounding blood vessels, but they were also found extravascularly in inflamed connective tissue stroma. Type VII collagen and laminin-5 (also known as kalinin, epiligrin, or nicein) were poorly preserved in the basement membrane zone, but both were found in unusual streak-like distributions in the subepithelial connective tissue stroma in inflamed tissue. Both fibronectin and tenascin were substantially decreased in chronically inflamed connective tissue, showing only punctate staining at the basement membrane zone. Integrins of the beta 1 family showed two distinct staining patterns in epithelial cells during chronic inflammation; focal losses of beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1) were found in most areas, while in other areas the entire pocket epithelium was found to be strongly positive for beta 1 integrins. No members of the alpha v integrin family were found in any epithelia studied. Expression of the alpha 6 beta 4 integrin was high in basal cells of healthy tissue, but weak in epithelium associated with chronic inflammation. Chronic inflammation therefore involves alterations in both adhesion proteins and integrins expressed by epithelial cells. Basement membrane components found at abnormal sites in stroma in chronic inflammation might serve as new adhesive ligands for various cell types in inflamed stroma. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:7541610

  4. The postnatal accumulation of junctional E-cadherin is inversely correlated with the capacity for supporting cells to convert directly into sensory hair cells in mammalian balance organs.

    PubMed

    Collado, Maria Sol; Thiede, Benjamin R; Baker, Wendy; Askew, Charles; Igbani, Lisa M; Corwin, Jeffrey T

    2011-08-17

    Mammals experience permanent impairments from hair cell (HC) losses, but birds and other non-mammals quickly recover hearing and balance senses after supporting cells (SCs) give rise to replacement HCs. Avian HC epithelia express little or no E-cadherin, and differences in the thickness of F-actin belts at SC junctions strongly correlate with different species' capacities for HC replacement, so we investigated junctional cadherins in human and murine ears. We found strong E-cadherin expression at SC-SC junctions that increases more than sixfold postnatally in mice. When we cultured utricles from young mice with γ-secretase inhibitors (GSIs), striolar SCs completely internalized their E-cadherin, without affecting N-cadherin. Hes and Hey expression also decreased and the SCs began to express Atoh1. After 48 h, those SCs expressed myosins VI and VIIA, and by 72 h, they developed hair bundles. However, some scattered striolar SCs retained E-cadherin and the SC phenotype. In extrastriolar regions, the vast majority of SCs also retained E-cadherin and failed to convert into HCs even after long GSI treatments. Microscopic measurements revealed that the junctions between extrastriolar SCs were more developed than those between striolar SCs. In GSI-treated utricles as old as P12, differentiated striolar SCs converted into HCs, but such responses declined with age and ceased by P16. Thus, temporal and spatial differences in postnatal SC-to-HC phenotype conversion capacity are linked to the structural attributes of E-cadherin containing SC junctions in mammals, which differ substantially from their counterparts in non-mammalian vertebrates that readily recover from hearing and balance deficits through hair cell regeneration.

  5. The Postnatal Accumulation of Junctional E-cadherin Is Inversely Correlated with the Capacity for Supporting Cells to Convert Directly into Sensory Hair Cells in Mammalian Balance Organs

    PubMed Central

    Collado, Maria Sol; Thiede, Benjamin R.; Baker, Wendy; Askew, Charles; Igbani, Lisa M.; Corwin, Jeffrey T.

    2011-01-01

    Mammals experience permanent impairments from hair cell (HC) losses, but birds and other non-mammals quickly recover hearing and balance senses after supporting cells (SCs) give rise to replacement HCs. Avian HC epithelia express little or no E-cadherin, and differences in the thickness of F-actin belts at SC junctions strongly correlate with different species’ capacities for HC replacement, so we investigated junctional cadherins in human and murine ears. We found strong E-cadherin expression at SC-SC junctions that increases >6-fold postnatally in mice. When we cultured utricles from young mice with γ-secretase inhibitors (GSIs), striolar SCs completely internalized their E-cadherin, without affecting N-cadherin. Hes and Hey expression also decreased and the SCs began to express Atoh1. After 48 h, those SCs expressed myosins VI and VIIA, and by 72 h they developed hair bundles. However, some scattered striolar SCs retained E-cadherin and the SC phenotype. In extrastriolar regions the vast majority of SCs also retained E-cadherin and failed to convert into HCs even after long GSI treatments. Microscopic measurements revealed that the junctions between extrastriolar SCs were more developed than those between striolar SCs. In GSI-treated utricles as old as P12, differentiated striolar SCs converted into HCs, but such responses declined with age and ceased by P16. Thus, temporal and spatial differences in postnatal SC-to-HC phenotype conversion capacity are linked to the structural attributes of E-cadherin containing SC junctions in mammals, which differ substantially from their counterparts in non-mammalian vertebrates that readily recover from hearing and balance deficits through hair cell regeneration. PMID:21849546

  6. Boronate Complex Formation with Dopa Containing Mussel Adhesive Protein Retards pH-Induced Oxidation and Enables Adhesion to Mica

    PubMed Central

    Israelachvili, Jacob N.; Chen, Yunfei; Waite, J. Herbert

    2014-01-01

    The biochemistry of mussel adhesion has inspired the design of surface primers, adhesives, coatings and gels for technological applications. These mussel-inspired systems often focus on incorporating the amino acid 3,4-dihydroxyphenyl-L-alanine (Dopa) or a catecholic analog into a polymer. Unfortunately, effective use of Dopa is compromised by its susceptibility to auto-oxidation at neutral pH. Oxidation can lead to loss of adhesive function and undesired covalent cross-linking. Mussel foot protein 5 (Mfp-5), which contains ∼30 mole % Dopa, is a superb adhesive under reducing conditions but becomes nonadhesive after pH-induced oxidation. Here we report that the bidentate complexation of borate by Dopa to form a catecholato-boronate can be exploited to retard oxidation. Although exposure of Mfp-5 to neutral pH typically oxidizes Dopa, resulting in a>95% decrease in adhesion, inclusion of borate retards oxidation at the same pH. Remarkably, this Dopa-boronate complex dissociates upon contact with mica to allow for a reversible Dopa-mediated adhesion. The borate protection strategy allows for Dopa redox stability and maintained adhesive function in an otherwise oxidizing environment. PMID:25303409

  7. Affixing plant sections without protein based adhesives for protease histochemistry.

    PubMed

    Jona, R; Griglione, R

    1999-01-01

    To submit a section of plant tissue to histochemical analysis using protease, the protein based adhesives which keep the slices attached to the slides must be replaced because they are attacked by the enzyme and the slices are washed off the slides. We devised a method to keep the slices attached to the slides during histochemical extractions and subsequent staining. Slides are frosted on two lateral zones by spreading on them a fluoride paste composed of 15 g barium sulfate, 15 g ammonium difluoride, 8 g oxalic acid, 40 ml glycerine and 12 ml deionized water using a thin paint brush. After removing the paste with tap water and drying the slides, the sections are placed on the central clear zone of the slide and covered with an ethyl-cellulose film that keeps the slices in place and allows the reagents to act through it. To do this, the slides are dipped into 0.5% ethyl cellulose (ETC) prepared in a 4:1 mixture of toluene and absolute ethanol. The ETC coating is layered three times to improve its firmness and its ability to retain the slices on the slides. To obtain perfect adhesion, the slide should be oven dried (40-50 C for 10-15 min) to remove any trace of humidity before applying each layer of ETC. Subsequently the sections can be extracted and stained without undue loss of material. PMID:10190256

  8. Adhesions

    MedlinePlus

    ... surfaces so they can shift easily as the body moves. Adhesions cause tissues and organs to stick together. They might connect the loops of the intestines to each other, to nearby ... can occur anywhere in the body. But they often form after surgery on the ...

  9. Bmi-1 promotes the invasion and migration of colon cancer stem cells through the downregulation of E-cadherin.

    PubMed

    Zhang, Zefeng; Bu, Xiaoling; Chen, Hao; Wang, Qiyi; Sha, Weihong

    2016-10-01

    Metastasis and recurrence are the challenges of cancer therapy. Recently, mounting evidence has suggested that cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) are critical factors in tumor metastasis and recurrence. The oncogene, Bmi-1, promotes the development of hematologic malignancies and many solid tumors. The aim of the present study was to elucidate the mechanisms through which Bmi-1 promotes the invasion and migration of colon CSCs (CCSCs) using the HCT116 colon cancer cell line. Sphere formation medium and magnetic‑activated cell sorting were used to enrich and screen the CCSCs. CD133 and CD44 were regarded as markers of CCSCs and they were found to be co-expressed in the HCT116 colon cancer cell line. Colony formation assay, cell proliferation assay and viability assay using the Cell Counting Kit-8, and transplantation assay using nude mice injected with CCSCs were used to examine the CCSCs. The CD133+CD44+ HCT116 cells exhibited greater cloning efficiency, an enhanced proliferative ability, increased cell viability and stronger tumorigenicity; these cells were used as the CCSCs for subsequent experiments. In addition, the invasive and migratory abilities of the CD133+CD44+ HCT116 cells were markedly decreased when Bmi-1 was silenced by small interfering RNA (siRNA). The results of RT-qPCR and western blot analysis suggested that Bmi-1 had a negative effect on E-cadherin expression. On the whole, our findings suggest that Bmi-1 promotes the invasion and migration of CCSCs through the downregulation of E-cadherin, possibly by inducing EMT. Our findings thus indicate that Bmi-1 may be a novel therapeutic target for the treatment of colon cancer. PMID:27600678

  10. Bmi-1 promotes the invasion and migration of colon cancer stem cells through the downregulation of E-cadherin

    PubMed Central

    Zhang, Zefeng; Bu, Xiaoling; Chen, Hao; Wang, Qiyi; Sha, Weihong

    2016-01-01

    Metastasis and recurrence are the challenges of cancer therapy. Recently, mounting evidence has suggested that cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) are critical factors in tumor metastasis and recurrence. The oncogene, Bmi-1, promotes the development of hematologic malignancies and many solid tumors. The aim of the present study was to elucidate the mechanisms through which Bmi-1 promotes the invasion and migration of colon CSCs (CCSCs) using the HCT116 colon cancer cell line. Sphere formation medium and magnetic-activated cell sorting were used to enrich and screen the CCSCs. CD133 and CD44 were regarded as markers of CCSCs and they were found to be co-expressed in the HCT116 colon cancer cell line. Colony formation assay, cell proliferation assay and viability assay using the Cell Counting Kit-8, and transplantation assay using nude mice injected with CCSCs were used to examine the CCSCs. The CD133+CD44+ HCT116 cells exhibited greater cloning efficiency, an enhanced proliferative ability, increased cell viability and stronger tumorigenicity; these cells were used as the CCSCs for subsequent experiments. In addition, the invasive and migratory abilities of the CD133+CD44+ HCT116 cells were markedly decreased when Bmi-1 was silenced by small interfering RNA (siRNA). The results of RT-qPCR and western blot analysis suggested that Bmi-1 had a negative effect on E-cadherin expression. On the whole, our findings suggest that Bmi-1 promotes the invasion and migration of CCSCs through the downregulation of E-cadherin, possibly by inducing EMT. Our findings thus indicate that Bmi-1 may be a novel therapeutic target for the treatment of colon cancer. PMID:27600678

  11. Protein-based underwater adhesives and the prospects for their biotechnological production.

    PubMed

    Stewart, Russell J

    2011-01-01

    Biotechnological approaches to practical production of biological protein-based adhesives have had limited success over the last several decades. Broader efforts to produce recombinant adhesive proteins may have been limited by early disappointments. More recent synthetic polymer approaches have successfully replicated some aspects of natural underwater adhesives. For example, synthetic polymers, inspired by mussels, containing the catecholic functional group of 3,4-L-dihydroxyphenylalanine adhere strongly to wet metal oxide surfaces. Synthetic complex coacervates inspired by the Sandcastle worm are water-borne adhesives that can be delivered underwater without dispersing. Synthetic approaches offer several advantages, including versatile chemistries and scalable production. In the future, more sophisticated mimetic adhesives may combine synthetic copolymers with recombinant or agriculture-derived proteins to better replicate the structural and functional organization of natural adhesives.

  12. Allosteric Coupling in the Bacterial Adhesive Protein FimH*

    PubMed Central

    Rodriguez, Victoria B.; Kidd, Brian A.; Interlandi, Gianluca; Tchesnokova, Veronika; Sokurenko, Evgeni V.; Thomas, Wendy E.

    2013-01-01

    The protein FimH is expressed by the majority of commensal and uropathogenic strains of Escherichia coli on the tips of type 1 fimbriae and mediates adhesion via a catch bond to its ligand mannose. Crystal structures of FimH show an allosteric conformational change, but it remains unclear whether all of the observed structural differences are part of the allosteric mechanism. Here we use the protein structural analysis tool RosettaDesign combined with human insight to identify and synthesize 10 mutations in four regions that we predicted would stabilize one of the conformations of that region. The function of each variant was characterized by measuring binding to the ligand mannose, whereas the allosteric state was determined using a conformation-specific monoclonal antibody. These studies demonstrated that each region investigated was indeed part of the FimH allosteric mechanism. However, the studies strongly suggested that some regions were more tightly coupled to mannose binding and others to antibody binding. In addition, we identified many FimH variants that appear locked in the low affinity state. Knowledge of regulatory sites outside the active and effector sites as well as the ability to make FimH variants locked in the low affinity state may be crucial to the future development of novel antiadhesive and antimicrobial therapies using allosteric regulation to inhibit FimH. PMID:23821547

  13. Surface functionalization of inorganic nano-crystals with fibronectin and E-cadherin chimera synergistically accelerates trans-gene delivery into embryonic stem cells.

    PubMed

    Kutsuzawa, K; Chowdhury, E H; Nagaoka, M; Maruyama, K; Akiyama, Y; Akaike, T

    2006-11-24

    Stem cells holding great promises in regenerative medicine have the potential to be differentiated to a specific cell type through genetic manipulation. However, conventional ways of gene transfer to such progenitor cells suffer from a number of disadvantages particularly involving safety and efficacy issues. Here, we report on the development of a bio-functionalized inorganic nano-carrier of DNA by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high affinity interactions with embryonic stem cell surface and accelerated trans-gene delivery for subsequent expression. While only apatite nano-particles were very inefficient in transfecting embryonic stem cells, fibronectin-anchored particles and to a more significant extent, fibronectin and E-cadherin-Fc-associated particles dramatically enhanced trans-gene delivery with a value notably higher than that of commercially available lipofection system. The involvement of both cell surface integrin and E-cadherin in mediating intracellular localization of the hybrid carrier was verified by blocking integrin binding site with excess free fibronectin and up-regulating both integrin and E-cadherin through PKC activation. Thus, the new establishment of a bio-functional hybrid gene-carrier would promote and facilitate development of stem cell-based therapy in regenerative medicine.

  14. Surface functionalization of inorganic nano-crystals with fibronectin and E-cadherin chimera synergistically accelerates trans-gene delivery into embryonic stem cells

    SciTech Connect

    Kutsuzawa, K.; Chowdhury, E.H.; Nagaoka, M.; Maruyama, K.; Akiyama, Y.; Akaike, T. . E-mail: takaike@bio.titech.ac.jp

    2006-11-24

    Stem cells holding great promises in regenerative medicine have the potential to be differentiated to a specific cell type through genetic manipulation. However, conventional ways of gene transfer to such progenitor cells suffer from a number of disadvantages particularly involving safety and efficacy issues. Here, we report on the development of a bio-functionalized inorganic nano-carrier of DNA by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high affinity interactions with embryonic stem cell surface and accelerated trans-gene delivery for subsequent expression. While only apatite nano-particles were very inefficient in transfecting embryonic stem cells, fibronectin-anchored particles and to a more significant extent, fibronectin and E-cadherin-Fc-associated particles dramatically enhanced trans-gene delivery with a value notably higher than that of commercially available lipofection system. The involvement of both cell surface integrin and E-cadherin in mediating intracellular localization of the hybrid carrier was verified by blocking integrin binding site with excess free fibronectin and up-regulating both integrin and E-cadherin through PKC activation. Thus, the new establishment of a bio-functional hybrid gene-carrier would promote and facilitate development of stem cell-based therapy in regenerative medicine.

  15. An hTERT/ZEB1 complex directly regulates E-cadherin to promote epithelial-to-mesenchymal transition (EMT) in colorectal cancer.

    PubMed

    Qin, Yong; Tang, Bo; Hu, Chang-Jiang; Xiao, Yu-Feng; Xie, Rui; Yong, Xin; Wu, Yu Yun; Dong, Hui; Yang, Shi-Ming

    2016-01-01

    In human cancer, high telomerase expression is correlated with tumor aggressiveness and metastatic potential. Telomerase activation occurs through telomerase reverse transcriptase (hTERT) induction, which contributes to malignant transformation by stabilizing telomeres. Previous studies have shown that hTERT can promote tumor invasion and metastasis of gastric cancer, liver cancer and esophageal cancer. Epithelial-to-mesenchymal transition (EMT), a requirement for tumor invasion and metastasis, plays a key role in cancer progression. Although hTERT promotes EMT through Wnt signaling in several cancers, it is unknown if other signaling pathways are involved. In the present study, we found that hTERT and ZEB1 form a complex, which directly binds to the E-cadherin promoter, and then inhibits E-cadherin expression and promots EMT in colorectal cancer cells. hTERT overexpression in HCT116 and SW480 cells could induce E-cadherin down-regulation. However, E-cadherin expression was recovered when ZEB1 function was impaired even during hTERT overexpression. Taken together, our findings suggest that hTERT can promote cancer metastasis by stimulating EMT through the ZEB1 pathway and therefore inhibiting them may prevent cancer progression.

  16. The lysyl oxidases LOX and LOXL2 are necessary and sufficient to repress E-cadherin in hypoxia: insights into cellular transformation processes mediated by HIF-1.

    PubMed

    Schietke, Ruth; Warnecke, Christina; Wacker, Ingrid; Schödel, Johannes; Mole, David R; Campean, Valentina; Amann, Kerstin; Goppelt-Struebe, Margarete; Behrens, Jürgen; Eckardt, Kai-Uwe; Wiesener, Michael S

    2010-02-26

    Hypoxia has been shown to promote tumor metastasis and lead to therapy resistance. Recent work has demonstrated that hypoxia represses E-cadherin expression, a hallmark of epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved. Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia. In addition to the previously demonstrated LOX, we characterize LOXL2 as a direct transcriptional target of HIF-1. We demonstrate that activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Our data support a molecular pathway from hypoxia to cellular transformation. It includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.

  17. PTEN Loss in E-Cadherin-Deficient Mouse Mammary Epithelial Cells Rescues Apoptosis and Results in Development of Classical Invasive Lobular Carcinoma.

    PubMed

    Boelens, Mirjam C; Nethe, Micha; Klarenbeek, Sjoerd; de Ruiter, Julian R; Schut, Eva; Bonzanni, Nicola; Zeeman, Amber L; Wientjens, Ellen; van der Burg, Eline; Wessels, Lodewyk; van Amerongen, Renée; Jonkers, Jos

    2016-08-23

    Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC), the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER) status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K) signaling as a potential therapeutic strategy for targeting CLC. PMID:27524621

  18. E-cadherin in non-tumor epithelium adjacent to oral cancer as risk marker for the development of multiple tumors.

    PubMed

    González-Moles, M A; Bravo, M; Ruiz-Avila, I; Gil-Montoya, J A; Acebal, F; Esteban, F

    2013-03-01

    Our aim was to find out whether the loss of E-cadherin is a risk factor for the development of multiple tumours in the oral cavity and whether it could serve as a diagnostic marker for oral premalignant fields. We studied 77 oral squamous cell carcinomas (SCC) with associated non-tumour epithelia from 61 patients. Immunohistochemical studies (antibody NHC-38) were used to investigate E-cadherin expression, which was completely lost in basal (48% of cases) and parabasal (43%) layers of non-tumour epithelia close to the tumour and in basal (47%) and parabasal (38%) layers of non-tumour epithelia distant from the tumour. In multiple tumours E-cadherin expression was significantly lower than in single tumours in the basal, parabasal layers, and the middle third of close (p=0.002, <0.001, <0.001) and distant (p=0.041, p<0.001, p=0.005) non-tumour epithelia, respectively. Downregulation of E-cadherin may be valuable as a risk marker for the development of multiple tumours in the oral cavity and for the diagnosis of premalignant fields.

  19. Aberrant Glycosylation of Plasma Proteins in Severe Preeclampsia Promotes Monocyte Adhesion

    PubMed Central

    Kazanjian, Avedis A.; Tinnemore, Deborah; Gafken, Philip R.; Ogata, Yuko; Napolitano, Peter G.; Stallings, Jonathan D.; Ippolito, Danielle L.

    2014-01-01

    Glycosylation of plasma proteins increases during pregnancy. Our objectives were to investigate an anti-inflammatory role of these proteins in normal pregnancies and determine whether aberrant protein glycosylation promotes monocyte adhesion in preeclampsia. Plasma was prospectively collected from nonpregnant controls and nulliparous patients in all 3 trimesters. Patients were divided into cohorts based on the applicable postpartum diagnosis. U937 monocytes were preconditioned with enzymatically deglycosylated plasma, and monocyte adhesion to endothelial cell monolayers was quantified by spectrophotometry. Plasma from nonpregnant controls, first trimester normotensives, and first trimester patients with mild preeclampsia inhibited monocyte–endothelial cell adhesion (P < .05), but plasma from first trimester patients with severe preeclampsia and second and third trimester normotensives did not. Deglycosylating plasma proteins significantly increased adhesion in all the cohorts. These results support a role of plasma glycoprotein interaction in monocyte–endothelial cell adhesion and could suggest a novel therapeutic target for severe preeclampsia. PMID:23757314

  20. p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion

    PubMed Central

    George, Margaret D.; Wine, Robert N.; Lackford, Brad; Kissling, Grace E.; Akiyama, Steven K.; Olden, Kenneth; Roberts, John D.

    2014-01-01

    Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading and that this association can be regulated by factors in the tumor microenvironment. PMID:24219282

  1. Interaction of alpha-actinin with the cadherin/catenin cell-cell adhesion complex via alpha-catenin

    PubMed Central

    1995-01-01

    Cadherins are Ca(2+)-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, transmembrane cadherins such as E- , P-, and N-cadherin self-associate, while intracellularly they interact indirectly with the actin-based cytoskeleton. Several intracellular proteins termed catenins, including alpha-catenin, beta- catenin, and plakoglobin, are tightly associated with these cadherins and serve to link them to the cytoskeleton. Here, we present evidence that in fibroblasts alpha-actinin, but not vinculin, colocalizes extensively with the N-cadherin/catenin complex. This is in contrast to epithelial cells where both cytoskeletal proteins colocalize extensively with E-cadherin and catenins. We further show that alpha- actinin, but not vinculin, coimmunoprecipitates specifically with alpha- and beta-catenin from N- and E-cadherin-expressing cells, but only if alpha-catenin is present. Moreover, we show that alpha-actinin coimmunoprecipitates with the N-cadherin/catenin complex in an actin- independent manner. We therefore propose that cadherin/catenin complexes are linked to the actin cytoskeleton via a direct association between alpha-actinin and alpha-catenin. PMID:7790378

  2. Monoclonal Antibody against the Ectodomain of E-Cadherin (DECMA-1) Suppresses Breast Carcinogenesis: Involvement of the HER/PI3K/Akt/mTOR and IAP Pathways

    PubMed Central

    Brouxhon, Sabine M.; Kyrkanides, Stephanos; Teng, Xiaofei; Raja, Veena; O’Banion, M. Kerry; Clarke, Robert; Byers, Stephen; Silberfeld, Andrew; Tornos, Carmen; Ma, Li

    2014-01-01

    Purpose Although targeted therapies against HER2 have been one of the most successful therapeutic strategies for breast cancer, patients eventually developed acquired resistance from compensatory upregulation of alternate HERs and mitogen-activated protein kinase–phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling. As we and others have shown that the soluble ectodomain fragment of E-cadherin exerts prooncogenic effects via HER1/2–mediated binding and activation of downstream prosurvival pathways, we explored whether targeting this ectodomain [DECMA-1 monoclonal antibody (mAb)] was effective in the treatment of HER2-positive (HER2+) breast cancers. Experimental Design MMTV-PyMT transgenic mice and HER2+/E-cadherin–positive MCF-7 and BT474 trastuzumab-resistant (TtzmR) cells were treated with the DECMA-1 mAb. Antitumor responses were assessed by bromodeoxyuridine incorporation, apoptosis, and necrosis. The underlying intracellular prooncogenic pathways were explored using subcellular fractionation, immunoprecipitation, fluorescence microscopy, and immunoblotting. Results Treatment with DECMA-1 mAb significantly delayed tumor onset and attenuated tumor burden in MMTV-PyMT mice by reducing tumor cell proliferation and inducing apoptosis without any detectable cytotoxicity to mice or end-organs. In vitro treatment of MCF-7 and BT474 TtzmR cells reduced proliferation and induced cancer cell apoptosis. Importantly, this inhibition of breast tumorigenesis was due to concomitant downregulation, via ubiquitin-mediated degradation through the lysosome and proteasome pathways, of all HER family members, components of downstream PI3K/Akt/mTOR prosurvival signaling and suppression of inhibitor of apoptosis proteins. Conclusions Our results establish that the E-cadherin ectodomain-specific mAb DECMA-1 inhibits Ecad+/HER2+ breast cancers by hindering tumor growth and inducing apoptosis via downregulation of key oncogenic pathways involved in trastuzumab resistance, thereby

  3. Molecular Architecture of a Complex between an Adhesion Protein from the Malaria Parasite and Intracellular Adhesion Molecule 1*

    PubMed Central

    Brown, Alan; Turner, Louise; Christoffersen, Stig; Andrews, Katrina A.; Szestak, Tadge; Zhao, Yuguang; Larsen, Sine; Craig, Alister G.; Higgins, Matthew K.

    2013-01-01

    The adhesion of Plasmodium falciparum-infected erythrocytes to human tissues or endothelium is central to the pathology caused by the parasite during malaria. It contributes to the avoidance of parasite clearance by the spleen and to the specific pathologies of cerebral and placental malaria. The PfEMP1 family of adhesive proteins is responsible for this sequestration by mediating interactions with diverse human ligands. In addition, as the primary targets of acquired, protective immunity, the PfEMP1s are potential vaccine candidates. PfEMP1s contain large extracellular ectodomains made from CIDR (cysteine-rich interdomain regions) and DBL (Duffy-binding-like) domains and show extensive variation in sequence, size, and domain organization. Here we use biophysical methods to characterize the entire ∼300-kDa ectodomain from IT4VAR13, a protein that interacts with the host receptor, intercellular adhesion molecule-1 (ICAM-1). We show through small angle x-ray scattering that IT4VAR13 is rigid, elongated, and monomeric. We also show that it interacts with ICAM-1 through the DBLβ domain alone, forming a 1:1 complex. These studies provide a first low resolution structural view of a PfEMP1 ectodomain in complex with its ligand. They show that it combines a modular domain arrangement consisting of individual ligand binding domains, with a defined higher order architecture that exposes the ICAM-1 binding surface to allow adhesion. PMID:23297413

  4. Protein Recovery from Secondary Paper Sludge and Its Potential Use as Wood Adhesive

    NASA Astrophysics Data System (ADS)

    Pervaiz, Muhammad

    Secondary sludge is an essential part of biosolids produced through the waste treatment plant of paper mills. Globally paper mills generate around 3.0 million ton of biosolids and in the absence of beneficial applications, the handling and disposal of this residual biomass poses a serious environmental and economic proposition. Secondary paper sludges were investigated in this work for recovery of proteins and their use as wood adhesive. After identifying extracellular polymeric substances as adhesion pre-cursors through analytical techniques, studies were carried out to optimize protein recovery from SS and its comprehensive characterization. A modified physicochemical protocol was developed to recover protein from secondary sludge in substantial quantities. The combined effect of French press and sonication techniques followed by alkali treatment resulted in significant improvement of 44% in the yield of solubilized protein compared to chemical methods. The characterization studies confirmed the presence of common amino acids in recovered sludge protein in significant quantities and heavy metal concentration was reduced after recovery process. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed the presence of both low and high molecular weight protein fractions in recovered sludge protein. After establishing the proof-of-concept in the use of recovered sludge protein as wood adhesive, the bonding mechanism of protein adhesives with cellulose substrate was further elucidated in a complementary protein-modification study involving soy protein isolate and its glycinin fractions. The results of this study validated the prevailing bonding theories by proving that surface wetting, protein structure, and type of wood play important role in determining final adhesive strength. Recovered sludge protein was also investigated for its compatibility to formulate hybrid adhesive blends with formaldehyde and bio-based polymers. Apart from chemical

  5. Effects of surface wettability and contact time on protein adhesion to biomaterial surfaces.

    PubMed

    Xu, Li-Chong; Siedlecki, Christopher A

    2007-08-01

    Atomic force microscopy (AFM) was used to directly measure the adhesion forces between three test proteins and low density polyethylene (LDPE) surfaces treated by glow discharge plasma to yield various levels of water wettability. The adhesion of proteins to the LDPE substrates showed a step dependence on the wettability of surfaces as measured by the water contact angle (theta). For LDPE surfaces with theta> approximately 60-65 degrees , stronger adhesion forces were observed for bovine serum albumin, fibrinogen and human FXII than for the surfaces with theta<60 degrees . Smaller adhesion forces were observed for FXII than for the other two proteins on all surfaces although trends were identical. Increasing the contact time from 0 to 50s for each protein-surface combination increased the adhesion force regardless of surface wettability. Time varying adhesion data was fit to an exponential model and free energies of protein unfolding were calculated. This data, viewed in light of previously published studies, suggests a 2-step model of protein denaturation, an early stage on the order of seconds to minutes where the outer surface of the protein interacts with the substrate and a second stage involving movement of hydrophobic amino acids from the protein core to the protein/surface interface. Impact statement: The work described in this manuscript shows a stark transition between protein adherent and protein non-adherent materials in the range of water contact angles 60-65 degrees , consistent with known changes in protein adsorption and activity. Time-dependent changes in adhesion force were used to calculate unfolding energies relating to protein-surface interactions. This analysis provides justification for a 2-step model of protein denaturation on surfaces. PMID:17466368

  6. Experimental strategies for the identification and characterization of adhesive proteins in animals: a review

    PubMed Central

    Hennebert, Elise; Maldonado, Barbara; Ladurner, Peter; Flammang, Patrick; Santos, Romana

    2015-01-01

    Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences. PMID:25657842

  7. Experimental strategies for the identification and characterization of adhesive proteins in animals: a review.

    PubMed

    Hennebert, Elise; Maldonado, Barbara; Ladurner, Peter; Flammang, Patrick; Santos, Romana

    2015-02-01

    Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences. PMID:25657842

  8. Embedded proteins and sacrificial bonds provide the strong adhesive properties of gastroliths

    NASA Astrophysics Data System (ADS)

    Thormann, Esben; MizunoPresent Address: Nihon L'Oreal, Research; Innovation Center, 3-2-1 Sakado, Takatsu, Kawasaki, Kanagawa, Japan., Hiroyasu; Jansson, Kjell; Hedin, Niklas; Fernández, M. Soledad; Arias, José Luis; Rutland, Mark W.; PaiPresent Address: CenterFunctional Nanomaterials, Brookhaven National Laboratory, 735 Brookhaven Avenue, Upton, New York 11973., Ranjith Krishna; Bergström, Lennart

    2012-06-01

    The adhesive properties of gastroliths from a freshwater crayfish (Cherax quadricarinatus) were quantified by colloidal probe atomic force microscopy (AFM) between heavily demineralized gastrolith microparticles and gastrolith substrates of different composition. Combined AFM and transmission electron microscopy studies demonstrated that the sequential detachment and large adhesion energies that characterise the adhesive behaviour of a native gastrolith substrate are dominated by sacrificial bonds between chitin fibres and between chitin fibres and CaCO3. The sacrificial bonds were shown to be strongly related to the gastrolith proteins and when the majority of these proteins were removed by ethylenediaminetetraacetic acid (EDTA), the sequential detachment disappeared and the adhesive energy was reduced by more than two orders of magnitude.The adhesive properties of gastroliths from a freshwater crayfish (Cherax quadricarinatus) were quantified by colloidal probe atomic force microscopy (AFM) between heavily demineralized gastrolith microparticles and gastrolith substrates of different composition. Combined AFM and transmission electron microscopy studies demonstrated that the sequential detachment and large adhesion energies that characterise the adhesive behaviour of a native gastrolith substrate are dominated by sacrificial bonds between chitin fibres and between chitin fibres and CaCO3. The sacrificial bonds were shown to be strongly related to the gastrolith proteins and when the majority of these proteins were removed by ethylenediaminetetraacetic acid (EDTA), the sequential detachment disappeared and the adhesive energy was reduced by more than two orders of magnitude. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr30536d

  9. Detecting cell-in-cell structures in human tumor samples by E-cadherin/CD68/CD45 triple staining.

    PubMed

    Huang, Hongyan; Chen, Ang; Wang, Ting; Wang, Manna; Ning, Xiangkai; He, Meifang; Hu, Yazhuo; Yuan, Long; Li, Shichong; Wang, Qiwei; Liu, Hong; Chen, Zhaolie; Ren, Jun; Sun, Qiang

    2015-08-21

    Although Cell-in-cell structures (CICs) had been documented in human tumors for decades, it is unclear what types of CICs were formed largely due to low resolution of traditional way such as H&E staining. In this work, we employed immunofluorescent method to stain a panel of human tumor samples simultaneously with antibodies against E-cadherin for Epithelium, CD68 for Macrophage and CD45 for Leukocytes, which we termed as "EML method" based on the cells detected. Detail analysis revealed four types of CICs, with tumor cells or macrophage engulfing tumor cells or leukocytes respectively. Interestingly, tumor cells seem to be dominant over macrophage (93% vs 7%) as the engulfer cells in all CICs detected, whereas the overall amount of internalized tumor cells is comparable to that of internalized CD45+ leukocytes (57% vs 43%). The CICs profiles vary from tumor to tumor, which may indicate different malignant stages and/or inflammatory conditions. Given the potential impacts different types of CICs might have on tumor growth, we therefore recommend EML analysis of tumor samples to clarify the correlation of CICs subtypes with clinical prognosis in future researches. PMID:26109430

  10. Deoxynivanelol and Fumonisin, Alone or in Combination, Induce Changes on Intestinal Junction Complexes and in E-Cadherin Expression

    PubMed Central

    Basso, Karina; Gomes, Fernando; Loureiro Bracarense, Ana Paula

    2013-01-01

    Fusariotoxins such as fumonisin B1 (FB1) and deoxynivalenol (DON) cause deleterious effects on the intestine of pigs. The aim of this study was to evaluate the effect of these mycotoxins, alone and in combination, on jejunal explants from piglets, using histological, immunohistochemical and ultrastructural assays. Five 24-day old pigs were used for sampling the explants. Forty-eight explants were sampled from each animal. Explants were incubated for 4 hours in culture medium and medium containing FB1 (100 µM), DON (10 µM) and both mycotoxins (100 µM FB1 plus 10 µM DON). Exposure to all treatments induced a significant decrease in the normal intestinal morphology and in the number of goblet cells, which were more severe in explants exposed to DON and both mycotoxins. A significant reduction in villus height occurred in groups treated with DON and with co-contamination. Expression of E-cadherin was significantly reduced in explants exposed to FB1 (40%), DON (93%) and FB1 plus DON (100%). The ultrastructural assay showed increased intercellular spaces and no junction complexes on enterocytes exposed to mycotoxins. The present data indicate that FB1 and DON induce changes in cell junction complexes that could contribute to increase paracellular permeability. The ex vivo model was adequate for assessing intestinal toxicity induced by exposure of isolated or associated concentrations of 100 µM of FB1 and 10 µM of DON. PMID:24287571

  11. Defects in the adherens junction complex (E-cadherin/ β-catenin) in inflammatory bowel disease.

    PubMed

    Mehta, Shameer; Nijhuis, Anke; Kumagai, Tomoko; Lindsay, James; Silver, Andrew

    2015-06-01

    The epithelial monolayer of the intestine is a selective barrier permitting nutrient and electrolyte absorption yet acting to protect the underlying tissue compartments and cellular components from attack and infiltration by antigens, bacteria and bacterial products present in the lumen. Disruption of this barrier has been associated with inflammatory bowel disease (IBD). The adherens junction (AJ), together with tight junctions (TJ) and desmosomes, form an apical junction complex that controls epithelial cell-to-cell adherence and barrier function as well as regulation of the actin cytoskeleton, intracellular signalling pathways and transcriptional regulation. Numerous studies and reviews highlight the responses of TJs to physiological and pathological stimuli. By comparison, the response of AJ proteins, and the subsequent consequences for barrier function, when exposed to the IBD inflammatory milieu, is less well studied. In this review, we will highlight the roles and responses of the AJ proteins in IBD and provide suggestions for future studies. We will also consider recently proposed therapeutic strategies to preserve or restore epithelial barrier functions to prevent and treat IBD. PMID:25238996

  12. Specific proteins mediate enhanced osteoblast adhesion on nanophase ceramics.

    PubMed

    Webster, T J; Ergun, C; Doremus, R H; Siegel, R W; Bizios, R

    2000-09-01

    Osteoblast, fibroblast, and endothelial cell adhesion on nanophase (that is, materials with grain sizes less than 100 nm) alumina, titania, and hydroxyapatite (HA) was investigated using in vitro cellular models. Osteoblast adhesion was significantly (p < 0.01) greater after 4 h on nanophase alumina, titania, and HA than it was on conventional formulations of the same ceramics. In contrast, compared to conventional alumina, titania, and HA, after 4 h fibroblast adhesion was significantly (p < 0.01) less on nanophase ceramics. Examination of the underlying mechanism(s) of cell adhesion on nanophase ceramics revealed that these ceramics adsorbed significantly (p < 0.01) greater quantities of vitronectin, which, subsequently, may have contributed to the observed select enhanced adhesion of osteoblasts. Select enhanced osteoblast adhesion was independent of surface chemistry and material phase but was dependent on the surface topography (specifically on grain and pore size) of nanophase ceramics. The capability of synthesizing and processing nanomaterials with tailored (through, for example, specific grain and pore size) structures and topographies to control select subsequent cell functions provides the possibility of designing the novel proactive biomaterials (that is, materials that elicit specific, timely, and desirable responses from surrounding cells and tissues) necessary for improved implant efficacy.

  13. Cloning and expression of recombinant adhesive protein Mefp-1 of the blue mussel, Mytilus edulis

    DOEpatents

    Silverman, Heather G.; Roberto, Francisco F.

    2006-01-17

    The present invention comprises a Mytilus edulis cDNA sequenc having a nucleotide sequence that encodes for the Mytilus edulis foot protein-1 (Mefp-1), an example of a mollusk foot protein. Mefp-1 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-1 gene will allow researchers to produce Mefp-1 protein using genetic engineering techniques. The discovery of Mefp-1 gene sequence will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

  14. Cloning and expression of recombinant adhesive protein MEFP-2 of the blue mussel, Mytilus edulis

    DOEpatents

    Silverman, Heather G.; Roberto, Francisco F.

    2006-02-07

    The present invention includes a Mytilus edulis cDNA having a nucleotide sequence that encodes for the Mytilus edulis foot protein-2 (Mefp-2), an example of a mollusk foot protein. Mefp-2 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-2 gene will allow researchers to produce Mefp-2 protein using genetic engineering techniques. The discovery of Mefp-2 gene sequences will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

  15. Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in Escherichia coli

    PubMed Central

    Hwang, Dong Soo; Yoo, Hyo Jin; Jun, Jong Hyub; Moon, Won Kyu; Cha, Hyung Joon

    2004-01-01

    Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments. PMID:15184131

  16. Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

    SciTech Connect

    Baeyens, Nicolas; Latrache, Iman; Yerna, Xavier; Noppe, Gauthier; Horman, Sandrine; Morel, Nicole

    2013-11-22

    Highlights: •The three ERM proteins are expressed in vascular smooth muscle cell. •ERM depletion inhibited PDGF-evoked migration redundantly. •ERM depletion increased cell adhesion redundantly. •ERM depletion did not affect PDGF-evoked Ca signal, Rac1 activation, proliferation. •ERM proteins control PDGF-induced migration by regulating adhesion. -- Abstract: Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca{sup 2+} signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.

  17. Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen.

    PubMed

    Yadav, Ashok Kumar; Tyagi, Ashish; Kaushik, Jai Kumar; Saklani, Asha Chandola; Grover, Sunita; Batish, Virender Kumar

    2013-12-14

    Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72 kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored.

  18. Expression of E-cadherin and β-catenin in basaloid and conventional squamous cell carcinoma of the oral cavity: are potential prognostic markers?

    PubMed Central

    2014-01-01

    Background Basaloid squamous cell carcinoma presents with a preference for the head and neck region, and shows a distinct aggressive behavior, with frequent local recurrences, regional and distant metastasis. The alterations in the cadherin-catenin complex are fundamental requirements for the metastasis process, and this is the first study to evaluate the immunostaining of E-cadherin and β-catenin in oral basaloid squamous cell carcinoma. Methods Seventeen cases of this tumor located exclusively in the mouth were compared to 26 cases of poorly differentiated squamous cell carcinoma and 28 cases of well to moderately differentiated squamous cell carcinoma matched by stage and tumor site. The immunostaining of E-cadherin and β-catenin were evaluated in the three groups and compared to their clinicopathological features and prognosis. Results For groups poorly differentiated squamous cell carcinoma and basaloid squamous cell carcinoma, reduction or absence of E-cadherin staining was observed in more than 80.0% of carcinomas, and it was statistically significant compared to well to moderately differentiated squamous cell carcinoma (p = .019). A strong expression of β-catenin was observed in 26.9% and 20.8% of well to moderately differentiated squamous cell carcinoma and poorly differentiated squamous cell carcinoma, respectively, and in 41.2% of basaloid squamous cell carcinoma. The 5-year and 10-year overall and disease-free survival rates demonstrated no significant differences among all three groups. Conclusions The clinical and biological behavior of three groups of the oral cavity tumors evaluated are similar. E-cadherin and β-catenin immunostaining showed no prognostic value for basaloid and conventional squamous cell carcinomas. PMID:24893577

  19. Strong underwater adhesives made by self-assembling multi-protein nanofibres.

    PubMed

    Zhong, Chao; Gurry, Thomas; Cheng, Allen A; Downey, Jordan; Deng, Zhengtao; Stultz, Collin M; Lu, Timothy K

    2014-10-01

    Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly and structure-function relationships of these natural amyloid fibres remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibres. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibres have an underwater adhesion energy approaching 20.9 mJ m(-2), which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibres taken on their own and exhibit better tolerance to auto-oxidation than Mfps at pH ≥ 7.0.

  20. Strong underwater adhesives made by self-assembling multi-protein nanofibres

    NASA Astrophysics Data System (ADS)

    Zhong, Chao; Gurry, Thomas; Cheng, Allen A.; Downey, Jordan; Deng, Zhengtao; Stultz, Collin M.; Lu, Timothy K.

    2014-10-01

    Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly and structure-function relationships of these natural amyloid fibres remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibres. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibres have an underwater adhesion energy approaching 20.9 mJ m-2, which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibres taken on their own and exhibit better tolerance to auto-oxidation than Mfps at pH ≥ 7.0.

  1. A new role of the Rac-GAP β2-chimaerin in cell adhesion reveals opposite functions in breast cancer initiation and tumor progression

    PubMed Central

    Casado-Medrano, Victoria; Barrio-Real, Laura; García-Rostán, Ginesa; Baumann, Matti; Rocks, Oliver; Caloca, María J.

    2016-01-01

    β2-chimaerin is a Rac1-specific negative regulator and a candidate tumor suppressor in breast cancer but its precise function in mammary tumorigenesis in vivo is unknown. Here, we study for the first time the role of β2-chimaerin in breast cancer using a mouse model and describe an unforeseen role for this protein in epithelial cell-cell adhesion. We demonstrate that expression of β2-chimaerin in breast cancer epithelial cells reduces E-cadherin protein levels, thus loosening cell-cell contacts. In vivo, genetic ablation of β2-chimaerin in the MMTV-Neu/ErbB2 mice accelerates tumor onset, but delays tumor progression. Finally, analysis of clinical databases revealed an inverse correlation between β2-chimaerin and E-cadherin gene expressions in Her2+ breast tumors. Furthermore, breast cancer patients with low β2-chimaerin expression have reduced relapse free survival but develop metastasis at similar times. Overall, our data redefine the role of β2-chimaerin as tumor suppressor and provide the first in vivo evidence of a dual function in breast cancer, suppressing tumor initiation but favoring tumor progression. PMID:27058424

  2. Adhesive proteins of stalked and acorn barnacles display homology with low sequence similarities.

    PubMed

    Jonker, Jaimie-Leigh; Abram, Florence; Pires, Elisabete; Varela Coelho, Ana; Grunwald, Ingo; Power, Anne Marie

    2014-01-01

    Barnacle adhesion underwater is an important phenomenon to understand for the prevention of biofouling and potential biotechnological innovations, yet so far, identifying what makes barnacle glue proteins 'sticky' has proved elusive. Examination of a broad range of species within the barnacles may be instructive to identify conserved adhesive domains. We add to extensive information from the acorn barnacles (order Sessilia) by providing the first protein analysis of a stalked barnacle adhesive, Lepas anatifera (order Lepadiformes). It was possible to separate the L. anatifera adhesive into at least 10 protein bands using SDS-PAGE. Intense bands were present at approximately 30, 70, 90 and 110 kilodaltons (kDa). Mass spectrometry for protein identification was followed by de novo sequencing which detected 52 peptides of 7-16 amino acids in length. None of the peptides matched published or unpublished transcriptome sequences, but some amino acid sequence similarity was apparent between L. anatifera and closely-related Dosima fascicularis. Antibodies against two acorn barnacle proteins (ab-cp-52k and ab-cp-68k) showed cross-reactivity in the adhesive glands of L. anatifera. We also analysed the similarity of adhesive proteins across several barnacle taxa, including Pollicipes pollicipes (a stalked barnacle in the order Scalpelliformes). Sequence alignment of published expressed sequence tags clearly indicated that P. pollicipes possesses homologues for the 19 kDa and 100 kDa proteins in acorn barnacles. Homology aside, sequence similarity in amino acid and gene sequences tended to decline as taxonomic distance increased, with minimum similarities of 18-26%, depending on the gene. The results indicate that some adhesive proteins (e.g. 100 kDa) are more conserved within barnacles than others (20 kDa).

  3. Adhesive Proteins of Stalked and Acorn Barnacles Display Homology with Low Sequence Similarities

    PubMed Central

    Jonker, Jaimie-Leigh; Abram, Florence; Pires, Elisabete; Varela Coelho, Ana; Grunwald, Ingo; Power, Anne Marie

    2014-01-01

    Barnacle adhesion underwater is an important phenomenon to understand for the prevention of biofouling and potential biotechnological innovations, yet so far, identifying what makes barnacle glue proteins ‘sticky’ has proved elusive. Examination of a broad range of species within the barnacles may be instructive to identify conserved adhesive domains. We add to extensive information from the acorn barnacles (order Sessilia) by providing the first protein analysis of a stalked barnacle adhesive, Lepas anatifera (order Lepadiformes). It was possible to separate the L. anatifera adhesive into at least 10 protein bands using SDS-PAGE. Intense bands were present at approximately 30, 70, 90 and 110 kilodaltons (kDa). Mass spectrometry for protein identification was followed by de novo sequencing which detected 52 peptides of 7–16 amino acids in length. None of the peptides matched published or unpublished transcriptome sequences, but some amino acid sequence similarity was apparent between L. anatifera and closely-related Dosima fascicularis. Antibodies against two acorn barnacle proteins (ab-cp-52k and ab-cp-68k) showed cross-reactivity in the adhesive glands of L. anatifera. We also analysed the similarity of adhesive proteins across several barnacle taxa, including Pollicipes pollicipes (a stalked barnacle in the order Scalpelliformes). Sequence alignment of published expressed sequence tags clearly indicated that P. pollicipes possesses homologues for the 19 kDa and 100 kDa proteins in acorn barnacles. Homology aside, sequence similarity in amino acid and gene sequences tended to decline as taxonomic distance increased, with minimum similarities of 18–26%, depending on the gene. The results indicate that some adhesive proteins (e.g. 100 kDa) are more conserved within barnacles than others (20 kDa). PMID:25295513

  4. Stromal Cells Positively and Negatively Modulate the Growth of Cancer Cells: Stimulation via the PGE2-TNFα-IL-6 Pathway and Inhibition via Secreted GAPDH-E-Cadherin Interaction

    PubMed Central

    Kawada, Manabu; Inoue, Hiroyuki; Ohba, Shun-ichi; Yoshida, Junjiro; Masuda, Tohru; Yamasaki, Manabu; Usami, Ihomi; Sakamoto, Shuichi; Abe, Hikaru; Watanabe, Takumi; Yamori, Takao; Shibasaki, Masakatsu; Nomoto, Akio

    2015-01-01

    Fibroblast-like stromal cells modulate cancer cells through secreted factors and adhesion, but those factors are not fully understood. Here, we have identified critical stromal factors that modulate cancer growth positively and negatively. Using a cell co-culture system, we found that gastric stromal cells secreted IL-6 as a growth and survival factor for gastric cancer cells. Moreover, gastric cancer cells secreted PGE2 and TNFα that stimulated IL-6 secretion by the stromal cells. Furthermore, we found that stromal cells secreted glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Extracellular GAPDH, or its N-terminal domain, inhibited gastric cancer cell growth, a finding confirmed in other cell systems. GAPDH bound to E-cadherin and downregulated the mTOR-p70S6 kinase pathway. These results demonstrate that stromal cells could regulate cancer cell growth through the balance of these secreted factors. We propose that negative regulation of cancer growth using GAPDH could be a new anti-cancer strategy. PMID:25785838

  5. Plant protein interactions studied using AFM force spectroscopy: nanomechanical and adhesion properties.

    PubMed

    Fahs, Ahmad; Louarn, Guy

    2013-07-21

    The present work was focused on the nanomechanical and adhesion properties of the napin (2S albumin) and cruciferin (12S globulin) rapeseed (Brassica napus L.) proteins, respectively, a low and high molecular weight seed protein. Using chemically modified AFM tips, force spectroscopy experiments demonstrated notable differences in the tip-protein interaction strength with regard to the nature of the protein and pH of the aqueous environment. The results clearly underline the role of residence time and electrostatic interactions in the protein-protein adhesion force. Although the nanomechanical experiments concerned more than a single molecule, unfolding length and force characteristics of the rapeseed proteins have been statistically found to be sensitive to the structural properties of the protein. This study provides insight into the characterization of rapeseed proteins and then a better knowledge of their interaction and assembling at the nanoscale range.

  6. Plant protein interactions studied using AFM force spectroscopy: nanomechanical and adhesion properties.

    PubMed

    Fahs, Ahmad; Louarn, Guy

    2013-07-21

    The present work was focused on the nanomechanical and adhesion properties of the napin (2S albumin) and cruciferin (12S globulin) rapeseed (Brassica napus L.) proteins, respectively, a low and high molecular weight seed protein. Using chemically modified AFM tips, force spectroscopy experiments demonstrated notable differences in the tip-protein interaction strength with regard to the nature of the protein and pH of the aqueous environment. The results clearly underline the role of residence time and electrostatic interactions in the protein-protein adhesion force. Although the nanomechanical experiments concerned more than a single molecule, unfolding length and force characteristics of the rapeseed proteins have been statistically found to be sensitive to the structural properties of the protein. This study provides insight into the characterization of rapeseed proteins and then a better knowledge of their interaction and assembling at the nanoscale range. PMID:23732983

  7. Synergistic roles for lipids and proteins in the permanent adhesive of barnacle larvae.

    PubMed

    Gohad, Neeraj V; Aldred, Nick; Hartshorn, Christopher M; Jong Lee, Young; Cicerone, Marcus T; Orihuela, Beatriz; Clare, Anthony S; Rittschof, Dan; Mount, Andrew S

    2014-07-11

    Thoracian barnacles rely heavily upon their ability to adhere to surfaces and are environmentally and economically important as biofouling pests. Their adhesives have unique attributes that define them as targets for bio-inspired adhesive development. With the aid of multi-photon and broadband coherent anti-Stokes Raman scattering microscopies, we report that the larval adhesive of barnacle cyprids is a bi-phasic system containing lipids and phosphoproteins, working synergistically to maximize adhesion to diverse surfaces under hostile conditions. Lipids, secreted first, possibly displace water from the surface interface creating a conducive environment for introduction of phosphoproteins while simultaneously modulating the spreading of the protein phase and protecting the nascent adhesive plaque from bacterial biodegradation. The two distinct phases are contained within two different granules in the cyprid cement glands, implying far greater complexity than previously recognized. Knowledge of the lipidic contribution will hopefully inspire development of novel synthetic bioadhesives and environmentally benign antifouling coatings.

  8. Cadherin transfection of Xenopus XTC cells downregulates expression of substrate adhesion molecules.

    PubMed

    Finnemann, S; Kühl, M; Otto, G; Wedlich, D

    1995-09-01

    Cadherins are discussed not in terms of their adhesive function but rather as morphoregulatory proteins. Changes in gene expression following cadherin transfection of cells in culture or by overexpression in embryos have, until now, not been reported. We established a protocol for stable transfection of Xenopus XTC cells and generated cells bearing high levels of membrane-integrated mouse uvomorulin (E-cadherin) or Xenopus XB-cadherin. These cell lines showed drastically impaired substrate adhesion on fibronectin and laminin. In immunoblot and radioimmunoprecipitation experiments, we found that fibronectin and alpha 3/beta 1 integrin are downregulated. The reduced amounts of proteins result from a decrease of the respective mRNAs as proven by RNase protection assays. Coprecipitations revealed that transfected cadherin molecules are complexed with alpha-catenin and beta-catenin at plasma membranes. However, the alpha-catenin present in the XB-cadherin complex differs immunologically from that found in the uvomorulin complex. When a truncated form of XB-cadherin lacking 38 of the most C-terminal amino acids was expressed in XTC cells, complex formation with endogenous catenins was abolished. In these transfectants, substrate adhesion was not affected. These results prove that complex formation of transfected cadherins in XTC cells with endogenous beta-catenin correlates with altered synthesis of certain substrate adhesion molecules.

  9. Anionic deep cavitands enable the adhesion of unmodified proteins at a membrane bilayer.

    PubMed

    Ghang, Yoo-Jin; Perez, Lizeth; Morgan, Melissa A; Si, Fang; Hamdy, Omar M; Beecher, Consuelo N; Larive, Cynthia K; Julian, Ryan R; Zhong, Wenwan; Cheng, Quan; Hooley, Richard J

    2014-12-28

    An anionic self-folding deep cavitand is capable of immobilizing unmodified proteins and enzymes at a supported lipid bilayer interface, providing a simple, soft bioreactive surface that allows enzymatic function under mild conditions. The adhesion is based on complementary charge interactions, and the hosts are capable of binding enzymes such as trypsin at the bilayer interface: the catalytic activity is retained upon adhesion, allowing selective reactions to be performed at the membrane surface. PMID:25366572

  10. Effects of ECM Protein Mimetics on Adhesion and Proliferation of Chorion Derived Mesenchymal Stem Cells

    PubMed Central

    Kim, Ji-Hyun; Jekarl, Dong Wook; Kim, Myungshin; Oh, Eun-Jee; Kim, Yonggoo; Park, In Yang; Shin, Jong Chul

    2014-01-01

    Background: We evaluated the effects of fibronectin, collagen, cadherin, and laminin based extracellular matrix (ECM) protein mimetics coated with mussel derived adhesive protein (MAP) on adhesion and proliferation of chorionic mesenchymal stem cells (cMSCs). Methods: Human placental chorionic tissues from term third-trimester pregnancies (n=3) were used. The cMSCs were cultured on rationally designed ECM protein mimetics coated with MAP on plastic surfaces with the addition of reduced fetal bovine serum (0.5%, 1% FBS). Adhesion capabilities were monitored by a real time cell analysis system (RTCA) utilizing an impedance method. Proliferation capabilities were monitored by RTCA and MTS assay. Results: Of the ECM protein mimetics tested, GRGDSP(FN) coated surfaces exhibited the highest adhesion and proliferation capabilities on RTCA at FBS concentration of 0.5% and 1%. When 0.5% FBS was added to ECM protein mimetics during the MTS assay, GRGDSP(FN), REDV(FN), and collagen mimetics, GPKGAAGEPGKP(ColI) showed higher cMSCs proliferation compared with the control. When 1% FBS was added, GRGDSP(FN) and TAIPSCPEGTVPLYS(ColIV) showed significant cMSCs proliferation capacity. Conclusions: Fibronectin mimetics, GRGDSP(FN) amino acid sequence showed the highest adhesion and proliferation capabilities. In addition, results from RTCA assessment of cell viability correlated well with the tetrazolium-based MTS assay. PMID:24516355

  11. A hot water extract of Curcuma longa inhibits adhesion molecule protein expression and monocyte adhesion to TNF-α-stimulated human endothelial cells.

    PubMed

    Kawasaki, Kengo; Muroyama, Koutarou; Yamamoto, Norio; Murosaki, Shinji

    2015-01-01

    The recruitment of arterial leukocytes to endothelial cells is an important step in the progression of various inflammatory diseases. Therefore, its modulation is thought to be a prospective target for the prevention or treatment of such diseases. Adhesion molecules on endothelial cells are induced by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), and contribute to the recruitment of leukocytes. In the present study, we investigated the effect of hot water extract of Curcuma longa (WEC) on the protein expression of adhesion molecules, monocyte adhesion induced by TNF-α in human umbilical vascular endothelial cells (HUVECs). Treatment of HUVECs with WEC significantly suppressed both TNF-α-induced protein expression of adhesion molecules and monocyte adhesion. WEC also suppressed phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) induced by TNF-α in HUVECs, suggesting that WEC inhibits the NF-κB signaling pathway.

  12. Rapid functional analysis in Xenopus oocytes of Po protein adhesive interactions.

    PubMed

    Yoshida, M; Colma, D R

    2001-06-01

    We have developed a coupled Xenopus oocyte expression system for evaluating the functional effects of mutations in known or suspected adhesion molecules, which allows for a very rapid assessment of intercellular adhesion. As a model protein, we first used Protein zero (Po), an adhesion molecule that mediates self-adhesion of the Schwann cell plasma membrane to form compact myelin in the mammalian PNS. A wide variety of mutations in Po cause certain human peripheral neuropathies, such as the Charcot-Marie-Tooth disease (CMT) type 1B and Dejerine-Sottas syndrome (DSS). After wild-type Po mRNA is injected, the protein is synthesized and correctly targeted to the oocyte cell surface. When two oocytes are paired, wild-type Po redistributes and concentrates at the cell-cell apposition region, and by electron microscopy, the oocyte pairs show close cell-cell appositions and are devoid of the microvilli that are observed in uninjected oocyte pairs. These are hallmark features of highly adhesive cell:cell interfaces. Several point mutations in Po were engineered, corresponding to the molecular defects in the CMT type 1B or DSS. The proteins encoded by these mutations reached the cell surface but failed to concentrate at the oocyte interface. Po carrying a point mutation that is found in DSS is not targeted on the plasma membrane and fail to accumulate at the cell-cell contact site. PMID:11519730

  13. Intraperitoneal administration of activated protein C prevents postsurgical adhesion band formation.

    PubMed

    Dinarvand, Peyman; Hassanian, Seyed Mahdi; Weiler, Hartmut; Rezaie, Alireza R

    2015-02-19

    Postsurgical peritoneal adhesion bands are the most important causes of intestinal obstruction, pelvic pain, and female infertility. In this study, we used a mouse model of adhesion and compared the protective effect of activated protein C (APC) to that of the Food and Drug Administration-approved antiadhesion agent, sodium hyaluronate/carboxymethylcellulose (Seprafilm) by intraperitoneal administration of either APC or Seprafilm to experimental animals. Pathological adhesion bands were graded on day 7, and peritoneal fluid concentrations of tissue plasminogen activator (tPA), d-dimer, thrombin-antithrombin complex, and cytokines (IL-1β, IL-6, interferon-γ, tumor necrosis factor-α, transforming growth factor-β1) were evaluated. Inflammation scores were also measured based on histologic data obtained from peritoneal tissues. Relative to Seprafilm, intraperitoneal administration of human APC led to significantly higher reduction of postsurgical adhesion bands. Moreover, a markedly lower inflammation score was obtained in the adhesive tissues of the APC-treated group, which correlated with significantly reduced peritoneal concentrations of proinflammatory cytokines and an elevated tPA level. Further studies using variants of human APC with or without protease-activated receptor 1 (PAR1) signaling function and mutant mice deficient for either endothelial protein C receptor (EPCR) or PAR1 revealed that the EPCR-dependent signaling activity of APC is primarily responsible for its protective activity in this model. These results suggest APC has therapeutic potential for preventing postsurgical adhesion bands. PMID:25575539

  14. Secreted Frizzled-related protein 1 (sFRP1) regulates spermatid adhesion in the testis via dephosphorylation of focal adhesion kinase and the nectin-3 adhesion protein complex

    PubMed Central

    Wong, Elissa W. P.; Lee, Will M.; Cheng, C. Yan

    2013-01-01

    Development of spermatozoa in adult mammalian testis during spermatogenesis involves extensive cell migration and differentiation. Spermatogonia that reside at the basal compartment of the seminiferous epithelium differentiate into more advanced germ cell types that migrate toward the apical compartment until elongated spermatids are released into the tubule lumen during spermiation. Apical ectoplasmic specialization (ES; a testis-specific anchoring junction) is the only cell junction that anchors and maintains the polarity of elongating/elongated spermatids (step 8–19 spermatids) in the epithelium. Little is known regarding the signaling pathways that trigger the disassembly of the apical ES at spermiation. Here, we show that secreted Frizzled-related protein 1 (sFRP1), a putative tumor suppressor gene that is frequently down-regulated in multiple carcinomas, is a crucial regulatory protein for spermiation. The expression of sFRP1 is tightly regulated in adult rat testis to control spermatid adhesion and sperm release at spermiation. Down-regulation of sFRP1 during testicular development was found to coincide with the onset of the first wave of spermiation at approximately age 45 d postpartum, implying that sFRP1 might be correlated with elongated spermatid adhesion conferred by the apical ES before spermiation. Indeed, administration of sFRP1 recombinant protein to the testis in vivo delayed spermiation, which was accompanied by down-regulation of phosphorylated (p)-focal adhesion kinase (FAK)-Tyr397 and retention of nectin-3 adhesion protein at the apical ES. To further investigate the functional relationship between p-FAK-Tyr397 and localization of nectin-3, we overexpressed sFRP1 using lentiviral vectors in the Sertoli-germ cell coculture system. Consistent with the in vivo findings, overexpression of sFRP1 induced down-regulation of p-FAK-Tyr397, leading to a decline in phosphorylation of nectin-3. In summary, this report highlights the critical role of s

  15. Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

    PubMed

    Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

    2015-01-01

    We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

  16. A self-assembled monolayer-based micropatterned array for controlling cell adhesion and protein adsorption.

    PubMed

    Kim, Dong Jin; Lee, Jong Min; Park, Jin-Goo; Chung, Bong Geun

    2011-05-01

    We developed a surface micropatterning technique to control the cell adhesion and protein adsorption. This micropatterned array system was fabricated by a photolithography technique and self-assembled monolayer (SAM) deposition. It was hypothesized that the wettability and functional terminal group would regulate cell adhesion and protein adsorption. To demonstrate this hypothesis, glass-based micropatterned arrays with various functional terminal groups, such as amine (NH(2)) group (3-aminopropyl-triethoxysilane, APT), methyl (CH(3)) group (trichlorovinylsilane, TVS), and fluorocarbon (CF(3)) group (trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane, FOTS), were used. The contact angle was measured to determine the hydrophilic and hydrophobic properties of materials, demonstrating that TVS and FOTS were hydrophobic, whereas APTs were relatively hydrophilic. The cell adhesion was significantly affected by the wettability, showing that the cells were not adhered to hydrophobic surfaces, such as TVS and FOTS. Thus, the cells were selectively adhered to glass substrates within TVS- and FOTS-based micropatterned arrays. However, the cells were randomly adhered to APTs-based micropatterned arrays due to hydrophilic property of APTs. Furthermore, the protein adsorption of the SAM-based micropatterned array was analyzed, showing that the protein was more absorbed to the TVS surface. The surface functional terminal group enabled the control of protein adsorption. Therefore, this SAM-based micropatterned array system enabled the control of cell adhesion and protein adsorption and could be a potentially powerful tool for regulating the cell-cell interactions in a well-defined microenvironment.

  17. Designed Stem Cell Aggregates: Enhanced Biological Functions of Human Mesenchymal Stem-Cell Aggregates Incorporating E-Cadherin-Modified PLGA Microparticles (Adv. Healthcare Mater. 15/2016).

    PubMed

    Zhang, Yan; Mao, Hongli; Gao, Chao; Li, Suhua; Shuai, Qizhi; Xu, Jianbin; Xu, Ke; Cao, Lei; Lang, Ren; Gu, Zhongwei; Akaike, Toshihiro; Yang, Jun

    2016-08-01

    E-cadherin-modified poly(lactic-co-glycolic acid) (hE-cad-PLGA) microparticles were fabricated and then mediated the 3D cell aggregates of human mesenchymal stem cells (MSCs) on page 1949 by Jun Yang and co-workers. The hE-cad-Fc matrix and the PLGA microparticles synergistically regulate the proliferation and bioactive factors secretions of MSCs by activating EGFR, AKT and ERK1/2 signaling pathways. The hE-cad-PLGA microparticles offer a novel route to expand multipotent stem cell-based clinical applications. PMID:27511954

  18. Corneal Cell Adhesion to Contact Lens Hydrogel Materials Enhanced via Tear Film Protein Deposition

    PubMed Central

    Elkins, Claire M.; Qi, Qin M.; Fuller, Gerald G.

    2014-01-01

    Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS), borate buffered saline (BBS), or Sensitive Eyes Plus Saline Solution (Sensitive Eyes), either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes) exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo. PMID:25144576

  19. Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin.

    PubMed

    Zuo, Jian-Hong; Zhu, Wei; Li, Mao-Yu; Li, Xin-Hui; Yi, Hong; Zeng, Gu-Qing; Wan, Xun-Xun; He, Qiu-Yan; Li, Jian-Huang; Qu, Jia-Quan; Chen, Yu; Xiao, Zhi-Qiang

    2011-09-01

    EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)-like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase-9 (MMP-9) and soluble E-cadherin (sE-cad), and knockdown of MMP-9 by siRNA inhibited sE-cad production induced by EGF in SCC10A. Moreover, both MMP-9 knockdown and E-cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP-9-mediated degradation of E-cadherin into sE-cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK-1/2 and AKT, production of MMP-9 and sE-cad, cell migration and invasion, and expressional changes of EMT markers (E-cadherin and N-cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK-1/2 and PI3K-regulated MMP-9/E-cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin into sE-cad related to activation of ERK-1/2 and PI3K signaling pathways.

  20. The Role of Crk Adaptor Proteins in T-Cell Adhesion and Migration

    PubMed Central

    Braiman, Alex; Isakov, Noah

    2015-01-01

    Crk adaptor proteins are key players in signal transduction from a variety of cell surface receptors. They are involved in early steps of lymphocyte activation through their SH2-mediated transient interaction with signal transducing effector molecules, such as Cbl, ZAP-70, CasL, and STAT5. In addition, they constitutively associate, via their SH3 domain, with effector molecules, such as C3G, that mediate cell adhesion and regulate lymphocyte extravasation and recruitment to sites of inflammation. Recent studies demonstrated that the conformation and function of CrkII is subjected to a regulation by immunophilins, which also affect CrkII-dependent T-cell adhesion to fibronectin and migration toward chemokines. This article addresses mechanisms that regulate CrkII conformation and function, in general, and emphasizes the role of Crk proteins in receptor-coupled signaling pathways that control T-lymphocyte adhesion and migration to inflammatory sites. PMID:26500649

  1. Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot for mechanosensitive proteins

    NASA Astrophysics Data System (ADS)

    Kirchenbüchler, David; Born, Simone; Kirchgeßner, Norbert; Houben, Sebastian; Hoffmann, Bernd; Merkel, Rudolf

    2010-05-01

    Mechanosensing is a vital prerequisite for dynamic remodeling of focal adhesions and cytoskeletal structures upon substrate deformation. For example, tissue formation, directed cell orientation or cell differentiation are regulated by such mechanosensing processes. Focal adhesions and the actin cytoskeleton are believed to be involved in these processes, but where mechanosensing molecules are located and how elastic substrate, focal adhesions and the cytoskeleton couple with each other upon substrate deformation still remains obscure. To approach these questions we have developed a sensitive method to apply defined spatially decaying deformation fields to cells cultivated on ultrasoft elastic substrates and to accurately quantify the resulting displacements of the actin cytoskeleton, focal adhesions, as well as the substrate. Displacement fields were recorded in live cell microscopy by tracking either signals from fluorescent proteins or marker particles in the substrate. As model cell type we used myofibroblasts. These cells are characterized by highly stable adhesion and force generating structures but are still able to detect mechanical signals with high sensitivity. We found a rigid connection between substrate and focal adhesions. Furthermore, stress fibers were found to be barely extendable almost over their whole lengths. Plastic deformation took place only at the very ends of actin filaments close to focal adhesions. As a result, this area became elongated without extension of existing actin filaments by polymerization. Both ends of the stress fibers were mechanically coupled with detectable plastic deformations on either site. Interestingly, traction force dependent substrate deformation fields remained mostly unaffected even when stress fiber elongations were released. These data argue for a location of mechanosensing proteins at the ends of actin stress fibers and describe, except for these domains, the whole system to be relatively rigid for tensile

  2. Intracellular uptake and intraspheroidal distribution of hypericin and hydrophilic analogues using E-cadherin transfected T-24 human bladder cancer cells

    NASA Astrophysics Data System (ADS)

    Crnolatac, Ivo; Huygens, Ann; de Witte, Peter A. M.

    2008-02-01

    Hypericin (HYP) is used after instillation as a diagnostic tool for the fluorescence detection of CIS in the human bladder. In this study the in vitro cellular accumulation and intraspheroidal distribution of HYP and three analogues (OH1, OH2, OH3) with gradually increasing hydrophilicity were studied. E-cadherin negative (T24-C1 -) and E-cadherin positive (T24-H3 ++) human bladder cancer cells were used. We report that in the presence of FBS all compounds were taken up by the monolayer cells to the same limited extent, whereas the overall intracellular accumulation was substantially higher when the incubation of the different dyes took place using cell medium not supplemented with FBS. The results of this study therefore confirm the competition between cellular uptake of HYP and analogues and binding to FBS constituents. Investigating the permeation of the compounds in spheroids, it was found that all HYP analogues diffused dramatically better through the three-dimensional cell layers than HYP itself. This enhanced ability of hydrophilic HYP analogues to permeate through the cell layers in the presence of FBS can be explained in terms of a preferred binding to HDL as compared to LDL. The results further show that all compounds, including LDL-binding HYP, substantially permeated better in T24-C1 - spheroids than in T24-H3 ++ spheroids. The data therefore support the hypothesis that a lowered cellular cohesion is the key to understand the selective uptake of hypericin and its analogues in malignant urothelial cells.

  3. Deciphering Seed Sequence Based Off-Target Effects in a Large-Scale RNAi Reporter Screen for E-Cadherin Expression

    PubMed Central

    Adams, Robert; Nicke, Barbara; Pohlenz, Hans-Dieter; Sohler, Florian

    2015-01-01

    Functional RNAi based screening is affected by large numbers of false positive and negative hits due to prevalent sequence based off-target effects. We performed a druggable genome targeting siRNA screen intended to identify novel regulators of E-cadherin (CDH1) expression, a known key player in epithelial mesenchymal transition (EMT). Analysis of primary screening results indicated a large number of false-positive hits. To address these crucial difficulties we developed an analysis method, SENSORS, which, similar to published methods, is a seed enrichment strategy for analyzing siRNA off-targets in RNAi screens. Using our approach, we were able to demonstrate that accounting for seed based off-target effects stratifies primary screening results and enables the discovery of additional screening hits. While traditional hit detection methods are prone to false positive results which are undetected, we were able to identify false positive hits robustly. Transcription factor MYBL1 was identified as a putative novel target required for CDH1 expression and verified experimentally. No siRNA pool targeting MYBL1 was present in the used siRNA library. Instead, MYBL1 was identified as a putative CDH1 regulating target solely based on the SENSORS off-target score, i.e. as a gene that is a cause for off-target effects down regulating E-cadherin expression. PMID:26361354

  4. Lysyl oxidase-like 2 (LOXL2) and E47 EMT factor: novel partners in E-cadherin repression and early metastasis colonization.

    PubMed

    Canesin, G; Cuevas, E P; Santos, V; López-Menéndez, C; Moreno-Bueno, G; Huang, Y; Csiszar, K; Portillo, F; Peinado, H; Lyden, D; Cano, A

    2015-02-19

    Epithelial-mesenchymal transition (EMT) has been associated with increased aggressiveness and acquisition of migratory properties providing tumor cells with the ability to invade into adjacent tissues. Downregulation of E-cadherin, a hallmark of EMT, is mediated by several transcription factors (EMT-TFs) that act also as EMT inducers, among them, Snail1 and the bHLH transcription factor E47. We previously described lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase family, as a Snail1 regulator and EMT inducer. Here we show that LOXL2 is also an E47-interacting partner and functionally collaborates in the repression of E-cadherin promoter. Loss and gain of function analyses combined with in vivo studies in syngeneic breast cancer models demonstrate the participation of LOXL2 and E47 in tumor growth and their requirement for lung metastasis. Furthermore, LOXL2 and E47 contribute to early steps of metastatic colonization by cell and noncell autonomous functions regulating the recruitment of bone marrow progenitor cells to the lungs and by direct transcriptional regulation of fibronectin and cytokines TNFα, ANG-1 and GM-CSF. Moreover, fibronectin and GM-CSF proved to be necessary for LOXL2/E47-mediated modulation of tumor growth and lung metastasis.

  5. Lysyl oxidase-like 2 (LOXL2) and E47 EMT factor: novel partners in E-cadherin repression and early metastasis colonization.

    PubMed

    Canesin, G; Cuevas, E P; Santos, V; López-Menéndez, C; Moreno-Bueno, G; Huang, Y; Csiszar, K; Portillo, F; Peinado, H; Lyden, D; Cano, A

    2015-02-19

    Epithelial-mesenchymal transition (EMT) has been associated with increased aggressiveness and acquisition of migratory properties providing tumor cells with the ability to invade into adjacent tissues. Downregulation of E-cadherin, a hallmark of EMT, is mediated by several transcription factors (EMT-TFs) that act also as EMT inducers, among them, Snail1 and the bHLH transcription factor E47. We previously described lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase family, as a Snail1 regulator and EMT inducer. Here we show that LOXL2 is also an E47-interacting partner and functionally collaborates in the repression of E-cadherin promoter. Loss and gain of function analyses combined with in vivo studies in syngeneic breast cancer models demonstrate the participation of LOXL2 and E47 in tumor growth and their requirement for lung metastasis. Furthermore, LOXL2 and E47 contribute to early steps of metastatic colonization by cell and noncell autonomous functions regulating the recruitment of bone marrow progenitor cells to the lungs and by direct transcriptional regulation of fibronectin and cytokines TNFα, ANG-1 and GM-CSF. Moreover, fibronectin and GM-CSF proved to be necessary for LOXL2/E47-mediated modulation of tumor growth and lung metastasis. PMID:24632622

  6. Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain

    PubMed Central

    1996-01-01

    The myelin Po protein is believed to hold myelin together via interactions of both its extracellular and cytoplasmic domains. We have already shown that the extracellular domains of Po can interact in a homophilic manner (Filbin, M.T., F.S. Walsh, B.D. Trapp, J.A. Pizzey, and G.I. Tennekoon. 1990. Nature (Lond.). 344:871-872). In addition, we have shown that for this homophilic adhesion to take place, the cytoplasmic domain of Po must be intact and most likely interacting with the cytoskeleton; Po proteins truncated in their cytoplasmic domains are not adhesive (Wong, M.H., and M.T. Filbin, 1994. J. Cell Biol. 126:1089-1097). To determine if the presence of these truncated forms of Po could have an effect on the functioning of the full-length Po, we coexpressed both molecules in CHO cells. The adhesiveness of CHO cells expressing both full-length Po and truncated Po was then compared to cells expressing only full-length Po. In these coexpressors, both the full-length and the truncated Po proteins were glycosylated. They reached the surface of the cell in approximately equal amounts as shown by an ELISA and surface labeling, followed by immunoprecipitation. Furthermore, the amount of full-length Po at the cell surface was equivalent to other cell lines expressing only full-length Po that we had already shown to be adhesive. Therefore, there should be sufficient levels of full-length Po at the surface of these coexpressors to measure adhesion of Po. However, as assessed by an aggregation assay, the coexpressors were not adhesive. By 60 min they had not formed large aggregates and were indistinguishable from the control transfected cells not expressing Po. In contrast, in the same time, the cells expressing only the full-length Po had formed large aggregates. This indicates that the truncated forms of Po have a dominant-negative effect on the adhesiveness of the full-length Po. Furthermore, from cross-linking studies, full-length Po, when expressed alone but not when

  7. A Regulatory Network Involving β-Catenin, e-Cadherin, PI3k/Akt, and Slug Balances Self-Renewal and Differentiation of Human Pluripotent Stem Cells In Response to Wnt Signaling.

    PubMed

    Huang, Tyng-Shyan; Li, Li; Moalim-Nour, Lilian; Jia, Deyong; Bai, Jian; Yao, Zemin; Bennett, Steffany A L; Figeys, Daniel; Wang, Lisheng

    2015-05-01

    The mechanisms underlying disparate roles of the canonical Wnt signaling pathway in maintaining self-renewal or inducing differentiation and lineage specification in embryonic stem cells (ESCs) are not clear. In this study, we provide the first demonstration that self-renewal versus differentiation of human ESCs (hESCs) in response to Wnt signaling is predominantly determined by a two-layer regulatory circuit involving β-catenin, E-cadherin, PI3K/Akt, and Slug in a time-dependent manner. Short-term upregulation of β-catenin does not lead to the activation of T-cell factor (TCF)-eGFP Wnt reporter in hESCs. Instead, it enhances E-cadherin expression on the cell membrane, thereby enhancing hESC self-renewal through E-cadherin-associated PI3K/Akt signaling. Conversely, long-term Wnt activation or loss of E-cadherin intracellular β-catenin binding domain induces TCF-eGFP activity and promotes hESC differentiation through β-catenin-induced upregulation of Slug. Enhanced expression of Slug leads to a further reduction of E-cadherin that serves as a β-catenin "sink" sequestering free cytoplasmic β-catenin. The formation of such a framework reinforces hESCs to switch from a state of temporal self-renewal associated with short-term Wnt/β-catenin activation to definitive differentiation. Stem Cells 2015;33:1419-1433.

  8. Nuclear magnetic resonance spectroscopy of mussel adhesive protein repeating peptide segment.

    PubMed

    Olivieri, M P; Wollman, R M; Alderfer, J L

    1997-12-01

    Mussel adhesive protein (MAP) is the adhesive agent used by the common blue sea mussel (Mytilus edulis) to attach the animal to various underwater surfaces. It is generally composed of 75 to 85 repeating decameric units with the reported primary sequence NH2-Ala(1)-Lyst(2)-Pro(3)-Ser(4)-Tyr(5)-Hyp(6)-Hyp(7)-Thr(8)-DOPA( 9)- Lys(10)-COOH. This study examines this peptide's solution-state conformation using proton nuclear magnetic resonance (NMR) spectroscopy. NMR and molecular modeling of the decamer before and after molecular dynamics calculations in water suggests a conformation that retains an overall bent helix.

  9. An integrated transcriptomic and proteomic analysis of sea star epidermal secretions identifies proteins involved in defense and adhesion.

    PubMed

    Hennebert, Elise; Leroy, Baptiste; Wattiez, Ruddy; Ladurner, Peter

    2015-10-14

    Sea stars rely on epidermal secretions to cope with their benthic life. Their integument produces a mucus, which represents the first barrier against invaders; and their tube feet produce adhesive secretions to pry open mussels and attach strongly but temporarily to rocks. In this study, we combined high-throughput sequencing of expressed mRNA and mass-spectrometry-based identification of proteins to establish the first proteome of mucous and adhesive secretions from the sea star Asterias rubens. We show that the two secretions differ significantly, the major adhesive proteins being only present in trace amounts in the mucus secretion. Except for 41 proteins which were present in both secretions, a total of 34 and 244 proteins were identified as specific of adhesive secretions and mucus, respectively. We discuss the role of some of these proteins in the adhesion of sea stars as well as in their protection against oxygen reactive species and microorganisms. In addition, 58% of the proteins identified in adhesive secretions did not present significant similarity to other known proteins, revealing a list of potential novel sea star adhesive proteins uncharacterized so far. The panel of proteins identified in this study offers unprecedented opportunities for the development of sea star-inspired biomimetic materials.

  10. The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.

    PubMed

    Lord, Megan S; Cheng, Bill; McCarthy, Simon J; Jung, MoonSun; Whitelock, John M

    2011-10-01

    Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by α(IIb)β(3) integrins, while α(2)β(1) integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of α(IIb)β(3) integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions.

  11. Corneal epithelial adhesion strength to tethered-protein/peptide modified hydrogel surfaces.

    PubMed

    Wallace, Christopher; Jacob, Jean T; Stoltz, Albert; Bi, Jingjing; Bundy, Kirk

    2005-01-01

    In this study, we investigated the suitability of microjet impingement for use on hydrogel materials to determine the cellular adhesion strength of corneal epithelial cells grown on novel hydrogels with extracellular matrix proteins (laminin and/or fibronectin) or a peptide sequence (fibronectin adhesion promoting peptide, FAP) tethered to their surface with poly(ethylene glycol) chains. The deformation of the hydrogel surface in response to the force of the microjet was analyzed both visually and mathematically. After the results of these experiments and calculations determined that no deformation occurred and that the pressure required for indentation (1.25 x 10(6) Pa) was three factors of 10 greater than the maximum pressure of the microjet, the relative mean adhesion strength of primary rabbit corneal epithelial cells grown on the novel poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogels was determined and compared with that of the same type of cells grown on control glass surfaces. Only confluent cell layers were tested. Cells grown on control glass surfaces adhered with a mean relative adhesion strength of 488 +/- 28 dynes/cm2. Under identical conditions, cells grown on laminin- and FAP-tethered hydrogel surfaces were unable to be removed, indicating an adhesion strength greater than 516 dynes/cm2. Cells grown on fibronectin- and fibronectin/laminin (1:1)-tethered surfaces showed significantly lower relative adhesion strengths (201 +/- 50 and 189 +/- 11 dynes/cm2, respectively), compared with laminin- and FAP-tethered surfaces (p = 0.001). Our results demonstrate that the microjet impingement method of cell adhesion analysis is applicable to hydrogel substrates. Additionally, analysis of our test surfaces indicates that fibronectin tethered to this hydrogel in the quantity and by the method used here does not induce stable ligand/receptor bonding to the epithelial cell membrane to the same degree as does laminin or FAP. PMID:15534866

  12. Mechanical and water soaking properties of medium density fiberboard with wood fiber and soybean protein adhesive.

    PubMed

    Li, Xin; Li, Yonghui; Zhong, Zhikai; Wang, Donghai; Ratto, Jo A; Sheng, Kuichuan; Sun, Xiuzhi Susan

    2009-07-01

    Soybean protein is a renewable and abundant material that offers an alternative to formaldehyde-based resins. In this study, soybean protein was modified with sodium dodecyl sulfate (SDS) as an adhesive for wood fiber medium density fiberboard (MDF) preparation. Second-order response surface regression models were used to study the effects and interactions of initial moisture content (IMC) of coated wood fiber, press time (PT) and temperature on mechanical and water soaking properties of MDF. Results showed that IMC of coated fiber was the dominant influencing factor. Mechanical and soaking properties improved as IMC increased and reached their highest point at an IMC of 35%. Press time and temperature also had a significant effect on mechanical and water soaking properties of MDF. Second-order regression results showed that there were strong relationships between mechanical and soaking properties of MDF and processing parameters. Properties of MDF made using soybean protein adhesive are similar to those of commercial board.

  13. The oxidase activity of vascular adhesion protein-1 (VAP-1) is essential for function.

    PubMed

    Noonan, Thomas; Lukas, Susan; Peet, Gregory W; Pelletier, Josephine; Panzenbeck, Mark; Hanidu, Adedayo; Mazurek, Suzanne; Wasti, Ruby; Rybina, Irina; Roma, Teresa; Kronkaitis, Anthony; Shoultz, Alycia; Souza, Donald; Jiang, Huiping; Nabozny, Gerald; Modis, Louise Kelly

    2013-01-01

    Vascular adhesion protein-1 (VAP-1) has been implicated in the pathogenesis of inflammatory diseases and is suggested to play a role in immune cell trafficking. It is not clear whether this effect is mediated by the oxidase activity or by other features of the protein such as direct adhesion. In order to study the role of VAP-1 oxidase activity in vivo, we have generated mice carrying an oxidase activity-null VAP-1 protein. We demonstrate that the VAP-1 oxidase null mutant mice have a phenotype similar to the VAP-1 null mice in animal models of sterile peritonitis and antibody induced arthritis suggesting that the oxidase activity is responsible for the inflammatory function of VAP-1.

  14. Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins.

    PubMed

    Seong, Jihye; Tajik, Arash; Sun, Jie; Guan, Jun-Lin; Humphries, Martin J; Craig, Susan E; Shekaran, Asha; García, Andrés J; Lu, Shaoying; Lin, Michael Z; Wang, Ning; Wang, Yingxiao

    2013-11-26

    Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.

  15. Adhesion of Fusobacterium necrophorum to bovine endothelial cells is mediated by outer membrane proteins.

    PubMed

    Kumar, Amit; Gart, Elena; Nagaraja, T G; Narayanan, Sanjeev

    2013-03-23

    Fusobacterium necrophorum, a Gram-negative anaerobe, is frequently associated with suppurative and necrotic infections of animals and humans. The organism is a major bovine pathogen, and in cattle, the common fusobacterial infections are hepatic abscesses, foot rot, and necrotic laryngitis. The species comprises two subspecies: F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme. Bacterial adhesion to the host cell surface is a critical initial step in the pathogenesis, and outer membrane proteins (OMP) play an important role in adhesion and establishment of certain Gram-negative bacterial infections. The means by which F. necrophorum attaches to epithelial or endothelial cells has not been determined. We evaluated whether OMP of F. necrophorum, isolated from a liver abscess, mediated adhesion to bovine endothelial cells (adrenal gland capillary endothelial cell line). The extent of binding of subsp. necrophorum to the endothelial cells was higher than that of F. necrophorum subsp. funduliforme. Trypsin treatment of bacterial cells decreased their binding to endothelial cells indicating the protein nature of adhesins. Preincubation of endothelial cells with OMP extracted from F. necrophorum decreased the binding of bacterial cells. In addition, binding of each subspecies to endothelial cells was inhibited by polyclonal antibodies raised against respective OMP and the antibody-mediated inhibition was subspecies specific. The western blot analysis of OMP bound to endothelial cells with anti-OMP antibodies showed four OMP of 17, 24, 40 and 74 kDa. We conclude that OMP of F. necrophorum play a role in adhesion of bacterial cells to the endothelial cells.

  16. Receptor protein tyrosine phosphatases are novel components of a polycystin complex

    PubMed Central

    Boucher, Catherine A.; Ward, Heather H.; Case, Ruth L.; Thurston, Katie S.; Li, Xiaohong; Needham, Andrew; Romero, Elsa; Hyink, Deborah; Qamar, Seema; Roitbak, Tamara; Powell, Samantha; Ward, Christopher; Wilson, Patricia D.; Wandinger-Ness, Angela; Sandford, Richard N.

    2011-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation of PKD1 and PKD2 that encode polycystin-1 and polycystin-2. Polycystin-1 is tyrosine phosphorylated and modulates multiple signaling pathways including AP-1, but the identity of the phosphatases regulating polycystin-1 are previously uncharacterized. Here we identify members of the LAR protein tyrosine phosphatase (RPTP) superfamily as members of the polycystin-1complex mediated through extra- and intracellular interactions. The first extracellular PKD1 domain of polycystin-1 interacts with the first Ig domain of RPTPσ, while the polycystin-1 C-terminus of polycystin-1 interacts with the regulatory D2 phosphatase domain of RPTPγ. Additional homo- and heterotypic interactions between RPTPs recruit RPTPδ The multimeric polycystin protein complex is found localised in cilia. RPTPσ and RPTPδ are also part of a polycystin-1/E-cadherin complex known to be important for early events in adherens junction stabilisation. The interaction between polycystin-1 and RPTPγ is disrupted in ADPKD cells, while RPTPσ and RPTPδ remain closely associated with E-cadherin, largely in an intracellular location. The polycystin-1 C-terminus is an in vitro substrate of RPTPγ, which dephosphorylates the c-Src phosphorylated Y4237 residue and activates AP1-mediated transcription. The data identify RPTPs as novel interacting partners of the polycystins both in cilia and at adhesion complexes and demonstrate RPTPγ phosphatase activity is central to the molecular mechanisms governing polycystin-dependent signaling. PMID:21126580

  17. Mussel adhesive protein provides cohesive matrix for collagen type-1α

    PubMed Central

    Martinez Rodriguez, Nadine R.; Das, Saurabh; Kaufman, Yair; Wei, Wei; Israelachvili, Jacob N.; Waite, J. Herbert

    2015-01-01

    Understanding the interactions between collagen and adhesive mussel foot proteins (mfps) can lead to improved medical and dental adhesives, particularly for collagen-rich tissues. Here we investigated interactions between collagen type-1, the most abundant loadbearing animal protein, and mussel foot protein-3 (mfp-3) using a quartz crystal microbalance and surface forces apparatus (SFA). Both hydrophilic and hydrophobic variants of mfp-3 were exploited to probe the nature of the interaction between the protein and collagen. Our chief findings are: 1) mfp-3 is an effective chaperone for tropocollagen adsorption to TiO2 and mica surfaces; 2) at pH 3, collagen addition between two mfp-3 films (Wc = 5.4 ± 0.2 mJ/m2) increased their cohesion by nearly 35%; 3) oxidation of Dopa in mfp-3 by periodate did not abolish the adhesion between collagen and mfp-3 films, and 4) collagen bridging between both hydrophilic and hydrophobic mfp-3 variant films is equally robust, suggesting that hydrophobic interactions play a minor role. Extensive H-bonding, π-cation and electrostatic interactions are more plausible to explain the reversible bridging of mfp-3 films by collagen. PMID:25770997

  18. Ubiquitous distribution of salts and proteins in spider glue enhances spider silk adhesion

    NASA Astrophysics Data System (ADS)

    Amarpuri, Gaurav; Chaurasia, Vishal; Jain, Dharamdeep; Blackledge, Todd A.; Dhinojwala, Ali

    2015-03-01

    Modern orb-weaving spiders use micron-sized glue droplets on their viscid silk to retain prey in webs. A combination of low molecular weight salts and proteins makes the glue viscoelastic and humidity responsive in a way not easily achieved by synthetic adhesives. Optically, the glue droplet shows a heterogeneous structure, but the spatial arrangement of its chemical components is poorly understood. Here, we use optical and confocal Raman microscopy to show that salts and proteins are present ubiquitously throughout the droplet. The distribution of adhesive proteins in the peripheral region explains the superior prey capture performance of orb webs as it enables the entire surface area of the glue droplet to act as a site for prey capture. The presence of salts throughout the droplet explains the recent Solid-State NMR results that show salts directly facilitate protein mobility. Understanding the function of individual glue components and the role of the droplet's macro-structure can help in designing better synthetic adhesives for humid environments.

  19. Antioxidant efficacy and adhesion rescue by a recombinant mussel foot protein-6.

    PubMed

    Nicklisch, Sascha C T; Das, Saurabh; Martinez Rodriguez, Nadine R; Waite, J Herbert; Israelachvili, Jacob N

    2013-01-01

    Mytilus foot protein type 6 (mfp-6) is crucial for maintaining the reducing conditions needed for optimal wet adhesion in marine mussels. In this report, we describe the expression and production of a recombinant Mytilus californianus foot protein type 6 variant 1 (rmfp-6.1) fused with a hexahistidine affinity tag in Escherichia coli and its purification by affinity chromatography. Recombinant mfp-6 showed high purification yields of 5-6 mg L(-1) cell culture and excellent solubility in low pH buffers that retard oxidation of its many thiol groups. Purified rmfp-6.1 protein showed high 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity when compared with vitamin C. Using the highly sensitive surface forces apparatus (SFA) technique to measure interfacial surface forces in the nano-Newton range, we show that rmfp-6.1 is also able to rescue the oxidation-dependent adhesion loss of mussel foot protein 3 (mfp-3) at pH 3. The adhesion rescue is related to a reduction of dopaquinone back to 3,4-dihydroxyphenyl-l-alanine in mfp-3, which is the reverse reaction observed during the detrimental enzymatic browning process in fruits and vegetables. Broadly viewed, rmfp-6.1 has potential as a versatile antioxidant for applications ranging from personal products to antispoilants for perishable foods during processing and storage. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1587-1593, 2013. PMID:24106182

  20. Ubiquitous distribution of salts and proteins in spider glue enhances spider silk adhesion.

    PubMed

    Amarpuri, Gaurav; Chaurasia, Vishal; Jain, Dharamdeep; Blackledge, Todd A; Dhinojwala, Ali

    2015-01-01

    Modern orb-weaving spiders use micron-sized glue droplets on their viscid silk to retain prey in webs. A combination of low molecular weight salts and proteins makes the glue viscoelastic and humidity responsive in a way not easily achieved by synthetic adhesives. Optically, the glue droplet shows a heterogeneous structure, but the spatial arrangement of its chemical components is poorly understood. Here, we use optical and confocal Raman microscopy to show that salts and proteins are present ubiquitously throughout the droplet. The distribution of adhesive proteins in the peripheral region explains the superior prey capture performance of orb webs as it enables the entire surface area of the glue droplet to act as a site for prey capture. The presence of salts throughout the droplet explains the recent Solid-State NMR results that show salts directly facilitate protein mobility. Understanding the function of individual glue components and the role of the droplet's macro-structure can help in designing better synthetic adhesives for humid environments. PMID:25761668

  1. Interactions of the Protein-tyrosine Phosphatase-α with the Focal Adhesion Targeting Domain of Focal Adhesion Kinase Are Involved in Interleukin-1 Signaling in Fibroblasts*

    PubMed Central

    Wang, Qin; Wang, Yongqiang; Fritz, Dominik; Rajshankar, Dhaarmini; Downey, Gregory P.; McCulloch, Christopher A.

    2014-01-01

    Interleukin-1 (IL-1) signaling in fibroblasts is mediated through focal adhesions, organelles that are enriched with adaptor and cytoskeletal proteins that regulate signal transduction. We examined interactions of the focal adhesion kinase (FAK) with protein-tyrosine phosphatase-α (PTP-α) in IL-1 signaling. In wild type and FAK knock-out mouse embryonic fibroblasts, we found by immunoblotting, immunoprecipitation, immunostaining, and gene silencing that FAK is required for IL-1-mediated sequestration of PTPα to focal adhesions. Immunoprecipitation and pulldown assays of purified proteins demonstrated a direct interaction between FAK and PTPα, which was dependent on the FAT domain of FAK and by an intact membrane-proximal phosphatase domain of PTPα. Recruitment of PTPα to focal adhesions, IL-1-induced Ca2+ release from the endoplasmic reticulum, ERK activation, and IL-6, MMP-3, and MMP-9 expression were all blocked in FAK knock-out fibroblasts. These processes were restored in FAK knock-out cells transfected with wild type FAK, FAT domain, and FRNK. Our data indicate that IL-1-induced signaling through focal adhesions involves interactions between the FAT domain of FAK and PTPα. PMID:24821720

  2. Interactions of the protein-tyrosine phosphatase-α with the focal adhesion targeting domain of focal adhesion kinase are involved in interleukin-1 signaling in fibroblasts.

    PubMed

    Wang, Qin; Wang, Yongqiang; Fritz, Dominik; Rajshankar, Dhaarmini; Downey, Gregory P; McCulloch, Christopher A

    2014-06-27

    Interleukin-1 (IL-1) signaling in fibroblasts is mediated through focal adhesions, organelles that are enriched with adaptor and cytoskeletal proteins that regulate signal transduction. We examined interactions of the focal adhesion kinase (FAK) with protein-tyrosine phosphatase-α (PTP-α) in IL-1 signaling. In wild type and FAK knock-out mouse embryonic fibroblasts, we found by immunoblotting, immunoprecipitation, immunostaining, and gene silencing that FAK is required for IL-1-mediated sequestration of PTPα to focal adhesions. Immunoprecipitation and pulldown assays of purified proteins demonstrated a direct interaction between FAK and PTPα, which was dependent on the FAT domain of FAK and by an intact membrane-proximal phosphatase domain of PTPα. Recruitment of PTPα to focal adhesions, IL-1-induced Ca(2+) release from the endoplasmic reticulum, ERK activation, and IL-6, MMP-3, and MMP-9 expression were all blocked in FAK knock-out fibroblasts. These processes were restored in FAK knock-out cells transfected with wild type FAK, FAT domain, and FRNK. Our data indicate that IL-1-induced signaling through focal adhesions involves interactions between the FAT domain of FAK and PTPα.

  3. Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell

    PubMed Central

    1993-01-01

    A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cell-cell adhesion (compaction) and in cell- substrate adhesion. We have previously shown that neither uvomorulin (E- cadherin) nor integrins are responsible for the mutant phenotype (Calogero, A., M. Samuels, T. Darland, S. A. Edwards, R. Kemler, and E. D. Adamson. 1991. Dev. Biol. 146:499-508). Several cytoskeleton proteins were assayed and only vinculin was found to be absent in mutant (5.51) cells. A chicken vinculin expression vector was transfected into the 5.51 cells together with a neomycin-resistance vector. Clones that were adherent to the substrate were selected in medium containing G418. Two clones, 5.51Vin3 and Vin4, were analyzed by Nomarski differential interference contrast and laser confocal microscopy as well as by biochemical and molecular biological techniques. Both clones adhered well to substrates and both exhibited F- actin stress fibers with vinculin localized at stress fiber tips in focal contacts. This was in marked contrast to 5.51 parental cells, which had no stress fibers and no vinculin. The mutant and complemented F9 cell lines will be useful models for examining the complex interactions between cytoskeletal and cell adhesion proteins. PMID:8491782

  4. Application of tung oil to improve adhesion strength and water resistance of cottonseed meal and protein adhesives on maple veneer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cottonseed meal-based products show promise in serving as environment-friendly wood adhesives. However, their practical utilization is currently limited due to low durability and water resistant properties. In this research, we tested the improvement of adhesion strength and water resistance of cott...

  5. Endocytosis Regulates Cell Soma Translocation and the Distribution of Adhesion Proteins in Migrating Neurons

    PubMed Central

    Shieh, Jennifer C.; Schaar, Bruce T.; Srinivasan, Karpagam; Brodsky, Frances M.; McConnell, Susan K.

    2011-01-01

    Newborn neurons migrate from their birthplace to their final location to form a properly functioning nervous system. During these movements, young neurons must attach and subsequently detach from their substrate to facilitate migration, but little is known about the mechanisms cells use to release their attachments. We show that the machinery for clathrin-mediated endocytosis is positioned to regulate the distribution of adhesion proteins in a subcellular region just proximal to the neuronal cell body. Inhibiting clathrin or dynamin function impedes the movement of migrating neurons both in vitro and in vivo. Inhibiting dynamin function in vitro shifts the distribution of adhesion proteins to the rear of the cell. These results suggest that endocytosis may play a critical role in regulating substrate detachment to enable cell body translocation in migrating neurons. PMID:21445347

  6. 'P-cadherin functional role is dependent on E-cadherin cellular context: a proof of concept using the breast cancer model'.

    PubMed

    2016-05-01

    This article corrects: P-cadherin functional role is dependent on E-cadherin cellular context: a proof of concept using the breast cancer model Volume 229, Issue 5, 705–718, Article first published online: 24 January 2013. By Ana Sofia Ribeiro, Bárbara Sousa, Laura Carreto, Nuno Mendes, Ana Rita Nobre, Sara Ricardo, André Albergaria, Jorge F Cameselle-Teijeiro, Rene Gerhard, Ola Söderberg, Raquel Seruca, Manuel A Santos, Fernando Schmitt and Joana Paredes, J Pathol 2013; 229: 708–718. DOI: 10.1002/path.4143. The above article, published online on 24 January 2013 on Wiley Online Library (wileyonlinelibrary.com). The funding information, “This work was also funded by FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE (FCOMP-01-0124-FEDER-021209).” was omitted from the Acknowledgements section. We apologise for any inconvenience caused. PMID:27071484

  7. 'P-cadherin functional role is dependent on E-cadherin cellular context: a proof of concept using the breast cancer model'.

    PubMed

    2016-05-01

    This article corrects: P-cadherin functional role is dependent on E-cadherin cellular context: a proof of concept using the breast cancer model Volume 229, Issue 5, 705–718, Article first published online: 24 January 2013. By Ana Sofia Ribeiro, Bárbara Sousa, Laura Carreto, Nuno Mendes, Ana Rita Nobre, Sara Ricardo, André Albergaria, Jorge F Cameselle-Teijeiro, Rene Gerhard, Ola Söderberg, Raquel Seruca, Manuel A Santos, Fernando Schmitt and Joana Paredes, J Pathol 2013; 229: 708–718. DOI: 10.1002/path.4143. The above article, published online on 24 January 2013 on Wiley Online Library (wileyonlinelibrary.com). The funding information, “This work was also funded by FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE (FCOMP-01-0124-FEDER-021209).” was omitted from the Acknowledgements section. We apologise for any inconvenience caused.

  8. Adhesion of phospholipid vesicles to Chinese hamster fibroblasts: Role of cell surface proteins

    PubMed Central

    Pagano, RE; Takeichi, M

    1977-01-01

    The adhesion of artificially generated lipid membrane vesicles to Chinese hamster V79 fibroblasts in suspension was used as a model system for studying membrane interactions. Below their gel-liquid crystalline phase transition temperature, vesicles comprised of dipalmitoyl lecithin (DPL) or dimyristoyl lecithin (DML) absorbed to the surfaces of EDTA- dissociated cells. These adherent vesicles could not be removed by repeated washings of the treated cells but could be released into the medium by treatment with trypsin. EM autoradiographic studies of cells treated with[(3)H]DML or [(3)H]DPL vesicles showed that most of the radioactive lipids were confined to the cell periphery. Scanning electron microscopy and fluorescence microscopy further confirmed the presence of adherent vesicles at the cell surface. Adhesion of DML or DPL vesicles to EDTA-dissociated cells modified the lactoperoxidase-catalyzed iodination pattern of the cell surface proteins; the inhibition of labeling of two proteins with an approximately 60,000- dalton mol wt was particularly evident. Incubation of cells wit h (3)H-lipid vesicles followed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis showed that some of the (3)H-lipid migrated preferentially with these approximately 60,000-mol wt proteins. Studies of the temperature dependence of vesicle uptake and subsequent release by trypsin showed that DML or DPL vesicle adhesion to EDTA- dissociated cells increased with decreasing temperatures. In contrast, cells trypsinized before incubation with vesicles showed practically no temperature dependence of vesicle uptake. These results suggest two pathways for adhesion of lipid vesicles to the cell surface-a temperature-sensitive one involving cell surface proteins, and a temperature-independent one. These findings are discussed in terms of current models for cell-cell interactions. PMID:407233

  9. Long noncoding RNA-EBIC promotes tumor cell invasion by binding to EZH2 and repressing E-cadherin in cervical cancer.

    PubMed

    Sun, Ning-xia; Ye, Chen; Zhao, Qian; Zhang, Qing; Xu, Chen; Wang, Shao-bing; Jin, Zhi-jun; Sun, Shu-han; Wang, Fang; Li, Wen

    2014-01-01

    In recent years, long noncoding RNAs (lncRNAs) have been demonstrated to play key roles in tumorgenesis. However, the contributions of lncRNAs to cervical cancer (CC) remain largely unknown. In this study, differentially expressed lncRNAs and mRNAs in cervical cancer and paired peritumoral tissues were detected by transcriptome microarray analysis. We found 708 probe sets of lncRNAs increased and 836 probe sets decreased in CC tissues, while 1288 mRNA differential probe sets increased and 901 mRNA probe sets decreased. The results were validated by quantitative real-time polymerase chain reaction (qPCR). Then, we found a specific differentially expressed lncRNA can physically bind to enhancer of zeste homolog2 (EZH2) by using RNA immunoprecipitation. We termed it as EZH2-binding lncRNA in cervical cancer [lncRNA-EBIC]. Wound healing assays and Matrigel invasion assays were used to determine the function of this lncRNA by silencing it. We observed that the migration and invasion of cervical cancer cells in vitro were inhibited upon suppression of lncRNA-EBIC by siRNA. We also found that the association between lncRNA-EBIC and EZH2 was required for the repression of E-cadherin, which was a key molecular in the metastasis of cervical cancer. Conclusion: These results demonstrated that lncRNA-EBIC was an oncogenic lncRNA, which could promote tumor cell invasion in CC by binding to EZH2 and inhibiting E-cadherin expression.

  10. Long Noncoding RNA-EBIC Promotes Tumor Cell Invasion by Binding to EZH2 and Repressing E-Cadherin in Cervical Cancer

    PubMed Central

    Zhao, Qian; Zhang, Qing; Xu, Chen; Wang, Shao-bing; Jin, Zhi-jun; Sun, Shu-han; Wang, Fang; Li, Wen

    2014-01-01

    In recent years, long noncoding RNAs (lncRNAs) have been demonstrated to play key roles in tumorgenesis. However, the contributions of lncRNAs to cervical cancer (CC) remain largely unknown. In this study, differentially expressed lncRNAs and mRNAs in cervical cancer and paired peritumoral tissues were detected by transcriptome microarray analysis. We found 708 probe sets of lncRNAs increased and 836 probe sets decreased in CC tissues, while 1288 mRNA differential probe sets increased and 901 mRNA probe sets decreased. The results were validated by quantitative real-time polymerase chain reaction (qPCR). Then, we found a specific differentially expressed lncRNA can physically bind to enhancer of zeste homolog2 (EZH2) by using RNA immunoprecipitation. We termed it as EZH2-binding lncRNA in cervical cancer [lncRNA-EBIC]. Wound healing assays and Matrigel invasion assays were used to determine the function of this lncRNA by silencing it. We observed that the migration and invasion of cervical cancer cells in vitro were inhibited upon suppression of lncRNA-EBIC by siRNA. We also found that the association between lncRNA-EBIC and EZH2 was required for the repression of E-cadherin, which was a key molecular in the metastasis of cervical cancer. Conclusion These results demonstrated that lncRNA-EBIC was an oncogenic lncRNA, which could promote tumor cell invasion in CC by binding to EZH2 and inhibiting E-cadherin expression. PMID:25007342

  11. NF-kappaB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2.

    PubMed

    Chua, H L; Bhat-Nakshatri, P; Clare, S E; Morimiya, A; Badve, S; Nakshatri, H

    2007-02-01

    The transcription factor nuclear factor kappa B (NF-kappaB) is constitutively active in both cancer cells and stromal cells of breast cancer; however, the precise role of activated NF-kappaB in cancer progression is not known. Using parental MCF10A cells and a variant that expresses the myoepithelial marker p63 stably overexpressing the constitutively active p65 subunit of NF-kappaB (MCF10A/p65), we show that NF-kappaB suppresses the expression of epithelial specific genes E-cadherin and desmoplakin and induces the expression of the mesenchymal specific gene vimentin. P65 also suppressed the expression of p63 and the putative breast epithelial progenitor marker cytokeratin 5/6. MCF10A/p65 cells were phenotypically similar to cells undergoing epithelial to mesenchymal transition (EMT). MCF10A/p65 cells failed to form characteristic acini in three-dimensional Matrigel. Analysis of parental and MCF10A/p65 cells for genes previously shown to be involved in EMT revealed elevated expression of ZEB-1 and ZEB-2 in MCF10A/p65 cells compared to parental cells. In transient transfection assays, p65 increased ZEB-1 promoter activity. Furthermore, MCF10A cells overexpressing ZEB-1 showed reduced E-cadherin and p63 expression and displayed an EMT phenotype. The siRNA against ZEB-1 or ZEB-2 reduced the number of viable MCF10A/p65 but not parental cells, suggesting the dependence of MCF10A/p65 cells to ZEB-1 and ZEB-2 for cell cycle progression or survival. MCF10A cells chronically exposed to tumor necrosis factor alpha (TNFalpha), a potent NF-kappaB inducer, also exhibited the EMT-like phenotype and ZEB-1/ZEB-2 induction, both of which were reversed following TNFalpha withdrawal.

  12. L1-CAM and N-CAM: From Adhesion Proteins to Pharmacological Targets.

    PubMed

    Colombo, Federico; Meldolesi, Jacopo

    2015-11-01

    L1 cell adhesion molecule (L1-CAM) and neural cell adhesion molecule (N-CAM), key members of the immunoglobulin-like CAM (Ig-CAM) family, were first recognized to play critical roles in surface interactions of neurons, by binding with each other and with extracellular matrix (ECM) proteins. Subsequently, adhesion was recognized to include signaling due to both activation of β-integrin, with the generation of intracellular cascades, and integration with the surface cytoskeleton. The importance of the two Ig-CAMs was revealed by their activation of the tyrosine kinase receptors of fibroblast growth factor (FGF), epidermal growth factor (EGF), and nerve growth factor (NGF). Based on these complex signaling properties, L1-CAM and N-CAM have become of great potential pharmacological interest in neurons and cancers. Treatment of neurodegenerative disorders and cognitive deficits of neurons is aimed to increase the cell Ig-CAM tone, possibly provided by synthetic/mimetic peptides. In cancer cells, where Ig-CAMs are often overexpressed, the proteins are employed for prognosis. The approaches to therapy are based on protein downregulation, antibodies, and adoptive immunotherapy. PMID:26478212

  13. Lectin Receptor Kinases Participate in Protein-Protein Interactions to Mediate Plasma Membrane-Cell Wall Adhesions in Arabidopsis1

    PubMed Central

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces. PMID:16361528

  14. The L1 family of cell adhesion molecules: a sickening number of mutations and protein functions.

    PubMed

    Hortsch, Michael; Nagaraj, Kakanahalli; Mualla, Rula

    2014-01-01

    L1-type proteins are transmembrane cell adhesion molecules with an evolutionary well-conserved protein domain structure of usually six immunoglobulin and five fibronectin type III domains. By engaging in many different protein-protein interactions they are involved in a multitude of molecular functions and are important players during the formation and maintenance of metazoan nervous systems. As a result, mutations in L1-type genes cause a great variety of phenotypes, most of which are neurological in nature. In humans, mutations in the L1CAM gene are responsible for L1 syndrome and other L1-type genes have been implicated in conditions as varied as mental retardation, autism, schizophrenia, multiple sclerosis, and other disorders. Equally, the overexpression of L1-type proteins appears to have deleterious effects in various types of human tumor cells, where they generally contribute to an increase in cell mobility and metastatic potential. PMID:25300138

  15. Cellular localization and trafficking of vascular adhesion protein-1 as revealed by an N-terminal GFP fusion protein.

    PubMed

    Weston, Chris J; Shepherd, Emma L; Adams, David H

    2013-06-01

    Recent studies of vascular adhesion protein-1 (VAP-1) have greatly advanced our understanding of the important role this protein plays in the establishment and progression of inflammatory disease. To facilitate more detailed studies on the function of VAP-1, we developed a GFP-fusion protein that enabled us to monitor the trafficking of the protein in three selected cell types: hepatic sinusoidal endothelial cells, liver myofibroblasts and an hepatic stellate cell line (LX-2). The fusion protein was detected as punctate cytoplasmic GFP staining, but was present only at low levels at the cell surface in all cell types studied. The subcellular distribution of the protein was not altered in a catalytically inactive mutant form of the protein (Tyr471Phe) or in the presence of exogenous VAP-1 substrate (methylamine) or inhibitor (semicarbazide). The GFP-VAP-1 protein was localized to the Golgi apparatus (GM-130), endoplasmic reticulum (GRP94) and early endosomes (EEA-1). Additional staining for VAP-1 revealed that the overexpressed protein was also present in vesicles that were negative for GFP fluorescent signal and did not express EEA-1. We propose that these vesicles are responsible for recycling the fusion protein and that the fluorescence of the GFP moiety is quenched at the low pH within these vesicles. This feature of the protein makes it well suited for live cell imaging studies where we wish to track protein that is being actively trafficked within the cell in preference to that which is being recycled.

  16. Ling Zhi-8 reduces lung cancer mobility and metastasis through disruption of focal adhesion and induction of MDM2-mediated Slug degradation.

    PubMed

    Lin, Tung-Yi; Hsu, Hsien-Yeh

    2016-06-01

    We recently reported that recombinant Ling Zhi-8 (rLZ-8), a medicinal mushroom Ganoderma lucidum recombinant protein, effectively prevents lung cancer cells proliferation in vivo mice model. In our current study, we demonstrated that rLZ-8 suppressed tumor metastasis and increased the survival rate in Lewis lung carcinoma cell-bearing mice. The epithelial to mesenchymal transition (EMT) process is regarded as the critical event in tumor metastasis. Herein, we showed that rLZ-8 effectively induced changes in EMT by interfering with cell adhesion and focal adhesion kinase (FAK) functions in lung cancer cells. Slug, a transcription factor, represses E-cadherin transcription and is regarded as a critical event in EMT and tumor metastasis. Functional studies revealed that downregulation of Slug as a result of rLZ-8-induced FAK inactivation enhanced E-cadherin expression and repressed cancer cell mobility. Moreover, we found that rLZ-8 enhanced the ubiquitination proteasome pathway (UPP)-mediated degradation of Slug in CL1-5 cells. Mechanistically, we demonstrated that rLZ-8 promoted the interaction between MDM2 and Slug, resulting in Slug degradation; however, MDM2-shRNA abolished rLZ-8-enhanced Slug degradation. This study is the first to determine anti-metastatic activity of rLZ-8 and its potential mechanism, with how the regulation of EMT and cell mobility is via the negative modulation of FAK, and thereby leading to the ubiquitination and degradation of Slug. Our findings suggest that the targets of FAK play a key role in metastasis. Moreover, rLZ-8 may be useful as a chemotherapeutic agent for treating lung cancer. PMID:26992741

  17. Signaling through the G-protein-coupled receptor Rickets is important for polarity, detachment, and migration of the border cells in Drosophila.

    PubMed

    Anllo, Lauren; Schüpbach, Trudi

    2016-06-15

    Cell migration plays crucial roles during development. An excellent model to study coordinated cell movements is provided by the migration of border cell clusters within a developing Drosophila egg chamber. In a mutagenesis screen, we isolated two alleles of the gene rickets (rk) encoding a G-protein-coupled receptor. The rk alleles result in border cell migration defects in a significant fraction of egg chambers. In rk mutants, border cells are properly specified and express the marker Slbo. Yet, analysis of both fixed as well as live samples revealed that some single border cells lag behind the main border cell cluster during migration, or, in other cases, the entire border cell cluster can remain tethered to the anterior epithelium as it migrates. These defects are observed significantly more often in mosaic border cell clusters, than in full mutant clusters. Reduction of the Rk ligand, Bursicon, in the border cell cluster also resulted in migration defects, strongly suggesting that Rk signaling is utilized for communication within the border cell cluster itself. The mutant border cell clusters show defects in localization of the adhesion protein E-cadherin, and apical polarity proteins during migration. E-cadherin mislocalization occurs in mosaic clusters, but not in full mutant clusters, correlating well with the rk border cell migration phenotype. Our work has identified a receptor with a previously unknown role in border cell migration that appears to regulate detachment and polarity of the border cell cluster coordinating processes within the cells of the cluster themselves.

  18. Bacterial adhesion to protein-coated surfaces: An AFM and QCM-D study

    NASA Astrophysics Data System (ADS)

    Strauss, Joshua; Liu, Yatao; Camesano, Terri A.

    2009-09-01

    Bacterial adhesion to biomaterials, mineral surfaces, or other industrial surfaces is strongly controlled by the way bacteria interact with protein layers or organic matter and other biomolecules that coat the materials. Despite this knowledge, many studies of bacterial adhesion are performed under clean conditions, instead of in the presence of proteins or organic molecules. We chose fetal bovine serum (FBS) as a model protein, and prepared FBS films on quartz crystals. The thickness of the FBS layer was characterized using atomic force microscopy (AFM) imaging under liquid and quartz crystal microbalance with dissipation (QCM-D). Next, we characterized how the model biomaterial surface would interact with the nocosomial pathogen Staphylococcus epidermidis. An AFM probe was coated with S. epidermidis cells and used to probe a gold slide that had been coated with FBS or another protein, fibronectin (FN). These experiments show that AFM and QCM-D can be used in complementary ways to study the complex interactions between bacteria, proteins, and surfaces.

  19. In vitro investigation of protein adsorption and platelet adhesion on inorganic biomaterial surfaces

    NASA Astrophysics Data System (ADS)

    Huang, Yan; Lü, Xiaoying; Jingwu, Ma; Huang, Nan

    2008-11-01

    The aim of this paper was to study the surface properties, protein adsorption and platelet adhesion behaviors of diamond-like carbon (DLC) and titanium (Ti) films. The surface energy and microstructures of these films were characterized by contact angle measurement and atomic force microscopy (AFM). A modified Coomassie brilliant blue (CBB) protein assay was used to study the amount of adsorbed proteins. Platelet adhesion was assessed by scanning electron microscopy (SEM). The AFM results show that the DLC film is smoother than Ti. Protein adsorption results from CBB protein assay show that the ratio of adsorbed albumin (Alb) to IgG ( RA/I) on DLC is larger than Ti, which coincide with the sequence of the ratio of interfacial tension between solid surface and Alb ( γS,Alb) to interfacial tension between surface and IgG ( γS,IgG) ( γS,Alb/ γS,IgG). The DLC film has a preferential adsorption for Alb. The results suggest that the ratio of γS,Alb/ γS,IgG may indicate an Alb/IgG affinity ratio of materials. More platelets adhere on Ti film than on DLC, which may correspond to the surface roughness of materials. The conclusion is the blood compatibility of DLC seems to be better than Ti.

  20. Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin

    PubMed Central

    Chamma, Ingrid; Letellier, Mathieu; Butler, Corey; Tessier, Béatrice; Lim, Kok-Hong; Gauthereau, Isabel; Choquet, Daniel; Sibarita, Jean-Baptiste; Park, Sheldon; Sainlos, Matthieu; Thoumine, Olivier

    2016-01-01

    The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures. PMID:26979420

  1. Promyelocytic Leukemia (PML) Protein Plays Important Roles in Regulating Cell Adhesion, Morphology, Proliferation and Migration

    PubMed Central

    Tang, Mei Kuen; Liang, Yong Jia; Chan, John Yeuk Hon; Wong, Sing Wan; Chen, Elve; Yao, Yao; Gan, Jingyi; Xiao, Lihai; Leung, Hin Cheung; Kung, Hsiang Fu; Wang, Hua; Lee, Kenneth Ka Ho

    2013-01-01

    PML protein plays important roles in regulating cellular homeostasis. It forms PML nuclear bodies (PML-NBs) that act like nuclear relay stations and participate in many cellular functions. In this study, we have examined the proteome of mouse embryonic fibroblasts (MEFs) derived from normal (PML+/+) and PML knockout (PML−/−) mice. The aim was to identify proteins that were differentially expressed when MEFs were incapable of producing PML. Using comparative proteomics, total protein were extracted from PML−/− and PML+/+ MEFs, resolved by two dimensional electrophoresis (2-DE) gels and the differentially expressed proteins identified by LC-ESI-MS/MS. Nine proteins (PML, NDRG1, CACYBP, CFL1, RSU1, TRIO, CTRO, ANXA4 and UBE2M) were determined to be down-regulated in PML−/− MEFs. In contrast, ten proteins (CIAPIN1, FAM50A, SUMO2 HSPB1 NSFL1C, PCBP2, YWHAG, STMN1, TPD52L2 and PDAP1) were found up-regulated. Many of these differentially expressed proteins play crucial roles in cell adhesion, migration, morphology and cytokinesis. The protein profiles explain why PML−/− and PML+/+ MEFs were morphologically different. In addition, we demonstrated PML−/− MEFs were less adhesive, proliferated more extensively and migrated significantly slower than PML+/+ MEFs. NDRG1, a protein that was down-regulated in PML−/− MEFs, was selected for further investigation. We determined that silencing NDRG1expression in PML+/+ MEFs increased cell proliferation and inhibited PML expression. Since NDRG expression was suppressed in PML−/− MEFs, this may explain why these cells proliferate more extensively than PML+/+ MEFs. Furthermore, silencing NDRG1expression also impaired TGF-β1 signaling by inhibiting SMAD3 phosphorylation. PMID:23555679

  2. Recombinant Probiotic Expressing Listeria Adhesion Protein Attenuates Listeria monocytogenes Virulence In Vitro

    PubMed Central

    Koo, Ok Kyung; Amalaradjou, Mary Anne Roshni; Bhunia, Arun K.

    2012-01-01

    Background Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection. Methodology/Principal Findings The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (LbpLAP) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to LbpLAP for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, LbpLAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. LbpLAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, LbpLAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h. Conclusions/Significance Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, LbpLAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant

  3. International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors.

    PubMed

    Hamann, Jörg; Aust, Gabriela; Araç, Demet; Engel, Felix B; Formstone, Caroline; Fredriksson, Robert; Hall, Randy A; Harty, Breanne L; Kirchhoff, Christiane; Knapp, Barbara; Krishnan, Arunkumar; Liebscher, Ines; Lin, Hsi-Hsien; Martinelli, David C; Monk, Kelly R; Peeters, Miriam C; Piao, Xianhua; Prömel, Simone; Schöneberg, Torsten; Schwartz, Thue W; Singer, Kathleen; Stacey, Martin; Ushkaryov, Yuri A; Vallon, Mario; Wolfrum, Uwe; Wright, Mathew W; Xu, Lei; Langenhan, Tobias; Schiöth, Helgi B

    2015-01-01

    The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.

  4. Role of the microtubule-targeting drug vinflunine on cell-cell adhesions in bladder epithelial tumour cells

    PubMed Central

    2014-01-01

    Background Vinflunine (VFL) is a microtubule-targeting drug that suppresses microtubule dynamics, showing anti-metastatic properties both in vitro and in living cancer cells. An increasing body of evidence underlines the influence of the microtubules dynamics on the cadherin-dependent cell-cell adhesions. E-cadherin is a marker of epithelial-to-mesenchymal transition (EMT) and a tumour suppressor; its reduced levels in carcinoma are associated with poor prognosis. In this report, we investigate the role of VFL on cell-cell adhesions in bladder epithelial tumour cells. Methods Human bladder epithelial tumour cell lines HT1376, 5637, SW780, T24 and UMUC3 were used to analyse cadherin-dependent cell-cell adhesions under VFL treatment. VFL effect on growth inhibition was measured by using a MTT colorimetric cell viability assay. Western blot, immunofluorescence and transmission electron microscopy analyses were performed to assess the roles of VFL effect on cell-cell adhesions, epithelial-to-mesenchymal markers and apoptosis. The role of the proteasome in controlling cell-cell adhesion was studied using the proteasome inhibitor MG132. Results We show that VFL induces cell death in bladder cancer cells and activates epithelial differentiation of the remaining living cells, leading to an increase of E-cadherin-dependent cell-cell adhesion and a reduction of mesenchymal markers, such as N-cadherin or vimentin. Moreover, while E-cadherin is increased, the levels of Hakai, an E3 ubiquitin-ligase for E-cadherin, were significantly reduced in presence of VFL. In 5637, this reduction on Hakai expression was blocked by MG132 proteasome inhibitor, indicating that the proteasome pathway could be one of the molecular mechanisms involved in its degradation. Conclusions Our findings underscore a critical function for VFL in cell-cell adhesions of epithelial bladder tumour cells, suggesting a novel molecular mechanism by which VFL may impact upon EMT and metastasis. PMID:25012153

  5. In Vivo Detection of Vascular Adhesion Protein-1 in Experimental Inflammation

    PubMed Central

    Jaakkola, Kimmo; Nikula, Tuomo; Holopainen, Riikka; Vähäsilta, Tommi; Matikainen, Marja-Terttu; Laukkanen, Marja-Leena; Huupponen, Risto; Halkola, Lauri; Nieminen, Lauri; Hiltunen, Jukka; Parviainen, Sakari; Clark, Michael R.; Knuuti, Juhani; Savunen, Timo; Kääpä, Pekka; Voipio-Pulkki, Liisa Maria; Jalkanen, Sirpa

    2000-01-01

    Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible endothelial glycoprotein which mediates leukocyte-endothelial cell interactions. To study the pathogenetic significance of VAP-1 in inflammatory disorders, an in vivo immunodetection method was used to detect the regulation of luminally expressed VAP-1 in experimental skin and joint inflammation in the pig and dog. Moreover, VAP-1 was studied as a potential target to localize inflammation by radioimmunoscintigraphy. Up-regulation of VAP-1 in experimental dermatitis and arthritis could be visualized by specifically targeted immunoscintigraphy. Moreover, the translocation of VAP-1 to the functional position on the endothelial surface was only seen in inflamed tissues. These results suggest that VAP-1 is both an optimal candidate for anti-adhesive therapy and a potential target molecule for imaging inflammation. PMID:10934150

  6. Mechanical Activation of a Multimeric Adhesive Protein Through Domain Conformational Change

    NASA Astrophysics Data System (ADS)

    Wijeratne, Sithara S.; Botello, Eric; Yeh, Hui-Chun; Zhou, Zhou; Bergeron, Angela L.; Frey, Eric W.; Patel, Jay M.; Nolasco, Leticia; Turner, Nancy A.; Moake, Joel L.; Dong, Jing-fei; Kiang, Ching-Hwa

    2013-03-01

    The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.

  7. Force Activation of a Multimeric Adhesive Protein through Domain Conformational Change

    NASA Astrophysics Data System (ADS)

    Wijeratne Sithara S

    The force-induced activation of adhesive proteins such as von Willebrand factor (VWF), which experience high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force induced functional change is manifested in the multimeric VWF's crucial role in blood coagulation, when high fluid shear stress activates pVWF multimers to bind platelets. Here we showed that a pathological level of high shear flow exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers.

  8. Flocculation protein structure and cell-cell adhesion mechanism in Saccharomyces cerevisiae.

    PubMed

    Goossens, Katty; Willaert, Ronnie

    2010-11-01

    Cell-cell adhesion occurs in a broad spectrum of biological processes, of which yeast flocculation is an area of interest for evolutionary scientists to brewers and winemakers. The flocculation mechanism is based on a lectin-carbohydrate interaction but is not yet fully understood, although the first model dates back to the 1950s. This review will update the current understanding of the complex mechanism behind yeast flocculation. Moreover, modern technologies to measure the forces involved in single carbohydrate-lectin interactions, are discussed. The Flo1 protein has been extensively described as the protein responsible for strong flocculation. Recently, more research has been directed to the detailed analysis of this flocculin. Due to the advances in the field of bioinformatics, more information about Flo1p could be obtained via structurally or functionally related proteins. Here, we review the current knowledge of the Flo1 protein, with a strong emphasis towards its structure.

  9. Expression of mutant amyloid precursor proteins decreases adhesion and delays differentiation of Hep-1 cells.

    PubMed

    Kusiak, J W; Lee, L L; Zhao, B

    2001-03-30

    The amyloid precursor protein (APP) is a type I integral membrane protein and is processed to generate several intra-cellular and secreted fragments. The physiological role of APP and its processed fragments is unclear. Several mutations have been discovered in APP, which are causative of early-onset, familial, neurological disease, including Alzheimer's disease (FAD). These mutations alter the processing of APP and lead to excess production and extra-cellular deposition of A-beta peptide (Abeta). We have examined the role of APP in a cell culture model of endothelial cell function. The endothelial cell line, Hep-1, was stably transfected with wild-type (wt) and FAD mutant forms of APP (mAPP). Secretion of sAPPalpha was reduced in cell lines over-expressing mAPP when these cells were grown on several different substrates. Levels of secreted Abeta were increased as measured by ELISA in the mutant cell lines. Cell adhesion to laminin-, fibronectin-, collagen I-, and collagen IV-coated culture flasks was reduced in all mAPP-expressing cell lines, while in lines over-expressing wt-APP, adhesiveness was slightly increased. Cell lines over-expressing mAPP differentiated more slowly into capillary network-like structures on Matrigel than those expressing wt-APP. No differences were detected among all cell lines in a migration/invasion assay. The results suggest that APP may have a role in cell adhesiveness and maturation of endothelial cells into capillary-like networks. The reduction in adhesion and differentiation in mutant cell lines may be due to reduced amounts of sAPPalpha released into the culture media or toxic effects of increased extracellular Abeta.

  10. Highly purified mussel adhesive protein to secure biosafety for in vivo applications

    PubMed Central

    2014-01-01

    Background Unique adhesive and biocompatibility properties of mussel adhesive proteins (MAPs) are known for their great potential in many tissue engineering and biomedical applications. Previously, it was successfully demonstrated that redesigned hybrid type MAP, fp-151, mass-produced in Gram-negative bacterium Escherichia coli, could be utilized as a promising adhesive biomaterial. However, purification of recombinant fp-151 has been unsatisfactory due to its adhesive nature and polarity which make separation of contaminants (especially, lipopolysaccharide, a toxic Gram-negative cell membrane component) very difficult. Results In the present work, we devised a high resolution purification approach to secure safety standards of recombinant fp-151 for the successful use in in vivo applications. Undesirable impurities were remarkably eliminated as going through sequential steps including treatment with multivalent ion and chelating agent for cell membrane washing, mechanical cell disruption, non-ionic surfactant treatment for isolated inclusion body washing, acid extraction of washed inclusion body, and ion exchange chromatography purification of acid extracted sample. Through various analyses, such as high performance liquid chromatographic purity assay, limulus amoebocyte lysate endotoxin assay, and in vitro mouse macrophage cell tests on inflammation, viability, cytotoxicity, and apoptosis, we confirmed the biological safety of bacterial-derived purified recombinant fp-151. Conclusions Through this purification design, recombinant fp-151 achieved 99.90% protein purity and 99.91% endotoxin reduction that nearly no inflammation response was observed in in vitro experiments. Thus, the highly purified recombinant MAP would be successfully used as a safety-secured in vivo bioadhesive for tissue engineering and biomedical applications. PMID:24725543

  11. Aire knockdown in medullary thymic epithelial cells affects Aire protein, deregulates cell adhesion genes and decreases thymocyte interaction.

    PubMed

    Pezzi, Nicole; Assis, Amanda Freire; Cotrim-Sousa, Larissa Cotrim; Lopes, Gabriel Sarti; Mosella, Maritza Salas; Lima, Djalma Sousa; Bombonato-Prado, Karina F; Passos, Geraldo Aleixo

    2016-09-01

    We demonstrate that even a partial reduction of Aire mRNA levels by siRNA-induced Aire knockdown (Aire KD) has important consequences to medullary thymic epithelial cells (mTECs). Aire knockdown is sufficient to reduce Aire protein levels, impair its nuclear location, and cause an imbalance in large-scale gene expression, including genes that encode cell adhesion molecules. These genes drew our attention because adhesion molecules are implicated in the process of mTEC-thymocyte adhesion, which is critical for T cell development and the establishment of central self-tolerance. Accordingly, we consider the following: 1) mTECs contribute to the elimination of self-reactive thymocytes through adhesion; 2) Adhesion molecules play a crucial role during physical contact between these cells; and 3) Aire is an important transcriptional regulator in mTECs. However, its role in controlling mTEC-thymocyte adhesion remains unclear. Because Aire controls adhesion molecule genes, we hypothesized that the disruption of its expression could influence mTEC-thymocyte interaction. To test this hypothesis, we used a murine Aire(+) mTEC cell line as a model system to reproduce mTEC-thymocyte adhesion in vitro. Transcriptome analysis of the mTEC cell line revealed that Aire KD led to the down-modulation of more than 800 genes, including those encoding for proteins involved in cell adhesion, i.e., the extracellular matrix constituent Lama1, the CAM family adhesion molecules Vcam1 and Icam4, and those that encode peripheral tissue antigens. Thymocytes co-cultured with Aire KD mTECs had a significantly reduced capacity to adhere to these cells. This finding is the first direct evidence that Aire also plays a role in controlling mTEC-thymocyte adhesion. PMID:27505711

  12. Aire knockdown in medullary thymic epithelial cells affects Aire protein, deregulates cell adhesion genes and decreases thymocyte interaction.

    PubMed

    Pezzi, Nicole; Assis, Amanda Freire; Cotrim-Sousa, Larissa Cotrim; Lopes, Gabriel Sarti; Mosella, Maritza Salas; Lima, Djalma Sousa; Bombonato-Prado, Karina F; Passos, Geraldo Aleixo

    2016-09-01

    We demonstrate that even a partial reduction of Aire mRNA levels by siRNA-induced Aire knockdown (Aire KD) has important consequences to medullary thymic epithelial cells (mTECs). Aire knockdown is sufficient to reduce Aire protein levels, impair its nuclear location, and cause an imbalance in large-scale gene expression, including genes that encode cell adhesion molecules. These genes drew our attention because adhesion molecules are implicated in the process of mTEC-thymocyte adhesion, which is critical for T cell development and the establishment of central self-tolerance. Accordingly, we consider the following: 1) mTECs contribute to the elimination of self-reactive thymocytes through adhesion; 2) Adhesion molecules play a crucial role during physical contact between these cells; and 3) Aire is an important transcriptional regulator in mTECs. However, its role in controlling mTEC-thymocyte adhesion remains unclear. Because Aire controls adhesion molecule genes, we hypothesized that the disruption of its expression could influence mTEC-thymocyte interaction. To test this hypothesis, we used a murine Aire(+) mTEC cell line as a model system to reproduce mTEC-thymocyte adhesion in vitro. Transcriptome analysis of the mTEC cell line revealed that Aire KD led to the down-modulation of more than 800 genes, including those encoding for proteins involved in cell adhesion, i.e., the extracellular matrix constituent Lama1, the CAM family adhesion molecules Vcam1 and Icam4, and those that encode peripheral tissue antigens. Thymocytes co-cultured with Aire KD mTECs had a significantly reduced capacity to adhere to these cells. This finding is the first direct evidence that Aire also plays a role in controlling mTEC-thymocyte adhesion.

  13. Redox Capacity of an Extracellular Matrix Protein Associated with Adhesion in Mytilus californianus.

    PubMed

    Nicklisch, Sascha C T; Spahn, Jamie E; Zhou, Hongjun; Gruian, Cristina M; Waite, J Herbert

    2016-04-01

    Adhesive mussel foot proteins (Mfps) rely in part on DOPA (3,4-dihydroxyphenyl-l-alanine) side chains to mediate attachment to mineral surfaces underwater. Oxidation of DOPA to Dopaquinone (Q) effectively abolishes the adsorption of Mfps to these surfaces. The thiol-rich mussel foot protein-6 (Mfp-6) rescues adhesion compromised by adventitious DOPA oxidation by reducing Q back to DOPA. The redox chemistry and kinetics of foot-extracted Mfp-6 were investigated by using a nonspecific chromogenic probe to equilibrate with the redox pool. Foot-extracted Mfp-6 has a reducing capacity of ~17 e(-) per protein; half of this comes from the cysteine residues, whereas the other half comes from other constituents, probably a cohort of four or five nonadhesive, redox-active DOPA residues in Mfp-6 with an anodic peak potential ~500 mV lower than that for oxidation of cysteine to cystine. At higher pH, DOPA redox reversibility is lost possibly due to Q scavenging by Cys thiolates. Analysis by one- and two-dimensional proton nuclear magnetic resonance identified a pronounced β-sheet structure with a hydrophobic core in foot-extracted Mfp-6 protein. The structure endows redox-active side chains in Mfp-6, i.e., cysteine and DOPA, with significant reducing power over a broad pH range, and this power is measurably diminished in recombinant Mfp-6. PMID:26998552

  14. Onion yellow phytoplasma P38 protein plays a role in adhesion to the hosts.

    PubMed

    Neriya, Yutaro; Maejima, Kensaku; Nijo, Takamichi; Tomomitsu, Tatsuya; Yusa, Akira; Himeno, Misako; Netsu, Osamu; Hamamoto, Hiroshi; Oshima, Kenro; Namba, Shigetou

    2014-12-01

    Adhesins are microbial surface proteins that mediate the adherence of microbial pathogens to host cell surfaces. In Mollicutes, several adhesins have been reported in mycoplasmas and spiroplasmas. Adhesins P40 of Mycoplasma agalactiae and P89 of Spiroplasma citri contain a conserved amino acid sequence known as the Mollicutes adhesin motif (MAM), whose function in the host cell adhesion remains unclear. Here, we show that phytoplasmas, which are plant-pathogenic mollicutes transmitted by insect vectors, possess an adhesion-containing MAM that was identified in a putative membrane protein, PAM289 (P38), of the 'Candidatus Phytoplasma asteris,' OY strain. P38 homologs and their MAMs were highly conserved in related phytoplasma strains. While P38 protein was expressed in OY-infected insect and plant hosts, binding assays showed that P38 interacts with insect extract, and weakly with plant extract. Interestingly, the interaction of P38 with the insect extract depended on MAM. These results suggest that P38 is a phytoplasma adhesin that interacts with the hosts. In addition, the MAM of adhesins is important for the interaction between P38 protein and hosts.

  15. Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation.

    PubMed

    Modjeski, Kristina L; Ture, Sara K; Field, David J; Cameron, Scott J; Morrell, Craig N

    2016-01-01

    Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium. PMID:27631377

  16. Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation

    PubMed Central

    Modjeski, Kristina L.; Ture, Sara K.; Field, David J.; Cameron, Scott J.; Morrell, Craig N.

    2016-01-01

    Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium. PMID:27631377

  17. Redox Capacity of an Extracellular Matrix Protein Associated with Adhesion in Mytilus californianus.

    PubMed

    Nicklisch, Sascha C T; Spahn, Jamie E; Zhou, Hongjun; Gruian, Cristina M; Waite, J Herbert

    2016-04-01

    Adhesive mussel foot proteins (Mfps) rely in part on DOPA (3,4-dihydroxyphenyl-l-alanine) side chains to mediate attachment to mineral surfaces underwater. Oxidation of DOPA to Dopaquinone (Q) effectively abolishes the adsorption of Mfps to these surfaces. The thiol-rich mussel foot protein-6 (Mfp-6) rescues adhesion compromised by adventitious DOPA oxidation by reducing Q back to DOPA. The redox chemistry and kinetics of foot-extracted Mfp-6 were investigated by using a nonspecific chromogenic probe to equilibrate with the redox pool. Foot-extracted Mfp-6 has a reducing capacity of ~17 e(-) per protein; half of this comes from the cysteine residues, whereas the other half comes from other constituents, probably a cohort of four or five nonadhesive, redox-active DOPA residues in Mfp-6 with an anodic peak potential ~500 mV lower than that for oxidation of cysteine to cystine. At higher pH, DOPA redox reversibility is lost possibly due to Q scavenging by Cys thiolates. Analysis by one- and two-dimensional proton nuclear magnetic resonance identified a pronounced β-sheet structure with a hydrophobic core in foot-extracted Mfp-6 protein. The structure endows redox-active side chains in Mfp-6, i.e., cysteine and DOPA, with significant reducing power over a broad pH range, and this power is measurably diminished in recombinant Mfp-6.

  18. Loss of miR-200b promotes invasion via activating the Kindlin-2/integrin β1/AKT pathway in esophageal squamous cell carcinoma: An E-cadherin-independent mechanism.

    PubMed

    Zhang, Hai-Feng; Alshareef, Abdulraheem; Wu, Chengsheng; Li, Shang; Jiao, Ji-Wei; Cao, Hui-Hui; Lai, Raymond; Xu, Li-Yan; Li, En-Min

    2015-10-01

    Our previous studies have shown that loss of miR-200b enhances the invasiveness of esophageal squamous cell carcinoma (ESCC) cells. However, whether the miR-200-ZEB1/2-E-cadherin regulatory cascade, a master regulator of epithelial-to-mesenchymal transition (EMT), is involved in the regulation of ESCC invasion remains elusive. Here, we show that miR-200b represses ESCC cell invasion in vivo without altering the expression of E-cadherin and vimentin, two surrogate markers of EMT. However, an inverse correlation was observed between the expression levels of miR-200b and ZEB1/2 in both ESCC cell lines (n = 7, P < 0.05) and ESCC tumor samples (n = 88, P < 0.05). Methylation of E-cadherin gene was found to block the regulation of E-cadherin by the miR-200b-ZEB1/2 axis, indicating that an E-cadherin-independent mechanism can mediate the biological function of miR-200b in ESCC. We revealed that miR-200b suppresses the integrin β1-AKT pathway via targeting Kindlin-2 to mitigate ESCC cell invasiveness. In two independent cohorts of ESCC samples (n = 20 and n = 53, respectively), Kindlin-2 expression positively correlated with the activation status of both the integrin signaling pathway and the PI3K-AKT signaling pathway (both P < 0.01). These data highlight that suppression of the Kindlin-2-integrin β1-AKT regulatory axis is an alternative mechanism underlying the tumor suppressor function of miR-200b in ESCC.

  19. Plakophilin 2 Affects Cell Migration by Modulating Focal Adhesion Dynamics and Integrin Protein Expression

    PubMed Central

    Koetsier, Jennifer L.; Amargo, Evangeline V.; Todorović, Viktor; Green, Kathleen J.; Godsel, Lisa M.

    2014-01-01

    Plakophilin 2 (PKP2), a desmosome component, modulates the activity and localization of the small GTPase RhoA at sites of cell–cell contact. PKP2 regulates cortical actin rearrangement during junction formation, and its loss is accompanied by an increase in actin stress fibers. We hypothesized that PKP2 may regulate focal adhesion dynamics and cell migration. Here we show that PKP2-deficient cells bind efficiently to the extracellular matrix, but upon spreading display total cell areas ~30% smaller than control cells. Focal adhesions in PKP2-deficient cells are ~2× larger and more stable than in control cells, and vinculin displays an increased time for fluorescence recovery after photobleaching. Furthermore, β4 and β1 integrin protein and mRNA expression is elevated in PKP2-silenced cells. Normal focal adhesion phenotypes can be restored in PKP2-null cells by dampening the RhoA pathway or silencing β1 integrin. However, integrin expression levels are not restored by RhoA signaling inhibition. These data uncover a potential role for PKP2 upstream of β1 integrin and RhoA in integrating cell–cell and cell–substrate contact signaling in basal keratinocytes necessary for the morphogenesis, homeostasis, and reepithelialization of the stratified epidermis. PMID:23884246

  20. Small heat shock proteins in cellular adhesion and migration: evidence from Plasmodium genetics.

    PubMed

    Montagna, Georgina N; Matuschewski, Kai; Buscaglia, Carlos A

    2012-01-01

    Cellular locomotion and adhesion critically depend on regulated turnover of filamentous actin. Biochemical data from diverse model systems support a role for the family of small heat shock proteins (HSPBs) in microfilament regulation. The small chaperones could either act directly, through competition with the motor myosin, or indirectly, through modulation of actin depolymerizing factor/cofilin activity. However, a direct link between HSPBs and actin-based cellular motility remained to be established. In a recent experimental genetics study, we provided evidence for regulation of Plasmodium motility by HSPB6/Hsp20. The infectious forms of malaria parasites, termed sporozoites, display fast and continuous substrate-dependent motility, which is largely driven by turnover of actin microfilaments. Sporozoite gliding locomotion is essential to avoid destruction by host defense mechanisms and to ultimately reach a hepatocyte, the target cell, where to transform and replicate. Genetic ablation of Plasmodium HSP20 dramatically changed sporozoite speed and substrate adhesion, resulting in impaired natural malaria transmission. In this article, we discuss the function of Hsp20 in this fast-moving unicellular protozoan and implications for the roles of HSPBs in adhesion and migration of eukaryotic cells.

  1. Nanometer polymer surface features: the influence on surface energy, protein adsorption and endothelial cell adhesion

    NASA Astrophysics Data System (ADS)

    Carpenter, Joseph; Khang, Dongwoo; Webster, Thomas J.

    2008-12-01

    Current small diameter (<5 mm) synthetic vascular graft materials exhibit poor long-term patency due to thrombosis and intimal hyperplasia. Tissue engineered solutions have yielded functional vascular tissue, but some require an eight-week in vitro culture period prior to implantation—too long for immediate clinical bedside applications. Previous in vitro studies have shown that nanostructured poly(lactic-co-glycolic acid) (PLGA) surfaces elevated endothelial cell adhesion, proliferation, and extracellular matrix synthesis when compared to nanosmooth surfaces. Nonetheless, these studies failed to address the importance of lateral and vertical surface feature dimensionality coupled with surface free energy; nor did such studies elicit an optimum specific surface feature size for promoting endothelial cell adhesion. In this study, a series of highly ordered nanometer to submicron structured PLGA surfaces of identical chemistry were created using a technique employing polystyrene nanobeads and poly(dimethylsiloxane) (PDMS) molds. Results demonstrated increased endothelial cell adhesion on PLGA surfaces with vertical surface features of size less than 18.87 nm but greater than 0 nm due to increased surface energy and subsequently protein (fibronectin and collagen type IV) adsorption. Furthermore, this study provided evidence that the vertical dimension of nanometer surface features, rather than the lateral dimension, is largely responsible for these increases. In this manner, this study provides key design parameters that may promote vascular graft efficacy.

  2. Host and Tissue Specificity of Trichomonas vaginalis Is Not Mediated by Its Known Adhesion Proteins

    PubMed Central

    Addis, Maria Filippa; Rappelli, Paola; Fiori, Pier Luigi

    2000-01-01

    Adhesion of Trichomonas vaginalis is believed to be dependent on four adhesion proteins, which are thought to bind to vaginal epithelial cells in a specific manner with a ligand-receptor type of interaction. However, the specific receptors on the host cell have not yet been identified. In this work, the ability of the T. vaginalis adhesins to bind to cells of different histologic derivations and from different species has been studied. HeLa, CHO, and Vero cell lines; erythrocytes from different species; and a prokaryote without a cell wall, Mycoplasma hominis, were employed in order to investigate the cell specificity of the T. vaginalis adhesins. We observed that the T. vaginalis adhesins are able to bind to the different cell types to the same extent, suggesting that the host and tissue specificity of T. vaginalis adhesion should not be due to specificity of the parasite adhesins. Our results suggest that the data published to date on the subject are probably artifactual and that the experiments reported in the literature are not appropriate for identification of protozoan adhesins. PMID:10858260

  3. A Multidomain Adhesion Protein Family Expressed in Plasmodium falciparum Is Essential for Transmission to the Mosquito

    PubMed Central

    Pradel, Gabriele; Hayton, Karen; Aravind, L.; Iyer, Lakshminarayan M.; Abrahamsen, Mitchell S.; Bonawitz, Annemarie; Mejia, Cesar; Templeton, Thomas J.

    2004-01-01

    The recent sequencing of several apicomplexan genomes has provided the opportunity to characterize novel antigens essential for the parasite life cycle that might lead to the development of new diagnostic and therapeutic markers. Here we have screened the Plasmodium falciparum genome sequence for genes encoding extracellular multidomain putative adhesive proteins. Three of these identified genes, named PfCCp1, PfCCp2, and PfCCp3, have multiple adhesive modules including a common Limulus coagulation factor C domain also found in two additional Plasmodium genes. Orthologues were identified in the Cryptosporidium parvum genome sequence, indicating an evolutionary conserved function. Transcript and protein expression analysis shows sexual stage–specific expression of PfCCp1, PfCCp2, and PfCCp3, and cellular localization studies revealed plasma membrane–associated expression in mature gametocytes. During gametogenesis, PfCCps are released and localize surrounding complexes of newly emerged microgametes and macrogametes. PfCCp expression markedly decreased after formation of zygotes. To begin to address PfCCp function, the PfCCp2 and PfCCp3 gene loci were disrupted by homologous recombination, resulting in parasites capable of forming oocyst sporozoites but blocked in the salivary gland transition. Our results describe members of a conserved apicomplexan protein family expressed in sexual stage Plasmodium parasites that may represent candidates for subunits of a transmission-blocking vaccine. PMID:15184503

  4. Amyloid precursor-like protein 1 (APLP1) exhibits stronger zinc-dependent neuronal adhesion than amyloid precursor protein and APLP2.

    PubMed

    Mayer, Magnus C; Schauenburg, Linda; Thompson-Steckel, Greta; Dunsing, Valentin; Kaden, Daniela; Voigt, Philipp; Schaefer, Michael; Chiantia, Salvatore; Kennedy, Timothy E; Multhaup, Gerhard

    2016-04-01

    The amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein 1 (APLP1) and APLP2, are metalloproteins with a putative role both in synaptogenesis and in maintaining synapse structure. Here, we studied the effect of zinc on membrane localization, adhesion, and secretase cleavage of APP, APLP1, and APLP2 in cell culture and rat neurons. For this, we employed live-cell microscopy techniques, a microcontact printing adhesion assay and ELISA for protein detection in cell culture supernatants. We report that zinc induces the multimerization of proteins of the amyloid precursor protein family and enriches them at cellular adhesion sites. Thus, zinc facilitates the formation of de novo APP and APLP1 containing adhesion complexes, whereas it does not have such influence on APLP2. Furthermore, zinc-binding prevented cleavage of APP and APLPs by extracellular secretases. In conclusion, the complexation of zinc modulates neuronal functions of APP and APLPs by (i) regulating formation of adhesion complexes, most prominently for APLP1, and (ii) by reducing the concentrations of neurotrophic soluble APP/APLP ectodomains. Earlier studies suggest a function of the amyloid precursor protein (APP) family proteins in neuronal adhesion. We report here that adhesive function of these proteins is tightly regulated by zinc, most prominently for amyloid precursor-like protein 1 (APLP1). Zinc-mediated APLP1 multimerization, which induced formation of new neuronal contacts and decreased APLP1 shedding. This suggests that APLP1 could function as a zinc receptor processing zinc signals to stabilized or new neuronal contacts.

  5. Amyloid precursor-like protein 1 (APLP1) exhibits stronger zinc-dependent neuronal adhesion than amyloid precursor protein and APLP2.

    PubMed

    Mayer, Magnus C; Schauenburg, Linda; Thompson-Steckel, Greta; Dunsing, Valentin; Kaden, Daniela; Voigt, Philipp; Schaefer, Michael; Chiantia, Salvatore; Kennedy, Timothy E; Multhaup, Gerhard

    2016-04-01

    The amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein 1 (APLP1) and APLP2, are metalloproteins with a putative role both in synaptogenesis and in maintaining synapse structure. Here, we studied the effect of zinc on membrane localization, adhesion, and secretase cleavage of APP, APLP1, and APLP2 in cell culture and rat neurons. For this, we employed live-cell microscopy techniques, a microcontact printing adhesion assay and ELISA for protein detection in cell culture supernatants. We report that zinc induces the multimerization of proteins of the amyloid precursor protein family and enriches them at cellular adhesion sites. Thus, zinc facilitates the formation of de novo APP and APLP1 containing adhesion complexes, whereas it does not have such influence on APLP2. Furthermore, zinc-binding prevented cleavage of APP and APLPs by extracellular secretases. In conclusion, the complexation of zinc modulates neuronal functions of APP and APLPs by (i) regulating formation of adhesion complexes, most prominently for APLP1, and (ii) by reducing the concentrations of neurotrophic soluble APP/APLP ectodomains. Earlier studies suggest a function of the amyloid precursor protein (APP) family proteins in neuronal adhesion. We report here that adhesive function of these proteins is tightly regulated by zinc, most prominently for amyloid precursor-like protein 1 (APLP1). Zinc-mediated APLP1 multimerization, which induced formation of new neuronal contacts and decreased APLP1 shedding. This suggests that APLP1 could function as a zinc receptor processing zinc signals to stabilized or new neuronal contacts. PMID:26801522

  6. MicroRNA-10b regulates epithelial-mesenchymal transition by modulating KLF4/Notch1/E-cadherin in cisplatin-resistant nasopharyngeal carcinoma cells

    PubMed Central

    Zhang, Pei; Hong, Haiyu; Sun, Xiaojin; Jiang, Hao; Ma, Shiyin; Zhao, Surong; Zhang, Mengxiao; Wang, Zhiwei; Jiang, Chenchen; Liu, Hao

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is an initiating event in tumor cell invasion and metastasis that contributes to therapeutic resistance to compounds including cisplatin. MicroRNAs (miRNAs) have been associated with EMT as well as resistance to standard therapies. However, the underlying mechanisms by which miRNAs control the development of resistance to cisplatin (DDP), and the accompanying EMT-like properties are required to elucidate. Here we show that microRNA-10b (miR-10b) is up-regulated in HNE1/DDP cells, and inhibition of miR-10b expression reversed the EMT phenotype. However, over-expression of miR-10b was able to promote the acquisition of an EMT phenotype in HNE1 cells. Additionally, we identified that miR-10b expression inversely correlates with KLF4, which then controls expression of Notch1. Knock-down of Notch1 inhibited cell migration, invasion, and reversed EMT in HNE1/DDP cells, which was dependent on miR-10b. In summary, our results reveal that miR-10b regulates EMT by modulating KLF4/Notch1/E-cadherin expression, which promotes invasion and migration of nasal pharyngeal carcinoma cells. PMID:27186392

  7. Concerted loss of TGFβ-mediated proliferation control and E-cadherin disrupts epithelial homeostasis and causes oral squamous cell carcinoma

    PubMed Central

    Andl, Thomas; Le Bras, Grégoire F.; Richards, Nicole F.; Allison, Gillian L.; Loomans, Holli A.; Washington, M.Kay; Revetta, Frank; Lee, Rebecca K.; Taylor, Chase; Moses, Harold L.; Andl, Claudia D.

    2014-01-01

    Although the etiology of squamous cell carcinomas of the oral mucosa is well understood, the cellular origin and the exact molecular mechanisms leading to their formation are not. Previously, we observed the coordinated loss of E-cadherin (CDH1) and transforming growth factor beta receptor II (TGFBR2) in esophageal squamous tumors. To investigate if the coordinated loss of Cdh1 and Tgfbr2 is sufficient to induce tumorigenesis in vivo, we developed two mouse models targeting ablation of both genes constitutively or inducibly in the oral–esophageal epithelium. We show that the loss of both Cdh1 and Tgfbr2 in both models is sufficient to induce squamous cell carcinomas with animals succumbing to the invasive disease by 18 months of age. Advanced tumors have the ability to invade regional lymph nodes and to establish distant pulmonary metastasis. The mouse tumors showed molecular characteristics of human tumors such as overexpression of Cyclin D1. We addressed the question whether TGFβ signaling may target known stem cell markers and thereby influence tumorigenesis. From our mouse and human models, we conclude that TGFβ signaling regulates key aspects of stemness and quiescence in vitro and in vivo. This provides a new explanation for the importance of TGFβ in mucosal homeostasis. PMID:25233932

  8. MicroRNA-421 regulated by HIF-1α promotes metastasis, inhibits apoptosis, and induces cisplatin resistance by targeting E-cadherin and caspase-3 in gastric cancer

    PubMed Central

    Li, Peng; Liu, Kaiyi; Geng, Ruixuan; Dai, Congqi; Lin, Ying; Tang, Wenbo; Wu, Zheng; Chang, Jinjia; Lu, Jianwei; Li, Jin

    2016-01-01

    Hypoxia and dysregulation of microRNAs (miRNAs) have been identified as crucial factors in carcinogenesis. However, the potential mechanisms of HIF-1α and miR-421 in gastric cancer have not been well elucidated. In this study, we found that miR-421 was up-regulated by HIF-1α. Overexpression of miR-421 promoted metastasis, inhibited apoptosis, and induced cisplatin resistance in gastric cancer in vivo and in vitro. E-cadherin and caspase-3 were identified as targets of miR-421. Besides, relative mRNA expression of miR-421 was significantly increased in gastric cancer tumor tissues compared with non-tumor tissues in a cohort of gastric cancer specimens (n=107). The expression of miR-421 was higher in advanced gastric cancers compared with localized ones. Moreover, Kaplan–Meier analysis illustrated that those patients with low levels of miR-421 had a significant longer overall survival (p = 0.006) and time to relapse (p = 0.007). Therefore, miR-421 could serve as an important prognostic marker and a potential molecular target for therapy in gastric cancer. PMID:27016414

  9. IGF-I Induces Epithelial-to-Mesenchymal Transition via the IGF-IR-Src-MicroRNA-30a-E-Cadherin Pathway in Nasopharyngeal Carcinoma Cells.

    PubMed

    Wang, Ruoyu; Li, Heming; Guo, Xuefen; Wang, Zhe; Liang, Shanshan; Dang, Chengxue

    2016-01-01

    Recurrence and distant metastasis are the most common cause of therapeutic failure in nasopharyngeal carcinoma (NPC) patients. Insulin-like growth factor I (IGF-I) can induce epithelial-to-mesenchymal transition (EMT) in many epithelial tumors; however, whether IGF-I can enhance NPC metastasis by EMT and the mechanisms remain unclear. Herein, we have identified that IGF-I could induce EMT and enhance migration ability in NPC cell lines. Furthermore, both Src inhibitor and microRNA-30a (miR-30a) inhibitor reversed IGF-I-induced EMT, suggesting the involvement of an IGF-IR-Src-miR-30a-E-cadherin pathway in IGF-I-induced EMT in NPC cell lines. Overall, the results of the present study may provide more useful information regarding the mechanisms of the IGF-IR signaling pathway in the regulation of NPC metastasis. Both Src kinase and miR-30a can be potential biomarkers for selecting high risk of metastasis in NPC patients. PMID:27656832

  10. Focal adhesion kinase regulates expression of thioredoxin-interacting protein (TXNIP) in cancer cells.

    PubMed

    Ho, Baotran; Huang, Grace; Golubovskaya, Vita M

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells. PMID:23387972

  11. Membrane and acto-myosin tension promote clustering of adhesion proteins

    PubMed Central

    Delanoë-Ayari, H.; Al Kurdi, R.; Vallade, M.; Gulino-Debrac, D.; Riveline, D.

    2004-01-01

    Physicists have studied the aggregation of adhesive proteins, giving a central role to the elastic properties of membranes, whereas cell biologists have put the emphasis on the cytoskeleton. However, there is a dramatic lack of experimental studies probing both contributions on cellular systems. Here, we tested both mechanisms on living cells. We compared, for the same cell line, the growth of cadherin-GFP patterns on recombinant cadherin-coated surfaces, with the growth of vinculin-GFP patterns on extracellular matrix protein-coated surfaces by using evanescent wave microscopy. In our setup, cadherins are not linked to actin, whereas vinculins are. This property allows us to compare formation of clusters with proteins linked or not to the cytoskeleton and thus study the role of membrane versus cytoskeleton in protein aggregation. Strikingly, the motifs we obtained on both surfaces share common features: they are both elongated and located at the cell edges. We showed that a local force application can impose this symmetry breaking in both cases. However, the origin of the force is different as demonstrated by drug treatment (butanedione monoxime) and hypotonic swelling. Cadherins aggregate when membrane tension is increased, whereas vinculins (cytoplasmic proteins of focal contacts) aggregate when acto-myosin stress fibers are pulling. We propose a mechanism by which membrane tension is localized at cell edges, imposing flattening of membrane and enabling aggregation of cadherins by diffusion. In contrast, cytoplasmic proteins of focal contacts aggregate by opening cryptic sites in focal contacts under acto-myosin contractility. PMID:14982992

  12. Effect of interfacial serum proteins on melanoma cell adhesion to biodegradable poly(l-lactic acid) microspheres coated with hydroxyapatite.

    PubMed

    Shinto, Hiroyuki; Hirata, Takuya; Fukasawa, Tomonori; Fujii, Syuji; Maeda, Hayata; Okada, Masahiro; Nakamura, Yoshinobu; Furuzono, Tsutomu

    2013-08-01

    We have measured the interaction forces between a murine melanoma cell and a poly(l-lactic acid) (PLLA) microsphere coated with/without hydroxyapatite (HAp) nanoparticles (i.e., an HAp/PLLA or a bare PLLA microsphere) in a serum-free culture medium, using atomic force microscopy (AFM) with colloid probe technique, in order to investigate how the HAp-nanoparticle coating as well as interfacial serum proteins influence the cell-microsphere adhesion. The cell adhesion force of the HAp/PLLA microspheres was 1.4-fold stronger than that of the bare PLLA microspheres. When the microspheres were pretreated with a culture medium supplemented with 10% fetal bovine serum, the cell adhesion force of the HAp/PLLA microspheres was increased by a factor of 2.1; in contrast, no change was observed in the cell adhesion force of the bare PLLA microspheres before/after the pretreatment. Indeed, the cell adhesion force of the HAp/PLLA was 2.8-fold larger than that of the bare PLLA after the pretreatment. Additionally, we have investigated the effect of interfacial serum proteins on the zeta potentials of these microspheres. On the basis of the obtained results, possible mechanism of cell adhesion to the HAp/PLLA and bare PLLA microspheres in the presence/absence of the interfacial serum proteins is discussed.

  13. Effect of interfacial serum proteins on melanoma cell adhesion to biodegradable poly(l-lactic acid) microspheres coated with hydroxyapatite.

    PubMed

    Shinto, Hiroyuki; Hirata, Takuya; Fukasawa, Tomonori; Fujii, Syuji; Maeda, Hayata; Okada, Masahiro; Nakamura, Yoshinobu; Furuzono, Tsutomu

    2013-08-01

    We have measured the interaction forces between a murine melanoma cell and a poly(l-lactic acid) (PLLA) microsphere coated with/without hydroxyapatite (HAp) nanoparticles (i.e., an HAp/PLLA or a bare PLLA microsphere) in a serum-free culture medium, using atomic force microscopy (AFM) with colloid probe technique, in order to investigate how the HAp-nanoparticle coating as well as interfacial serum proteins influence the cell-microsphere adhesion. The cell adhesion force of the HAp/PLLA microspheres was 1.4-fold stronger than that of the bare PLLA microspheres. When the microspheres were pretreated with a culture medium supplemented with 10% fetal bovine serum, the cell adhesion force of the HAp/PLLA microspheres was increased by a factor of 2.1; in contrast, no change was observed in the cell adhesion force of the bare PLLA microspheres before/after the pretreatment. Indeed, the cell adhesion force of the HAp/PLLA was 2.8-fold larger than that of the bare PLLA after the pretreatment. Additionally, we have investigated the effect of interfacial serum proteins on the zeta potentials of these microspheres. On the basis of the obtained results, possible mechanism of cell adhesion to the HAp/PLLA and bare PLLA microspheres in the presence/absence of the interfacial serum proteins is discussed. PMID:23524077

  14. Spontaneous unraveling of hagfish slime thread skeins is mediated by a seawater-soluble protein adhesive.

    PubMed

    Bernards, Mark A; Oke, Isdin; Heyland, Andreas; Fudge, Douglas S

    2014-04-15

    Hagfishes are known for their ability to rapidly produce vast quantities of slime when provoked. The slime is formed via the interaction between seawater and two components released by the slime glands: mucin vesicles from gland mucous cells, which swell and rupture in seawater to form a network of mucus strands, and intermediate filament-rich threads, which are produced within gland thread cells as tightly coiled bundles called skeins. A previous study showed that the unraveling of skeins from Atlantic hagfish (Myxine glutinosa) requires both the presence of mucins and hydrodynamic mixing. In contrast, skeins from Pacific hagfish (Eptatretus stoutii) unravel in the absence of both mucins and mixing. We tested the hypothesis that spontaneous unraveling of E. stoutii skeins is triggered by the dissolution of a seawater-soluble protein adhesive and the release of stored strain energy within the coiled thread. Here we show that, as predicted by this hypothesis, unraveling can be initiated by a protease under conditions in which unraveling does not normally occur. We also demonstrate, using high resolution scanning electron microscopy, that the treatment of skeins with solutions that cause unraveling also leads to the disappearance of surface and inter-thread features that remain when skeins are washed with stabilizing solutions. Our study provides a mechanism for the deployment of thread skeins in Pacific hagfish slime, and raises the possibility of producing novel biomimetic protein adhesives that are salt, temperature and kosmotrope sensitive.

  15. Protein adsorption and cell adhesion on polyurethane/Pluronic surface with lotus leaf-like topography.

    PubMed

    Zheng, Jun; Song, Wei; Huang, He; Chen, Hong

    2010-06-01

    Lotus leaf-like polyurethane/Pluronic F-127 surface was fabricated via replica molding using a natural lotus leaf as the template. Water contact angle measurements showed that both the hydrophobicity of the unmodified polyurethane (PU) surface and the hydrophilicity of the PU/Pluronic surface were enhanced by the construction of lotus leaf-like topography. Protein adsorption on the PU/Pluronic surface without topographic modification was significantly lower than on the PU surface. Adsorption was further reduced when lotus leaf-like topography was constructed on the PU/Pluronic surface. Cell culture experiments with L929 cells showed that adhesion on the PU/Pluronic surface with lotus leaf-like topography was low and adherent cells were spherical and of low viability. The PU/Pluronic surface with lotus leaf-like topography thus appears to be resistant to nonspecific protein adsorption and to cell adhesion, and these effects derive from the both chemical composition and topography. The results suggest a new strategy based on surface topography for the design of antifouling materials.

  16. Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy.

    PubMed

    Huang, Yujian; Wang, Yongzhong; Tan, Li; Sun, Leming; Petrosino, Jennifer; Cui, Mei-Zhen; Hao, Feng; Zhang, Mingjun

    2016-06-01

    Over 130 y have passed since Charles Darwin first discovered that the adventitious roots of English ivy (Hedera helix) exude a yellowish mucilage that promotes the capacity of this plant to climb vertical surfaces. Unfortunately, little progress has been made in elucidating the adhesion mechanisms underlying this high-strength adhesive. In the previous studies, spherical nanoparticles were observed in the viscous exudate. Here we show that these nanoparticles are predominantly composed of arabinogalactan proteins (AGPs), a superfamily of hydroxyproline-rich glycoproteins present in the extracellular spaces of plant cells. The spheroidal shape of the AGP-rich ivy nanoparticles results in a low viscosity of the ivy adhesive, and thus a favorable wetting behavior on the surface of substrates. Meanwhile, calcium-driven electrostatic interactions among carboxyl groups of the AGPs and the pectic acids give rise to the cross-linking of the exuded adhesive substances, favor subsequent curing (hardening) via formation of an adhesive film, and eventually promote the generation of mechanical interlocking between the adventitious roots of English ivy and the surface of substrates. Inspired by these molecular events, a reconstructed ivy-mimetic adhesive composite was developed by integrating purified AGP-rich ivy nanoparticles with pectic polysaccharides and calcium ions. Information gained from the subsequent tensile tests, in turn, substantiated the proposed adhesion mechanisms underlying the ivy-derived adhesive. Given that AGPs and pectic polysaccharides are also observed in bioadhesives exuded by other climbing plants, the adhesion mechanisms revealed by English ivy may forward the progress toward understanding the general principles underlying diverse botanic adhesives. PMID:27217558

  17. Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy

    NASA Astrophysics Data System (ADS)

    Huang, Yujian; Wang, Yongzhong; Tan, Li; Sun, Leming; Petrosino, Jennifer; Cui, Mei-Zhen; Hao, Feng; Zhang, Mingjun

    2016-06-01

    Over 130 y have passed since Charles Darwin first discovered that the adventitious roots of English ivy (Hedera helix) exude a yellowish mucilage that promotes the capacity of this plant to climb vertical surfaces. Unfortunately, little progress has been made in elucidating the adhesion mechanisms underlying this high-strength adhesive. In the previous studies, spherical nanoparticles were observed in the viscous exudate. Here we show that these nanoparticles are predominantly composed of arabinogalactan proteins (AGPs), a superfamily of hydroxyproline-rich glycoproteins present in the extracellular spaces of plant cells. The spheroidal shape of the AGP-rich ivy nanoparticles results in a low viscosity of the ivy adhesive, and thus a favorable wetting behavior on the surface of substrates. Meanwhile, calcium-driven electrostatic interactions among carboxyl groups of the AGPs and the pectic acids give rise to the cross-linking of the exuded adhesive substances, favor subsequent curing (hardening) via formation of an adhesive film, and eventually promote the generation of mechanical interlocking between the adventitious roots of English ivy and the surface of substrates. Inspired by these molecular events, a reconstructed ivy-mimetic adhesive composite was developed by integrating purified AGP-rich ivy nanoparticles with pectic polysaccharides and calcium ions. Information gained from the subsequent tensile tests, in turn, substantiated the proposed adhesion mechanisms underlying the ivy-derived adhesive. Given that AGPs and pectic polysaccharides are also observed in bioadhesives exuded by other climbing plants, the adhesion mechanisms revealed by English ivy may forward the progress toward understanding the general principles underlying diverse botanic adhesives.

  18. Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy.

    PubMed

    Huang, Yujian; Wang, Yongzhong; Tan, Li; Sun, Leming; Petrosino, Jennifer; Cui, Mei-Zhen; Hao, Feng; Zhang, Mingjun

    2016-06-01

    Over 130 y have passed since Charles Darwin first discovered that the adventitious roots of English ivy (Hedera helix) exude a yellowish mucilage that promotes the capacity of this plant to climb vertical surfaces. Unfortunately, little progress has been made in elucidating the adhesion mechanisms underlying this high-strength adhesive. In the previous studies, spherical nanoparticles were observed in the viscous exudate. Here we show that these nanoparticles are predominantly composed of arabinogalactan proteins (AGPs), a superfamily of hydroxyproline-rich glycoproteins present in the extracellular spaces of plant cells. The spheroidal shape of the AGP-rich ivy nanoparticles results in a low viscosity of the ivy adhesive, and thus a favorable wetting behavior on the surface of substrates. Meanwhile, calcium-driven electrostatic interactions among carboxyl groups of the AGPs and the pectic acids give rise to the cross-linking of the exuded adhesive substances, favor subsequent curing (hardening) via formation of an adhesive film, and eventually promote the generation of mechanical interlocking between the adventitious roots of English ivy and the surface of substrates. Inspired by these molecular events, a reconstructed ivy-mimetic adhesive composite was developed by integrating purified AGP-rich ivy nanoparticles with pectic polysaccharides and calcium ions. Information gained from the subsequent tensile tests, in turn, substantiated the proposed adhesion mechanisms underlying the ivy-derived adhesive. Given that AGPs and pectic polysaccharides are also observed in bioadhesives exuded by other climbing plants, the adhesion mechanisms revealed by English ivy may forward the progress toward understanding the general principles underlying diverse botanic adhesives.

  19. Characterization and binding analysis of a microneme adhesive repeat domain-containing protein from Toxoplasma gondii.

    PubMed

    Gong, Haiyan; Kobayashi, Kyousuke; Sugi, Tatsuki; Takemae, Hitoshi; Ishiwa, Akiko; Recuenco, Frances C; Murakoshi, Fumi; Xuan, Xuenan; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

    2014-04-01

    The intracellular parasite Toxoplasma gondii invades almost all nucleated cells, and has infected approximately 34% of the world's population to date. In order to develop effective vaccines against T. gondii infection, understanding of the role of the molecules that are involved in the invasion process is important. For this purpose, we characterized T. gondii proteins that contain microneme adhesive repeats (MARs), which are common in moving junction proteins. T. gondii MAR domain-containing protein 4a (TgMCP4a), which contains repeats of 17-22 amino acid segments at the N-terminus and three putative MAR domains at the C-terminus, is localized near the rhoptry of extracellular parasites. Following infection, TgMCP4a was detected in the parasitophorous vacuole. The recombinant Fc-TgMCP4a N-terminus protein (rTgMCP4a-1/Fc) showed binding activity to the surface proteins of Vero, 293T, and CHO cells. The recombinant GST-TgMCP4a N-terminus protein (rTgMCP4a-1/GST), which exhibited binding activity, was used to pull down the interacting factors from 293T cell lysate, and subsequent mass spectrometry analysis revealed that three types of heat shock proteins (HSPs) interacted with TgMCP4a. Transfection of a FLAG fusion protein of TgMCP4a-1 (rTgMCP4a-1/FLAG) into 293T cell and the following immunoprecipitation with anti-FLAG antibody confirmed the interactions of HSC70 with TgMCP4a. The addition of rTgMCP4a-1/GST into the culture medium significantly affected the growth of the parasite. This study hints that T. gondii may employ HSP proteins of host cell to facilitate their growth.

  20. A standardized bamboo leaf extract inhibits monocyte adhesion to endothelial cells by modulating vascular cell adhesion protein-1

    PubMed Central

    Choi, Sunga; Park, Myoung Soo; Lee, Yu Ran; Lee, Young Chul; Kim, Tae Woo; Do, Seon-Gil; Kim, Dong Seon

    2013-01-01

    Bamboo leaves (Phyllostachys pubescens Mazel ex J. Houz (Poacea)) have a long history of food and medical applications in Asia, including Japan and Korea. They have been used as a traditional medicine for centuries. We investigated the mechanism of anti-inflammatory activity of a bamboo leaf extract (BLE) on tumor necrosis factor-alpha (TNF-α)-induced monocyte adhesion in human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to BLE did not inhibit cell viability or cause morphological changes at concentrations ranging from 1 µg/ml to 1 mg/ml. Treatment with 0.1 mg/ml BLE caused 63% inhibition of monocyte adhesion in TNF-α-activated HUVECs, which was associated with 38.4% suppression of vascular cell adhesion molecule-1 expression. Furthermore, TNF-α-induced reactive oxygen species generation was decreased to 47.9% in BLE treated TNF-α-activated HUVECs. BLE (0.05 mg/ml) also caused about 50% inhibition of interleukin-6 secretion from lipopolysaccharide-stimulated monocyte. The results indicate that BLE may be clinically useful as an anti-inflammatory or anti-oxidant for human cardiovascular disease including atherosclerosis. PMID:23422838

  1. Understanding Marine Mussel Adhesion

    PubMed Central

    Roberto, Francisco F.

    2007-01-01

    In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion. PMID:17990038

  2. Understanding Marine Mussel Adhesion

    SciTech Connect

    H. G. Silverman; F. F. Roberto

    2007-12-01

    In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are waterimpervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion.

  3. High affinity neurexin binding to cell adhesion G-protein-coupled receptor CIRL1/latrophilin-1 produces an intercellular adhesion complex.

    PubMed

    Boucard, Antony A; Ko, Jaewon; Südhof, Thomas C

    2012-03-16

    The G-protein-coupled receptor CIRL1/latrophilin-1 (CL1) and the type-1 membrane proteins neurexins represent distinct neuronal cell adhesion molecules that exhibit no similarities except for one common function: both proteins are receptors for α-latrotoxin, a component of black widow spider venom that induces massive neurotransmitter release at synapses. Unexpectedly, we have now identified a direct binding interaction between the extracellular domains of CL1 and neurexins that is regulated by alternative splicing of neurexins at splice site 4 (SS4). Using saturation binding assays, we showed that neurexins lacking an insert at SS4 bind to CL1 with nanomolar affinity, whereas neurexins containing an insert at SS4 are unable to bind. CL1 competed for neurexin binding with neuroligin-1, a well characterized neurexin ligand. The extracellular sequences of CL1 contain five domains (lectin, olfactomedin-like, serine/threonine-rich, hormone-binding, and G-protein-coupled receptor autoproteolysis-inducing (GAIN) domains). Of these domains, the olfactomedin-like domain mediates neurexin binding as shown by deletion mapping. Cell adhesion assays using cells expressing neurexins and CL1 revealed that their interaction produces a stable intercellular adhesion complex, indicating that their interaction can be trans-cellular. Thus, our data suggest that CL1 constitutes a novel ligand for neurexins that may be localized postsynaptically based on its well characterized interaction with intracellular SH3 and multiple ankyrin repeats adaptor proteins (SHANK) and could form a trans-synaptic complex with presynaptic neurexins.

  4. Protein Kinase D1 regulates focal adhesion dynamics and cell adhesion through Phosphatidylinositol-4-phosphate 5-kinase type-l γ

    PubMed Central

    Durand, Nisha; Bastea, Ligia I.; Long, Jason; Döppler, Heike; Ling, Kun; Storz, Peter

    2016-01-01

    Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs’ lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process. PMID:27775029

  5. Inhibition of E-cadherin/catenin complex formation by O-linked N-acetylglucosamine transferase is partially independent of its catalytic activity.

    PubMed

    Liu, Haiyan; Gu, Yuchao; Qi, Jieqiong; Han, Cuifang; Zhang, Xinling; Bi, Chuanlin; Yu, Wengong

    2016-02-01

    p120-catenin (p120) contains a large central armadillo repeat domain, via which it binds to E‑cadherin to stabilize the latter, thereby regulating cell‑to‑cell adhesion. A previous study by our group demonstrated that O‑linked N‑acetylglucosamine (O‑GlcNAc) is involved in the regulation of the interaction between p120 and E‑cadherin. As O‑GlcNAc transferase (OGT) is able to directly bind to the majority of its target proteins, the present study hypothesized that OGT may additionally regulate the formation of the E‑cadherin/catenin complex independent of its catalytic activity. To verify this hypothesis, a catalytically inactive OGT mutant was expressed in H1299 cells, and its effects on the formation of the E‑cadherin/catenin complex were assessed. A cytoskeleton‑binding protein extraction assay confirmed that OGT inhibited the formation of the E‑cadherin/catenin complex independent of its catalytic activity. In addition, co‑immunoprecipitation and pull‑down assays were used to evaluate the interaction between OGT and p120. Immunoblotting indicated that OGT was able to directly bind to p120. To determine the domain of p120 involved in binding to OGT, a series of deletion mutants of p120 were constructed and subjected to protein binding assays by pull‑down assays. Immunoblotting showed that OGT bound to the regulatory and armadillo domains of p120, which might interfere with the interaction between p120 and E‑cadherin. Finally, OGT, p120 and E‑cadherin cytoplasmic domains (ECD) were recombinantly expressed in BL21 (DE3) recombinant E. coli cells, and a glutathione S‑transferase (GST) pull‑down assay was performed to assess the interactions among the purified recombinant proteins. Immunoblotting indicated that maltose‑binding protein (MBP)‑OGT inhibited the binding of His‑p120 to GST‑ECD in a dose‑dependent manner. All of these results suggested that OGT inhibited the formation of the E‑cadherin/catenin complex

  6. Nanostructured Biointerfaces: Nanoarchitectonics of Thermoresponsive Polymer Brushes Impact Protein Adsorption and Cell Adhesion.

    PubMed

    Psarra, Evmorfia; König, Ulla; Ueda, Yuichiro; Bellmann, Cornelia; Janke, Andreas; Bittrich, Eva; Eichhorn, Klaus-J; Uhlmann, Petra

    2015-06-17

    Controlling the reversibility, quantity, and extent of biomolecule interaction at interfaces has a significant relevance for biomedical and biotechnological applications, because protein adsorption is always the first step when a solid surface gets in contact with a biological fluid. Polymer brushes, composed of end-tethered linear polymers with sufficient grafting density, are very promising to control and alter interactions with biological systems because of their unique structure and distinct collaborative response to environmental changes. We studied protein adsorption and cell adhesion at polymer brush substrates which consisted of poly(N-isopropylacrylamide) (PNIPAAm), having a lower critical solution temperature (LCST), to control bioadsorptive processes by changing the environmental temperature. Preparing the PNIPAAm brushes by the "grafting-to"-method two differently synthesized PNIPAAm polymers were used, at which one possessed an additional hydrophobic terminal headgroup. It is known that hydrophobic moieties can influence protein adsorption significantly. The films were comprehensively analyzed by in situ spectroscopic ellipsometry, contact angle measurements, streaming potential, and atomic force microscopy. Our study was mainly focused on the investigation of the fibrinogen (FGN) adsorption responsiveness both on homo polymer PNIPAAm brushes with and without the hydrophobic terminal functionalization, and further on binary brushes made of the polyelectrolyte poly(acrylic acid) (PAA) and one of the prior described two PNIPAAm species. The results show that the terminal hydrophobic modification of PNIPAAm has a considerable impact on wettability, LCST, and morphology of the homo and the binary brush systems, which consequently led to an alteration of FGN adsorption. By using binary PNIPAAm-PAA brushes with different composition it was possible to induce stimuli dependent FGN adsorption with a considerable amplified switching effect by introducing a

  7. Unraveling the Role of Surface Mucus-Binding Protein and Pili in Muco-Adhesion of Lactococcus lactis

    PubMed Central

    Duviau, Marie-Pierre; Meyrand, Mickael; Guérardel, Yann; Castelain, Mickaël; Loubière, Pascal; Chapot-Chartier, Marie-Pierre; Dague, Etienne; Mercier-Bonin, Muriel

    2013-01-01

    Adhesion of bacteria to mucus may favor their persistence within the gut and their beneficial effects to the host. Interactions between pig gastric mucin (PGM) and a natural isolate of Lactococcus lactis (TIL448) were measured at the single-cell scale and under static conditions, using atomic force microscopy (AFM). In parallel, these interactions were monitored at the bacterial population level and under shear flow. AFM experiments with a L. lactis cell-probe and a PGM-coated surface revealed a high proportion of specific adhesive events (60%) and a low level of non-adhesive ones (2%). The strain muco-adhesive properties were confirmed by the weak detachment of bacteria from the PGM-coated surface under shear flow. In AFM, rupture events were detected at short (100−200 nm) and long distances (up to 600−800 nm). AFM measurements on pili and mucus-binding protein defective mutants demonstrated the comparable role played by these two surface proteinaceous components in adhesion to PGM under static conditions. Under shear flow, a more important contribution of the mucus-binding protein than the pili one was observed. Both methods differ by the way of probing the adhesion force, i.e. negative force contact vs. sedimentation and normal-to-substratum retraction vs. tangential detachment conditions, using AFM and flow chamber, respectively. AFM blocking assays with free PGM or O-glycan fractions purified from PGM demonstrated that neutral oligosaccharides played a major role in adhesion of L. lactis TIL448 to PGM. This study dissects L. lactis muco-adhesive phenotype, in relation with the nature of the bacterial surface determinants. PMID:24260308

  8. Blockade of Vascular Adhesion Protein-1 Inhibits Lymphocyte Infiltration in Rat Liver Allograft Rejection

    PubMed Central

    Martelius, Timi; Salaspuro, Ville; Salmi, Marko; Krogerus, Leena; Höckerstedt, Krister; Jalkanen, Sirpa; Lautenschlager, Irmeli

    2004-01-01

    Vascular adhesion protein-1 (VAP-1) has been shown to mediate lymphocyte adhesion to endothelia at sites of inflammation, but its functional role in vivo has not been tested in any rodent model. Here we report the effects of VAP-1 blockade on rat liver allograft rejection. BN recipients of PVG liver allografts (known to develop acute rejection by day 7) were treated with 2 mg/kg anti-VAP-1 (a new anti-rat VAP-1 mAb 174–5) or isotype-matched irrelevant antibody (NS1) every other day (n = 6/group) and one group with anti-VAP-1 2 mg/kg daily (n = 7). On day 7, samples were collected for transplant aspiration cytology, histology, and immunohistochemistry. Lymphocyte infiltration to the graft was clearly affected by VAP-blockade. The total inflammation, mainly the number of active lymphoid cells, in transplant aspiration cytology was significantly decreased in animals treated with anti-VAP-1 (4.7 ± 1.0 and 2.4 ± 1.0 corrected increment units, respectively) compared to control (6.6 ± 1.0) (P < 0.05). In histology, the intensity of portal inflammation was significantly decreased (P < 0.05). The amount of T cells expressing activation markers diminished. This is the first demonstration in any prolonged in vivo model that VAP-1 plays an important role in lymphocyte infiltration to sites of inflammation, and, in particular, liver allograft rejection. PMID:15579442

  9. Leukocyte trafficking-associated vascular adhesion protein 1 is expressed and functionally active in atherosclerotic plaques

    PubMed Central

    Silvola, Johanna M. U.; Virtanen, Helena; Siitonen, Riikka; Hellberg, Sanna; Liljenbäck, Heidi; Metsälä, Olli; Ståhle, Mia; Saanijoki, Tiina; Käkelä, Meeri; Hakovirta, Harri; Ylä-Herttuala, Seppo; Saukko, Pekka; Jauhiainen, Matti; Veres, Tibor Z.; Jalkanen, Sirpa; Knuuti, Juhani; Saraste, Antti; Roivainen, Anne

    2016-01-01

    Given the important role of inflammation and the potential association of the leukocyte trafficking-associated adhesion molecule vascular adhesion protein 1 (VAP-1) with atherosclerosis, this study examined whether functional VAP-1 is expressed in atherosclerotic lesions and, if so, whether it could be targeted by positron emission tomography (PET). First, immunohistochemistry revealed that VAP-1 localized to endothelial cells of intra-plaque neovessels in human carotid endarterectomy samples from patients with recent ischemic symptoms. In low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 (LDLR−/−ApoB100/100), VAP-1 was expressed on endothelial cells lining inflamed atherosclerotic lesions; normal vessel walls in aortas of C57BL/6N control mice were VAP-1-negative. Second, we discovered that the focal uptake of VAP-1 targeting sialic acid-binding immunoglobulin-like lectin 9 based PET tracer [68Ga]DOTA-Siglec-9 in atherosclerotic plaques was associated with the density of activated macrophages (r = 0.58, P = 0.022). As a final point, we found that the inhibition of VAP-1 activity with small molecule LJP1586 decreased the density of macrophages in inflamed atherosclerotic plaques in mice. Our results suggest for the first time VAP-1 as a potential imaging target for inflamed atherosclerotic plaques, and corroborate VAP-1 inhibition as a therapeutic approach in the treatment of atherosclerosis. PMID:27731409

  10. Vascular adhesion protein-1 promotes liver inflammation and drives hepatic fibrosis

    PubMed Central

    Weston, Chris J.; Shepherd, Emma L.; Claridge, Lee C.; Rantakari, Pia; Curbishley, Stuart M.; Tomlinson, Jeremy W.; Hubscher, Stefan G.; Reynolds, Gary M.; Aalto, Kristiina; Anstee, Quentin M.; Jalkanen, Sirpa; Salmi, Marko; Smith, David J.; Day, Christopher P.; Adams, David H.

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) encompasses a range of manifestations, including steatosis and cirrhosis. Progressive disease is characterized by hepatic leukocyte accumulation in the form of steatohepatitis. The adhesion molecule vascular adhesion protein-1 (VAP-1) is a membrane-bound amine oxidase that promotes leukocyte recruitment to the liver, and the soluble form (sVAP-1) accounts for most circulating monoamine oxidase activity, has insulin-like effects, and can initiate oxidative stress. Here, we determined that hepatic VAP-1 expression is increased in patients with chronic liver disease and that serum sVAP-1 levels are elevated in patients with NAFLD compared with those in control individuals. In 4 murine hepatic injury models, an absence or blockade of functional VAP-1 reduced inflammatory cell recruitment to the liver and attenuated fibrosis. Moreover, disease was reduced in animals expressing a catalytically inactive form of VAP-1, implicating enzyme activity in the disease pathogenesis. Within the liver, hepatic stromal cells expressed functional VAP-1, and evaluation of cultured cells revealed that sVAP-1 promotes leukocyte migration through catalytic generation of ROS, which depended on VAP-1 enzyme activity. VAP-1 enhanced stromal cell spreading and wound closure and modulated expression of profibrotic genes. Together, these results link the amine oxidase activity of VAP-1 with hepatic inflammation and fibrosis and suggest that targeting VAP-1 has therapeutic potential for NAFLD and other chronic fibrotic liver diseases. PMID:25562318

  11. Anterior Gradient Protein-2 Is a Regulator of Cellular Adhesion in Prostate Cancer

    PubMed Central

    Chanda, Diptiman; Lee, Joo Hyoung; Sawant, Anandi; Hensel, Jonathan A.; Isayeva, Tatyana; Reilly, Stephanie D.; Siegal, Gene P.; Smith, Claire; Grizzle, William; Singh, Raj; Ponnazhagan, Selvarangan

    2014-01-01

    Anterior Gradient Protein (AGR-2) is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s) has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis. PMID:24587138

  12. Calcium- and integrin-binding protein 1 regulates megakaryocyte ploidy, adhesion, and migration

    PubMed Central

    Kostyak, John C.; Naik, Meghna U.

    2012-01-01

    Megakaryocytes are large, polyploid cells that produce platelets. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in megakaryopoiesis, we used a Cib1−/− mouse model. Cib1−/− mice have more platelets and BM megakaryocytes than wild-type (WT) controls (P < .05). Furthermore, subsequent analysis of megakaryocyte-CFU production revealed an increase with Cib1 deletion compared with WT (P < .05). In addition, BM from Cib1−/− mice, cultured with thrombopoietin (TPO) for 24 hours, produced more highly polyploid megakaryocytes than WT BM (P < .05). Subsequent analysis of TPO signaling revealed enhanced Akt and ERK1/2 phosphorylation, whereas FAKY925 phosphorylation was reduced in Cib1−/− megakaryocytes treated with TPO. Conversely, platelet recovery in Cib1−/− mice after platelet depletion was attenuated compared with WT (P < .05). This could be the result of impaired adhesion and migration, as adhesion to fibrinogen and fibronectin and migration toward an SDF-1α gradient were reduced in Cib1−/− megakaryocytes compared with WT (P < .05). In addition, Cib1−/− megakaryocytes formed fewer proplatelets compared with WT (P < .05), when plated on fibrinogen. These data suggest that CIB1 plays a dual role in megakaryopoiesis, initially by negatively regulating TPO signaling and later by augmenting proplatelet production. PMID:22128142

  13. Evaluation of Serum Vascular Adhesion Protein-1 as a Potential Biomarker in Thyroid Cancer

    PubMed Central

    Zhao, Pengxin; Zhang, Kaili

    2016-01-01

    Vascular adhesion protein-1 (VAP-1) is a glycoprotein that mediates tissue-selective lymphocyte adhesion. The prognostic value of VAP-1 has been determined in gastric cancer. The aim of this study was to evaluate the changes and the predictive value of serum VAP-1 in patients with thyroid cancer. A total of 126 patients with thyroid nodules and 53 healthy controls participated in this study. The patients were further divided into subgroup 1 (69 cases with benign thyroid nodules) and subgroup 2 (57 cases with thyroid cancer). Serum VAP-1 was measured by time-resolved immunofluorometric assay. Diagnostic value of presurgical VAP-1 for thyroid cancer was conducted by receiver operating characteristic (ROC) curves. Serum levels of VAP-1 were significantly lower in thyroid cancer group than in healthy control and benign thyroid nodule groups. VAP-1 concentrations negatively correlated with serum thyroglobulin (Tg) levels in thyroid cancer patients (r = −0.81; p < 0.001). The optimum cut-off value of VAP-1 was 456.6 ng/mL with a 77.4% specificity and 66.7% sensitivity for thyroid cancer diagnosis. Serum VAP-1 decreased in thyroid cancer patients and VAP-1 could be a potential useful adjunct biomarker in the diagnosis of thyroid cancer. PMID:27446209

  14. Fabrication of three-dimensional multi-protein microstructures for cell migration and adhesion enhancement

    PubMed Central

    Da Sie, Yong; Li, Yi-Cheng; Chang, Nan-Shan; Campagnola, Paul J.; Chen, Shean-Jen

    2015-01-01

    In this study, three-dimensional (3D) multi-component microstructures were precisely fabricated via multiphoton excited photochemistry using a femtosecond laser direct-writing system with proposed repetition positioning and vector scanning techniques. Extracellular matrix (ECM) proteins, such as fibronectin (FN), are difficult to stack and form 3D structures larger than several-hundred microns in height due to the nature of their protein structure. Herein, to fabricate complex 3D microstructures with FN, a 3D scaffold was designed and formed from bovine serum albumin (BSA), after which human FN was inserted at specific locations on the BSA scaffold; in this manner, the fabricated ECM microstructure can guide cells in a 3D environment. A human breast cancer cell line, MDA-MB-231, was used to investigate the behavior of cell migration and adhesion on the fabricated human FN and BSA protein structures. Experimental results indicate that many cells are not able to attach or climb on a 3D structure’s inclined plane without FN support; hence, the influence of cell growth in a 3D context with FN should being taken into consideration. This 3D multi-protein fabrication technique holds potential for cell studies in designed complex 3D ECM scaffolds. PMID:25780738

  15. Adhesion and fusion efficiencies of human immunodeficiency virus type 1 (HIV-1) surface proteins

    NASA Astrophysics Data System (ADS)

    Dobrowsky, Terrence M.; Rabi, S. Alireza; Nedellec, Rebecca; Daniels, Brian R.; Mullins, James I.; Mosier, Donald E.; Siliciano, Robert F.; Wirtz, Denis

    2013-10-01

    In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones to tensile strengths of the individual Env-receptor bonds of Env proteins obtained from a HIV-1 infected patient prior to and during coreceptor switching. The results suggest that the genetic determinants of viral entry were predominantly enriched in the C3, HR1 and CD regions rather than V3. Env proteins can better mediate entry into cells after coreceptor switch; this effective entry capacity does not correlate with the bond strengths between viral Env and cellular receptors.

  16. RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells

    PubMed Central

    Liu, Xiaoxia; Sun, Guiling; Sun, Xiuju

    2016-01-01

    This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP) gene on renal cell cancer (RCC) cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA)-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte–macrophage colony-stimulating factor and E-cadherin was significantly increased (P<0.05). The relevant signaling pathways were the integrin-mediated cell surface interactions pathway and extracellular matrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial growth factor receptor, matrix metallopeptidase-9, and vascular cell adhesion molecule, which are related to the integrin-mediated cell surface interactions and extracellular

  17. RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells.

    PubMed

    Liu, Xiaoxia; Sun, Guiling; Sun, Xiuju

    2016-01-01

    This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP) gene on renal cell cancer (RCC) cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA)-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte-macrophage colony-stimulating factor and E-cadherin was significantly increased (P<0.05). The relevant signaling pathways were the integrin-mediated cell surface interactions pathway and extracellular matrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial growth factor receptor, matrix metallopeptidase-9, and vascular cell adhesion molecule, which are related to the integrin-mediated cell surface interactions and extracellular

  18. Redistribution of microfilament-associated proteins during the formation of focal contacts and adhesions in chick fibroblasts.

    PubMed

    Couchman, J R; Badley, R A; Rees, D A

    1983-12-01

    The roles of the microfilament-associated proteins vinculin, alpha-actinin, myosin and filamin have been studied by immunofluorescence and double fluorescence in conjunction with interference reflection microscopy (IRM), during the development of focal contacts and focal adhesions in a chick fibroblast system which initially has no such adhesion specializations but then develops them sequentially over a 48 h period. Without exception, all focal contacts and focal adhesions contain both vinculin and alpha-actinin at every stage that we can detect by IRM or by double staining to reveal the associated microfilament bundles. Indeed the appearance of small bodies containing alpha-actinin and vinculin is shown to precede focal contact formation in our model system and such structures (not visible by IRM) are proposed to be the precursors of focal contacts and adhesions. Myosin and filamin are distributed generally with some reticular patterning in the early motile cells which lack the focal specializations, but as focal contacts and adhesions form these proteins become progressively recruited into the associated microfilament bundles. Only then do we see the marked depletion that has been reported earlier of diffusely distributed myosin and filamin in the leading lamella. Although this is not initially associated with any change in the motile status of the cells, the recruitment of these microfilament-associated proteins into stress fibres is proposed to occur in preparation for anchorage and bracing of cells to the substratum when they later become stationary. PMID:6421873

  19. Characterization of the protein fraction of the temporary adhesive secreted by the tube feet of the sea star Asterias rubens.

    PubMed

    Hennebert, Elise; Wattiez, Ruddy; Waite, J Herbert; Flammang, Patrick

    2012-01-01

    Sea stars are able to make firm but temporary attachments to various substrata by secretions released by their tube feet. After tube foot detachment, the adhesive secretions remain on the substratum as a footprint. Proteins presumably play a key role in sea star adhesion, as evidenced by the removal of footprints from surfaces after a treatment with trypsin. However, until now, characterisation was hampered by their high insolubility. In this study, a non-hydrolytic method was used to render most of the proteins constituting the adhesive footprints soluble. After analysis by SDS-PAGE, the proteins separated into about 25 bands, which ranged from 25 to 450 kDa in apparent molecular weight. Using mass spectrometry and a homology-database search, it was shown that several of the proteins are known intracellular proteins, presumably resulting from contamination of footprint material with tube foot epidermal cells. However, 11 protein bands, comprising the most abundant proteins, were not identified and might correspond to novel adhesive proteins. They were named 'Sea star footprint proteins' (Sfps). Tandem mass spectrometry analysis of the protein bands yielded 43 de novo-generated peptide sequences. Most of them were shared by several, if not all, Sfps. Polyclonal antibodies were raised against one of the peptides (HEASGEYYR from Sfp-115) and were used in immunoblotting. They specifically labelled Sfp-115 and other bands with lower apparent molecular weights. The different results suggest that all Sfps might belong to a single family of related proteins sharing similar motifs or, alternatively, they are the products of polymerization and/or degradation processes.

  20. Vimentin contributes to epithelial-mesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation

    PubMed Central

    Liu, Ching-Yi; Lin, Hsi-Hui; Tang, Ming-Jer; Wang, Yang-Kao

    2015-01-01

    Modulations of cytoskeletal organization and focal adhesion turnover correlate to tumorigenesis and epithelial-mesenchymal transition (EMT), the latter process accompanied by the loss of epithelial markers and the gain of mesenchymal markers (e.g., vimentin). Clinical microarray results demonstrated that increased levels of vimentin mRNA after chemotherapy correlated to a poor prognosis of breast cancer patients. We hypothesized that vimentin mediated the reorganization of cytoskeletons to maintain the mechanical integrity in EMT cancer cells. By using knockdown strategy, the results showed reduced cell proliferation, impaired wound healing, loss of directional migration, and increased large membrane extension in MDA-MB 231 cells. Vimentin depletion also induced reorganization of cytoskeletons and reduced focal adhesions, which resulted in impaired mechanical strength because of reduced cell stiffness and contractile force. In addition, overexpressing vimentin in MCF7 cells increased cell stiffness, elevated cell motility and directional migration, reoriented microtubule polarity, and increased EMT phenotypes due to the increased β1-integrin and the loss of junction protein E-cadherin. The EMT-related transcription factor slug was also mediated by vimentin. The current study demonstrated that vimentin serves as a regulator to maintain intracellular mechanical homeostasis by mediating cytoskeleton architecture and the balance of cell force generation in EMT cancer cells. PMID:25965826

  1. Quantum dot-based quantification revealed differences in subcellular localization of EGFR and E-cadherin between EGFR-TKI sensitive and insensitive cancer cells

    NASA Astrophysics Data System (ADS)

    Huang, Dong-hai; Su, Ling; Peng, Xiang-hong; Zhang, Hongzheng; Khuri, Fadlo R.; Shin, Dong M.; Chen, Zhuo

    2009-06-01

    Nanoparticle quantum dots (QDs) provide sharper and more photostable fluorescent signals than organic dyes, allowing quantification of multiple biomarkers simultaneously. In this study, we quantified the expression of epidermal growth factor receptor (EGFR) and E-cadherin (E-cad) in the same cells simultaneously by using secondary antibody-conjugated QDs with two different emission wavelengths (QD605 and QD565) and compared the cellular distribution of EGFR and E-cad between EGFR-tyrosine kinase inhibitor (TKI)-insensitive and -sensitive lung and head and neck cancer cell lines. Relocalization of EGFR and E-cad upon treatment with the EGFR-TKI erlotinib in the presence of EGF was visualized and analyzed quantitatively. Our results showed that QD-immunocytochemistry (ICC)-based technology can not only quantify basal levels of multiple biomarkers but also track the localization of the biomarkers upon biostimulation. With this new technology we found that in EGFR-TKI-insensitive cells, EGFR and E-cad were located mainly in the cytoplasm; while in sensitive cells, they were found mainly on the cell membrane. After induction with EGF, both EGFR and E-cad internalized to the cytoplasm, but the internalization capability in sensitive cells was greater than that in insensitive cells. Quantification also showed that inhibition of EGF-induced EGFR and E-cad internalization by erlotinib in the sensitive cells was stronger than that in the insensitive cells. These studies demonstrate substantial differences between EGFR-TKI-insensitive and -sensitive cancer cells in EGFR and E-cad expression and localization both at the basal level and in response to EGF and erlotinib. QD-based analysis facilitates the understanding of the features of EGFR-TKI-insensitive versus -sensitive cancer cells and may be used in the prediction of patient response to EGFR-targeted therapy.

  2. The adhesion of mussel foot protein-3 to TiO2 surfaces: the effect of pH

    PubMed Central

    Yu, Jing; Wei, Wei; Menyo, Matthew S.; Masic, Admir; Waite, J. Herbert; Israelachvili, Jacob N.

    2013-01-01

    The underwater adhesion of marine mussels relies on mussel foot proteins (mfps) rich in the catecholic amino acid 3, 4-dihydroxyphenylalanine (Dopa). As a side-chain, Dopa is capable of strong bidentate interactions with a variety of surfaces, including many minerals and metal oxides. Titanium is among the most widely used medical implant material and quickly forms a TiO2 passivation layer under physiological conditions. Understanding the binding mechanism of Dopa to TiO2 surfaces is therefore of considerable theoretical and practical interest. Using a surface forces apparatus, we explored the force-distance profiles and adhesion energies of mussel foot protein 3 (mfp-3) to TiO2 surfaces at three different pHs (pH3, 5.5 and 7.5). At pH3, mfp-3 showed the strongest adhesion force on TiO2, with an adhesion energy of ~ −7.0 mJ/m2. Increasing the pH gives rise to two opposing effects: (1) increased oxidation of Dopa, thus decreasing availability for the Dopa-mediated adhesion, and (2) increased bidentate Dopa-Ti coordination, leading to the further stabilization of the Dopa group and thus an increasing of adhesion force. Both effects were reflected in the resonance-enhanced Raman spectra obtained at the three deposition pHs. The two competing effects give rise to a higher adhesion force of mfp-3 on TiO2 surface at pH 7.5 than at pH 5.5. Our results suggest that Dopa-containing proteins and synthetic polymers have great potential as coating materials for medical implant materials, particularly if redox activity can be controlled. PMID:23452271

  3. Novel pyridazinone inhibitors for vascular adhesion protein-1 (VAP-1): old target-new inhibition mode.

    PubMed

    Bligt-Lindén, Eva; Pihlavisto, Marjo; Szatmári, István; Otwinowski, Zbyszek; Smith, David J; Lázár, László; Fülöp, Ferenc; Salminen, Tiina A

    2013-12-27

    Vascular adhesion protein-1 (VAP-1) is a primary amine oxidase and a drug target for inflammatory and vascular diseases. Despite extensive attempts to develop potent, specific, and reversible inhibitors of its enzyme activity, the task has proven challenging. Here we report the synthesis, inhibitory activity, and molecular binding mode of novel pyridazinone inhibitors, which show specificity for VAP-1 over monoamine and diamine oxidases. The crystal structures of three inhibitor-VAP-1 complexes show that these compounds bind reversibly into a unique binding site in the active site channel. Although they are good inhibitors of human VAP-1, they do not inhibit rodent VAP-1 well. To investigate this further, we used homology modeling and structural comparison to identify amino acid differences, which explain the species-specific binding properties. Our results prove the potency and specificity of these new inhibitors, and the detailed characterization of their binding mode is of importance for further development of VAP-1 inhibitors.

  4. Adhesive Properties of YapV and Paralogous Autotransporter Proteins of Yersinia pestis

    PubMed Central

    Nair, Manoj K. M.; De Masi, Leon; Yue, Min; Galván, Estela M.; Chen, Huaiqing; Wang, Fang

    2015-01-01

    Yersinia pestis is the causative agent of plague. This bacterium evolved from an ancestral enteroinvasive Yersinia pseudotuberculosis strain by gene loss and acquisition of new genes, allowing it to use fleas as transmission vectors. Infection frequently leads to a rapidly lethal outcome in humans, a variety of rodents, and cats. This study focuses on the Y. pestis KIM yapV gene and its product, recognized as an autotransporter protein by its typical sequence, outer membrane localization, and amino-terminal surface exposure. Comparison of Yersinia genomes revealed that DNA encoding YapV or each of three individual paralogous proteins (YapK, YapJ, and YapX) was present as a gene or pseudogene in a strain-specific manner and only in Y. pestis and Y. pseudotuberculosis. YapV acted as an adhesin for alveolar epithelial cells and specific extracellular matrix (ECM) proteins, as shown with recombinant Escherichia coli, Y. pestis, or purified passenger domains. Like YapV, YapK and YapJ demonstrated adhesive properties, suggesting that their previously related in vivo activity is due to their capacity to modulate binding properties of Y. pestis in its hosts, in conjunction with other adhesins. A differential host-specific type of binding to ECM proteins by YapV, YapK, and YapJ suggested that these proteins participate in broadening the host range of Y. pestis. A phylogenic tree including 36 Y. pestis strains highlighted an association between the gene profile for the four paralogous proteins and the geographic location of the corresponding isolated strains, suggesting an evolutionary adaption of Y. pestis to specific local animal hosts or reservoirs. PMID:25690102

  5. Effect of dispersion method and CNT loading on the quality and performance of nanocomposite soy protein/CNTs adhesive for wood application

    NASA Astrophysics Data System (ADS)

    Afolabi, Ayo Samuel; Oluwafolakemi Sadare, Olawumi; Olawale Daramola, Michael

    2016-09-01

    In this article the effect of dispersion method and carbon nanotubes (CNTs) loading on the quality and performance of a nanocomposite adhesive is reported. The nanocomposite soy protein isolate adhesive was successfully developed by incorporating CNTs into the soy protein isolate (SPI) for enhanced bond strength and water resistance. Dispersion methods, namely mechanical (shear) mixing and mechanical/sonication were employed to aid good dispersion and interfacial interaction between soy protein matrix and the carbon nanofillers during the preparation of the adhesive. The concentration of the CNT was varied from 0.1–0.7 wt% in the nanocomposite adhesive. The morphology and the surface chemistry of the adhesives were checked with SEM and FTIR, respectively. The shear strength of the developed adhesives was investigated according to European standard (EN-204) for interior wood application on a tensile testing machine. The morphological structure of the nanocomposite adhesive obtained from SEM images showed homogeneous dispersion of CNTs in SPI using the two dispersion methods; shear mixing and sonication/shear mixing. Fourier transform infrared spectra showed chemical functionalities and successful interaction between CNTs and SPI adhesive. Thermogravimetric profile of the adhesive samples showed that the newly developed nanocomposite adhesive was thermally stable at a temperature up to about 600 °C at a higher percentage loading of 0.5 wt% CNTs. The result showed that sonication method of dispersion of CNTs into the SPI adhesive had a higher shear strength compared to the mechanical method of dispersion both at dry and wet state.

  6. Effect of dispersion method and CNT loading on the quality and performance of nanocomposite soy protein/CNTs adhesive for wood application

    NASA Astrophysics Data System (ADS)

    Afolabi, Ayo Samuel; Oluwafolakemi Sadare, Olawumi; Olawale Daramola, Michael

    2016-09-01

    In this article the effect of dispersion method and carbon nanotubes (CNTs) loading on the quality and performance of a nanocomposite adhesive is reported. The nanocomposite soy protein isolate adhesive was successfully developed by incorporating CNTs into the soy protein isolate (SPI) for enhanced bond strength and water resistance. Dispersion methods, namely mechanical (shear) mixing and mechanical/sonication were employed to aid good dispersion and interfacial interaction between soy protein matrix and the carbon nanofillers during the preparation of the adhesive. The concentration of the CNT was varied from 0.1-0.7 wt% in the nanocomposite adhesive. The morphology and the surface chemistry of the adhesives were checked with SEM and FTIR, respectively. The shear strength of the developed adhesives was investigated according to European standard (EN-204) for interior wood application on a tensile testing machine. The morphological structure of the nanocomposite adhesive obtained from SEM images showed homogeneous dispersion of CNTs in SPI using the two dispersion methods; shear mixing and sonication/shear mixing. Fourier transform infrared spectra showed chemical functionalities and successful interaction between CNTs and SPI adhesive. Thermogravimetric profile of the adhesive samples showed that the newly developed nanocomposite adhesive was thermally stable at a temperature up to about 600 °C at a higher percentage loading of 0.5 wt% CNTs. The result showed that sonication method of dispersion of CNTs into the SPI adhesive had a higher shear strength compared to the mechanical method of dispersion both at dry and wet state.

  7. Diamagnetic levitation causes changes in the morphology, cytoskeleton, and focal adhesion proteins expression in osteocytes.

    PubMed

    Qian, A R; Wang, L; Gao, X; Zhang, W; Hu, L F; Han, J; Li, J B; Di, S M; Shang, Peng

    2012-01-01

    Diamagnetic levitation technology is a novel simulated weightless technique and has recently been applied in life-science research. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels, namely, μg (diamagnetic levitation), 1g, and 2g for diamagnetic materials. In this study, the effects of LG-HMF on the activity, morphology, and cytoskeleton (actin filament, microtubules, and vimentin intermediate filaments) in osteocyte - like cell line MLO-Y4 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods, hematoxylin-eosin (HE) staining, and laser scanning confocal microscopy (LSCM), respectively. The changes induced by LG-HMF in distribution and expression of focal adhesion (FA) proteins, including vinculin, paxillin, and talin in MLO-Y4 were determined by LSCM and Western blotting. The results showed that LG-HMF produced by superconducting magnet had no lethal effects on MLO-Y4. Compared to control, diamagnetic levitation (μg) affected MLO-Y4 morphology, nucleus size, cytoskeleton architecture, and FA proteins distribution and expression. The study indicates that osteocytes are sensitive to altered gravity and FA proteins (vinculin, paxillin, and talin) may be involved in osteocyte mechanosensation. The diamagnetic levitation may be a novel ground-based space-gravity simulator and can be used for biological experiment at cellular level. PMID:21216704

  8. Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist

    PubMed Central

    Stoveken, Hannah M.; Hajduczok, Alexander G.; Xu, Lei; Tall, Gregory G.

    2015-01-01

    The large class of adhesion G protein-coupled receptors (aGPCRs) bind extracellular matrix or neighboring cell-surface ligands to regulate organ and tissue development through an unknown activation mechanism. We examined aGPCR activation using two prototypical aGPCRs, GPR56 and GPR110. Active dissociation of the noncovalently bound GPR56 or GPR110 extracellular domains (ECDs) from the respective seven-transmembrane (7TM) domains relieved an inhibitory influence and permitted both receptors to activate defined G protein subtypes. After ECD displacement, the newly revealed short N-terminal stalk regions of the 7TM domains were found to be essential for G protein activation. Synthetic peptides comprising these stalks potently activated GPR56 or GPR110 in vitro or in cells, demonstrating that the stalks comprise a tethered agonist that was encrypted within the ECD. Establishment of an aGPCR activation mechanism provides a rational platform for the development of aGPCR synthetic modulators that could find clinical utility toward aGPCR-directed disease. PMID:25918380

  9. The Terminal A Domain of the Fibrillar Accumulation-Associated Protein (Aap) of Staphylococcus epidermidis Mediates Adhesion to Human Corneocytes▿

    PubMed Central

    Macintosh, Robin L.; Brittan, Jane L.; Bhattacharya, Ritwika; Jenkinson, Howard F.; Derrick, Jeremy; Upton, Mathew; Handley, Pauline S.

    2009-01-01

    The opportunistic pathogen Staphylococcus epidermidis colonizes indwelling medical devices by biofilm formation but is primarily a skin resident. In many S. epidermidis strains biofilm formation is mediated by a cell wall-anchored protein, the accumulation-associated protein (Aap). Here, we investigate the role of Aap in skin adhesion. Aap is an LPXTG protein with a domain architecture including a terminal A domain and a B-repeat region. S. epidermidis NCTC 11047 expresses Aap as localized, lateral tufts of fibrils on one subpopulation of cells (Fib+), whereas a second subpopulation does not express these fibrils of Aap (Fib−). Flow cytometry showed that 72% of NCTC 11047 cells expressed Aap and that 28% of cells did not. Aap is involved in the adhesion of Fib+ cells to squamous epithelial cells from the hand (corneocytes), as the recombinant A-domain protein partially blocked binding to corneocytes. To confirm the role of the Aap A domain in corneocyte attachment, Aap was expressed on the surface of Lactococcus lactis MG1363 as sparsely distributed, peritrichous fibrils. The expression of Aap increased corneocyte adhesion 20-fold compared to L. lactis carrying Aap without an A domain. S. epidermidis isolates from catheters, artificial joints, skin, and the nose also used the A domain of Aap to adhere to corneocytes, emphasizing the role of Aap in skin adhesion. In addition, L. lactis expressing Aap with different numbers of B repeats revealed a positive correlation between the number of B repeats and adhesion to corneocytes, suggesting an additional function for the B region in enhancing A-domain-dependent attachment to skin. Therefore, in addition to its established role in biofilm formation, Aap can also promote adhesion to corneocytes and is likely to be an important adhesin in S. epidermidis skin colonization. PMID:19749046

  10. The re-expression of the epigenetically silenced e-cadherin gene by a polyamine analogue lysine-specific demethylase-1 (LSD1) inhibitor in human acute myeloid leukemia cell lines

    PubMed Central

    Murray-Stewart, Tracy; Woster, Patrick M.; Casero, Robert A.

    2013-01-01

    Aberrant epigenetic silencing of tumor suppressor genes is a common feature observed during the transformation process of many cancers, including those of hematologic origin. Histone modifications, including acetylation, phosphorylation, and methylation, collaborate with DNA CpG island methylation to regulate gene expression. The dynamic process of histone methylation is the latest of these epigenetic modifications to be described, and the identification and characterization of LSD1 as a demethylase of lysine 4 of histone H3 (H3K4) has confirmed that both the enzyme and the modified histone play important roles as regulators of gene expression. LSD1 activity contributes to the suppression of gene expression by demethylating promoter-region mono- and dimethyl- H3K4 histone marks that are associated with active gene expression. As most posttranslational modifications are reversible, the enzymes involved in the modification of histones have become targets for chemotherapeutic intervention. In this study, we examined the effects of the polyamine analogue LSD1 inhibitor 2d (1,15-bis{N5-[3,3-(diphenyl)propyl]-N1-biguanido}-4,12-diazapentadecane) in human acute myeloid leukemia (AML) cell lines. In each line studied, 2d evoked cytotoxicity and inhibited LSD1 activity, as evidenced by increases in the global levels of mono- and di-methylated H3K4 proteins. Global increases in other chromatin modifications were also observed following exposure to 2d, suggesting a broad response to this compound with respect to chromatin regulation. On a gene-specific level, treatment with 2d resulted in the reexpression of e-cadherin, a tumor suppressor gene frequently silenced by epigenetic modification in AML. Quantitative chromatin immunoprecipitation analysis of the ecadherin promoter further confirmed that this re-expression was concurrent with changes in both active and repressive histone marks that were consistent with LSD1 inhibition. As hematologic malignancies have demonstrated

  11. Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein-Coupled Receptor Antagonist.

    PubMed

    Stoveken, Hannah M; Bahr, Laura L; Anders, M W; Wojtovich, Andrew P; Smrcka, Alan V; Tall, Gregory G

    2016-09-01

    Adhesion G protein-coupled receptors (aGPCRs) have emerging roles in development and tissue maintenance and is the most prevalent GPCR subclass mutated in human cancers, but to date, no drugs have been developed to target them in any disease. aGPCR extracellular domains contain a conserved subdomain that mediates self-cleavage proximal to the start of the 7-transmembrane domain (7TM). The two receptor protomers, extracellular domain and amino terminal fragment (NTF), and the 7TM or C-terminal fragment remain noncovalently bound at the plasma membrane in a low-activity state. We recently demonstrated that NTF dissociation liberates the 7TM N-terminal stalk, which acts as a tethered-peptide agonist permitting receptor-dependent heterotrimeric G protein activation. In many cases, natural aGPCR ligands are extracellular matrix proteins that dissociate the NTF to reveal the tethered agonist. Given the perceived difficulty in modifying extracellular matrix proteins to create aGPCR probes, we developed a serum response element (SRE)-luciferase-based screening approach to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000-compound library comprising known drugs and natural products was screened for GPR56-dependent SRE activation inhibitors that did not inhibit constitutively active Gα13-dependent SRE activation. Dihydromunduletone (DHM), a rotenoid derivative, was validated using cell-free aGPCR/heterotrimeric G protein guanosine 5'-3-O-(thio)triphosphate binding reconstitution assays. DHM inhibited GPR56 and GPR114/ADGRG5, which have similar tethered agonists, but not the aGPCR GPR110/ADGRF1, M3 muscarinic acetylcholine, or β2 adrenergic GPCRs. DHM inhibited tethered peptide agonist-stimulated and synthetic peptide agonist-stimulated GPR56 but did not inhibit basal activity, demonstrating that it antagonizes the peptide agonist. DHM is a novel aGPCR antagonist and potentially useful chemical probe that may be developed as a future aGPCR therapeutic. PMID:27338081

  12. Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

    PubMed Central

    Takeshita, S; Kikuno, R; Tezuka, K; Amann, E

    1993-01-01

    A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8363580

  13. In vitro adhesion and invasion inhibition of Shigella dysenteriae, Shigella flexneri and Shigella sonnei clinical strains by human milk proteins

    PubMed Central

    Willer, Emerson da Motta; Lima, Renato de Lourenço; Giugliano, Loreny Gimenes

    2004-01-01

    Background Shigella is the etiological agent of shigellosis, a disease responsible for more than 500,000 deaths of children per year, in developing countries. These pathogens colonize the intestinal colon, invade, spreading to the other enterocytes. Breastfeeding plays a very important role in protecting infants from intestinal infections. Amongst milk compounds, glycosylated proteins prevent the adhesion of many enteropathogens in vitro. The aim of this work was to determine the effect of human milk proteins on the colonization potential of Shigella dysenteriae, S. flexneri and S. sonnei. To fulfill this purpose, pooled milk samples from five donors, were fractionated by gel filtration and affinity chromatography. Using tissue culture, the milk fractions obtained were tested in Shigella adhesion and invasion assays. Results Our revealed showed that both adhesion and invasion of Shigella species were inhibited by low concentration of secretory immunoglobulin A, lactoferrin and free secretory component. This work also showed that, these proteins bind to superficial and whole-cell Shigella proteins. Conclusions Our findings suggest that human milk may act inhibiting adhesion and, consequently, invasion of Shigella, thereafter preventing shigellosis in infants. PMID:15115555

  14. Adsorption and adhesion of common serum proteins to nanotextured gallium nitride

    NASA Astrophysics Data System (ADS)

    Bain, Lauren E.; Hoffmann, Marc P.; Bryan, Isaac; Collazo, Ramón; Ivanisevic, Albena

    2015-01-01

    , particularly when considering biological systems. It is well known that thin films and nanostructures feature different optical, electrical, and mechanical properties from their bulk composites; however, interactions taking place at the interface between nanomaterials and their surroundings are less understood. Here, we explore interactions between common serum proteins - serum albumin, fibrinogen, and immunoglobulin G - and a nanotextured gallium nitride surface. Atomic force microscopy with a carboxyl-terminated colloid tip is used to probe the `activity' of proteins adsorbed onto the surface, including both the accessibility of the terminal amine to the tip as well as the potential for protein extension. By evaluating the frequency of tip-protein interactions, we can establish differences in protein behaviour on the basis of both the surface roughness as well as morphology, providing an assessment of the role of surface texture in dictating protein-surface interactions. Unidirectional surface features - either the half-unit cell steppes of as-grown GaN or those produced by mechanical polishing - appear to promote protein accessibility, with a higher frequency of protein extension events taking place on these surfaces when compared with less ordered surface features. Development of a full understanding of the factors influencing surface-biomolecule interactions can pave the way for specific surface modification to tailor the bio-material interface, offering a new path for device optimization. Electronic supplementary information (ESI) available: Additional figures demonstrating the adhesion force magnitude (Fig. S1) and lateral steppe surface topography (Fig. S2). See DOI: 10.1039/c4nr06353h

  15. Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins.

    PubMed

    Jamerson, Melissa; da Rocha-Azevedo, Bruno; Cabral, Guy A; Marciano-Cabral, Francine

    2012-03-01

    Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis.

  16. Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins

    PubMed Central

    Jamerson, Melissa; da Rocha-Azevedo, Bruno; Cabral, Guy A.

    2012-01-01

    Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis. PMID:22222499

  17. Multifunctional and Redundant Roles of Borrelia burgdorferi Outer Surface Proteins in Tissue Adhesion, Colonization, and Complement Evasion

    PubMed Central

    Caine, Jennifer A.; Coburn, Jenifer

    2016-01-01

    Borrelia burgdorferi is the causative agent of Lyme disease in the U.S., with at least 25,000 cases reported to the CDC each year. B. burgdorferi is thought to enter and exit the bloodstream to achieve rapid dissemination to distal tissue sites during infection. Travel through the bloodstream requires evasion of immune surveillance and pathogen clearance in the host, a process at which B. burgdorferi is adept. B. burgdorferi encodes greater than 19 adhesive outer surface proteins many of which have been found to bind to host cells or components of the extracellular matrix. Several others bind to host complement regulatory factors, in vitro. Production of many of these adhesive proteins is tightly regulated by environmental cues, and some have been shown to aid in vascular interactions and tissue colonization, as well as survival in the blood, in vivo. Recent work has described multifaceted and redundant roles of B. burgdorferi outer surface proteins in complement component interactions and tissue targeted adhesion and colonization, distinct from their previously identified in vitro binding capabilities. Recent insights into the multifunctional roles of previously well-characterized outer surface proteins such as BBK32, DbpA, CspA, and OspC have changed the way we think about the surface proteome of these organisms during the tick–mammal life cycle. With the combination of new and old in vivo models and in vitro techniques, the field has identified distinct ligand binding domains on BBK32 and DbpA that afford tissue colonization or blood survival to B. burgdorferi. In this review, we describe the multifunctional and redundant roles of many adhesive outer surface proteins of B. burgdorferi in tissue adhesion, colonization, and bloodstream survival that, together, promote the survival of Borrelia spp. throughout maintenance in their multi-host lifestyle.

  18. Immunohistochemistry of adhesion molecules, metalloproteinases and NO-synthases in extravillous trophoblast of tubal pregnancy.

    PubMed

    Dubernard, G; Galtier-Fougairolles, M; Cortez, A; Uzan, S; Challier, J C

    2005-12-12

    Trophoblast invasion in uterine pregnancy is fine-tuned for the remodelling of the uterine wall and its vascularization. Tubal pregnancy, which occurs in a limited number of patients, involves a dramatic trophoblast invasion in a context of a poor decidualization. By studying the histology of the extravillous trophoblast (EVC) in the anchoring villi, the Ki67 labelling, the location of several adhesion markers (cytokeratin-7, alpha1, alpha6, alphaV, beta1, beta4 integrin subunits and E-cadherin, V/E-cadherin), metalloproteinases (MMP-2, 9 and11), NOS2 and 3, we aimed to detect the specificity of tubal compared to intrauterine pregnancies. No difference could be observed between meso or anti-salpingial trophoblast proliferation or invasion using Ki67. Cytokeratin-7 allowed detection of spindle-shape EVCs and we identified some decidualized stromal cells. Integrins alpha1, beta1 and alphaV, and V/E-cadherin were expressed mainly in the distal EVC correspondingly to intrauterine pregnancy, with a poor expression of alpha1. Integrins alpha6 and beta4, E-cadherin were detected in the distal EVC in contrast to uterine pregnancy. MMP-2, 9, 11 were also shown in distal EVC. NOS2 and 3 labelled the perivascular EVC and NOS3 the endothelial cells of the tubal vessels. These changed distributions of adhesion molecules and MMP together with that of the basic and inducible NOS expressions could be related to mechanical effects in superficial implantation or to a failure of decidualization in tubal pregnancies.

  19. Induction of the neural cell adhesion molecule and neuronal aggregation by osteogenic protein 1.

    PubMed Central

    Perides, G; Safran, R M; Rueger, D C; Charness, M E

    1992-01-01

    The neural cell adhesion molecule (N-CAM) plays a fundamental role in nervous system development and regeneration, yet the regulation of the expression of N-CAM in different brain regions has remained poorly understood. Osteogenic protein 1 (OP-1) is a member of the transforming growth factor beta superfamily that is expressed in the nervous system. Treatment of the neuroblastoma-glioma hybrid cell line NG108-15 for 1-4 days with recombinant human OP-1 (hOP-1) induced alterations in cell shape, formation of epithelioid sheets, and aggregation of cells into multilayered clusters. Immunofluorescence studies and Western blots demonstrated a striking differential induction of the three N-CAM isoforms in hOP-1-treated cells. hOP-1 caused a 6-fold up-regulation of the 140-kDa N-CAM, the isoform showing the highest constitutive expression, and a 29-fold up-regulation of the 180-kDa isoform. The 120-kDa isoform was not detected in control NG108-15 cells but was readily identified in hOP-1-treated cells. Incubation of NG108-15 cells with an antisense N-CAM oligonucleotide reduced the induction of N-CAM by hOP-1 and decreased the formation of multilayered cell aggregates. Anti-N-CAM monoclonal antibodies also diminished the formation of multilayered cell aggregates by hOP-1 and decreased cell-cell adhesion when hOP-1-treated NG108-15 cells were dispersed and replated. Thus, hOP-1 produces morphologic changes in NG108-15 cells, at least in part, by inducing N-CAM. These observations suggest that OP-1 or a homologue may participate in the regulation of N-CAM during nervous system development and regeneration. Images PMID:1438217

  20. Overexpression of vascular adhesion protein-1 is associated with poor prognosis of astrocytomas.

    PubMed

    Kostoro, Joanna; Chang, Shu-Jyuan; Clark Lai, Yen-Chang; Wu, Chun-Chieh; Chai, Chee-Yin; Kwan, Aij-Lie

    2016-06-01

    Vascular adhesion protein-1 (VAP-1) is one of the endothelial adhesion molecules that is believed to play a role in tumor progression and metastasis, supporting cancer cell extravasation. Very few studies have been performed on analyzing the contribution of VAP-1 in brain tumor. Astrocytomas are the most common type of brain tumors, which are classified by World Health Organization (WHO) into four grades according to the degree of malignancy. This study was designed to investigate VAP-1 expression level in different astrocytoma grades and its correlation with clinicopathological features as well as prognosis of astrocytoma patients. Eighty-seven patients with different grades of astrocytoma (WHO Grade I-Grade IV) were enrolled in this study. The expression of VAP-1 was assayed by immunohistochemistry. The correlation between VAP-1 expression and clinicopathological features was evaluated by Chi-square test, and overall survival was analyzed by Kaplan-Meier method. Cox regression analysis was applied to analyze the independent influence of each parameter on overall survival. The expression level of VAP-1 was significantly higher in diffuse astrocytoma than those of pilocytic astrocytoma (p < 0.0001). In the subgroup analysis, upregulated VAP-1 expression was frequently found in older age patients (≥50 years). The VAP-1 expression was found to be significantly correlated with the overall survival (p = 0.0002). There was a statistical correlation between VAP-1(high) tumors in diffuse astrocytoma and VAP-1(low) tumors in pilocytic astrocytoma (p < 0.0001). Multivariate Cox analysis indicated VAP-1 was an independent predictive marker for poorer prognosis (p = 0.0036). Therefore, VAP-1 could be a promising prognostic biomarker in astrocytoma.

  1. Scanning-force techniques to monitor time-dependent changes in topography and adhesion force of proteins on surfaces.

    PubMed

    Mondon, M; Berger, S; Ziegler, C

    2003-04-01

    Scanning-force microscopy (SFM) investigations were conducted to probe the influences of the interactions of proteins with surfaces relevant in medicine. These interactions are an important feature in the area of biofilm formation. The adsorption of proteins leads to changes in topography, which was monitored for the build up of protein layers of hen egg-white lysozyme and bovine serum albumin (BSA) on mica in real time in phosphate-buffered aqueous solution over a time period of 10 min. Phase imaging was additionally applied to compare material contrasts and to evaluate this method for further application in this field. The adhesion forces that develop on a time scale below 20 s between a protein-modified SFM tip and titanium surfaces (TiO(2), TiAl6V4 and TiAl6Nb7) were investigated. The influences of the parameters loading force and interaction time between the protein and the surface were monitored as well as the influence of protein structure. The interaction time dependency of the adhesion force could be described with a kinetic model of two consecutive first-order reactions. For the maximal adhesion force a correlation to the ratio of the amino acids cysteine, proline and glycine has been proposed.

  2. Surface conjugation of zwitterionic polymers to inhibit cell adhesion and protein adsorption.

    PubMed

    Chien, Hsiu-Wen; Tsai, Chih-Chi; Tsai, Wei-Bor; Wang, Meng-Jiy; Kuo, Wei-Hsuan; Wei, Ta-Chin; Huang, Sheng-Tung

    2013-07-01

    Non-fouling surfaces that resist non-specific protein adsorption and cell adhesion are desired for many biomedical applications such as blood-contact devices and biosensors. Therefore, surface conjugation of anti-fouling molecules has been the focus of many studies. In this study, layer-by-layer polyelectrolyte deposition was applied to create an amine-rich platform for conjugation of zwitterionic polymers. A tri-layer polyelectrolyte (TLP) coating representing poly(ethylene imine) (PEI), poly(acrylic acid)-g-azide and PEI was deposited on various polymeric substrates via layer-by-layer deposition and then crosslinked via UV irradiation. Carboxyl-terminated poly(sulfobetaine methacrylate) p(SBMA) or poly(carboxybetaine methacrylate) p(CBMA) was then conjugated onto TLP coated substrates via a carbodiimide reaction. Our results demonstrate that the zwitterionic polymers could be easily conjugated over a wide pH range except under alkaline conditions, and almost completely block protein adsorption and the attachment of L929 cells and platelets. Therefore, this method has outstanding potential in biomedical applications that require low-fouling surfaces. PMID:23500725

  3. Interfacial tension of complex coacervated mussel adhesive protein according to the Hofmeister series.

    PubMed

    Lim, Seonghye; Moon, Dustin; Kim, Hyo Jeong; Seo, Jeong Hyun; Kang, In Seok; Cha, Hyung Joon

    2014-02-01

    Complex coacervation is a liquid-liquid phase separation in a colloidal system of two oppositely charged polyelectrolytes or colloids. The interfacial tension of the coacervate phase is the key parameter for micelle formation and interactions with the encapsulating material. However, the relationship between interfacial tensions and various salt solutions is poorly understood in complex coacervation. In the present work, the complex coacervate dynamics of recombinant mussel adhesive protein (MAP) with hyaluronic acid (HA) were determined in the presence of Hofmeister series salt ions. Using measurements of absorbance, hydrodynamic diameter, capillary force, and receding contact angle in the bulk phase, the interfacial tensions of complex coacervated MAP/HA were determined to be 0.236, 0.256, and 0.287 mN/m in 250 mM NaHCOO, NaCl, and NaNO3 solutions, respectively. The sequences of interfacial tensions and contact angles of the complex coacervates in the presence of three sodium salts with different anions were found to follow the Hofmeister ordering. The tendency of interfacial tension between the coacervate and dilute phases in the presence of different types of Hofmeister salt ions could provide a better understanding of Hofmeister effects on complex coacervated materials based on the protein-polysaccharide system. This information can also be utilized for microencapsulation and adsorption by controlling intramolecular interactions. In addition, the injection molding dynamics of mussel byssus formation was potentially explained based on the measured interfacial tension of coacervated MAP. PMID:24490867

  4. Myelin basic protein cleaves cell adhesion molecule L1 and promotes neuritogenesis and cell survival.

    PubMed

    Lutz, David; Loers, Gabriele; Kleene, Ralf; Oezen, Iris; Kataria, Hardeep; Katagihallimath, Nainesh; Braren, Ingke; Harauz, George; Schachner, Melitta

    2014-05-01

    The cell adhesion molecule L1 is a Lewis(x)-carrying glycoprotein that plays important roles in the developing and adult nervous system. Here we show that myelin basic protein (MBP) binds to L1 in a Lewis(x)-dependent manner. Furthermore, we demonstrate that MBP is released by murine cerebellar neurons as a sumoylated dynamin-containing protein upon L1 stimulation and that this MBP cleaves L1 as a serine protease in the L1 extracellular domain at Arg(687) yielding a transmembrane fragment that promotes neurite outgrowth and neuronal survival in cell culture. L1-induced neurite outgrowth and neuronal survival are reduced in MBP-deficient cerebellar neurons and in wild-type cerebellar neurons in the presence of an MBP antibody or L1 peptide containing the MBP cleavage site. Genetic ablation of MBP in shiverer mice and mutagenesis of the proteolytically active site in MBP or of the MBP cleavage site within L1 as well as serine protease inhibitors and an L1 peptide containing the MBP cleavage site abolish generation of the L1 fragment. Our findings provide evidence for novel functions of MBP in the nervous system. PMID:24671420

  5. Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective

    PubMed Central

    Chagnot, Caroline; Zorgani, Mohamed A.; Astruc, Thierry; Desvaux, Mickaël

    2013-01-01

    Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates) is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative), monoderm (archetypal Gram-positive) and diderm-mycolate (archetypal acid-fast) bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors) involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field. PMID:24133488

  6. Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion.

    PubMed

    Montanari, Paolo; Bozza, Giuseppe; Capecchi, Barbara; Caproni, Elena; Barrile, Riccardo; Norais, Nathalie; Capitani, Mirco; Sallese, Michele; Cecchini, Paola; Ciucchi, Laura; Gao, Zhenai; Rappuoli, Rino; Pizza, Mariagrazia; Aricò, Beatrice; Merola, Marcello

    2012-03-01

    NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA.

  7. Composites containing albumin protein or cyanoacrylate adhesives and biodegradable scaffolds: II. In vivo wound closure study in a rat model

    NASA Astrophysics Data System (ADS)

    McNally-Heintzelman, Karen M.; Heintzelman, Douglas L.; Duffy, Mark T.; Bloom, Jeffrey N.; Soller, Eric C.; Gilmour, Travis M.; Hoffman, Grant T.; Edward, Deepak

    2004-07-01

    Our Scaffold-Enhanced Biological Adhesive (SEBA) system was investigated as an alternative to sutures or adhesives alone for repair of wounds. Two scaffold materials were investigated: (i) a synthetic biodegradable material fabricated from poly(L-lactic-co-glycolic acid); and (ii) a biologic material, small intestinal submucosa, manufactured by Cook BioTech. Two adhesive materials were also investigated: (i) a biologic adhesive composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser; and (ii) Ethicon"s Dermabond, a 2-octyl-cyanoacrylate. The tensile strength and time-to-failure of skin incisions repaired in vivo in a rat model were measured at seven days postoperative. Incisions closed by protein solder alone, by Dermabond alone, or by suture, were also tested for comparison. The tensile strength of repairs formed using the SEBA system were 50% to 65% stronger than repairs formed by suture or either adhesive alone, with significantly less variations within each experimental group (average standard deviations of 15% for SEBA versus 38% for suture and 28% for adhesive alone). In addition, the time-to-failure curves showed a longevity not previously seen with the suture or adhesive alone techniques. The SEBA system acts to keep the dermis in tight apposition during the critical early phase of wound healing when tissue gaps are bridged by scar and granulation tissue. It has the property of being more flexible than either of the adhesives alone and may allow the apposed edges to move in conjunction with each other as a unit for a longer period of time and over a greater range of stresses than adhesives alone. This permits more rapid healing and establishment of integrity since the microgaps between the dermis edges are significantly reduced. By the time the scaffolds are sloughed from the wound site, there is greater strength and healing than that produced by adhesive alone or

  8. Facilitation of cell adhesion by immobilized dengue viral nonstructural protein 1 (NS1): arginine-glycine-aspartic acid structural mimicry within the dengue viral NS1 antigen.

    PubMed

    Chang, Hsin-Hou; Shyu, Huey-Fen; Wang, Yo-Ming; Sun, Der-Shan; Shyu, Rong-Hwa; Tang, Shiao-Shek; Huang, Yao-Shine

    2002-09-15

    Dengue virus infection causes life-threatening hemorrhagic fever. Increasing evidence implies that dengue viral nonstructural protein 1 (NS1) exhibits a tendency to elicit potentially hazardous autoantibodies, which show a wide spectrum of specificity against extracellular matrix and platelet antigens. How NS1 elicits autoantibodies remains unclear. To address the hypothesis that NS1 and matrix proteins may have structural and functional similarity, cell-matrix and cell-NS1 interactions were evaluated using a cell-adhesion assay. The present study showed that dengue NS1 immobilized on coverslips resulted in more cell adhesion than did the control proteins. This cell adhesion was inhibited by peptides containing arginine-glycine-aspartic acid (RGD), a motif important for integrin-mediated cell adhesion. In addition, anti-NS1 antibodies blocked RGD-mediated cell adhesion. Although there is no RGD motif in the NS1 protein sequence, these data indicate that RGD structural mimicry exists within the NS1 antigen.

  9. Interleukin-1 receptor accessory protein organizes neuronal synaptogenesis as a cell adhesion molecule.

    PubMed

    Yoshida, Tomoyuki; Shiroshima, Tomoko; Lee, Sung-Jin; Yasumura, Misato; Uemura, Takeshi; Chen, Xigui; Iwakura, Yoichiro; Mishina, Masayoshi

    2012-02-22

    Interleukin-1 receptor accessory protein (IL-1RAcP) is the essential component of receptor complexes mediating immune responses to interleukin-1 family cytokines. IL-1RAcP in the brain exists in two isoforms, IL-1RAcP and IL-1RAcPb, differing only in the C-terminal region. Here, we found robust synaptogenic activities of IL-1RAcP in cultured cortical neurons. Knockdown of IL-1RAcP isoforms in cultured cortical neurons suppressed synapse formation as indicated by decreases of active zone protein Bassoon puncta and dendritic protrusions. IL-1RAcP recovered the accumulation of presynaptic Bassoon puncta, while IL-1RAcPb rescued both Bassoon puncta and dendritic protrusions. Consistently, the expression of IL-1RAcP in cortical neurons enhances the accumulation of Bassoon puncta and that of IL-1RAcPb stimulated both Bassoon puncta accumulation and spinogenesis. IL-1RAcP interacted with protein tyrosine phosphatase (PTP) δ through the extracellular domain. Mini-exon peptides in the Ig-like domains of PTPδ splice variants were critical for their efficient binding to IL-1RAcP. The synaptogenic activities of IL-1RAcP isoforms were diminished in cortical neurons from PTPδ knock-out mice. Correspondingly, PTPδ required IL-1RAcPb to induce postsynaptic differentiation. Thus, IL-1RAcPb bidirectionally regulated synapse formation of cortical neurons. Furthermore, the spine densities of cortical and hippocampal pyramidal neurons were reduced in IL-1RAcP knock-out mice lacking both isoforms. These results suggest that IL-1RAcP isoforms function as trans-synaptic cell adhesion molecules in the brain and organize synapse formation. Thus, IL-1RAcP represents an interesting molecular link between immune systems and synapse formation in the brain.

  10. Lymphocyte binding to vascular endothelium in inflamed skin revisited: a central role for vascular adhesion protein-1 (VAP-1).

    PubMed

    Arvilommi, A M; Salmi, M; Kalimo, K; Jalkanen, S

    1996-04-01

    The binding of leukocytes to vascular endothelium and their migration into tissues is mediated by adhesion molecules on the endothelial cells and leukocytes. Vascular adhesion protein-1 (VAP-1) is a 170-180/90-kDa endothelial molecule expressed most prominently in high endothelial venules in peripheral lymph node (PLN) type lymphatic tissues. VAP-1 mediates lymphocyte binding to PLN, tonsil and synovium. The expression of VAP-1 is induced in inflammatory diseases such as arthritis and gut inflammation. We examined the expression, structure and function of VAP-1 in normal and inflamed skin and compared it to those of other adhesion molecules implicated in skin homing. In psoriasis lichen ruber planus, pemphigoid and allergic lesions, VAP-1 was markedly upregulated. The expression of VAP-1 was also increased in biopsies of healthy skin of the patients. The VAP-1 molecule induced in skin is decorated with abundant sialic acids. VAP-1 inflamed skin is functional, since inhibition with anti-VAP-1 monoclonal antibodies caused a 60% reduction in lymphocytes adhesion to vascular endothelium. Antibodies against E-selectin, which has been regarded as the major vascular addressin directing cutaneous lymphocyte traffic, and, surprisingly, against peripheral lymph node addressin (PNAd), caused inhibitions of 30% and 60%, respectively, in the frozen section adhesion assay. These findings suggest important roles also for VAP-1 and PNAd in lymphocyte homing into inflamed skin. PMID:8625974

  11. Low density lipoprotein receptor-related protein 1 mediated endocytosis of β1-integrin influences cell adhesion and cell migration.

    PubMed

    Rabiej, Verena K; Pflanzner, Thorsten; Wagner, Timo; Goetze, Kristina; Storck, Steffen E; Eble, Johannes A; Weggen, Sascha; Mueller-Klieser, Wolfgang; Pietrzik, Claus U

    2016-01-01

    The low density lipoprotein receptor-related protein 1 (LRP1) has been shown to interact with β1-integrin and regulate its surface expression. LRP1 knock-out cells exhibit altered cytoskeleton organization and decreased cell migration. Here we demonstrate coupled endocytosis of LRP1 and β1-integrin and the involvement of the intracellular NPxY2 motif of LRP1 in this process. Mouse embryonic fibroblasts harboring a knock in replacement of the NPxY2 motif of LRP1 by a multiple alanine cassette (AAxA) showed elevated surface expression of β1-integrin and decreased β1-integrin internalization rates. As a consequence, cell spreading was altered and adhesion rates were increased in our cell model. Cells formed more focal adhesion complexes, whereby in vitro cell migration rates were decreased. Similar results could be observed in a corresponding mouse model, the C57Bl6 LRP1 NPxYxxL knock in mice, therefore, the biochemistry of cellular adhesion was altered in primary cortical neurons. In vivo cell migration experiments demonstrated a disturbance of neuroblast cell migration along the rostral migratory stream. In summary, our results indicate that LRP1 interacts with β1-integrin mediating integrin internalization and thus correlates with downstream signaling of β1-integrin such as focal adhesion dynamics. Consequently, the disturbance of this interaction resulted in a dysfunction in in vivo and in vitro cell adhesion and cell migration.

  12. Exchange of adsorbed serum proteins during adhesion of Staphylococcus aureus to an abiotic surface and Candida albicans hyphae--an AFM study.

    PubMed

    Ovchinnikova, Ekaterina S; van der Mei, Henny C; Krom, Bastiaan P; Busscher, Henk J

    2013-10-01

    Staphylococcus aureus and Candida albicans are the second and third most commonly isolated microorganisms in hospital-related-infections, that are often multi-species in nature causing high morbidity and mortality. Here, adhesion forces between a S. aureus strain and abiotic (tissue-culture-polystyrene, TCPS) or partly biotic (TCPS with adhering hyphae of C. albicans) surfaces were investigated in presence of fetal-bovine-serum or individual serum proteins and related with staphylococcal adhesion. Atomic-force-microscopy was used to measure adhesion forces between S. aureus and the abiotic and biotic surfaces. Adsorption of individual serum proteins like albumin and apo-transferrin to abiotic TCPS surfaces during 60min, impeded development of strong adhesion forces as compared to fibronectin, while 60min adsorption of proteins from fetal-bovine-serum yielded a decrease in adhesion force from -5.7nN in phosphate-buffered-saline to -0.6nN. Adsorption of albumin and apo-transferrin also decreased staphylococcal adhesion forces to hyphae as compared with fibronectin. During 60min exposure to fetal-bovine-serum however, initial (5min protein adsorption) staphylococcal adhesion forces were low (-1.6nN), but strong adhesion forces of around -5.5nN were restored within 60min. This suggests for the first time that in whole fetal-bovine-serum exchange of non-adhesive proteins by fibronectin occurs on biotic C. albicans hyphal surfaces. No evidence was found for such protein exchange on abiotic TCPS surfaces. Staphylococcal adhesion of abiotic and biotic surfaces varied in line with the adhesion forces and was low on TCPS in presence of fetal-bovine-serum. On partly biotic TCPS, staphylococci aggregated in presence of fetal-bovine-serum around adhering C. albicans hyphae.

  13. Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins

    PubMed Central

    Lopez-Sanchez, Inmaculada; Kalogriopoulos, Nicholas; Lo, I-Chung; Kabir, Firooz; Midde, Krishna K.; Wang, Honghui; Ghosh, Pradipta

    2015-01-01

    GIV/Girdin is a multimodular signal transducer and a bona fide metastasis-related protein. As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi. Here we report that mechanical signals triggered by the extracellular matrix (ECM) also converge on GIV-GEF via β1 integrins and that focal adhesions (FAs) serve as the major hubs for mechanochemical signaling via GIV. GIV interacts with focal adhesion kinase (FAK) and ligand-activated β1 integrins. Phosphorylation of GIV by FAK enhances PI3K-Akt signaling, the integrity of FAs, increases cell–ECM adhesion, and triggers ECM-induced cell motility. Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs. Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis. Thus GIV-GEF serves as a unifying platform for integration and amplification of adhesion (mechanical) and growth factor (chemical) signals during cancer progression. PMID:26446841

  14. Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins.

    PubMed

    Lopez-Sanchez, Inmaculada; Kalogriopoulos, Nicholas; Lo, I-Chung; Kabir, Firooz; Midde, Krishna K; Wang, Honghui; Ghosh, Pradipta

    2015-12-01

    GIV/Girdin is a multimodular signal transducer and a bona fide metastasis-related protein. As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi. Here we report that mechanical signals triggered by the extracellular matrix (ECM) also converge on GIV-GEF via β1 integrins and that focal adhesions (FAs) serve as the major hubs for mechanochemical signaling via GIV. GIV interacts with focal adhesion kinase (FAK) and ligand-activated β1 integrins. Phosphorylation of GIV by FAK enhances PI3K-Akt signaling, the integrity of FAs, increases cell-ECM adhesion, and triggers ECM-induced cell motility. Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs. Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis. Thus GIV-GEF serves as a unifying platform for integration and amplification of adhesion (mechanical) and growth factor (chemical) signals during cancer progression.

  15. Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

    PubMed

    Lee, Young Ah; Saito-Nakano, Yumiko; Kim, Kyeong Ah; Min, Arim; Nozaki, Tomoyoshi; Shin, Myeong Heon

    2015-02-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.

  16. Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

    PubMed

    Lee, Young Ah; Saito-Nakano, Yumiko; Kim, Kyeong Ah; Min, Arim; Nozaki, Tomoyoshi; Shin, Myeong Heon

    2015-02-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis. PMID:25500214

  17. Interspecific variations in adhesive protein sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus.

    PubMed

    Inoue, K; Waite, J H; Matsuoka, M; Odo, S; Harayama, S

    1995-12-01

    Variation in the adhesive protein gene sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus collected in Delaware, Kamaishi (Japan), and Alaska, respectively, was analyzed by the polymerase chain reaction (PCR) using two sets of oligonucleotide primers. The first set, Me 13 and Me 14, was designed to amplify the repetitive region. The length of the amplified fragments was highly variable, even among samples of the same species. Another set, Me 15 and Me 16, was designed to amplify a part of the nonrepetitive region. The length of the amplified fragments was uniform in each species and differed interspecifically; 180, 168, and 126 bp for M. edulis, M. trossulus, and M. galloprovincialis, respectively. The amplified sequence of M. trossulus resembled that of M. edulis. Mussels from other sites were also examined by PCR using Me 15 and Me 16. Wild mussels from Tromsö (Norway)