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Sample records for ezrin radixin moesin

  1. Human Ezrin-Moesin-Radixin Proteins Modulate Hepatitis C Virus Infection

    OpenAIRE

    Bukong, Terence N; Kodys, Karen; Szabo, Gyongyi

    2013-01-01

    Host cytoskeletal proteins of the ezrin-moesin-radixin (EMR) family have been shown to modulate single-stranded RNA virus infection through regulating stable microtubule formation. Antibody engagement of CD81, a key receptor for HCV entry, induces ezrin phosphorylation. Here we tested the role of EMR proteins in regulating HCV infection and explored potential therapeutic targets. We show that HCV E2 protein induces rapid ezrin phosphorylation and its cellular redistribution with F-actin via s...

  2. Ezrin, Radixin, and Moesin (ERM) proteins function as pleiotropic regulators of human immunodeficiency virus type 1 infection.

    OpenAIRE

    2008-01-01

    Ezrin, radixin, and moesin (ERM) proteins supply functional linkage between integral membrane proteins and cytoskeleton in mammalian cells to regulate membrane protein dynamisms and cytoskeleton rearrangement. To assess potential role of the ERM proteins in HIV-1 lifecycle, we examined if suppression of ERM function in human cells expressing HIV-1 infection receptors influences HIV-1 envelope (Env)-mediated HIV-1-vector transduction and cell-cell fusion. Expression of an ezrin dominant negati...

  3. Ezrin, Radixin, and Moesin (ERM) proteins function as pleiotropic regulators of human immunodeficiency virus type 1 infection.

    Science.gov (United States)

    Kubo, Yoshinao; Yoshii, Hiroaki; Kamiyama, Haruka; Tominaga, Chika; Tanaka, Yuetsu; Sato, Hironori; Yamamoto, Naoki

    2008-05-25

    Ezrin, radixin, and moesin (ERM) proteins supply functional linkage between integral membrane proteins and cytoskeleton in mammalian cells to regulate membrane protein dynamisms and cytoskeleton rearrangement. To assess potential role of the ERM proteins in HIV-1 lifecycle, we examined if suppression of ERM function in human cells expressing HIV-1 infection receptors influences HIV-1 envelope (Env)-mediated HIV-1-vector transduction and cell-cell fusion. Expression of an ezrin dominant negative mutant or knockdown of ezrin, radixin, or moesin with siRNA uniformly decreased transduction titers of HIV-1 vectors having X4-tropic Env. In contrast, transduction titers of R5-tropic Env HIV-1 vectors were decreased only by radixin knockdown: ezrin knockdown had no detectable effects and moesin knockdown rather increased transduction titer. Each of the ERM suppressions had no detectable effects on cell surface expression of CD4, CCR5, and CXCR4 or VSV-Env-mediated HIV-1 vector transductions. Finally, the individual knockdown of ERM mRNAs uniformly decreased efficiency of cell-cell fusion mediated by X4- or R5-tropic Env and HIV-1 infection receptors. These results suggest that (i) the ERM proteins function as positive regulators of infection by X4-tropic HIV-1, (ii) moesin additionally functions as a negative regulator of R5-tropic HIV-1 virus infection at the early step(s) after the membrane fusion, and (iii) receptor protein dynamisms are regulated differently in R5- and X4-tropic HIV-1 infections.

  4. Model membranes to shed light on the biochemical and physical properties of ezrin/radixin/moesin

    Science.gov (United States)

    Picart, Catherine

    2014-01-01

    Ezrin, radixin and moesin (ERM) proteins are now more and more recognized to play a key role in a large number of important physiological processes such as morphogenesis, cancer metastasis and virus infection. Several recent reviews extensively discuss their biological functions [1-4]. In this review, we will first remind the main features of this family of proteins, which are now known as linkers and regulators of the plasma membrane/cytoskeleton linkage. We will then briefly review their implication in pathological processes such as cancer and viral infection. In a second part, we will focus on biochemical and biophysical approaches to study ERM interaction with lipid membranes and conformational change in well-defined environments. In vitro studies using biomimetic lipid membranes, especially large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) and recombinant proteins help to understand the molecular mechanism of conformational activation of ERM proteins. These tools are aimed to decorticate the different steps of the interaction, to simplify the experiments performed in vivo in much more complex biological environments. PMID:23041444

  5. Unfurling of the band 4.1, ezrin, radixin, moesin (FERM) domain of the merlin tumor suppressor

    Energy Technology Data Exchange (ETDEWEB)

    Yogesha, S.D.; Sharff, Andrew J.; Giovannini, Marco; Bricogne, Gerard; Izard, Tina (House Ear); (Globel Phasing); (Scripps)

    2014-10-02

    The merlin-1 tumor suppressor is encoded by the Neurofibromatosis-2 (Nf2) gene and loss-of-function Nf2 mutations lead to nervous system tumors in man and to several tumor types in mice. Merlin is an ERM (ezrin, radixin, moesin) family cytoskeletal protein that interacts with other ERM proteins and with components of cell-cell adherens junctions (AJs). Merlin stabilizes the links of AJs to the actin cytoskeleton. Thus, its loss destabilizes AJs, promoting cell migration and invasion, which in Nf2{sup +/-} mice leads to highly metastatic tumors. Paradoxically, the 'closed' conformation of merlin-1, where its N-terminal four-point-one, ezrin, radixin, moesin (FERM) domain binds to its C-terminal tail domain, directs its tumor suppressor functions. Here we report the crystal structure of the human merlin-1 head domain when crystallized in the presence of its tail domain. Remarkably, unlike other ERM head-tail interactions, this structure suggests that binding of the tail provokes dimerization and dynamic movement and unfurling of the F2 motif of the FERM domain. We conclude the 'closed' tumor suppressor conformer of merlin-1 is in fact an 'open' dimer whose functions are disabled by Nf2 mutations that disrupt this architecture.

  6. Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway

    Institute of Scientific and Technical Information of China (English)

    Heping Yang; Dapeng Wu; Xiaojie Zhang; Xiang Wang; Yi Peng; Zhiping Hu

    2012-01-01

    Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU.In this study,we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process.Western blot analysis demonstrated that telencephalin,phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid,while they were expressed in PAJU cells transfected with a telencephalin expression plasmid.After treatment with 1.0 nM amyloid beta protein 42,expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished,while levels of phosphorylated ezrin/radixin/moesin increased.In addition,the high levels of telencephalin,phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002.These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

  7. Phox homology band 4.1/ezrin/radixin/moesin-like proteins function as molecular scaffolds that interact with cargo receptors and Ras GTPases.

    Science.gov (United States)

    Ghai, Rajesh; Mobli, Mehdi; Norwood, Suzanne J; Bugarcic, Andrea; Teasdale, Rohan D; King, Glenn F; Collins, Brett M

    2011-05-10

    Following endocytosis, the fates of receptors, channels, and other transmembrane proteins are decided via specific endosomal sorting pathways, including recycling to the cell surface for continued activity. Two distinct phox-homology (PX)-domain-containing proteins, sorting nexin (SNX) 17 and SNX27, are critical regulators of recycling from endosomes to the cell surface. In this study we demonstrate that SNX17, SNX27, and SNX31 all possess a novel 4.1/ezrin/radixin/moesin (FERM)-like domain. SNX17 has been shown to bind to Asn-Pro-Xaa-Tyr (NPxY) sequences in the cytoplasmic tails of cargo such as LDL receptors and the amyloid precursor protein, and we find that both SNX17 and SNX27 display similar affinities for NPxY sorting motifs, suggesting conserved functions in endosomal recycling. Furthermore, we show for the first time that all three proteins are able to bind the Ras GTPase through their FERM-like domains. These interactions place the PX-FERM-like proteins at a hub of endosomal sorting and signaling processes. Studies of the SNX17 PX domain coupled with cellular localization experiments reveal the mechanistic basis for endosomal localization of the PX-FERM-like proteins, and structures of SNX17 and SNX27 determined by small angle X-ray scattering show that they adopt non-self-assembling, modular structures in solution. In summary, this work defines a novel family of proteins that participate in a network of interactions that will impact on both endosomal protein trafficking and compartment specific Ras signaling cascades.

  8. Inhibitory Effects of Hydrogen on Proliferation and Migration of Vascular Smooth Muscle Cells via Down-Regulation of Mitogen/Activated Protein Kinase and Ezrin-Radixin-Moesin Signaling Pathways.

    Science.gov (United States)

    Zhang, Ya-Xing; Xu, Jing-Ting; You, Xin-Chao; Wang, Chen; Zhou, Ke-Wen; Li, Ping; Sun, Peng; Wang, Ling; Wang, Ting-Huai

    2016-02-29

    Molecular hydrogen (H₂) has recently attracted considerable attention for the prevention of oxidative stress-related vascular diseases. The purpose of this study is to evaluate the effects of hydrogen on proliferation and migration of vascular smooth muscle cells (VSMCs) stimulated by angiotensin II (Ang II) in vitro, and on vascular hypertrophy induced by abdominal aortic coarctation (AAC) in vivo. Hydrogen-rich medium (0.6~0.9 ppm) was added 30 min before 10⁻⁷ M Ang II administration, then the proliferation and migration index were determined 24 h after Ang II stimulation. Hydrogen gas (99.999%) was given by intraperitoneal injection at the dose of 1 ml/100 g/day consecutively for one week before AAC and lasted for 6 weeks in vivo. Hydrogen inhibited proliferation and migration of VSMCs with Ang II stimulation in vitro, and improved the vascular hypertrophy induced by AAC in vivo. Treatment with hydrogen reduced Ang II- or AAC-induced oxidative stress, which was reflected by diminishing the induction of reactive oxygen species (ROS) in Ang II-stimulated VSMCs, inhibiting the levels of 3-nitrotyrosine (3-NT) in vascular and serum malondialdehyde (MDA). Hydrogen treatment also blocked Ang II-induced phosphorylation of the extracellular signal-regulated kinase1/2 (ERK1/2), p38 MAPK, c-Jun NH₂-terminal kinase (JNK) and the ezrin/radixin/moesin (ERM) in vitro. Taken together, our studies indicate that hydrogen prevents AAC-induced vascular hypertrophy in vivo, and inhibits Ang II-induced proliferation and migration of VSMCs in vitro possibly by targeting ROS-dependent ERK1/2, p38 MAPK, JNK and ERM signaling. It provides the molecular basis of hydrogen on inhibiting the abnormal proliferation and migration of VSMCs and improving vascular remodeling diseases.

  9. Immunohistochemical staining of radixin and moesin in prostatic adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Becich Michael J

    2011-01-01

    Full Text Available Abstract Background Some members of the Protein 4.1 superfamily are believed to be involved in cell proliferation and growth, or in the regulation of these processes. While the expression levels of two members of this family, radixin and moesin, have been studied in many tumor types, to our knowledge they have not been investigated in prostate cancer. Methods Tissue microarrays were immunohistochemically stained for either radixin or moesin, with the staining intensities subsequently quantified and statistically analyzed using One-Way ANOVA or nonparametric equivalent with subsequent Student-Newman-Keuls tests for multiple comparisons. There were 11 cases of normal donor prostates (NDP, 14 cases of benign prostatic hyperplasia (BPH, 23 cases of high-grade prostatic intraepithelial neoplasia (HGPIN, 88 cases of prostatic adenocarcinoma (PCa, and 25 cases of normal tissue adjacent to adenocarcinoma (NAC analyzed in the microarrays. Results NDP, BPH, and HGPIN had higher absolute staining scores for radixin than PCa and NAC, but with a significant difference observed between only HGPIN and PCa (p = Conclusions To our knowledge, these studies represent the first reports on the expression profiles of radixin and moesin in prostatic adenocarcinoma. The current study has shown that there were statistically significant differences observed between HGPIN and PCa and HGPIN and NAC in terms of radixin expression. The differences in the moesin profiles by tissue type were not statistically significant. Additional larger studies with these markers may further elucidate their potential roles in prostatic neoplasia progression.

  10. Ezrin is highly expressed in early thymocytes, but dispensable for T cell development in mice.

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    Meredith H Shaffer

    Full Text Available BACKGROUND: Ezrin/radixin/moesin (ERM proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: We characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin(-/- mice likely arise as a consequence of nutritional stress. CONCLUSIONS/SIGNIFICANCE: We conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin.

  11. [Rat brain cells containing ezrin (cytovillin)].

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    Korzhevskiĭ, D E; Kirik, O V; Giliarov, A V

    2011-01-01

    Ezrin (cytovillin or p81 protein) is an actin-binding protein, a member of ERM (ezrin, radixin and moesin) family, which species contribute to stabilization of the plasma membrane-formed structures. The aim of the present study was to demonstrate the ezrin-containing cells in the rat brain and to describe their topography and morphological features. The most pronounced immunohistochemical reaction to ezrin was found in the epithelium of the choroid plexus, cells of the subcommissural organ and ventricular ependyma. Moreover, ezrin staining was also detected in the unidentifiable cells in the subventricular zone, rostral migration pathway and astrocytes in various brain areas. Preferential ezrin localization in the brain cells contributing to formation of barrier structures suggests its involvement in transport processes in the CNS.

  12. Hypotonicity causes actin reorganization and recruitment of the actin-binding ERM protein moesin in membrane protrusions in collecting duct principal cells

    NARCIS (Netherlands)

    Tamma, G.; Procino, G.; Svelto, M.; Valenti, G.

    2007-01-01

    Hypotonicity-induced cell swelling is characterized by a modification in cell architecture associated with actin cytoskeleton remodeling. The ezrin/radixin/moesin (ERM) family proteins are important signal transducers during actin reorganization regulated by the monomeric G proteins of the Rho famil

  13. Hypotonicity causes actin reorganization and recruitment of the actin-binding ERM protein moesin in membrane protrusions in collecting duct principal cells

    NARCIS (Netherlands)

    Tamma, G.; Procino, G.; Svelto, M.; Valenti, G.

    2007-01-01

    Hypotonicity-induced cell swelling is characterized by a modification in cell architecture associated with actin cytoskeleton remodeling. The ezrin/radixin/moesin (ERM) family proteins are important signal transducers during actin reorganization regulated by the monomeric G proteins of the Rho famil

  14. Moesin is required for HIV-1-induced CD4-CXCR4 interaction, F-actin redistribution, membrane fusion and viral infection in lymphocytes.

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    Barrero-Villar, Marta; Cabrero, José Román; Gordón-Alonso, Mónica; Barroso-González, Jonathan; Alvarez-Losada, Susana; Muñoz-Fernández, M Angeles; Sánchez-Madrid, Francisco; Valenzuela-Fernández, Agustín

    2009-01-01

    The human immunodeficiency virus 1 (HIV-1) envelope regulates the initial attachment of viral particles to target cells through its association with CD4 and either CXCR4 or CCR5. Although F-actin is required for CD4 and CXCR4 redistribution, little is known about the molecular mechanisms underlying this fundamental process in HIV infection. Using CD4(+) CXCR4(+) permissive human leukemic CEM T cells and primary lymphocytes, we have investigated whether HIV-1 Env might promote viral entry and infection by activating ERM (ezrin-radixin-moesin) proteins to regulate F-actin reorganization and CD4/CXCR4 co-clustering. The interaction of the X4-tropic protein HIV-1 gp120 with CD4 augments ezrin and moesin phosphorylation in human permissive T cells, thereby regulating ezrin-moesin activation. Moreover, the association and clustering of CD4-CXCR4 induced by HIV-1 gp120 requires moesin-mediated anchoring of actin in the plasma membrane. Suppression of moesin expression with dominant-negative N-moesin or specific moesin silencing impedes reorganization of F-actin and HIV-1 entry and infection mediated by the HIV-1 envelope protein complex. Therefore, we propose that activated moesin promotes F-actin redistribution and CD4-CXCR4 clustering and is also required for efficient X4-tropic HIV-1 infection in permissive lymphocytes.

  15. Ezrin Inhibition Up-regulates Stress Response Gene Expression*

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    Çelik, Haydar; Bulut, Gülay; Han, Jenny; Graham, Garrett T.; Minas, Tsion Z.; Conn, Erin J.; Hong, Sung-Hyeok; Pauly, Gary T.; Hayran, Mutlu; Li, Xin; Özdemirli, Metin; Ayhan, Ayşe; Rudek, Michelle A.; Toretsky, Jeffrey A.; Üren, Aykut

    2016-01-01

    Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that links cortical cytoskeleton to the plasma membrane. High expression of ezrin correlates with poor prognosis and metastasis in osteosarcoma. In this study, to uncover specific cellular responses evoked by ezrin inhibition that can be used as a specific pharmacodynamic marker(s), we profiled global gene expression in osteosarcoma cells after treatment with small molecule ezrin inhibitors, NSC305787 and NSC668394. We identified and validated several up-regulated integrated stress response genes including PTGS2, ATF3, DDIT3, DDIT4, TRIB3, and ATF4 as novel ezrin-regulated transcripts. Analysis of transcriptional response in skin and peripheral blood mononuclear cells from NSC305787-treated mice compared with a control group revealed that, among those genes, the stress gene DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of ezrin inhibitor compound activity. In addition, we validated the anti-metastatic effects of NSC305787 in reducing the incidence of lung metastasis in a genetically engineered mouse model of osteosarcoma and evaluated the pharmacokinetics of NSC305787 and NSC668394 in mice. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive protein, has important functions in regulating gene expression that may result in down-regulation of stress response genes. PMID:27137931

  16. Structure of the active N-terminal domain of Ezrin. Conformational and mobility changes identify keystone interactions.

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    Smith, William James; Nassar, Nicolas; Bretscher, Anthony; Cerione, Richard A; Karplus, P Andrew

    2003-02-14

    Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins that cross-link the actin cytoskeleton to the plasma membrane and also may function in signaling cascades that regulate the assembly of actin stress fibers. Here, we report a crystal structure for the free (activated) FERM domain (residues 2-297) of recombinant human ezrin at 2.3 A resolution. Structural comparison among the dormant moesin FERM domain structure and the three known active FERM domain structures (radixin, moesin, and now ezrin) allows the clear definition of regions that undergo structural changes during activation. The key regions affected are residues 135-150 and 155-180 in lobe F2 and residues 210-214 and 235-267 in lobe F3. Furthermore, we show that a large increase in the mobilities of lobes F2 and F3 accompanies activation, suggesting that their integrity is compromised. This leads us to propose a new concept that we refer to as keystone interactions. Keystone interactions occur when one protein (or protein part) contributes residues that allow another protein to complete folding, meaning that it becomes an integral part of the structure and would rarely dissociate. Such interactions are well suited for long-lived cytoskeletal protein interactions. The keystone interactions concept leads us to predict two specific docking sites within lobes F2 and F3 that are likely to bind target proteins.

  17. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC.

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    Ren, L; Hong, S H; Cassavaugh, J; Osborne, T; Chou, A J; Kim, S Y; Gorlick, R; Hewitt, S M; Khanna, C

    2009-02-12

    Ezrin is a member of the ERM (ezrin, radixin, moesin) protein family and links F-actin to the cell membrane following phosphorylation. Ezrin has been associated with tumor progression and metastasis in several cancers including the pediatric solid tumors, osteosarcoma and rhabdomyosarcoma. In this study, we were surprised to find that ezrin was not constitutively phosphorylated but rather was dynamically regulated during metastatic progression in osteosarcoma. Metastatic osteosarcoma cells expressed phosphorylated ERM early after their arrival in the lung, and then late in progression, only at the invasive front of larger metastatic lesions. To pursue mechanisms for this regulation, we found that inhibitors of PKC (protein kinase C) blocked phosphorylation of ezrin, and that ezrin coimmunoprecipitated in cells with PKCalpha, PKCiota and PKCgamma. Furthermore, phosphorylated forms of ezrin and PKC had identical expression patterns at the invasive front of pulmonary metastatic lesions in murine and human patient samples. Finally, we showed that the promigratory effects of PKC were linked to ezrin phosphorylation. These data are the first to suggest a dynamic regulation of ezrin phosphorylation during metastasis and to connect the PKC family members with this regulation.

  18. A Novel Human Radixin Peptide Inhibits Hepatitis C Virus Infection at the Level of Cell Entry.

    Science.gov (United States)

    Bukong, Terence N; Kodys, Karen; Szabo, Gyongyi

    2014-09-01

    Hepatitis C virus infection of hepatocytes is a multistep process involving the interaction between viral and host cell molecules. Recently, we identified ezrin-moesin-radixin proteins and spleen tyrosine kinase (SYK) as important host therapeutic targets for HCV treatment development. Previously, an ezrin hinge region peptide (Hep1) has been shown to exert anti-HCV properties in vivo, though its mechanism of action remains limited. In search of potential novel inhibitors of HCV infection and their functional mechanism we analyzed the anti-HCV properties of different human derived radixin peptides. Sixteen different radixin peptides were derived, synthesized and tested. Real-time quantitative PCR, cell toxicity assay, immuno-precipitation/western blot analysis and computational resource for drug discovery software were used for experimental analysis. We found that a human radixin hinge region peptide (Peptide1) can specifically block HCV J6/JFH-1 infection of Huh7.5 cells. Peptide 1 had no cell toxicity or intracellular uptake into Huh7.5 cells. Mechanistically, the anti-HCV activity of Peptide 1 extended to disruption of HCV engagement of CD81 thereby blocking downstream SYK activation, which we have recently demonstrated to be important for effective HCV infection of target hepatocytes. Our findings highlight a novel functional class of anti-HCV agents that can inhibit HCV infection, most likely by disrupting vital viral-host signaling interactions at the level of virus entry.

  19. The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

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    Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter

    2017-10-01

    Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Ezrin is required for the functional regulation of the epithelial sodium proton exchanger, NHE3.

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    Hisayoshi Hayashi

    Full Text Available The sodium hydrogen exchanger isoform 3 (NHE3 mediates absorption of sodium, bicarbonate and water from renal and intestinal lumina. This activity is fundamental to the maintenance of a physiological plasma pH and blood pressure. To perform this function NHE3 must be present in the apical membrane of renal tubular and intestinal epithelia. The molecular determinants of this localization have not been conclusively determined, although linkage to the apical actin cytoskeleton through ezrin has been proposed. We set out to evaluate this hypothesis. Functional studies of NHE3 activity were performed on ezrin knockdown mice (Vil2(kd/kd and NHE3 activity similar to wild-type animals detected. Interpretation of this finding was difficult as other ERM (ezrin/radixin/moesin proteins were present. We therefore generated an epithelial cell culture model where ezrin was the only detectable ERM. After knockdown of ezrin expression with siRNA, radixin and moesin expression remained undetectable. Consistent with the animal ultrastructural data, cells lacking ezrin retained an epithelial phenotype but had shortened and thicker microvilli. NHE3 localization was identical to cells transfected with non-targeting siRNA. The attachment of NHE3 to the apical cytoskeleton was unaltered as assessed by fluorescent recovery after photobleaching (FRAP and the solubility of NHE3 in Triton X-100. Baseline NHE3 activity was unaltered, however, cAMP-dependent inhibition of NHE3 was largely lost even though NHE3 was phosphorylated at serines 552 and 605. Thus, ezrin is not necessary for the apical localization, attachment to the cytoskeleton, baseline activity or cAMP induced phosphrylation of NHE3, but instead is required for cAMP mediated inhibition.

  1. Lipid raft association restricts CD44-ezrin interaction and promotion of breast cancer cell migration.

    LENUS (Irish Health Repository)

    Donatello, Simona

    2012-12-01

    Cancer cell migration is an early event in metastasis, the main cause of breast cancer-related deaths. Cholesterol-enriched membrane domains called lipid rafts influence the function of many molecules, including the raft-associated protein CD44. We describe a novel mechanism whereby rafts regulate interactions between CD44 and its binding partner ezrin in migrating breast cancer cells. Specifically, in nonmigrating cells, CD44 and ezrin localized to different membranous compartments: CD44 predominantly in rafts, and ezrin in nonraft compartments. After the induction of migration (either nonspecific or CD44-driven), CD44 affiliation with lipid rafts was decreased. This was accompanied by increased coprecipitation of CD44 and active (threonine-phosphorylated) ezrin-radixin-moesin (ERM) proteins in nonraft compartments and increased colocalization of CD44 with the nonraft protein, transferrin receptor. Pharmacological raft disruption using methyl-β-cyclodextrin also increased CD44-ezrin coprecipitation and colocalization, further suggesting that CD44 interacts with ezrin outside rafts during migration. Conversely, promoting CD44 retention inside lipid rafts by pharmacological inhibition of depalmitoylation virtually abolished CD44-ezrin interactions. However, transient single or double knockdown of flotillin-1 or caveolin-1 was not sufficient to increase cell migration over a short time course, suggesting complex crosstalk mechanisms. We propose a new model for CD44-dependent breast cancer cell migration, where CD44 must relocalize outside lipid rafts to drive cell migration. This could have implications for rafts as pharmacological targets to down-regulate cancer cell migration.

  2. Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro.

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    Rohini Shrivastava

    Full Text Available Ezrin, a member of the ERM (Ezrin/Radixin/Moesin protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2 or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.

  3. Knockdown of ezrin suppresses the migration and angiogenesis of human umbilical vein endothelial cells in vitro.

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    Zhao, Liang-ping; Huang, Lei; Tian, Xun; Liang, Feng-qi; Wei, Jun-cheng; Zhang, Xian; Li, Sha; Zhang, Qing-hua

    2016-04-01

    Progressive tumor growth is dependent on angiogenesis. The mechanisms by which endothelial cells (ECs) are incorporated to develop new blood vessels are not well understood. Recent studies reveal that the ezrin radixin moesin (ERM) family members are key regulators of cellular activities such as adhesion, morphogenetic change, and migration. We hypothesized that ezrin, one of the ERM family members, may play important roles in ECs organization during angiogenesis, and new vessels formation in preexisting tissues. To test this hypothesis, in this study, we investigated the effects of ezrin gene silencing on the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs were transfected with plasmids with ezrin-targeting short hairpin RNA by using the lipofectamine-2000 system. Wound assay in vitro and three-dimensional culture were used to detect the migration and angiogenesis capacity of HUVECs. The morphological changes of transfected cells were observed by confocal and phase contrast microscopy. Our results demonstrated that the decreased expression of ezrin in HUVECs significantly induced the morphogenetic changes and cytoskeletal reorganization of the transfected cells, and also reduced cell migration and angiogenesis capacity in vitro, suggesting that ezrin play an important role in the process of HUVECs migration and angiogenesis.

  4. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    Energy Technology Data Exchange (ETDEWEB)

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  5. Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of adhesion molecule CD44

    Energy Technology Data Exchange (ETDEWEB)

    Mori, Tomoyuki; Kitano, Ken; Terawaki, Shin-ichi; Maesaki, Ryoko; Hakoshima, Toshio, E-mail: hakosima@bs.naist.jp [Structural Biology Laboratory, Nara Institute of Science and Technology, Keihanna Science City, Nara 630-0192 (Japan)

    2007-10-01

    The radixin FERM domain complexed with the CD44 cytoplasmic tail peptide has been crystallized. A diffraction data set from the complex was collected to 2.1 Å. CD44 is an important adhesion molecule that specifically binds hyaluronic acid and regulates cell–cell and cell–matrix interactions. Increasing evidence has indicated that CD44 is assembled in a regulated manner into the membrane–cytoskeletal junction, a process that is mediated by ERM (ezrin/radixin/moesin) proteins. Crystals of a complex between the radixin FERM domain and the C-terminal cytoplasmic region of CD44 have been obtained. The crystal of the radixin FERM domain bound to the CD44 cytoplasmic tail peptide belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.70, b = 66.18, c = 86.22 Å, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.1 Å.

  6. Moesin controls clathrin-mediated S1PR1 internalization in T cells.

    Directory of Open Access Journals (Sweden)

    Akira Nomachi

    Full Text Available The lipid mediator sphingosine 1-phosphate (S1P regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5, S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs and early endosomes, and S1PR1 internalization was blocked when clathrin was pharmacologically inhibited. Stimulating moesin-deficient T cells with S1P failed to induce S1PR1 internalization and CCV formation. Furthermore, treating moesin-deficient mice with FTY720, an S1P receptor agonist known to internalize S1PR1, caused delayed lymphopenia, and lymphocytes isolated from FTY720-treated moesin-deficient mice still responded to S1P ex vivo in chemotaxis assays. These results reveal a novel role for moesin in regulating clathrin-dependent S1PR1 internalization through CCV formation.

  7. LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson's disease mutants affect kinase activity

    Science.gov (United States)

    Jaleel, Mahaboobi; Nichols, R. Jeremy; Deak, Maria; Campbell, David G.; Gillardon, Frank; Knebel, Axel; Alessi, Dario R.

    2007-01-01

    Mutations in the LRRK2 (leucine-rich repeat kinase-2) gene cause late-onset PD (Parkinson's disease). LRRK2 contains leucine-rich repeats, a GTPase domain, a COR [C-terminal of Roc (Ras of complex)] domain, a kinase and a WD40 (Trp-Asp 40) motif. Little is known about how LRRK2 is regulated, what its physiological substrates are or how mutations affect LRRK2 function. Thus far LRRK2 activity has only been assessed by autophosphorylation and phosphorylation of MBP (myelin basic protein), which is catalysed rather slowly. We undertook a KESTREL (kinase substrate tracking and elucidation) screen in rat brain extracts to identify proteins that were phosphorylated by an activated PD mutant of LRRK2 (G2019S). This led to the discovery that moesin, a protein which anchors the actin cytoskeleton to the plasma membrane, is efficiently phosphorylated by LRRK2, at Thr558, a previously identified in-vivo-phosphorylation site that regulates the ability of moesin to bind actin. LRRK2 also phosphorylated ezrin and radixin, which are related to moesin, at the residue equivalent to Thr558, as well as a peptide (LRRKtide: RLGRDKYKTLRQIRQ) encompassing Thr558. We exploited these findings to determine how nine previously reported PD mutations of LRRK2 affected kinase activity. Only one of the mutations analysed, namely G2019S, stimulated kinase activity. Four mutations inhibited LRRK2 kinase activity (R1941H, I2012T, I2020T and G2385R), whereas the remainder (R1441C, R1441G, Y1699C and T2356I) did not influence activity. Therefore the manner in which LRRK2 mutations induce PD is more complex than previously imagined and is not only caused by an increase in LRRK2 kinase activity. Finally, we show that the minimum catalytically active fragment of LRRK2 requires an intact GTPase, COR and kinase domain, as well as a WD40 motif and a C-terminal tail. The results of the present study suggest that moesin, ezrin and radixin may be LRRK2 substrates, findings that have been exploited to develop

  8. Changes in PtdIns(4,5)P2 induced by etoposide treatment modulates small intestinal P-glycoprotein via radixin.

    Science.gov (United States)

    Kobori, Takuro; Harada, Shinichi; Nakamoto, Kazuo; Tokuyama, Shogo

    2014-01-01

    Previously, we reported that repeated oral administration of etoposide (ETP) increases P-glycoprotein (P-gp) expression in association with activation of ezrin/radixin/moesin (ERM) via Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase (ROCK) signaling in the small intestine. However, the detailed mechanisms of this pathway have yet to be fully elucidated. Recently, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], one of the most abundant phosphoinositides in the plasma membrane, has attracted attention regarding its involvement in the plasma membrane localization of various membrane proteins. PtdIns(4,5)P2 is an essential factor in the dissociation and subsequent membrane translocation (activation) of ERM, and its synthetic pathway is known to be highly regulated by RhoA/ROCK signaling. Here, we examined the involvement of PtdIns(4,5)P2 in the mechanism by which ETP treatment increases small intestinal P-gp levels, and we determined which protein within ERM contributes to this phenomenon. Repeated oral treatment with ETP (10 mg/kg/d) over 5 d significantly increased PtdIns(4,5)P2 expression in the ileal membrane as measured by dot blot. Furthermore, this increase was suppressed by co-administration of a RhoA inhibitor, rosuvastatin (5 mg/kg/d, per os (p.o.)), or a ROCK inhibitor, fasudil (5 mg/kg/d, p.o.). In immunoprecipitation assays, radixin (but not ezrin or moesin) binding to PtdIns(4,5)P2 was observed to increase in association with the up-regulation of P-gp in the same fraction, and immunofluorescence studies indicated that radixin co-localized with PtdIns(4,5)P2 in the ileal tissue. In conclusion, ETP treatment appears to up-regulate PtdIns(4,5)P2 expression via RhoA/ROCK signaling, leading to the activation of ERM, presumably through the physical interaction of radixin with PtdIns(4,5)P2. This in turn increases the expression of ileal P-gp.

  9. Effects of transplantation sites on tumour growth, pulmonary metastasis and ezrin expression of canine osteosarcoma cell lines in nude mice.

    Science.gov (United States)

    Jaroensong, T; Endo, Y; Lee, S-J; Kamida, A; Mochizuki, M; Nishimura, R; Sasaki, N; Nakagawa, T

    2012-12-01

    To determine the influence of the transplantation site of canine osteosarcoma (OS) cell lines on tumour growth and pulmonary metastasis, three OS cell lines were transplanted into nude mice via subcutaneous (SC), intratibial (IT) or intravenous (IV) injection. IT-xenografts exhibited greater potential for developing primary masses and pulmonary metastasis than SC-xenografts. In IT and IV xenografts, lung micrometastases along with phosphorylated ezrin-radixin-moesin (p-ERM) overexpression were found in mice xenografted with HMPOS and OOS cells after 1 week and metastasis was found with decreased p-ERM expression at later time points. The expression of ezrin and p-ERM in the primary tumours of IT-xenografted mice was higher than those in SC-xenografted mice with HMPOS and OOS cells. The results suggest that the orthotopic transplantation site plays an important role in the spontaneous metastasis of canine OS and that ezrin phosphorylation may be involved in the early metastatic mechanism of canine OS cells. © 2011 Blackwell Publishing Ltd.

  10. Structural characterization suggests models for monomeric and dimeric forms of full-length ezrin.

    Science.gov (United States)

    Phang, Juanita M; Harrop, Stephen J; Duff, Anthony P; Sokolova, Anna V; Crossett, Ben; Walsh, James C; Beckham, Simone A; Nguyen, Cuong D; Davies, Roberta B; Glöckner, Carina; Bromley, Elizabeth H C; Wilk, Krystyna E; Curmi, Paul M G

    2016-09-15

    Ezrin is a member of the ERM (ezrin-radixin-moesin) family of proteins that have been conserved through metazoan evolution. These proteins have dormant and active forms, where the latter links the actin cytoskeleton to membranes. ERM proteins have three domains: an N-terminal FERM [band Four-point-one (4.1) ERM] domain comprising three subdomains (F1, F2, and F3); a helical domain; and a C-terminal actin-binding domain. In the dormant form, FERM and C-terminal domains form a stable complex. We have determined crystal structures of the active FERM domain and the dormant FERM:C-terminal domain complex of human ezrin. We observe a bistable array of phenylalanine residues in the core of subdomain F3 that is mobile in the active form and locked in the dormant form. As subdomain F3 is pivotal in binding membrane proteins and phospholipids, these transitions may facilitate activation and signaling. Full-length ezrin forms stable monomers and dimers. We used small-angle X-ray scattering to determine the solution structures of these species. As expected, the monomer shows a globular domain with a protruding helical coiled coil. The dimer shows an elongated dumbbell structure that is twice as long as the monomer. By aligning ERM sequences spanning metazoan evolution, we show that the central helical region is conserved, preserving the heptad repeat. Using this, we have built a dimer model where each monomer forms half of an elongated antiparallel coiled coil with domain-swapped FERM:C-terminal domain complexes at each end. The model suggests that ERM dimers may bind to actin in a parallel fashion.

  11. Sequence Classification: 778590 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|17505420|ref|NP_491560.1| ezrin..., Radixin and Moesin family, Ezrin/Radixin/Moesin (66.0 kD) (erm-1) || http://www.ncbi.nlm.nih.gov/protein/17505420 ...

  12. Sequence Classification: 778589 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|17505422|ref|NP_491559.1| ezrin..., Radixin and Moesin family, Ezrin/Radixin/Moesin (66.0 kD) (erm-1) || http://www.ncbi.nlm.nih.gov/protein/17505422 ...

  13. A Novel Human Radixin Peptide Inhibits Hepatitis C Virus Infection at the Level of Cell Entry

    OpenAIRE

    Bukong, Terence N; Kodys, Karen; Szabo, Gyongyi

    2014-01-01

    Hepatitis C virus infection of hepatocytes is a multistep process involving the interaction between viral and host cell molecules. Recently, we identified ezrin–moesin–radixin proteins and spleen tyrosine kinase (SYK) as important host therapeutic targets for HCV treatment development. Previously, an ezrin hinge region peptide (Hep1) has been shown to exert anti-HCV properties in vivo, though its mechanism of action remains limited. In search of potential novel inhibitors of HCV infection and...

  14. An in vivo EGF receptor localization screen in C. elegans Identifies the Ezrin homolog ERM-1 as a temporal regulator of signaling.

    Science.gov (United States)

    Haag, Andrea; Gutierrez, Peter; Bühler, Alessandra; Walser, Michael; Yang, Qiutan; Langouët, Maeva; Kradolfer, David; Fröhli, Erika; Herrmann, Christina J; Hajnal, Alex; Escobar-Restrepo, Juan M

    2014-05-01

    The subcellular localization of the epidermal growth factor receptor (EGFR) in polarized epithelial cells profoundly affects the activity of the intracellular signaling pathways activated after EGF ligand binding. Therefore, changes in EGFR localization and signaling are implicated in various human diseases, including different types of cancer. We have performed the first in vivo EGFR localization screen in an animal model by observing the expression of the EGFR ortholog LET-23 in the vulval epithelium of live C. elegans larvae. After systematically testing all genes known to produce an aberrant vulval phenotype, we have identified 81 genes regulating various aspects of EGFR localization and expression. In particular, we have found that ERM-1, the sole C. elegans Ezrin/Radixin/Moesin homolog, regulates EGFR localization and signaling in the vulval cells. ERM-1 interacts with the EGFR at the basolateral plasma membrane in a complex distinct from the previously identified LIN-2/LIN-7/LIN-10 receptor localization complex. We propose that ERM-1 binds to and sequesters basolateral LET-23 EGFR in an actin-rich inactive membrane compartment to restrict receptor mobility and signaling. In this manner, ERM-1 prevents the immediate activation of the entire pool of LET-23 EGFR and permits the generation of a long-lasting inductive signal. The regulation of receptor localization thus serves to fine-tune the temporal activation of intracellular signaling pathways.

  15. Identification of moesin as NKCC2-interacting protein and analysis of its functional role in the NKCC2 apical trafficking.

    Science.gov (United States)

    Carmosino, Monica; Rizzo, Federica; Procino, Giuseppe; Zolla, Lello; Timperio, Anna Maria; Basco, Davide; Barbieri, Claudia; Torretta, Silvia; Svelto, Maria

    2012-11-01

    The renal Na(+) -K(+) -2Cl(-) co-transporter (NKCC2) is expressed in kidney thick ascending limb cells, where it mediates NaCl re-absorption regulating body salt levels and blood pressure. In this study, we used a well-characterised NKCC2 construct (c-NKCC2) to identify NKCC2-interacting proteins by an antibody shift assay coupled with blue native/SDS-PAGE and mass spectrometry. Among the interacting proteins, we identified moesin, a protein belonging to ezrin, eadixin and moesin family. Co-immunoprecipitation experiments confirmed that c-NKCC2 interacts with the N-terminal domain of moesin in LLC-PK1 cells. Moreover, c-NKCC2 accumulates in intracellular and sub-apical vesicles in cells transfected with a moesin dominant negative green fluorescent protien (GFP)-tagged construct. In addition, moesin knock-down by short interfering RNA decreases by about 50% c-NKCC2 surface expression. Specifically, endocytosis and exocytosis assays showed that moesin knock-down does not affect c-NKCC2 internalisation but strongly reduces exocytosis of the co-transporter. Our data clearly demonstrate that moesin plays a critical role in apical membrane insertion of NKCC2, suggesting a possible involvement of moesin in regulation of Na(+) and Cl(-) absorption in the kidney. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

  16. EST Table: CK489003 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CK489003 rswab0_006693.y1 10/09/29 64 %/101 aa ref|XP_001868590.1| ezrin-radixin-mo...esin-binding phosphoprotein 50 [Culex quinquefasciatus] gb|EDS27176.1| ezrin-radixin-moesin-binding phosphop

  17. Sorafenib blocks tumour growth, angiogenesis and metastatic potential in preclinical models of osteosarcoma through a mechanism potentially involving the inhibition of ERK1/2, MCL-1 and ezrin pathways

    Directory of Open Access Journals (Sweden)

    Ferrari Stefano

    2009-12-01

    Full Text Available Abstract Background Osteosarcoma (OS is the most common primary bone tumour in children and young adults. Despite improved prognosis, metastatic or relapsed OS remains largely incurable and no significant improvement has been observed in the last 20 years. Therefore, the search for alternative agents in OS is mandatory. Results We investigated phospho-ERK 1/2, MCL-1, and phospho-Ezrin/Radixin/Moesin (P-ERM as potential therapeutic targets in OS. Activation of these pathways was shown by immunohistochemistry in about 70% of cases and in all OS cell lines analyzed. Mutational analysis revealed no activating mutations in KRAS whereas BRAF gene was found to be mutated in 4/30 OS samples from patients. Based on these results we tested the multi-kinase inhibitor sorafenib (BAY 43-9006 in preclinical models of OS. Sorafenib inhibited OS cell line proliferation, induced apoptosis and downregulated P-ERK1/2, MCL-1, and P-ERM in a dose-dependent manner. The dephosphorylation of ERM was not due to ERK inhibition. The downregulation of MCL-1 led to an increase in apoptosis in OS cell lines. In chick embryo chorioallantoic membranes, OS supernatants induced angiogenesis, which was blocked by sorafenib and it was also shown that sorafenib reduced VEGF and MMP2 production. In addition, sorafenib treatment dramatically reduced tumour volume of OS xenografts and lung metastasis in SCID mice. Conclusion In conclusion, ERK1/2, MCL-1 and ERM pathways are shown to be active in OS. Sorafenib is able to inhibit their signal transduction, both in vitro and in vivo, displaying anti-tumoural activity, anti-angiogenic effects, and reducing metastatic colony formation in lungs. These data support the testing of sorafenib as a potential therapeutic option in metastatic or relapsed OS patients unresponsive to standard treatments.

  18. Radixin expression in microglia after cortical stroke lesion.

    Science.gov (United States)

    Persson, Åsa; Osman, Ahmed; Bolouri, Hayde; Mallard, Carina; Kuhn, H Georg

    2013-05-01

    Stroke induces extensive tissue remodeling, resulting in the activation of several cell types in the brain as well as recruitment of blood-borne leucocytes. Radixin is part of a cytoskeleton linker protein family with the ability to connect transmembrane proteins to the actin cytoskeleton, promoting cell functions involving a dynamic cytoskeleton such as morphological changes, cell division and migration which are common events of different cell types after stroke. In the healthy adult brain radixin is expressed in Olig2(+) cells throughout the brain and in neural progenitor cells in the subventricular zone. In the current study, we detected a 2.5 fold increase in the number of radixin positive cells in the peri-infarct cortex two weeks after the induction of cortical stroke by photothrombosis. Similarly, the number of Olig2(+) cells increased in the peri-infarct area after stroke; however, the number of radixin(+)/Olig2(+) cells was unchanged. Neural progenitor cells maintained radixin expression on their route to the infarct. More surprising however, was the expression of radixin in activated microglia in the peri-infarct cortex. Seventy percent of Iba1(+) cells expressed radixin after stroke, a population which was not present in the control brain. Furthermore, activation of radixin was predominantly detected in the peri-infarct region of oligodendrocyte progenitors and microglia. The specific location of radixin(+) cells in the peri-infarct region and in microglia suggests a role for radixin in microglial activation after stroke.

  19. STRIPAK components determine mode of cancer cell migration and metastasis

    DEFF Research Database (Denmark)

    Madsen, Chris D; Hooper, Steven; Tozluoglu, Melda;

    2015-01-01

    and MST4 kinases, which promote the co-localization of the contractile actomyosin machinery with the Ezrin/Radixin/Moesin family proteins by phosphorylating the inhibitors of PPP1CB, PPP1R14A-D. Using computational modelling, in vitro cell migration assays and in vivo breast cancer metastasis assays we...

  20. Ezrin expression predicts local recurrence and development of metastases in soft tissue sarcomas

    DEFF Research Database (Denmark)

    Carneiro, Ana; Bendahl, Pär-Ola; Åkerman, Måns;

    2011-01-01

    Ezrin is a cytoskeletal protein involved in tumour growth and invasion. Ezrin expression has been suggested to play a role in metastasis in paediatric osteosarcoma and rhabdomyosarcoma.......Ezrin is a cytoskeletal protein involved in tumour growth and invasion. Ezrin expression has been suggested to play a role in metastasis in paediatric osteosarcoma and rhabdomyosarcoma....

  1. merlin分子生物学特性及其与肿瘤关系的研究进展

    Institute of Scientific and Technical Information of China (English)

    江忠清; 钟莉娉; 戴丽玉

    2010-01-01

    @@ ERM(ezrin/radixin/moesin/merlin)家族有4个成员,分别为埃兹蛋白(ezrin)、根蛋白(radixin)、膜突蛋白(moesin)及merlin.其中merlin是最后一个被发现的ERM成员.merlin是Ⅱ型神经纤维瘤病(neurofibromatosis Ⅱ,NF2)基因的编码蛋白,也是一种肿瘤抑制蛋白.NF2基因突变或表达缺失可能导致肿瘤发生,而merlin过度表达可能阻止癌细胞增殖和癌基因诱导的恶性转化.但近来也有研究提示,merlin可能也是一种癌蛋白.

  2. Ezrin Is Associated with Disease Progression in Ovarian Carcinoma

    Science.gov (United States)

    Horwitz, Vered; Davidson, Ben; Stern, Dganit; Tropé, Claes G.; Tavor Re’em, Tali; Reich, Reuven

    2016-01-01

    Objective Ezrin and p130Cas are structural proteins with an important role in signaling pathways and have been shown to promote cancer dissemination. We previously reported on overexpression of both ezrin and p130Cas in breast carcinoma effusions compared to primary carcinomas. Since ovarian and breast carcinomas share the ability to disseminate by forming malignant effusions, we sought to study the role of these molecules in ovarian carcinoma (OC). Methods OC cell lines were cultured in two different 3-dimensional conditions, on alginate scaffolds and as spheroids, which served as models for solid tumor and malignant effusions, respectively. shRNA was used to reduce protein expression in the cells. The malignant potential was evaluated by chemo-invasion assay, branching capacity on Matrigel and rate of proliferation. Subsequently, clinical specimens of high-grade serous carcinoma effusions, ovarian tumors and solid metastases were analyzed for ezrin and p130Cas expression. Results Higher ezrin expression was found in cells composing the spheroids compared to their counterparts cultured on alginate scaffold and in clinical samples of malignant effusions compared to solid tumors. In addition, reduced Ezrin expression impaired the invasion ability and the branching capacity of OC cells to a greater extent than reduced p130Cas expression. However, ezrin and p130Cas expression in effusions was unrelated to clinical outcome. Conclusions The 3-dimensional cell cultures were found to mimic the different tumor sites and be applicable as a model. The in vitro results concur with the clinical specimen analysis, suggesting that in OC, the role of ezrin in disease progression is more pronounced than that of p130Cas. PMID:27622508

  3. Correlation of Ezrin Expression Pattern and Clinical Outcomes in Ewing Sarcoma

    Directory of Open Access Journals (Sweden)

    Thomas Cash

    2017-01-01

    Full Text Available Background. Ezrin is a membrane-cytoskeleton linker protein that has been associated with metastasis and poor outcomes in osteosarcoma and high-grade soft tissue sarcomas. The prognostic value of ezrin expression in Ewing sarcoma is unknown. Methods. The relationship between ezrin expression and outcome was analyzed in a cohort of 53 newly diagnosed Ewing sarcoma patients treated between 2000 and 2011. The intensity and proportion of cells with ezrin immunoreactivity were assessed in diagnostic tumor tissue using a semiquantitative scoring system to yield intensity and positivity scores for each tumor. Results. Ezrin expression was detected in 72% (38/53 of tumor samples. The proportion of patients with metastatic disease was equal in the positive and negative ezrin expression groups. There was no significant difference in the 5-year event-free survival (EFS between patients with positive versus negative ezrin expression. Patients whose tumor sample showed high ezrin intensity had significantly better 5-year EFS when compared to patients with low/no ezrin intensity (78% versus 55%; P=0.03. Conclusions. Ezrin expression can be detected in the majority of Ewing sarcoma tumor samples. Intense ezrin expression may be correlated with a favorable outcome; however further investigation with a larger cohort is needed to validate this finding.

  4. The Ezrin Metastatic Phenotype Is Associated with the Initiation of Protein Translation

    Directory of Open Access Journals (Sweden)

    Joseph W. Briggs

    2012-04-01

    Full Text Available We previously associated the cytoskeleton linker protein, Ezrin, with the metastatic phenotype of pediatric sarcomas, including osteosarcoma and rhabdomyosarcoma. These studies have suggested that Ezrin contributes to the survival of cancer cells after their arrival at secondary metastatic locations. To better understand this role in metastasis, we undertook two noncandidate analyses of Ezrin function including a microarray subtraction of high-and low-Ezrin-expressing cells and a proteomic approach to identify proteins that bound the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis, we found Ezrin to be part of the ribonucleoprotein complex to facilitate the expression of complex messenger RNA in cells and to bind with poly A binding protein 1 (PABP1; PABPC1. The relevance of these findings was supported by our identification of Ezrin and components of the translational machinery in pseudopodia of highly metastatic cells during the process of cell invasion. Finally, two small molecule inhibitors recently shown to inhibit the Ezrin metastatic phenotype disrupted the Ezrin/PABP1 association. Taken together, these results provide a novel mechanistic basis by which Ezrin may contribute to metastasis.

  5. ExpressionofEzrin,HGF,C-metinpancreatic cancerandnon-cancerouspancreatic tissuesofrats

    Institute of Scientific and Technical Information of China (English)

    Xing-Guo Tan; Zhu-Lin Yang

    2010-01-01

    BACKGROUND: Recent studies have conifrmed that the expression of Ezrin, hepatocyte growth factor (HGF) and its receptor (C-met) is related to the genesis, progress, invasion and metastasis of some malignant tumors. Researches have also found that the biological function of Ezrin is closely related to HGF/C-met in malignant tumors. However, there is no report on the expression levels of Ezrin, HGF and C-met in rat pancreatic cancer induced by dimethylbenzanthracene (DMBA). This study aimed to detect the expression of Ezrin, HGF and C-met in rat pancreatic cancer and non-cancerous pancreatic tissues, and assess its effect in cancer induction by DMBA. METHODS: Ninety Sprague-Dawley rats were divided into 3 groups randomly: 40 in a pancreatic cancer model group (group A), 40 in a trichostatin A (TSA) intervention group (group B), and 10 in a control group (group C). DMBA was directly implanted into the parenchyma of rat pancreas in group A+group B. The rats of group B were treated with 1 ml of TSA saline solution (1μg/ml) via intraperitoneal injection weekly. The carcinogenesis of rats executed within 3-5 months in groups A and B was observed by macrograph and microscopy. Meanwhile, the rats in group C were executed within 5 months. The EnVisionTM immunohistochemistry for detecting the expression levels of Ezrin, HGF and C-met was used in parafifn-embedded sections of the pancreatic specimens. RESULTS: The incidence of pancreatic cancer in group A was 48.6%and in group B 33.3%. The maximal diameter of tumor mass was signiifcantly larger in group A than that in group B (P in the pancreas of group C and other main organs of groups A and B. The positive rates of Ezrin, HGF and C-met were signiifcantly higher in ductal adenocarcinoma than in non-cancerous pancreatic tissues of groups A and B (P0.05). The positive rates of Ezrin, HGF and C-met in non-cancerous pancreatic tissues proved mild to severe atypical hyperplasia of the ductal epithelia. The pancreas of group

  6. The role of the membrane cytoskeleton cross-linker ezrin in medulloblastoma cells.

    Science.gov (United States)

    Osawa, Hirokatsu; Smith, Christian A; Ra, Young Shin; Kongkham, Paul; Rutka, James T

    2009-08-01

    Medulloblastoma is a highly malignant brain tumor that occurs predominantly in children. The molecular pathogenesis of medulloblastoma is under investigation. Previously, we used complementary DNA microarray analysis to compare patterns of gene expression in medulloblastoma samples versus normal cerebellum. The cytoskeletal protein ezrin was found to be overexpressed in medulloblastoma compared with normal cerebellum, an observation that was further validated by immunohistochemistry and real-time PCR analysis. To assess the role of ezrin in medulloblastoma, we studied ezrin's role in medulloblastoma migration, invasion, and adhesion. Western blotting and immunofluorescence showed high expression of ezrin in four medulloblastoma cell lines, and ezrin was primarily localized to filopodia. Ezrin-specific small interfering RNA suppressed the formation of filopodia and in vitro migration, invasion, and adhesion. We also used a stably transfected medulloblastoma cell line to study the effect of ezrin overexpression. We showed that high expression of ezrin promotes filopodia formation and in vitro invasion. Finally, athymic mice implanted with ezrin-overexpressing DAOY medullo-blastoma cell clones in the cerebellum showed shortened survival compared with controls. These findings suggest that, in addition to other cytoskeletal proteins, ezrin plays an important role in medulloblastoma adhesion, migration, and invasion.

  7. Epigenetic drugs can stimulate metastasis through enhanced expression of the pro-metastatic Ezrin gene.

    Directory of Open Access Journals (Sweden)

    Yanlin Yu

    Full Text Available Ezrin has been reported to be upregulated in many tumors and to participate in metastatic progression. No study has addressed epigenetic modification in the regulation of Ezrin gene expression, the importance of which is unknown. Here, we report that highly metastatic rhabdomyosarcoma (RMS cells with high levels of Ezrin have elevated acetyl-H3-K9 and tri-methyl-H3-K4 as well as reduced DNA methylation at the Ezrin gene promoter. Conversely, poorly metastatic RMS cells with low levels of Ezrin have reduced acetyl-H3-K9 and elevated methylation. Thus epigenetic covalent modifications to histones within nucleosomes of the Ezrin gene promoter are linked to Ezrin expression, which in fact can be regulated by epigenetic mechanisms. Notably, treatment with histone deacetylase (HDAC inhibitors or DNA demethylating agents could restore Ezrin expression and stimulate the metastatic potential of poorly metastatic RMS cells characterized by low Ezrin levels. However, the ability of epigenetic drugs to stimulate metastasis in RMS cells was inhibited by expression of an Ezrin-specific shRNA. Our data demonstrate the potential risk associated with clinical application of broadly acting covalent epigenetic modifiers, and highlight the value of combination therapies that include agents specifically targeting potent pro-metastatic genes.

  8. Ezrin promotes invasion and migration of the MG63 osteosarcoma cell

    Institute of Scientific and Technical Information of China (English)

    Zhang Jian; Zuo Jianhong; Lei Mingsheng; Wu Song; Zang Xiaofang; Zhang Chaoyue

    2014-01-01

    Background Evidence shows that ezrin plays an important role in the development of some human malignancies.But the mechanism by which ezrin may affect tumor cell invasion and metastasis remains unclear.Methods In this study,the expression of ezrin was verified in osteosarcoma (OS) cells and tissues by comparison with normal bone cells and tissues using Western blotting.OS-MG63 were transfected with pcDNA3.1-ezrin or pGenesil-1/ shRNA-ezrin and the stably transfected cells were selected with G418 to yield the ezrin cell line.The OS-MG63 tumor cells were delivered by tail vein to female BALB/c to develop pulmonary metastasis model in vivo.Ezrin was identified as a direct target of miR-183 via a luciferase reporter carrying the 3'-untranslated region of ezrin.Migration assays and invasion assays were done with the transwells.Signaling pathway was studied by Western blotting and/or inhibitor.Results Ectopic overexpression of ezrin in OS cell line MG63 promoted tumor cell invasion and migration.Consistent with this,knockdown of ezrin inhibited tumor cell invasion and migration.Similar results were obtained in the experimental metastasis model in vivo.We identified ezrin as a direct target of miR-183.What is more,ectopic expression of ezrin could induce the expression of N-cadherin and enhance the activity of extracellular signal-regulated kinase (ERK) signaling.Conclusion Collectively,these results suggest that ezrin as a direct target of miR-183 promotes the aggressiveness of OS via increased N-cadherin and activating ERK signaling.

  9. PP1-mediated moesin dephosphorylation couples polar relaxation to mitotic exit.

    Science.gov (United States)

    Kunda, Patricia; Rodrigues, Nelio T L; Moeendarbary, Emadaldin; Liu, Tao; Ivetic, Aleksandar; Charras, Guillaume; Baum, Buzz

    2012-02-01

    Animal cells undergo dramatic actin-dependent changes in shape as they progress through mitosis; they round up upon mitotic entry and elongate during chromosome segregation before dividing into two [1-3]. Moesin, the sole Drosophila ERM-family protein [4], plays a critical role in this process, through the construction of a stiff, rounded metaphase cortex [5-7]. At mitotic exit, this rigid cortex must be dismantled to allow for anaphase elongation and cytokinesis through the loss of the active pool of phospho-Thr559moesin from cell poles. Here, in an RNA interference (RNAi) screen for phosphatases involved in the temporal and spatial control of moesin, we identify PP1-87B RNAi as having elevated p-moesin levels and reduced cortical compliance. In mitosis, RNAi-induced depletion of PP1-87B or depletion of a conserved noncatalytic PP1 phosphatase subunit Sds22 leads to defects in p-moesin clearance from cell poles at anaphase, a delay in anaphase elongation, together with defects in bipolar anaphase relaxation and cytokinesis. Importantly, similar cortical defects are seen at anaphase following the expression of a constitutively active, phosphomimetic version of moesin. These data reveal a new role for the PP1-87B/Sds22 phosphatase, an important regulator of the metaphase-anaphase transition, in coupling moesin-dependent cell shape changes to mitotic exit.

  10. Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis

    Science.gov (United States)

    Roubinet, Chantal; Decelle, Barbara; Chicanne, Gaëtan; Dorn, Jonas F.; Payrastre, Bernard; Payre, François; Carreno, Sébastien

    2011-01-01

    The cortical mechanisms that drive the series of mitotic cell shape transformations remain elusive. In this paper, we identify two novel networks that collectively control the dynamic reorganization of the mitotic cortex. We demonstrate that Moesin, an actin/membrane linker, integrates these two networks to synergize the cortical forces that drive mitotic cell shape transformations. We find that the Pp1-87B phosphatase restricts high Moesin activity to early mitosis and down-regulates Moesin at the polar cortex, after anaphase onset. Overactivation of Moesin at the polar cortex impairs cell elongation and thus cytokinesis, whereas a transient recruitment of Moesin is required to retract polar blebs that allow cortical relaxation and dissipation of intracellular pressure. This fine balance of Moesin activity is further adjusted by Skittles and Pten, two enzymes that locally produce phosphoinositol 4,5-bisphosphate and thereby, regulate Moesin cortical association. These complementary pathways provide a spatiotemporal framework to explain how the cell cortex is remodeled throughout cell division. PMID:21969469

  11. Ezrin ubiquitylation by the E3 ubiquitin ligase, WWP1, and consequent regulation of hepatocyte growth factor receptor activity.

    Directory of Open Access Journals (Sweden)

    Rania F Zaarour

    Full Text Available The membrane cytoskeleton linker ezrin participates in several functions downstream of the receptor Met in response to Hepatocyte Growth Factor (HGF stimulation. Here we report a novel interaction of ezrin with a HECT E3 ubiquitin ligase, WWP1/Aip5/Tiul1, a potential oncogene that undergoes genomic amplification and overexpression in human breast and prostate cancers. We show that ezrin binds to the WW domains of WWP1 via the consensus motif PPVY(477 present in ezrin's C-terminus. This association results in the ubiquitylation of ezrin, a process that requires an intact PPVY(477 motif. Interestingly ezrin ubiquitylation does not target the protein for degradation by the proteasome. We find that ezrin ubiquitylation by WWP1 in epithelial cells leads to the upregulation of Met level in absence of HGF stimulation and increases the response of Met to HGF stimulation as measured by the ability of the cells to heal a wound. Interestingly this effect requires ubiquitylated ezrin since it can be rescued, after depletion of endogenous ezrin, by wild type ezrin but not by a mutant of ezrin that cannot be ubiquitylated. Taken together our data reveal a new role for ezrin in Met receptor stability and activity through its association with the E3 ubiquitin ligase WWP1. Given the role of Met in cell proliferation and tumorigenesis, our results may provide a mechanistic basis for understanding the role of ezrin in tumor progression.

  12. Correlations of Ezrin Expression with Pathological Characteristics and Prognosis of Osteosarcoma: A Meta-Analysis

    Directory of Open Access Journals (Sweden)

    Da-Hang Zhao

    2014-01-01

    Full Text Available We conducted a meta-analysis to comprehensively evaluate the correlations of ezrin expression with pathological characteristics and the prognosis of osteosarcoma. The MEDLINE (1966–2013, the Cochrane Library Database, EMBASE, CINAHL, Web of Science (1945–2013, and the Chinese Biomedical Database were searched without language restrictions. Meta-analyses conducted using STATA software were calculated. Ten studies met the inclusion criteria, including 459 patients with osteosarcoma. Meta-analysis results illustrated that ezrin expression may be closely associated with the recurrence of osteosarcoma or metastasis in osteosarcoma. Our findings also demonstrated that patients with grade III-IV osteosarcoma showed a higher frequency of ezrin expression than those with histological grade I-II osteosarcoma. Furthermore, we found that patients with positive expression of ezrin exhibited a shorter overall survival than those with negative ezrin expression. The results also indicated that positive ezrin expression was strongly correlated with poorer metastasis-free survival. Nevertheless, no significant relationships were observed between ezrin expression and clinical variables (age and gender. In the current meta-analysis, our results illustrated significant relationships of ezrin expression with pathological characteristics and prognosis of osteosarcoma. Thus, ezrin expression could be a promising marker in predicting the clinical outcome of patients with osteosarcoma.

  13. Moesin and stress-induced phosphoprotein-1 are possible sero-diagnostic markers of psoriasis.

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    Hideki Maejima

    Full Text Available To identify diagnostic markers for psoriasis vulgaris and psoriatic arthritis, autoantibodies in sera from psoriasis vulgaris and psoriatic arthritis patients were screened by two-dimensional immunoblotting (2D-IB. Based on 2D-IB and MADLI TOF/TOF-MS analyses, eleven proteins each in psoriasis vulgaris and psoriatic arthritis were identified as autoantigens. Furthermore, serum levels of moesin, keratin 17 (K17, annexin A1 (ANXA1, and stress-induced phophoprotein-1 (STIP1, which were detected as autoantigens, were studied by dot blot analysis with psoriasis patients and healthy controls. The levels of moesin and STIP1 were significantly higher in sera from patients with psoriasis vulgaris than in the controls (moesin: P<0.05, STIP1: P<0.005. The area under the curve (AUC for moesin and STIP1 between patients with psoraisis vulgaris and controls was 0.747 and 0.792, respectively. STIP1 and K17 levels were significantly higher in sera from patients with psoriatic arthritis than in those with psoriasis vulgaris (P<0.05 each. The AUC for STIP1 and K17 between patients with psoriatic arthritis and psoriasis vulgaris was 0.69 and 0.72, respectively. The STIP1 or moesin, CK17 serum level was not correlated with disease activity of psoriasis patients. These data suggest that STIP1 and moesin may be novel and differential sero-diagnostic markers for psoriasis vulgaris and psoriatic arthritis.

  14. Myosin Vb and Rab11a regulate phosphorylation of ezrin in enterocytes

    NARCIS (Netherlands)

    Dhekne, Herschel S.; Hsiao, Nai-Hua; Roelofs, Pieter; Kumari, Meena; Slim, Christiaan L.; Rings, Edmond H. H. M.; van IJzendoorn, Sven C. D.

    2014-01-01

    Microvilli at the apical surface of enterocytes allow the efficient absorption of nutrients in the intestine. Ezrin activation by its phosphorylation at T567 is important for microvilli development, but how such ezrin phosphorylation is controlled is not well understood. We demonstrate that a subset

  15. Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level.

    Science.gov (United States)

    Çelik, Haydar; Sajwan, Kamal P; Selvanathan, Saravana P; Marsh, Benjamin J; Pai, Amrita V; Kont, Yasemin Saygideger; Han, Jenny; Minas, Tsion Z; Rahim, Said; Erkizan, Hayriye Verda; Toretsky, Jeffrey A; Üren, Aykut

    2015-09-01

    Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5' untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane.

  16. Association of ezrin expression in intestinal and diffuse gastric carcinoma with clinicopathological parameters and tumor type

    Institute of Scientific and Technical Information of China (English)

    Nebil Bal; Sedat Yildirim; Tarik Z Nursal; Filiz Bolat; Fazilet Kayaselcuk

    2007-01-01

    AIM: To investigate the correlation between ezrin expression and types of gastric carcinoma and clinicopathological variables.METHODS: We examined ezrin protein expression in 75 gastric carcinoma (53 intestinal types of adenocarcinoma, 22 diffuse types of carcinoma) tissues by immunohistochemistry. The results were compared with clinicopathological parameters such as tumor type,grade of tumor, clinical stage, presence of metastatic lymph node, and depth of invasion.RESULTS: Ezrin immunostaining was positive in 43 cases (81.1%) of intestinal type and in 9 (40.9%) cases of diffuse type adenocarcinomas (P<0.001). In gastric carcinomas, the expression of ezrin protein correlated with the status of H pylori and survival. There was no correlation between expression of ezrin with TNM stage and histological grade of gastric carcinomas (P>0.05).CONCLUSION: The low expression of ezrin implicates the loss of adhesion in diffuse carcinomas. Furthermore,overexpression of ezrin in carcinomas with H pylori infection may be a genuine specific pathway in which H pylori may cause/initiate gastric carcinoma.

  17. Fetal growth retardation and lack of hypotaurine in ezrin knockout mice.

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    Tomohiro Nishimura

    Full Text Available Ezrin is a membrane-associated cytoplasmic protein that serves to link cell-membrane proteins with the actin-based cytoskeleton, and also plays a role in regulation of the functional activities of some transmembrane proteins. It is expressed in placental trophoblasts. We hypothesized that placental ezrin is involved in the supply of nutrients from mother to fetus, thereby influencing fetal growth. The aim of this study was firstly to clarify the effect of ezrin on fetal growth and secondly to determine whether knockout of ezrin is associated with decreased concentrations of serum and placental nutrients. Ezrin knockout mice (Ez(-/- were confirmed to exhibit fetal growth retardation. Metabolome analysis of fetal serum and placental extract of ezrin knockout mice by means of capillary electrophoresis-time-of-flight mass spectrometry revealed a markedly decreased concentration of hypotaurine, a precursor of taurine. However, placental levels of cysteine and cysteine sulfinic acid (precursors of hypotaurine and taurine were not affected. Lack of hypotaurine in Ez(-/- mice was confirmed by liquid chromatography with tandem mass spectrometry. Administration of hypotaurine to heterogenous dams significantly decreased the placenta-to-maternal plasma ratio of hypotaurine in wild-type fetuses but only slightly decreased it in ezrin knockout fetuses, indicating that the uptake of hypotaurine from mother to placenta is saturable and that disruption of ezrin impairs the uptake of hypotaurine by placental trophoblasts. These results indicate that ezrin is required for uptake of hypotaurine from maternal serum by placental trophoblasts, and plays an important role in fetal growth.

  18. Relationship of RhoA signaling activity with ezrin expression and its significance in the prognosis for breast cancer patients

    Institute of Scientific and Technical Information of China (English)

    MA Li; LIU Yue-ping; ZHANG Xiang-hong; GENG Cui-zhi; LI Zeng-huai

    2013-01-01

    Background We have recently reported that RhoA may regulate the invasion and metastasis of breast cancer cells as an upstream signal of ezrin in vitro.In this study,we examined the relationship of RhoA signaling activity with ezrin expression in breast cancer and its prognostic significance in patients with breast cancer.Methods Paraffin tumor sections of breast cancer were collected retrospectively from 487 patients diagnosed between 2001 and 2004.Immunohistochemical methods were used to detect the expression of RhoA,phosphorylated (activated) RhoA,and ezrin.Results Ezrin overexpression was detectable in 15.2% of 487 invasive breast cancers.The majority (85.1%) of ezrin-overexpressing tumors coexpressed phosphorylated RhoA; 78.8% of tumors with phosphorylated RhoA cooverexpressed ezrin.Patients whose cancers showed overexpression of ezrin or expression of phosphorylated RhoA had shorter survival rates.Conclusions RhoA activation is important in human breast cancer due to its upregulation of ezrin; thus,agents that target phosphorylated RhoA may be useful in the treatment of tumors with ezrin overexpression.

  19. Ezrin蛋白在皮肤鳞癌中的表达及临床意义%Expression of Ezrin and phos-ezrin in skin squamous carcinomas and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    谭功军; 周珊; 伍斌; 黄大毛; 唐发清

    2014-01-01

    目的 探讨Ezrin蛋白及磷酸化Ezrin (phos-Ezrin)在皮肤鳞癌中的表达及临床意义.方法 采用免疫组化法检测20例皮肤正常组织(NS)、31例脂溢性角化组织(SK)、36例基底细胞癌组织(BCC)、37例皮肤鳞癌(SCC)中Ezrin、phosEzrin蛋白的表达,分析Ezrin、phos-Ezrin表达与皮肤癌病理组织类型、临床分期和肿瘤转移的相关性.结果 (1)Ezrin蛋白在NS、SK、BCC、SCC中的阳性表达率分别为20.0%、25.8%、66.7%和89.2%,phos-Ezrin蛋白阳性率分别为10.0%、22.6%、77.8%、94.6%,癌组织中的Ezrin和phos-Ezrin表达显著高于非癌组织(P<0.01).(2)Ezrin,phos-Ezrin蛋白的表达与皮肤肿瘤组织类型(R1 =0.87,0.89)、SCC病理分级(R2 =0.80,0.86)及淋巴转移(R3 =0.89,0.91)密切相关(P<0.01).结论 Ezrin,phos-Ezrin表达与皮肤肿瘤的类型、恶性程度(病理分级)和淋巴结转移有关.Ezrin,phos-Ezrin联合检测可为预测皮肤恶性肿瘤转移及预后提供参考.%Objective To investigate the expressions of Ezrin and phos-ezrin in squamous cell carcinomas (SCC),and to an alyze their clinic significance.Methods Immunohistochemistry was used to detect the expressions of Ezrin and phos-Ezrin in 20 cases of normal skin,31 cases of seborrheic Keratosis (SK),36 cases of basal cell carcinoma (BCC),and 37 cases of SCC.Results (1) The positive rates of Ezrin in normal skin (NS),SK,BCC,and SCC were 20.0%,25.8%,66.7%,and 89.2%,respectively.The positive reates of phos-Ezrin in NS,SK,BCC,and SCC were 10.0 %,22.6%,77.8%,and 94.6%,respectively.Ezrin and phos ezrin in cancers were higher than that in noncancer tissues (P < 0.01).(2) The expressions of Ezrin,and phos-Ezrin were closely correlated with the cutaneous tumor's type(R1 =0.87,r1 =0.89,P < 0.01),malignant degree (patho-grading of SCC) (R2 =0.80,r2=0.86,P < 0.01),metastasis via lymph node (R3 =0.89,r3 =0.91,P < 0.01).Conclusions Ezrin and phos-Ezrin expressions were closely

  20. Effect of Radix Isatidis on the Expression of Moesin mRNA Induced by LPS in the Tissues of Mice

    Institute of Scientific and Technical Information of China (English)

    LI Jing; LIU Yunhai; FANG Jianguo; CHEN Xin; XIE Wei

    2007-01-01

    To investigate the effect of the anti-endotoxic part of Radix lsatidis on the expression of moesin mRNA in murine tissues induced by lipopolysaccharide (LPS), the sample solution of F022 part from Radix lsatidis was intraperitoneally administered to experimental mice, and the lipopolysaccharide (LPS) were injected into the tail vein, and then the tissues of liver, kidney and spleen were colleted and cut into slices. The mRNA was detected by moesin mRNA hybridization in situ. The staining results were observed under microscope. It was found that moesin mRNA expression was increased in the tissues of liver, kidndy and spleen in mice treated with LPS, while in the mice pre-treated with F022 part from Radix Isatidis, the LPS-induced moesin mRNA expressions in these tissues were inhibited in a dose-dependant manner. Our study showed that F022 part from Radix Isatidis can inhibit the LPS-induced expression of moesin mRNA in the tissues of liver, kidney and spleen in mice.

  1. The influence of hyaluronic acid on vascular endothelial cell proliferation and the relationship with ezrin/merlin expression

    Institute of Scientific and Technical Information of China (English)

    Wei Mo; Cuixia Yang; Yiwen Liu; Yiqing He; Yingzhi Wang; Feng Gao

    2011-01-01

    It has been established that hyaluronic acid (HA) glycans (nHA) and oligosaccharide (oHA) exert different effects on the biological function of the vascular endothelial cell (EC),resulting in altered regulation of angiogenesis.However,the specific mechanism is still unclear.Our study focused on the effects of nHA and oHA on the ezrin and merlin proteins in EC.The expression of ezrin and merlin was silenced by siRNA,and the regulation on EC growth as well as the mRNA expression and activation (phosphorylation) of ezrin and merlin stimulated by oHA and nHA was investigated.The results revealed that when treated with nHA,there was no significant change in ezrin expression or activation.After being treated with oHA,the expression and activation of ezrin were definitively increased whereas there were no obvious changes in merlin expression (including its phosphorylation).With ezrin expression silenced,the expression of merlin as well as its phosphorylation levels in nHA-stimulated human umbilical vein endothelial cells were notably elevated,while there was no significant change induced by oHA. With merlin expression silenced,no obvious change was found in the expression of ezrin (including its phosphorylation)induced by nHA.Conversely,the expression of ezrin and its activation was significantly improved after being treated with oHA.The results suggest that the mechanism for the promotion of EC proliferation by oHA is likely related to the expression and activation of ezrin,and the inhibition of EC proliferation by nHA is likely related to the expression and activation of merlin.

  2. Ezrin expression in rectal cancer predicts time to development of local recurrence

    DEFF Research Database (Denmark)

    Jörgren, Fredrik; Nilbert, Mef; Rambech, Eva

    2012-01-01

    : Immunohistochemical expression of ezrin was analysed in 104 primary rectal cancers from patients who developed local recurrences despite being treated with R0 major abdominal surgery. Time to local recurrence and distant metastasis as well as 5-year overall and cancer-specific survival were used as end points...

  3. Ezrin interacts with the SARS coronavirus Spike protein and restrains infection at the entry stage.

    Directory of Open Access Journals (Sweden)

    Jean Kaoru Millet

    Full Text Available BACKGROUND: Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S. There are still many unknowns on the implication of cellular factors that regulate the entry process. METHODOLOGY/PRINCIPAL FINDINGS: We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion. CONCLUSIONS/SIGNIFICANCE: Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.

  4. Ezrin Interacts with the SARS Coronavirus Spike Protein and Restrains Infection at the Entry Stage

    Science.gov (United States)

    Millet, Jean Kaoru; Kien, François; Cheung, Chung-Yan; Siu, Yu-Lam; Chan, Wing-Lim; Li, Huiying; Leung, Hiu-Lan; Jaume, Martial; Bruzzone, Roberto; Malik Peiris, Joseph S.; Altmeyer, Ralf Marius; Nal, Béatrice

    2012-01-01

    Background Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process. Methodology/Principal Findings We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion. Conclusions/Significance Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection. PMID:23185364

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

    Energy Technology Data Exchange (ETDEWEB)

    Baeyens, Nicolas; Latrache, Iman; Yerna, Xavier [Laboratory of Cell Physiology, IoNS, Université Catholique de Louvain (Belgium); Noppe, Gauthier; Horman, Sandrine [Pôle de Recherche Cardiovasculaire, IREC, Université Catholique de Louvain (Belgium); Morel, Nicole, E-mail: nicole.morel@uclouvain.be [Laboratory of Cell Physiology, IoNS, Université Catholique de Louvain (Belgium)

    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.

  6. Gastrointestinal Hormone Cholecystokinin Increases P-Glycoprotein Membrane Localization and Transport Activity in Caco-2 Cells.

    Science.gov (United States)

    Yano, Kentaro; Shimizu, Saori; Tomono, Takumi; Ogihara, Takuo

    2017-09-01

    It was reported that stimulation of taste receptor type 2 member 38 by a bitter substance, phenylthiocarbamide (PTC), increased P-glycoprotein (P-gp) mRNA level and transport activity via release of the gastrointestinal hormone cholecystokinin-8 (CCK-8) at 9 h. Therefore, we hypothesized that CCK-8 and PTC might also regulate P-gp activity more rapidly via a different mechanism. As a result, we found that the pretreatment of human colon adenocarcinoma (Caco-2) cells with 10-mM PTC significantly decreased the intracellular accumulation of P-gp substrate rhodamine 123 (Rho123) compared with the control after 90-min incubation. Moreover, CCK-8 treatments significantly reduced the accumulation of Rho123 within 30 min, compared with the control. On the other hand, when Caco-2 cells were pretreated with PTC, the efflux ratio of Rho123 was significantly increased compared with control. The efflux ratio of Rho123 in CCK-8 treatment cells was also significantly increased compared with control. Furthermore, CCK-8 increased the phosphorylation of the scaffold proteins ezrin, radixin, and moesin, which regulate translocation of P-gp to the plasma membrane. Therefore, our results indicate that PTC induced release of CCK-8, which in turn induced the phosphorylation of ezrin, radixin, and moesin proteins, leading to upregulation of P-gp transport activity via increased membrane localization of P-gp. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  7. Resistin, a fat-derived secretory factor, promotes metastasis of MDA-MB-231 human breast cancer cells through ERM activation.

    Science.gov (United States)

    Lee, Jung Ok; Kim, Nami; Lee, Hye Jeong; Lee, Yong Woo; Kim, Su Jin; Park, Sun Hwa; Kim, Hyeon Soo

    2016-01-05

    Resistin, an adipocyte-secreted factor, is known to be elevated in breast cancer patients. However, the molecular mechanism by which resistin acts is not fully understood. The aim of this study was to investigate whether resistin could stimulate invasion and migration of breast cancer cells. Here, we report that resistin stimulated invasion and migration of breast cancer cells as well as phosphorylation of c-Src. Inhibition of c-Src blocked resistin-induced breast cancer cell invasion. Resistin increased intracellular calcium concentration, and chelation of intracellular calcium blocked resistin-mediated activation of Src. Resistin also induced phosphorylation of protein phosphatase 2A (PP2A). Inhibition of c-Src blocked resistin-mediated PP2A phosphorylation. In addition, resistin increased phosphorylation of PKCα. Inhibition of PP2A enhanced resistin-induced PKCα phosphorylation, demonstrating that PP2A activity is critical for PKCα phosphorylation. Resistin also increased phosphorylation of ezrin, radixin, and moesin (ERM). Additionally, ezrin interacted with PKCα, and resistin promoted co-localization of ezrin and PKCα. Either inhibition of c-Src and PKCα or knock-down of ezrin blocked resistin-induced breast cancer cells invasion. Moreover, resistin increased expression of vimentin, a key molecule for cancer cell invasion. Knock-down of ezrin abrogated resistin-induced vimentin expression. These results suggest that resistin play as a critical regulator of breast cancer metastasis.

  8. Upregulated expression of Ezrin and invasive phenotype in malignantly transformed esophageal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Zhong-Ying Shen; Li-Yan Xu; Ming-Hua Chen; En-Min Li; Jin-Tao Li; Xian-Ying Wu; Yi Zeng

    2003-01-01

    AIM: To investigate the correlation between ezrin expression and invasive phenotype formation in malignantly transformed esophageal epithelial cells. METHODS: The experimental cell line employed in the present study was originated form the progressive induction of a human embryonic esophageal epithelial cell line (SHEE)by the E6E7 genes of human papillomavirus (HPV) type 18.The cells at the 35th passage after induction called SHEEIMM were in a state of immortalized phase and used as the control,while that of the 85th passage denominated as SHEEMT represented the status of cells that were malignantly transformed. The expression changes of ezrin and its mRNA in both cell passages were respectively analyzed by RT-PCR and Western blot. Invasive phenotype was assessed in vivo by inoculating these cells into the severe combined immunodeficient (SCID) mice via subcutaneous and intraperitoneal injection, and in vitro by inoculating them on the surface of the amnion membranes, which then was determined by light microscopy and scanning electron microscopy. RESULTS: Upregulated expression of ezrin protein and its mRNA was observed in SHEEMT compared with that in SHEEIMM cells. The SHEEMT cells inoculated in SCID mice were observed forming tumor masses in both visceral organs and soft tissues in a period of 40 days with a special propensity to invading mesentery and pancreas, but did not exhibit hepatic metastases. Pathologically, these tumor cells harboring larger nucleus, nucleolus and less cytoplasm could infiltrate and destroy adjacent tissues. In the in vitro study,the inoculated SHEEMT cells could grow in cluster on the amniotic epithelial surface and intrude into the amniotic stroma. In contrast, unrestricted growth and invasiveness were not found in SHEEIMM cells in both in vivo and in vitroexperiment. CONCLUSION: The upregulated ezrin expression is one of the important factors that are possibly associated with the invasive phenotype formation in malignantly

  9. PPI Network Analysis of mRNA Expression Profile of Ezrin Knockdown in Esophageal Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Bingli Wu

    2014-01-01

    Full Text Available Ezrin, coding protein EZR which cross-links actin filaments, overexpresses and involves invasion, metastasis, and poor prognosis in various cancers including esophageal squamous cell carcinoma (ESCC. In our previous study, Ezrin was knock down and analyzed by mRNA expression profile which has not been fully mined. In this study, we applied protein-protein interactions (PPI network knowledge and methods to explore our understanding of these differentially expressed genes (DEGs. PPI subnetworks showed that hundreds of DEGs interact with thousands of other proteins. Subcellular localization analyses found that the DEGs and their directly or indirectly interacting proteins distribute in multiple layers, which was applied to analyze the shortest paths between EZR and other DEGs. Gene ontology annotation generated a functional annotation map and found hundreds of significant terms, especially those associated with cytoskeleton organization of Ezrin protein, such as “cytoskeleton organization,” “regulation of actin filament-based process,” and “regulation of actin cytoskeleton organization.” The algorithm of Random Walk with Restart was applied to prioritize the DEGs and identified several cancer related DEGs ranked closest to EZR. These analyses based on PPI network have greatly expanded our comprehension of the mRNA expression profile of Ezrin knockdown for future examination of the roles and mechanisms of Ezrin.

  10. Interactions between the L1 cell adhesion molecule and ezrin support traction-force generation and can be regulated by tyrosine phosphorylation.

    Science.gov (United States)

    Sakurai, Takeshi; Gil, Orlando D; Whittard, John D; Gazdoiu, Mihaela; Joseph, Todd; Wu, James; Waksman, Adam; Benson, Deanna L; Salton, Stephen R; Felsenfeld, Dan P

    2008-09-01

    An Ig superfamily cell-adhesion molecule, L1, forms an adhesion complex at the cell membrane containing both signaling molecules and cytoskeletal proteins. This complex mediates the transduction of extracellular signals and generates actin-mediated traction forces, both of which support axon outgrowth. The L1 cytoplasmic region binds ezrin, an adapter protein that interacts with the actin cytoskeleton. In this study, we analyzed L1-ezrin interactions in detail, assessed their role in generating traction forces by L1, and identified potential regulatory mechanisms controlling ezrin-L1 interactions. The FERM domain of ezrin binds to the juxtamembrane region of L1, demonstrated by yeast two-hybrid interaction traps and protein binding analyses in vitro. A lysine-to-leucine substitution in this domain of L1 (K1147L) shows reduced binding to the ezrin FERM domain. Additionally, in ND7 cells, the K1147L mutation inhibits retrograde movement of L1 on the cell surface that has been linked to the generation of the traction forces necessary for axon growth. A membrane-permeable peptide consisting of the juxtamembrane region of L1 that can disrupt endogenous L1-ezrin interactions inhibits neurite extension of cerebellar cells on L1 substrates. Moreover, the L1-ezrin interactions can be modulated by tyrosine phosphorylation of the L1 cytoplasmic region, namely, Y1151, possibly through Src-family kinases. Replacement of this tyrosine together with Y1176 with either aspartate or phenylalanine changes ezrin binding and alters colocalization with ezrin in ND7 cells. Collectively, these data suggest that L1-ezrin interactions mediated by the L1 juxtamembrane region are involved in traction-force generation and can be regulated by the phosphorylation of L1. (c) 2008 Wiley-Liss, Inc.

  11. Guidance of subcellular tubulogenesis by actin under the control of a synaptotagmin-like protein and Moesin.

    Science.gov (United States)

    JayaNandanan, N; Mathew, Renjith; Leptin, Maria

    2014-01-01

    Apical membranes in many polarized epithelial cells show specialized morphological adaptations that fulfil distinct physiological functions. The air-transporting tubules of Drosophila tracheal terminal cells represent an extreme case of membrane specialization. Here we show that Bitesize (Btsz), a synaptotagmin-like protein family member, is needed for luminal membrane morphogenesis. Unlike in multicellular tubes and other epithelia, where it influences apical integrity by affecting adherens junctions, Btsz here acts at a distance from junctions. Localized at the luminal membrane through its tandem C2 domain, it recruits activated Moesin. Both proteins are needed for the integrity of the actin cytoskeleton at the luminal membrane, but not for other pools of F-actin in the cell, nor do actin-dependent processes at the outer membrane, such as filopodial activity or membrane growth depend on Btsz. Btsz and Moesin guide luminal membrane morphogenesis through organizing actin and allowing the incorporation of membrane containing the apical determinant Crumbs.

  12. ANDROGENS REGULATE T47D CELLS MOTILITY AND INVASION THROUGH ACTIN CYTOSKELETON REMODELLING

    Directory of Open Access Journals (Sweden)

    Maria Magdalena Montt-Guevara

    2016-09-01

    Full Text Available The relationship between androgens and breast cancer is controversial. Androgens have complex effects on breast cancer progression and metastasis. Moreover, androgens receptor (AR is expressed in approximately 70% to 90% of invasive breast carcinomas, which has prognostic relevance in basal-like cancers and in triple negative breast cancers. Recent studies have associated the actin-binding proteins of the Ezrin-Radixin-Moesin (ERM family with metastasis in endocrine-sensitive cancers. We studied on T47D breast cancer cells whether androgens with different characteristics, such as testosterone (T, dihydrotestosterone (DHT and dehydroepiandrosterone (DHEA may regulate breast cancer cell motility and invasion through the control of actin remodelling. We demonstrate that androgens promote migration and invasion in T47D via Moesin activation. We show that T and DHEA exert their actions via the AR and estrogen receptor (ER, while the non aromatizable androgen – DHT only recruits AR. We further report that androgen induced significant changes in actin organization with pseudopodia along with membrane ruffles formation, and this process is mediated by Moesin. Our work identifies novel mechanisms of action of androgens on breast cancer cells. Through the modulation of Moesin, androgens alter the architecture of cytoskeleton in T47D breast cancer cell and promote cell migration and invasion. These results could help to understand the biological actions of androgens on breast cancer, and eventually to develop new strategies for treatment of breast cancer.

  13. Vitamin D Receptor, Retinoid X Receptor, Ki-67, Survivin, and Ezrin Expression in Canine Osteosarcoma

    Directory of Open Access Journals (Sweden)

    John Davies

    2012-01-01

    Full Text Available Canine osteosarcoma (OS is an aggressive malignant bone tumor. Prognosis is primarily determined by clinical parameters. Vitamin D has been postulated as a novel therapeutic option for many malignancies. Upon activation, vitamin D receptors (VDRs combine with retinoid receptor (RXR forming a heterodimer initiating a cascade of events. Vitamin D's antineoplastic activity and its mechanism of action in OS remain to be clearly established. Expression of VDR, RXR, Ki-67, survivin, and ezrin was studied in 33 archived, canine OS specimens. VDR, RXR, survivin, and ezrin were expressed in the majority of cases. There was no statistically significant difference in VDR expression in relationship with tumor grade, type, or locations or animal breed, age, and/or sex. No significant association (p=0.316 between tumor grade and Ki-67 expression was found; in particular, no difference in Ki-67 expression between grades 2 and 3 OSs was found, while a negative correlation was noted between Ki-67 and VDR expression (ρ=−0.466, a positive correlation between survivin and RXR expression was found (p=0.374. A significant relationship exists between VDR and RXR expression in OSs and proliferative/apoptosis markers. These results establish a foundation for elucidating mechanisms by which vitamin D induces antineoplastic activity in OS.

  14. Ezrin, maspin, peroxiredoxin 2, and heat shock protein 27: potential targets of a streptococcal-induced autoimmune response in psoriasis.

    Science.gov (United States)

    Besgen, Petra; Trommler, Paul; Vollmer, Sigrid; Prinz, Joerg Christoph

    2010-05-01

    Psoriasis is an HLA-Cw6-associated T cell-mediated autoimmune disease of the skin that is often triggered by streptococcal angina. To identify keratinocyte proteins, which may become psoriatic autoantigens as the result of an immune response against streptococci, rabbits were immunized with heat-killed Streptococcus pyogenes. Streptococcal immunization induced Ab formation against various human keratinocyte proteins. Sera from psoriasis patients reacted against several of these proteins as well. Common serologic reactivities of rabbits and patients included the proteins ezrin, maspin, peroxiredoxin 2 (PRDX2), heat shock protein (hsp)27, and keratin 6. When used for stimulation of blood lymphocytes, ezrin, maspin, PRDX2, and hsp27 induced increased T cell activation in psoriasis patients, which was particularly evident for HLA-Cw6(+) individuals. Ag-specific T cell lines generated with these proteins consisted predominantly of CD8(+) T cells and used TCR beta-chain rearrangements, which were highly homologous to those expanded within the corresponding skin lesion. Several immunodominant epitopes on the different proteins could be defined according to sequence alignments with the whole genome of S. pyogenes. Our data indicate that maspin, ezrin, PRDX2, hsp27, and potentially keratin 6 could act as autoantigens of a streptococcal-induced autoimmune response and represent targets of the exaggerated T cell response in psoriasis. Additionally, ezrin and hsp27 might constitute antigenic links between psoriasis and inflammatory bowel disease, uveitis, or arteriosclerosis, which are clinically associated.

  15. miR-183 inhibits the metastasis of osteosarcoma via downregulation of the expression of Ezrin in F5M2 cells.

    Science.gov (United States)

    Zhao, Haien; Guo, Mingjun; Zhao, Guangyi; Ma, Qiong; Ma, Baoan; Qiu, Xiuchun; Fan, Qingyu

    2012-11-01

    Osteosarcoma is the most common primary malignancy of bone in teenagers and approximately 30% of patients develop lung metastasis, which is the leading cause of mortality. Recent studies suggest that the Ezrin protein is correlated with the metastatic potential of several malignant tumors. In our study, ectopic overexpression of miR-183 repressed the expression levels of Ezrin and significantly inhibited the motility and invasion of osteosarcoma cells. This suggests that miR-183 may possibly play a tumor suppressor role in the metastasis of osteosarcoma by downregulating Ezrin expression levels. These findings show that through inhibition of Ezrin expression levels, miR-183 is significantly involved in cell migration and invasion of osteosarcoma.

  16. Potential transcriptional regulatory regions exist upstream of the human ezrin gene promoter in esophageal carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Shuying Gao; Yanpeng Dai; Meijun Yin; Jing Ye; Gang Li; Jie Yu

    2011-01-01

    We previously demonstrated that the region -87/+ 134 of the human ezrin gene (VIL2) exhibited promoter activity in human esophageal carcinoma EC109 cells, and a further upstream region -1324/-890 positively regulated transcription.In this study, to identify the transcriptional regulatory regions upstream of the VIL2 promoter, we cloned VIL2 - 1541/- 706 segment containing the -1324/-890, and investigated its transcriptional regulatory properties via luciferase assays in transiently transfected cells.In EC109 cells, it was found that VIL2 -1541/-706 possessed promoter and enhancer activities.We also localized transcriptional regulatory regions by fusing 5′- or 3′-deletion segments of VIL2 -1541/-706 to a luciferase reporter.We found that there were three positive and one negative transcriptional regulatory regions ithin VIL2 -1541/-706 in EC109 cells.When these regions were separately located upstream of the luciferase gene without promoter, or located upstream of the VIL2 promoter or SV40 promoter directing the luciferase gene, only VIL2 -1297/-1186 exhibited considerable promoter and enhancer activities, which were lower than those of -1541/-706.In addition, transient expression of Sp1 increased ezrin expression and the transcriptional activation of VIL2 -1297/-1186.Other three regions,although exhibiting significantly positive or negative transcriptional regulation in deletion experiments, showed a weaker or absent regulation.These data suggested that more than one region upstream of the VIL2 promoter participated in VIL2 transcription, and the VIL2 -1297/-1186, probably as a key transcriptional regulatory region, regulated VIL2 transcription in company with other potential regulatory regions.

  17. 晚期糖基化终产物诱导人脐静脉内皮细胞ERM蛋白磷酸化的机制%Mechanism of advanced glycation end products-stimulated phosphorylation of ERM protein in human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    王占华; 郭晓华; 陈波; 李强; 王吉萍; 朱平; 王陵军; 吴炜; 黄巧冰

    2010-01-01

    采用免疫荧光细胞化学和Western印迹法,探讨晚期糖基化终产物(AGE)对人脐静脉内皮细胞的ERM(ezrin/radixint/moesin)蛋白磷酸化水平和定位的影响及其机制.AGE以时间和剂量依赖性地增加ERM蛋白磷酸化水平(均P<0.05),并促进其转位至胞浆内.AGE通过与其受体结合刺激ERM蛋白的磷酸化,Rho激酶及p38丝裂原活化蛋白激酶通路参与了此过程.%The effects of advanced glycation end products(AGE)on phosphorylation of ezrin/radixin/ moesin(ERM)protein in human umbilical vein endothelial ceils were detected by immunofluorescence cytochemistry and its mechanism was explored.AGE stimulated the phosphorylation of ERM protein in dose-and time-dependent manners(all P<0.05),which was involved in AGE receptor,Rho kinase,and p38 mitogen-activated protein kinase pathways.

  18. Internalization of NK cells into tumor cells requires ezrin and leads to programmed cell-in-cell death

    Institute of Scientific and Technical Information of China (English)

    Shan Wang; Zhen Guo; Peng Xia; Tingting Liu; Jufang Wang; Shan Li; Lihua Sun; Jianxin Lu; Qian Wen; Mingqian Zhou; Li Ma; Xia Ding; Xiaoning Wang; Xuebiao Yao

    2009-01-01

    Cytotoxic lymphocytes are key players in the orchestration of immune response and elimination of defective cells. We have previously reported that natural killer (NK) cells enter target tumor cells, leading to either target cell death or self-destruction within tumor cells. However, it has remained elusive as to the fate of NK cells after internaliza-tion and whether the heterotypic cell-in-cell process is different from that of the homotypic cell-in-cell event recently named entosis. Here, we show that NK cells undergo a cell-in-cell process with the ultimate fate of apoptosis within tumor cells and reveal that the internalization process requires the actin cytoskeletal regulator, ezrin. To visualize how NK cells enter into tumor cells, we carried out real-time dual color imaging analyses of NK cell internalization into tumor cells. Surprisingly, most NK cells commit to programmed cell death after their entry into tumor cells, which is distinctively different from entosis observed in the homotypic cell-in-cell process. The apoptotic cell death of the internalized NK cells was evident by activation of caspase 3 and DNA fragmentation. Furthermore, NK cell death after internalization is attenuated by the caspase inhibitor, Z-VAD-FMK, confirming apoptosis as the mode of NK cell death within tumor cells. To determine protein factors essential for the entry of NK cells into tumor cells, we car-ried out siRNA-based knockdown analysis and discovered a critical role of ezrin in NK cell internalization. Impor-tantly, PKA-mediated phosphorylation of ezrin promotes the NK cell internalization process. Our findings suggest a novel regulatory mechanism by which ezrin governs NK cell internalization into tumor cells.

  19. A Cell-Based High-Throughput Assay for Gap Junction Communication Suitable for Assessing Connexin 43-Ezrin Interaction Disruptors Using IncuCyte ZOOM.

    Science.gov (United States)

    Dukic, Aleksandra R; McClymont, David W; Taskén, Kjetil

    2017-01-01

    Connexin 43 (Cx43), the predominant gap junction (GJ) protein, directly interacts with the A-kinase-anchoring protein (AKAP) Ezrin in human cytotrophoblasts and a rat liver epithelial cells (IAR20). The Cx43-Ezrin-protein kinase (PKA) complex facilitates Cx43 phosphorylation by PKA, which triggers GJ opening in cytotrophoblasts and IAR20 cells and may be a general mechanism regulating GJ intercellular communication (GJIC). Considering the importance of Cx43 GJs in health and disease, they are considered potential pharmaceutical targets. The Cx43-Ezrin interaction is a protein-protein interaction that opens possibilities for targeting with peptides and small molecules. For this reason, we developed a high-throughput cell-based assay in which GJIC can be assessed and new compounds characterized. We used two pools of IAR20 cells, calcein loaded and unloaded, that were mixed and allowed to attach. Next, GJIC was monitored over time using automated imaging via the IncuCyte imager. The assay was validated using known GJ inhibitors and anchoring peptide disruptors, and we further tested new peptides that interfered with the Cx43-Ezrin binding region and reduced GJIC. Although an AlphaScreen assay can be used to screen for Cx43-Ezrin interaction inhibitors, the cell-based assay described is an ideal secondary screen for promising small-molecule hits to help identify the most potent compounds.

  20. Amaranthus leucocarpus lectin recognizes a moesin-like O-glycoprotein and costimulates murine CD3-activated CD4+ T cells

    Science.gov (United States)

    Arenas-Del Ángel, Maria; Legorreta-Herrera, Martha; Mendoza-Hernández, Guillermo; Garfias, Yonathan; Chávez, Raul; Zenteno, Edgar; Lascurain, Ricardo

    2015-01-01

    The Galβ1,3GalNAcα1,O-Ser/Thr specific lectin from Amaranthus leucocarpus (ALL) binds a ∼70 kDa glycoprotein on murine T cell surface. We show that in the absence of antigen presenting cells, murine CD4+ T cells activated by an anti-CD3 antibody plus ALL enhanced cell proliferation similar to those cells activated via CD3/CD28 at 48 h of culture. Moreover, ALL induced the production of IL-4, IL-10, TNF-alpha, and TGF-beta in CD3-activated cells. Proteomic assay using two-dimensional electrophoresis and far-Western blotting, ALL recognized two prominent proteins associated to the lipid raft microdomains in CD3/CD28-activated CD4+ T cells. By mass spectrometry, the peptide fragments from ALL-recognized proteins showed sequences with 33% homology to matricin (gi|347839 NCBInr) and 41% identity to an unnamed protein related to moesin (gi|74186081 NCBInr). Confocal microscopy analysis of CD3/CD28-activated CD4+ T cells confirmed that staining by ALL colocalized with anti-moesin FERM domain antibody along the plasma membrane and in the intercellular contact sites. Our findings suggest that a moesin-like O-glycoprotein is the ALL-recognized molecule in lipid rats, which induces costimulatory signals on CD4+ T cells. PMID:26417436

  1. Distribution dynamics and functional importance of NHERF1 in regulation of Mrp-2 trafficking in hepatocytes.

    Science.gov (United States)

    Karvar, Serhan; Suda, Jo; Zhu, Lixin; Rockey, Don C

    2014-10-15

    Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) is a multifunctional scaffolding protein that interacts with receptors and ion transporters in its PDZ domains and with the ezrin-radixin-moesin (ERM) family of proteins in its COOH terminus. The role of NHERF1 in hepatocyte function remains largely unknown. We examine the distribution and physiological significance of NHERF1 and multidrug resistance-associated protein 2 (Mrp-2) in hepatocytes. A WT radixin binding site mutant (F355R) and NHERF1 PDZ1 and PDZ2 domain adenoviral mutant constructs were tagged with yellow fluorescent protein and expressed in polarized hepatocytes to study localization and function of NHERF1. Cellular distribution of NHERF1 and radixin was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate (CMFDA) assay was used to characterize Mrp-2 function. Similar to Mrp-2, WT NHERF1 and the NHERF1 PDZ2 deletion mutant were localized to the canalicular membrane. In contrast, the radixin binding site mutant (F355R) and the NHERF1 PDZ1 deletion mutant, which interacts poorly with Mrp-2, were rarely associated with the canalicular membrane. Knockdown of NHERF1 led to dramatically impaired CMFDA secretory response. Use of CMFDA showed that the NHERF1 PDZ1 and F355R mutants were devoid of a secretory response, while WT NHERF1-infected cells exhibited increased secretion of glutathione-methylfluorescein. The data indicate that NHERF1 interacts with Mrp-2 via the PDZ1 domain of NHERF1 and, furthermore, that NHERF1 is essential for maintaining the localization and function of Mrp-2.

  2. Ezrin蛋白在子宫内膜异位症中的表达及意义%Expression of ezrin in endometriosis and its clinicopathological significance

    Institute of Scientific and Technical Information of China (English)

    丁海钢; 华凯; 包磊

    2011-01-01

    目的 研究细胞骨架相关蛋白ezrin在子宫内膜异位症(endometriosis,EM)患者中的表达情况,探讨ezrin蛋白在EM发生、发展中的作用.方法 选择卵巢EM患者异位内膜标本60例作为异位内膜组,其在位内膜标本30例作为在位内膜组;正常子宫内膜20例作为正常内膜组,应用免疫组化EnvisionTM二步法检测ezrin蛋白在卵巢EM异位内膜、在位内膜和正常内膜的表达情况.结果 在位内膜组ezrin表达值高于正常内膜组,异位内膜组ezrin表达高于在位内膜组,在位内膜组及异位内膜重度组ezrin表达水平高于轻度组,差异均有统计学意义(均P<0.05).结论 ezrin蛋白可能在EM的发病机制中起着重要作用,ezrin蛋白的表达水平和EM的临床分期密切相关,提示ezrin可能在EM的发生、发展中起重要作用.%Objective To investigate the expression of ezrin in endometriosis and its clinicopathological significance.Methods Ninety specimens from patients with endometriosis, including 60 specimens of ovarian endometriosis and 30 specimens of eutopic endometrium were collected; and 20 specimens of normal endometrium served as a controls.The expression of ezrin in ectopic and eutopic endometrium of endometriosis and in normal endometrium was detected by immunohistochemistry Envision TM method.Results Immunohistochemistry showed that the expression of ezrin in eutopic from women with endometriosis was higher than that in normal endometrium.The expression of ezrin in ectopic was higher than that in eutopic endometrium of endometriosis.The levels of ezrin expression in ectopic and eutopic endometrium from woman with severe endometriosis were much higher than those with mild endometriosis (all P<0.05).Conclusion The expression levels of ezrin were significantly correlated with the clinical stage of endometriosis, which suggests that ezrin might play an important role in development of endometriosis.

  3. Effects of 1alpha, 25-dihydroxyvitamin D3 on programmed cell death of Ishikawa endometrial cancer cells through ezrin phosphorylation.

    Science.gov (United States)

    Kim, Tae-Hee; Park, Junsik; Lee, Jeong-Sang; Lee, Hae-Hyeog

    2017-05-01

    This study investigated the effects of 1α, 25-dihydroxyvitamin D3-induced cell death and its underlying molecular mechanisms in Ishikawa endometrial carcinoma cells. The effects of 1α, 25-dihydroxyvitamin D3 on Ishikawa cells were examined by 3-[4,5-dimethylthiazol-2-yl]-2.5-diphenyl-tetrazolium bromide, thiazolyl blue (MTT) assay. 1α, 25-dihydroxyvitamin D3 was shown to induce programmed cell death in Ishikawa endometrial carcinoma cells by activation of caspase-3 and caspase-9, along with elevation of Bcl-2 and Bcl-xL. Cell viability was reduced by 1α, 25-dihydroxyvitamin D3 in a concentration-dependent manner up to 2.5 μM. In addition, ezrin phosphorylation increased with the 1α, 25-dihydroxyvitamin D3 concentration (0-0.5 μM). The protein level of caspase-9 was increased by 1α, 25-dihydroxyvitamin D3 up to 0.5 μM. This is the first report regarding the efficacy and molecular mechanisms underlying the effects of 1α, 25-dihydroxyvitamin D3 in endometrial cancer cells. Our findings indicate that 1α, 25-dihydroxyvitamin D3 induces endometrial cancer cell death in a concentration-dependent manner. Impact statement Up to date, there is no report about the efficacy and molecular underlying mechanisms on the effect of vitamin D3 in endometrial cancer cells. Our findings indicate that 1α, 25-dihydroxyvitamin D3. which is an active metabolite of vitamin D3, induces Ishikawa endometrial cancer cell death in a concentration-dependent manner by activation of caspase-3 and -9, along with elevation of Bcl-2 and Bcl-xL. In addition, the same concentration of 1α, 25-dihydroxyvitamin D3 that provoked apoptotic signals caused phosphorylation of ezrin at threonine 567 in a VDR-dependent manner. This study suggests that 1α, 25-dihydroxyvitamin D3 within the optimal range (0.5 uM) would induce apoptosis through Fas-ezrin-caspase-3, -8, -9 signalling axis which may be a critical cell death regulator in Ishikawa endometrial cancer cell. Further study will be more

  4. CD99 suppresses osteosarcoma cell migration through inhibition of ROCK2 activity.

    Science.gov (United States)

    Zucchini, C; Manara, M C; Pinca, R S; De Sanctis, P; Guerzoni, C; Sciandra, M; Lollini, P-L; Cenacchi, G; Picci, P; Valvassori, L; Scotlandi, K

    2014-04-10

    CD99, a transmembrane protein encoded by MIC2 gene is involved in multiple cellular events including cell adhesion and migration, apoptosis, cell differentiation and regulation of protein trafficking either in physiological or pathological conditions. In osteosarcoma, CD99 is expressed at low levels and functions as a tumour suppressor. The full-length protein (CD99wt) and the short-form harbouring a deletion in the intracytoplasmic domain (CD99sh) have been associated with distinct functional outcomes with respect to tumour malignancy. In this study, we especially evaluated modulation of cell-cell contacts, reorganisation of the actin cytoskeleton and modulation of signalling pathways by comparing osteosarcoma cells characterised by different metastasis capabilities and CD99 expression, to identify molecular mechanisms responsible for metastasis. Our data indicate that forced expression of CD99wt induces recruitment of N-cadherin and β-catenin to adherens junctions. In addition, transfection of CD99wt inhibits the expression of several molecules crucial to the remodelling of the actin cytoskeleton, such as ACTR2, ARPC1A, Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) as well as ezrin, an ezrin/radixin/moesin family member that has been clearly associated with tumour progression and metastatic spread in osteosarcoma. Functional studies point to ROCK2 as a crucial intracellular mediator regulating osteosarcoma migration. By maintaining c-Src in an inactive conformation, CD99wt inhibits ROCK2 signalling and this leads to ezrin decrease at cell membrane while N-cadherin and β-catenin translocate to the plasma membrane and function as main molecular bridges for actin cytoskeleton. Taken together, we propose that the re-expression of CD99wt, which is generally present in osteoblasts but lost in osteosarcoma, through inhibition of c-Src and ROCK2 activity, manages to increase contact strength and reactivate stop-migration signals that counteract the

  5. Noise-induced cochlear F-actin depolymerization is mediated via ROCK2/p-ERM signaling.

    Science.gov (United States)

    Han, Yu; Wang, Xianren; Chen, Jun; Sha, Su-Hua

    2015-06-01

    Our previous work has suggested that traumatic noise activates Rho-GTPase pathways in cochlear outer hair cells (OHCs), resulting in cell death and noise-induced hearing loss (NIHL). In this study, we investigated Rho effectors, Rho-associated kinases (ROCKs), and the targets of ROCKs, the ezrin-radixin-moesin (ERM) proteins, in the regulation of the cochlear actin cytoskeleton using adult CBA/J mice under conditions of noise-induced temporary threshold shift (TTS) and permanent threshold shift (PTS) hearing loss, which result in changes to the F/G-actin ratio. The levels of cochlear ROCK2 and p-ERM decreased 1 h after either TTS- or PTS-noise exposure. In contrast, ROCK2 and p-ERM in OHCs decreased only after PTS-, not after TTS-noise exposure. Treatment with lysophosphatidic acid, an activator of the Rho pathway, resulted in significant reversal of the F/G-actin ratio changes caused by noise exposure and attenuated OHC death and NIHL. Conversely, the down-regulation of ROCK2 by pretreatment with ROCK2 siRNA reduced the expression of ROCK2 and p-ERM in OHCs, exacerbated TTS to PTS, and worsened OHC loss. Additionally, pretreatment with siRNA against radixin, an ERM protein, aggravated TTS to PTS. Our results indicate that a ROCK2-mediated ERM-phosphorylation signaling cascade modulates noise-induced hair cell loss and NIHL by targeting the cytoskeleton. We propose the following cascade following noise trauma leading to alteration of the F-actin arrangement in the outer hair cell cytoskeleton: Noise exposure reduces the levels of GTP-RhoA and subsequently diminishes levels of RhoA effector ROCK2 (Rho-associated kinase 2). Phosphorylation of ezrin-radixin-moesin (ERM) by ROCK2 normally allows ERM to cross-link actin filaments with the plasma membrane. Noise-decreased levels of ROCK results in reduction of phosphorylation of ERM that leads to depolymerization of actin filaments. Lysophosphatidic acid (LPA), an agonist of RhoA, binds to the G-protein-coupled receptor

  6. Ezrin基因敲低及过表达对脑胶质瘤细胞U87迁移的影响%Effect of Ezrin gene knockdown and over-expression on invasion of glioma U87 cells

    Institute of Scientific and Technical Information of China (English)

    刘乃杰; 秦治刚; 孙利波; 金星一; 叶保国; 张金男; 朱庆三

    2013-01-01

    Objective To study the relationship between Ezrin gene and infiltrative growth of glioma through Ezrin gene knockdown and over-expression. Methods According to Ezrin gene sequence in GenBank, primers were designed using Prime Primer 5. 0 software, with which the gene fragment encoding CDS region of Ezrin gene was amplified from U87 cells and cloned into expression vector pEGFP-1. U87 cells were transfected with the constructed recombinant plasmid pEGFP-C 1/Ezrin as well as plasmids shRNA-EZrin-2 and pEGFP-C 1 in mediation of LipofectimineTM 2000 respectively, then deter-mined for expression of Ezrin protein by Western blot, and for migration by scarification test. Results The homologies of mRNAs of cloned Ezrin gene were 99% to those of homo sapiens ezrin (EZR) and transcript variant 1 reported in Gen-Bank. The homologies of amino acids encoding by the cloned gene was 99% to that of ezrin [Homo sapiens] (Sequence ID: ref-NP_003370. 2-, Length:586), with variations of S66P, K258R, P265L and K577R, while the opening read frame was correct. The relative expression level (1. 17) of Ezrin protein in U87 cells transfected with recombinant plasmid pEGFP-C1 /Ezrin was higher those transfected with shRNA-Ezrin-2 (0. 47) and pEGFP-C1 (0. 82). Scarification test showed migration of a small quantity of U87 cells transfected with shRNA-Ezrin-2 to the wound. However, in pEGFP-C1/ Ezrin transfection group, the wound was almost filled with cells. Conclusion Ezrin gene knockdown blocked , while the over-expression promoted the migration of U87 cells, indicating that Ezrin gene involved in the invasive growth of U87 cells.%目的 分析Ezrin基因敲低及过表达对脑胶质瘤细胞U87迁移的影响,以探讨脑胶质瘤浸润性生长的机理.方法 从U87细胞中扩增Ezrin基因CDS区片段,克隆至表达载体pEGFP-C1中,构建Ezrin基因表达质粒pEGFP-C1/Ezrin.将pEGFP-C1/Ezrin、Ezrin基因shRNA质粒shRNA-Ezrin-2和pEGFP-C1以脂质体LipofectimineTM 2000

  7. Rac activation by the T-cell receptor inhibits T cell migration.

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    Eva Cernuda-Morollón

    Full Text Available BACKGROUND: T cell migration is essential for immune responses and inflammation. Activation of the T-cell receptor (TCR triggers a migration stop signal to facilitate interaction with antigen-presenting cells and cell retention at inflammatory sites, but the mechanisms responsible for this effect are not known. METHODOLOGY/PRINCIPAL FINDINGS: Migrating T cells are polarized with a lamellipodium at the front and uropod at the rear. Here we show that transient TCR activation induces prolonged inhibition of T-cell migration. TCR pre-activation leads to cells with multiple lamellipodia and lacking a uropod even after removal of the TCR signal. A similar phenotype is induced by expression of constitutively active Rac1, and TCR signaling activates Rac1. TCR signaling acts via Rac to reduce phosphorylation of ezrin/radixin/moesin proteins, which are required for uropod formation, and to increase stathmin phosphorylation, which regulates microtubule stability. T cell polarity and migration is partially restored by inhibiting Rac or by expressing constitutively active moesin. CONCLUSIONS/SIGNIFICANCE: We propose that transient TCR signaling induces sustained inhibition of T cell migration via Rac1, increased stathmin phosphorylation and reduced ERM phosphorylation which act together to inhibit T-cell migratory polarity.

  8. Dystroglycan versatility in cell adhesion: a tale of multiple motifs

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    Winder Steve J

    2010-02-01

    Full Text Available Abstract Dystroglycan is a ubiquitously expressed heterodimeric adhesion receptor. The extracellular α-subunit makes connections with a number of laminin G domain ligands including laminins, agrin and perlecan in the extracellular matrix and the transmembrane β-subunit makes connections to the actin filament network via cytoskeletal linkers including dystrophin, utrophin, ezrin and plectin, depending on context. Originally discovered as part of the dystrophin glycoprotein complex of skeletal muscle, dystroglycan is an important adhesion molecule and signalling scaffold in a multitude of cell types and tissues and is involved in several diseases. Dystroglycan has emerged as a multifunctional adhesion platform with many interacting partners associating with its short unstructured cytoplasmic domain. Two particular hotspots are the cytoplasmic juxtamembrane region and at the very carboxy terminus of dystroglycan. Regions which between them have several overlapping functions: in the juxtamembrane region; a nuclear localisation signal, ezrin/radixin/moesin protein, rapsyn and ERK MAP Kinase binding function, and at the C terminus a regulatory tyrosine governing WW, SH2 and SH3 domain interactions. We will discuss the binding partners for these motifs and how their interactions and regulation can modulate the involvement of dystroglycan in a range of different adhesion structures and functions depending on context. Thus dystroglycan presents as a multifunctional scaffold involved in adhesion and adhesion-mediated signalling with its functions under exquisite spatio-temporal regulation.

  9. Sodium loading changes urinary protein excretion: a proteomic analysis.

    Science.gov (United States)

    Thongboonkerd, Visith; Klein, Jon B; Pierce, William M; Jevans, Anthony W; Arthur, John M

    2003-06-01

    Plasma sodium concentration is maintained even when sodium intake is altered. Sodium homeostasis may involve changes in renal tubular protein expression that are reflected in the urine. We used proteomic analysis to investigate changes in urinary protein excretion in response to acute sodium loading. Rats were given deionized water followed by hypertonic (2.7%) saline for 28 h each. Urinary protein expression was determined during the final 4 h of each treatment. Acute sodium loading increased urinary sodium excretion (4.53 +/- 1.74 vs. 1.70 +/- 0.27 mmol/day, P = 0.029). Urinary proteins were separated by two-dimensional PAGE and visualized by Sypro ruby staining. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry followed by peptide mass fingerprinting. The abundance of a total of 45 protein components was changed after acute sodium loading. Neutral endopeptidase, solute carrier family 3, meprin 1alpha, diphor-1, chaperone heat shock protein 72, vacuolar H(+)-ATPase, ezrin, ezrin/radixin/moesin-binding protein, glutamine synthetase, guanine nucleotide-binding protein, Rho GDI-1, and chloride intracellular channel protein 1 were decreased, whereas albumin and alpha-2u globulin were increased. Some of these proteins have previously been shown to be associated with tubular transport. These data indicate that alterations in the excretion of several urinary proteins occur during acute sodium loading.

  10. Co-Expression of Ezrin-CLIC5-Podocalyxin Is Associated with Migration and Invasiveness in Hepatocellular Carcinoma.

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    Teresita N J Flores-Téllez

    Full Text Available Prognostic markers are important for predicting the progression and staging of hepatocellular carcinoma (HCC. Ezrin (EZR and Podocalyxin (PODXL are proteins associated with invasion, migration and poor prognosis in various types of cancer. Recently, it has been observed that chloride intracellular channel 5 (CLIC5 forms a complex with EZR and PODXL and that it is required for podocyte structure and function. In this study, we evaluated the overexpression of EZR, PODXL and CLIC5 in HCC.The modified resistant hepatocyte model (MRHR, human biopsies and HCC cell lines (HepG2, Huh7 and SNU387 were used in this study. Gene and protein expression levels were evaluated in the MRHR by qRT-PCR, Western blot and immunohistochemistry analyses, and protein expression in the human biopsies was evaluated by immunohistochemistry. Protein expression in the HCC cell lines was evaluated by immunofluorescence and Western blot, also the migration and invasive abilities of Huh7 cells were evaluated using shRNA-mediated inhibition.Our results indicated that these genes and proteins were overexpressed in HCC. Moreover, when the expression of CLIC5 and PODXL was inhibited in Huh7 cells, we observed decreased migration and invasion.This study suggested that EZR, CLIC5 and PODXL could be biological markers to predict the prognosis of HCC and that these proteins participate in migration and invasion processes.

  11. Expression of metastasis-associated protein 1 and Ezrin in bladder urothelial carcinoma%转移相关蛋白1和埃兹蛋白在膀胱尿路上皮癌中的表达

    Institute of Scientific and Technical Information of China (English)

    潘兆君; 黄伟佳; 瞿虎; 邹自灏; 高兴成

    2010-01-01

    Objective To detect the expression of metastasis- associated protein 1 (MTA- 1) and Ezrin in bladder urothelial carcinoma (BUC) and normal bladder mucosa, and to investigate their correlation with clinical pathology and prognosis. Methods The expression of MTA- 1 and Ezrin proteins was detected in 65 specimens of surgically resected BUC tissue and 30 of normal bladder mucosa with immunohistochemistry. The correlation of MTA- 1 and Ezrin expressions in BUC with clinicopathological parameters, the correlation between MTA-1 and Ezrin protein expressions and their effects on prognosis were also analyzed. Results In 65 specimens of BUC tissue, the positive rates of MTA- 1 and Ezrin expression were 72.3% (n=47) and 89.2% (n=58) , respectively, in contrast to 0% for both MTA- 1 and Ezrin expressions in 30 specimens of normal bladder mucosa, which reached the level of statistical difference (all P<0.01). The expression of MTA- 1 in BUC tissue was closely related to the clinical stages, tumor pathological grading, metastasis and recurrence (all P<0.01 ) , while the expression of Ezrin was closely related to the clinical stages, metastasis and recurrence (all P<0.01 ), and the expression of MTA-1 was positively correlated with Ezrin expression (r=0.742, P<0.01 ). The disease free survival (DFS) in patients with combined expression was significantly lower than that in those with negative expression of MTA- 1 and Ezrin (P<0.01). Conclusion The high expression of MTA-1 and Ezrin is related to the occurrence,progression, as well as invasion and metastasis of BUC.%目的 检测转移相关蛋白1(MTA-1)、埃兹蛋白(Ezrin)在膀胱尿路上皮癌(BUC)组织及正常膀胱黏膜组织中的表达,探讨两者与患者临床病理及预后之间的关系.方法 采用免疫组化法检测65例手术切除的BUC组织和30例正常膀胱黏膜组织中MTA-1和Ezrin蛋白表达,分析MTA-1和Ezrin在BUC中的表达与患者临床病理参数的关系,分析MTA-1和Ezrin

  12. 埃兹蛋白在骨巨细胞瘤中的表达及意义%Expression of Ezrin and its significance in giant-cell tumor of bone

    Institute of Scientific and Technical Information of China (English)

    龚骏; 李平生; 胡海波; 林伟龙

    2015-01-01

    Objective To detect the expression of Ezrin in giant-cell tumor of bone,and to investigate its cilincal significance. Methods 60 cases of biopsy which had been confirmed as bone giant-cell tumors in our hospital from January 2008 to December 2013 were set as observation group;tumor tissues from 8 cases of reactive new bone in nonmalignant bone diseases,12 cases of osteoid osteoma and 11 cases of osteoblastoma in the corresponding period were set as control group. Protein and gene levels of Ezrin were tested with Western blotting method and real-time PCR detection,simultaneously proceeded the corresponding analysis combined with the clinical data of patients;60 cases of bone giant-cell tumor patients accepted tumor resection and pros-thesis replacement,2 courses of preoperative chemotherapy;mitochondria morphological changes of tumor tissue and Ezrin protein and genetic changes were observed before and after chemotherapy. Results In the giant-cell tumors of bone,the Ezrin protein mainly located in the cytoplasm,and its expression positive rate was much higher than that in reactive new bone of nonmalignant bone diseases(19. 7% ),osteoid osteoma(21. 2% )and osteoblastoma(20. 7% );the difference was statistically significant(χ2 = 4. 18,P = 0. 024),but no statistical difference in the Ezrin expression among the groups of osteosarcoma,osteoid osteoma and osteblastoma(χ2 =6. 18,P = 0. 087). In the giant-cell tumors of bone tissue after chemotherapy,mitochondria pyknosis and the phenomenon of liquid cavitation was less than that before the treatment,and Ezrin protein expression decreased and gene levels reduced[(23. 99 ± 1. 49)vs(20. 11 ± 1. 11),t = 5. 03,P = 0. 018)]. Conclusion The expression of Ezrin in giant-cell tumor of bone is much higher than other benign bone tumor,and it could be a biological marker for differentiating benign and malignant bone tumor. Early intervention in Ezrin may be helpful for reatment of giant-cell tumor of bone.%目的:检测埃兹

  13. A proteomic and cellular analysis of uropods in the pathogen Entamoeba histolytica.

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    Jacques Marquay Markiewicz

    Full Text Available Exposure of Entamoeba histolytica to specific ligands induces cell polarization via the activation of signalling pathways and cytoskeletal elements. The process leads to formation of a protruding pseudopod at the front of the cell and a retracting uropod at the rear. In the present study, we show that the uropod forms during the exposure of trophozoites to serum isolated from humans suffering of amoebiasis. To investigate uropod assembly, we used LC-MS/MS technology to identify protein components in isolated uropod fractions. The galactose/N-acetylgalactosamine lectin, the immunodominant antigen M17 (which is specifically recognized by serum from amoeba-infected persons and a few other cells adhesion-related molecules were primarily involved. Actin-rich cytoskeleton components, GTPases from the Rac and Rab families, filamin, α-actinin and a newly identified ezrin-moesin-radixin protein were the main factors found to potentially interact with capped receptors. A set of specific cysteine proteases and a serine protease were enriched in isolated uropod fractions. However, biological assays indicated that cysteine proteases are not involved in uropod formation in E. histolytica, a fact in contrast to the situation in human motile immune cells. The surface proteins identified here are testable biomarkers which may be either recognized by the immune system and/or released into the circulation during amoebiasis.

  14. STRIPAK components determine mode of cancer cell migration and metastasis

    Science.gov (United States)

    Madsen, Chris D.; Hooper, Steven; Tozluoglu, Melda; Bruckbauer, Andreas; Fletcher, Georgina; Erler, Janine T.; Bates, Paul A.; Thompson, Barry; Sahai, Erik

    2017-01-01

    The contractile actomyosin cytoskeleton and its connection to the plasma membrane are critical for control of cell shape and migration. We identify three STRIPAK complex components, FAM40A, FAM40B, and STRN3, as regulators of the actomyosin cortex. We show that FAM40A negatively regulates the MST3 and MST4 kinases, which promote the co-localisation of the contractile actomyosin machinery with the Ezrin/Radixin/Moesin family proteins by phosphorylating the inhibitors of PPP1CB, PPP1R14A-D. Using computational modelling, in vitro cell migration assays and in vivo breast cancer metastasis assays we demonstrate that co-localisation of contractile activity and actin-plasma membrane linkage reduces cell speed on planar surfaces, but favours migration in confined environments similar to those observed in vivo. We further show that FAM40B mutations found in human tumours uncouple it from PP2A and enable it to drive a contractile phenotype, which may underlie its role in human cancer. PMID:25531779

  15. Regulation of cell survival by Na+/H+ exchanger-1.

    Science.gov (United States)

    Schelling, Jeffrey R; Abu Jawdeh, Bassam G

    2008-09-01

    Na(+)/H(+) exchanger-1 (NHE1) is a ubiquitous plasma membrane Na(+)/H(+) exchanger typically associated with maintenance of intracellular volume and pH. In addition to the NHE1 role in electroneutral Na(+)/H(+) transport, in renal tubular epithelial cells in vitro the polybasic, juxtamembrane NHE1 cytosolic tail domain acts as a scaffold, by binding with ezrin/radixin/moesin (ERM) proteins and phosphatidylinositol 4,5-bisphosphate, which initiates formation of a signaling complex that culminates in Akt activation and opposition to initial apoptotic stress. With robust apoptotic stimuli renal tubular epithelial cell NHE1 is a caspase substrate, and proteolytic cleavage may permit progression to apoptotic cell death. In vivo, genetic or pharmacological NHE1 loss of function causes renal tubule epithelial cell apoptosis and renal dysfunction following streptozotocin-induced diabetes, ureteral obstruction, and adriamycin-induced podocyte toxicity. Taken together, substantial in vivo and in vitro data demonstrate that NHE1 regulates tubular epithelial cell survival. In contrast to connotations of NHE1 as an unimportant "housekeeping" protein, this review highlights that NHE1 activity is critical for countering tubular atrophy and chronic renal disease progression.

  16. FERM family proteins and their importance in cellular movements and wound healing (review).

    Science.gov (United States)

    Bosanquet, David C; Ye, Lin; Harding, Keith G; Jiang, Wen G

    2014-07-01

    Motility is a requirement for a number of biological processes, including embryonic development, neuronal development, immune responses, cancer progression and wound healing. Specific to wound healing is the migration of endothelial cells, fibroblasts and other key cellular players into the wound space. Aberrations in wound healing can result in either chronic wounds or abnormally healed wounds. The protein 4.1R, ezrin, radixin, moesin (FERM) superfamily consists of over 40 proteins all containing a three lobed N-terminal FERM domain which binds a variety of cell-membrane associated proteins and lipids. The C-terminal ends of these proteins typically contain an actin-binding domain (ABD). These proteins therefore mediate the linkage between the cell membrane and the actin cytoskeleton, and are involved in cellular movements and migration. Certain FERM proteins have been shown to promote cancer metastasis via this very mechanism. Herein we review the effects of a number of FERM proteins on wound healing and cancer. We show how these proteins typically aid wound healing through their effects on increasing cellular migration and movements, but also typically promote metastasis in cancer. We conclude that FERM proteins play important roles in cellular migration, with markedly different outcomes in the context of cancer and wound healing.

  17. A previously unidentified deletion in G protein-coupled receptor 143 causing X-linked congenital nystagmus in a Chinese family

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    Jing Liu

    2016-01-01

    Full Text Available Background: Congenital nystagmus (CN is characterized by conjugated, spontaneous, and involuntary ocular oscillations. It is an inherited disease and the most common inheritance pattern is X-linked CN. In this study, our aim is to identify the disease-causing mutation in a large sixth-generation Chinese family with X-linked CN. Methods: It has been reported that mutations in four-point-one, ezrin, radixin, moesin domain-containing 7 gene (FRMD7 and G protein-coupled receptor 143 gene (GPR143 account for the majority patients of X-linked nystagmus. We collected 8 ml blood samples from members of a large sixth-generation pedigree with X-linked CN and 100 normal controls. FRMD7 and GPR143 were scanned by polymerase chain reaction (PCR-based DNA sequencing assays, and multiplex PCR assays were applied to detect deletions. Results: We identified a previously unreported deletion covering 7 exons in GPR143 in a Chinese family. The heterozygous deletion from exon 3 to exon 9 of GPR143 was detected in all affected males in the family, while it was not detected in other unaffected relatives or 100 normal controls. Conclusions: This is the first report of molecular characterization in GPR143 gene in the CN family. Our results expand the spectrum of GPR143 mutations causing CN and further confirm the role of GPR143 in the pathogenesis of CN.

  18. A Role for CD81 and Hepatitis C Virus in Hepatoma Mobility

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    Claire L. Brimacombe

    2014-03-01

    Full Text Available Tetraspanins are a family of small proteins that interact with themselves, host transmembrane and cytosolic proteins to form tetraspanin enriched microdomains (TEMs that regulate important cellular functions. Several tetraspanin family members are linked to tumorigenesis. Hepatocellular carcinoma (HCC is an increasing global health burden, in part due to the increasing prevalence of hepatitis C virus (HCV associated HCC. The tetraspanin CD81 is an essential receptor for HCV, however, its role in hepatoma biology is uncertain. We demonstrate that antibody engagement of CD81 promotes hepatoma spread, which is limited by HCV infection, in an actin-dependent manner and identify an essential role for the C-terminal interaction with Ezrin-Radixin-Moesin (ERM proteins in this process. We show enhanced hepatoma migration and invasion following expression of CD81 and a reduction in invasive potential upon CD81 silencing. In addition, we reveal poorly differentiated HCC express significantly higher levels of CD81 compared to adjacent non-tumor tissue. In summary, these data support a role for CD81 in regulating hepatoma mobility and propose CD81 as a tumour promoter.

  19. A role for CD81 and hepatitis C virus in hepatoma mobility.

    Science.gov (United States)

    Brimacombe, Claire L; Wilson, Garrick K; Hübscher, Stefan G; McKeating, Jane A; Farquhar, Michelle J

    2014-03-24

    Tetraspanins are a family of small proteins that interact with themselves, host transmembrane and cytosolic proteins to form tetraspanin enriched microdomains (TEMs) that regulate important cellular functions. Several tetraspanin family members are linked to tumorigenesis. Hepatocellular carcinoma (HCC) is an increasing global health burden, in part due to the increasing prevalence of hepatitis C virus (HCV) associated HCC. The tetraspanin CD81 is an essential receptor for HCV, however, its role in hepatoma biology is uncertain. We demonstrate that antibody engagement of CD81 promotes hepatoma spread, which is limited by HCV infection, in an actin-dependent manner and identify an essential role for the C-terminal interaction with Ezrin-Radixin-Moesin (ERM) proteins in this process. We show enhanced hepatoma migration and invasion following expression of CD81 and a reduction in invasive potential upon CD81 silencing. In addition, we reveal poorly differentiated HCC express significantly higher levels of CD81 compared to adjacent non-tumor tissue. In summary, these data support a role for CD81 in regulating hepatoma mobility and propose CD81 as a tumour promoter.

  20. A Proteomic and Cellular Analysis of Uropods in the Pathogen Entamoeba histolytica

    Science.gov (United States)

    Marquay Markiewicz, Jacques; Syan, Sylvie; Hon, Chung-Chau; Weber, Christian; Faust, Daniela; Guillen, Nancy

    2011-01-01

    Exposure of Entamoeba histolytica to specific ligands induces cell polarization via the activation of signalling pathways and cytoskeletal elements. The process leads to formation of a protruding pseudopod at the front of the cell and a retracting uropod at the rear. In the present study, we show that the uropod forms during the exposure of trophozoites to serum isolated from humans suffering of amoebiasis. To investigate uropod assembly, we used LC-MS/MS technology to identify protein components in isolated uropod fractions. The galactose/N-acetylgalactosamine lectin, the immunodominant antigen M17 (which is specifically recognized by serum from amoeba-infected persons) and a few other cells adhesion-related molecules were primarily involved. Actin-rich cytoskeleton components, GTPases from the Rac and Rab families, filamin, α-actinin and a newly identified ezrin-moesin-radixin protein were the main factors found to potentially interact with capped receptors. A set of specific cysteine proteases and a serine protease were enriched in isolated uropod fractions. However, biological assays indicated that cysteine proteases are not involved in uropod formation in E. histolytica, a fact in contrast to the situation in human motile immune cells. The surface proteins identified here are testable biomarkers which may be either recognized by the immune system and/or released into the circulation during amoebiasis. PMID:21483708

  1. Activation of Yes-Associated Protein in Low-Grade Meningiomas Is Regulated by Merlin, Cell Density, and Extracellular Matrix Stiffness.

    Science.gov (United States)

    Tanahashi, Kuniaki; Natsume, Atsushi; Ohka, Fumiharu; Motomura, Kazuya; Alim, Adiljan; Tanaka, Ichidai; Senga, Takeshi; Harada, Ichiro; Fukuyama, Ryuichi; Sumiyoshi, Naoyuki; Sekido, Yoshitaka; Wakabayashi, Toshihiko

    2015-07-01

    The NF2 gene product Merlin is a protein containing ezrin, radixin, and moesin domains; it is a member of the 4.1 protein superfamily associated with the membrane cytoskeleton and also interacts with cell surface molecules. The mammalian Hippo cascade, a downstream signaling cascade of merlin, inactivates the Yes-associated protein (YAP). Yes-associated protein is activated by loss of the NF2 gene and functions as an oncogene in meningioma cells; however, the factors controlling YAP expression, phosphorylation, and subcellular localization in meningiomas have not been fully elucidated. Here, we demonstrate that merlin expression is heterogeneous in 1 NF2 gene-negative and 3 NF2 gene-positive World Health Organization grade I meningiomas. In the NF2 gene-positive meningiomas, regions with low levels of merlin (tumor rims) had greater numbers of cells with nuclear YAP versus regions with high merlin levels (tumor cores). Merlin expression and YAP phosphorylation were also affected by cell density in the IOMM-Lee and HKBMM human meningioma cell lines; nuclear localization of YAP was regulated by cell density and extracellular matrix (ECM) stiffness in IOMM-Lee cells. These results suggest that cell density and ECM stiffness may contribute to the heterogeneous loss of merlin and increased nuclear YAP expression in human meningiomas.

  2. Dynamic recruitment of protein tyrosine phosphatase PTPD1 to EGF stimulation sites potentiates EGFR activation.

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    Pedro Roda-Navarro

    Full Text Available Balanced activity of protein tyrosine kinases and phosphatases (PTPs controls tyrosine phosphorylation levels and, consequently, is needed to prevent pathologies like cancer. Phosphatase activity is tightly regulated in space and time. Thus, in order to understand how phospho-tyrosine signalling is regulated, the intracellular dynamics of PTPs should be investigated. Here, we have studied the intracellular dynamics of PTPD1, a FERM (four-point-one, ezrin, radixin, moesin domain-containing PTP that is over expressed in cancer cells and potentiates EGFR signalling. Whereas PTPD1 was excluded from E-cadherin rich cell-cell adhesions in epithelial cell monolayers, it diffused from the cytoplasm to those membranes in contact with the extracellular medium. Localisation of PTPD1 at the plasma membrane was mediated by its FERM domain and enabled the formation of EGFR/PTPD1-containing signalling complexes that pre-existed at the plasma membrane before EGF stimulation. PTPD1 and EGFR transiently co-localised at EGF stimulation sites until the formation of macropinosomes containing active species of EGFR. Interference of PTPD1 expression caused a decrease in EGFR phosphorylated species at the periphery of the cell. Presented data suggest that the transient formation of dynamic PTPD1/EGFR signalling complexes strengthens EGF signalling by promoting the spatial propagation of EGFR phosphorylated species.

  3. LKB1 Is Required for the Development and Maintenance of Stereocilia in Inner Ear Hair Cells in Mice.

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    Yuqin Men

    Full Text Available The LKB1 gene, which encodes a serine/threonine kinase, was discovered to play crucial roles in cell differentiation, proliferation, and the establishment of cell polarity. In our study, LKB1 conditional knockout mice (Atoh1-LKB1-/- mice were generated to investigate LKB1 function in the inner ear. Tests of auditory brainstem response and distortion product otoacoustic emissions revealed significant decreases in the hearing sensitivities of the Atoh1-LKB1-/- mice. In Atoh1-LKB1-/- mice, malformations of hair cell stereocilliary bundles were present as early as postnatal day 1 (P1, a time long before the maturation of the hair cell bundles. In addition, we also observed outer hair cell (OHC loss starting at P14. The impaired stereocilliary bundles occurred long before the presence of hair cell loss. Stereociliary cytoskeletal structure depends on the core actin-based cytoskeleton and several actin-binding proteins. By Western blot, we examined actin-binding proteins, specifically ERM (ezrin/radixin/moesin proteins involved in the regulation of the actin cytoskeleton of hair cell stereocilia. Our results revealed that the phosphorylation of ERM proteins (pERM was significantly decreased in mutant mice. Thus, we propose that the decreased pERM may be a key factor for the impaired stereocillia function, and the damaged stereocillia may induce hair cell loss and hearing impairments. Taken together, our data indicates that LKB1 is required for the development and maintenance of stereocilia in the inner ear.

  4. A novel nonsense mutation of the GPR143 gene identified in a Chinese pedigree with ocular albinism.

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    Naihong Yan

    Full Text Available BACKGROUND: The purpose of this study was to elucidate the molecular basis of ocular albinism type I in a Chinese pedigree. METHODOLOGY/PRINCIPAL FINDINGS: Complete ophthalmologic examinations were performed on 4 patients, 7 carriers and 17 unaffected individuals in this five-generation family. All coding exons of four-point-one (4.1, ezrin, radixin, moesin (FERM domain-containing 7 (FRMD7 and G protein-coupled receptor 143 (GPR143 genes were amplified by polymerase chain reaction (PCR, sequenced and compared with a reference database. Ocular albinism and nystagmus were found in all patients of this family. Macular hypoplasia was present in the patients including the proband. A novel nonsense hemizygous mutation c.807T>A in the GPR143 gene was identified in four patients and the heterozygous mutation was found in seven asymptomatic individuals. This mutation is a substitution of tyrosine for adenine which leads to a premature stop codon at position 269 (p.Y269X of GPR143. CONCLUSIONS/SIGNIFICANCE: This is the first report that p.Y269X mutation of GPR143 gene is responsible for the pathogenesis of familial ocular albinism. These results expand the mutation spectrum of GPR143, and demonstrate the clinical characteristics of ocular albinism type I in Chinese population.

  5. EFFECTS OF ESTETROL ON MIGRATION AND INVASION IN T47-D BREAST CANCER CELLS THROUGH THE ACTIN CYTOSKELETON

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    Maria Silvia eGiretti

    2014-05-01

    Full Text Available Estetrol (E4 is a natural human estrogen present at high concentrations during pregnancy. Due to its high oral bioavailability and long plasma half-life, E4 is particularly suitable for therapeutic applications. E4 acts as a selective estrogen receptor modulator, exerting estrogenic actions on the endometrium or the central nervous system, while antagonizing the actions of estradiol in the breast. We tested the effects of E4 on its own or in the presence of 17β-estradiol (E2 on T47-D estrogen receptor (ER positive breast cancer cell migration and invasion of three-dimensional matrices. E4 administration to T47-D cells weakly stimulated migration and invasion. However, E4 decreased the extent of movement and invasion induced by E2. Breast cancer cell movement requires a remodeling of the actin cytoskeleton. During exposure to E4, a weak, concentration-dependent, redistribution of actin fibers towards the cell membrane was observed. However, when E4 was added to E2, a inhibition of actin remodeling induced by E2 was seen. Estrogens stimulate ER+ breast cancer cell movement through the ezrin-radixin-moesin (ERM family of actin regulatory proteins, inducing actin and cell membrane remodeling. E4 was a weak inducer of moesin phosphorylation on Thr558, which accounts for its functional activation. In co-treatment with E2, E4 blocked the activation of this actin controller in a concentration-related fashion. These effects were obtained through recruitment of ERα. In conclusion, E4 acted as a weak estrogen on breast cancer cell cytoskeleton remodeling and movement. However, when E2 was present, E4 counteracted the stimulatory actions of E2. This contributes to the emerging hypothesis that E4 may be a naturally occurring estrogen receptor modulator in the breast.

  6. Expression of RHOGTPase regulators in human myometrium

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    Morrison John J

    2008-01-01

    Full Text Available Abstract Background RHOGTPases play a significant role in modulating myometrial contractility in uterine smooth muscle. They are regulated by at least three families of proteins, RHO guanine nucleotide exchange factors (RHOGEFs, RHOGTPase-activating proteins (RHOGAPs and RHO guanine nucleotide inhibitors (RHOGDIs. RHOGEFs activate RHOGTPases from the inactive GDP-bound to the active GTP-bound form. RHOGAPs deactivate RHOGTPases by accelerating the intrinsic GTPase activity of the RHOGTPases, converting them from the active to the inactive form. RHOGDIs bind to GDP-bound RHOGTPases and sequester them in the cytosol, thereby inhibiting their activity. Ezrin-Radixin-Moesin (ERM proteins regulate the cortical actin cytoskeleton, and an ERM protein, moesin (MSN, is activated by and can also activate RHOGTPases. Methods We therefore investigated the expression of various RHOGEFs, RHOGAPs, a RHOGDI and MSN in human myometrium, by semi-quantitative reverse transcription PCR, real-time fluorescence RT-PCR, western blotting and immunofluorescence microscopy. Expression of these molecules was also examined in myometrial smooth muscle cells. Results ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN mRNA and protein expression was confirmed in human myometrium at term pregnancy, at labour and in the non-pregnant state. Furthermore, their expression was detected in myometrial smooth muscle cells. It was determined that ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labour state. Conclusion This study demonstrated for the first time the expression of the RHOGTPase regulators ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN in human myometrium, at term pregnancy, at labour, in the non-pregnant state and also in myometrial smooth muscle cells. ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labouring state. Further investigation of these molecules may enable us

  7. Effects of Prisma® Skin dermal regeneration device containing glycosaminoglycans on human keratinocytes and fibroblasts.

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    Belvedere, Raffaella; Bizzarro, Valentina; Parente, Luca; Petrella, Francesco; Petrella, Antonello

    2017-08-10

    Prisma® Skin is a new pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. It includes alginates, hyaluronic acid and mainly mesoglycan. The latter is a natural glycosaminoglycan preparation containing chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin and it is used in the treatment of vascular disease. Glycosaminoglycans may contribute to the re-epithelialization in the skin wound healing, as components of the extracellular matrix. Here we describe, for the first time, the effects of Prisma® Skin in in vitro cultures of adult epidermal keratinocytes and dermal fibroblasts. Once confirmed the lack of cytotoxicity by mesoglycan and Prisma® Skin, we have shown the increase of S and G2 phases of fibroblasts cell cycle distribution. We further report the strong induction of cell migration rate and invasion capability on both cell lines, two key processes of wound repair. In support of these results, we found significant cytoskeletal reorganization, following the treatments with mesoglycan and Prisma® Skin, as confirmed by the formation of F-actin stress fibers. Additionally, together with a significant reduction of E-cadherin, keratinocytes showed an increase of CD44 expression and the translocation of ezrin to the plasma membrane, suggesting the involvement of CD44/ERM (ezrin-radixin-moesin) pathway in the induction of the analyzed processes. Furthermore, as showed by immunofluorescence assay, fibroblasts treated with mesoglycan and Prisma® Skin exhibited the increase of Fibroblast Activated Protein α and a remarkable change in shape and orientation, two common features of reactive stromal fibroblasts. In all experiments Prisma® Skin was slightly more potent than mesoglycan. In conclusion, based on these findings we suggest that Prisma® Skin may be able to accelerate the healing process in venous skin ulcers, principally enhancing re-epithelialization and granulation processes.

  8. LAT, EGFR -pY197, PCNL2, CDX2, HLA-DPDQDR, bromodeoxyuridine, JAM-A, and ezrin immunoreactants in a rubbed spongiotic dermatitis

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    Ana Maria Abreu Velez

    2011-10-01

    Full Text Available Background: Acute and subacute spongiotic dermatitides are among the most commonly diagnosed types of dermatitis. Many patients rub their lesions, with the lesions becoming clinically thickened. The precise immunologic mechanisms within the thickening process are not well defined. Case report: An 85 year old male presented with the sudden clinical appearance of erythematous patches and small blisters on the back of his legs, with pruritis. Methods: Skin biopsies, one from a rubbed lesion and one from a non-rubbed lesion were submitted for hematoxylin and eosin (H&E, immunohistochemistry (IHC, and for direct immunofluorescence (DIF analysis. Results: The H&E staining demonstrated classic features of a spongiotic dermatitis, but in the rubbed areas psoriasiform hyperplasia was also seen. The psoriasiform areas demonstrated positive, focal IHC staining with bromodeoxyuridine, LAT, EGFR-pY197, PCNL2, CDX2, and HLA-DPDQDR antibodies. DIF staining revealed positive staining of JAM-A and ezrin in the non-rubbed specimens in both the spongiotic epidermis and in the adjacent vessels; normal expression of these markers was appreciated in the rubbed biopsy. Conclusions: The immune response seems to be complex when a spongiotic dermatitis is converted from a non-rubbed to a rubbed lesion with histologic features of psoriasiform hyperplasia.

  9. 黄连素通过抑制磷酸化埃兹蛋白表达阻断鼻咽癌细胞侵袭和转移的机制研究%Berberine inhibits the invasion and metastasis of nasopharyngeal carcinoma cells through Ezrin phosphorylation

    Institute of Scientific and Technical Information of China (English)

    黄大毛; 王巍巍; Feng Zhu; 王雷; 陈宇; 谢春蕾; 孟菁菁; 唐发清

    2011-01-01

    Objective To determine the molecular mechanism of berberine (BBR) inhibiting the metastasis and invasion of nasopharyngeal carcinoma 5-8F cells, and identify whether berberine suppresses the tumor-invasive action through inhibiting Ezrin or phosphate-Ezrin. Methods The non-cytotoxic concentration of berberine was detected by MTT assay. Filopodia formation of 5-8F cells was observed by electron microscope. The invasion and motility of 5-8F cells with berberine treatment were measured with Trans-well assay. Western blot was used to investigate the Ezrin and phos-Ezrin expression in 5-8F cells treated by berberine. pcDNA3. l-Ezrin and pcDNA3. l-Ezrin M were transfected into 6-10B cells. The inhibitory effect of berberine on the motility and invasion of 6-10B-pcDNA3.1 -Ezrin and 6-10B-pcDNA3.1-Ezrin M was detected, respectively. Results Berberine non-cytotoxic concentration was 0-40 μmol/L. After being treated by berberine, filopodia of 5-8F cells obviously reduced, and the permeating artificial basement membrane cells largely decreased in both time- and concentration-dependent manner. There was significant difference compared with the control group (P <0.05 ). Berberine suppressed the phos-Ezrin expression of 5-8F cells in both time- and concentration-dependent manner (P < 0.05 ), but the effect of berberine was weaker on 6-10B-pcDNA3.1-Ezrin M than on 6-10B-pcDNA3.1-Ezrin. Conclusion Berberine inhibits nasopharyngeal carcinoma cell invasion through inhibiting phos-Ezrin expression and filopodia formation.%目的:探讨黄连素(berberine,BBR)抑制鼻咽癌细胞侵袭及移动的分子机制,明确BBR是否通过抑制Ezrin蛋白抑制鼻咽癌侵袭转移.方法:采用细胞增殖实验(MTT)测定BBR非毒性浓度(non-cytotoxic concentration,NCC),扫描电子显微镜观察BBR在NCC对鼻咽癌细胞5-8F细胞伪足形成的影响,Trans-well实验检测BBR处理后鼻咽癌细胞运动侵袭能力;Western印迹检测BBR对鼻咽癌细胞Ezrin蛋

  10. EST Table: DC536868 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available DC536868 E_FL_MFB-_14D05_F_0 10/09/28 64 %/101 aa ref|XP_001868590.1| ezrin-radixin...10 56 %/127 aa gi|189233930|ref|XP_973614.2| PREDICTED: similar to CG6688 CG6688-PA [Tribolium castaneum] FS930935 MFB- ...

  11. Myosin MyTH4-FERM structures highlight important principles of convergent evolution.

    Science.gov (United States)

    Planelles-Herrero, Vicente José; Blanc, Florian; Sirigu, Serena; Sirkia, Helena; Clause, Jeffrey; Sourigues, Yannick; Johnsrud, Daniel O; Amigues, Beatrice; Cecchini, Marco; Gilbert, Susan P; Houdusse, Anne; Titus, Margaret A

    2016-05-24

    Myosins containing MyTH4-FERM (myosin tail homology 4-band 4.1, ezrin, radixin, moesin, or MF) domains in their tails are found in a wide range of phylogenetically divergent organisms, such as humans and the social amoeba Dictyostelium (Dd). Interestingly, evolutionarily distant MF myosins have similar roles in the extension of actin-filled membrane protrusions such as filopodia and bind to microtubules (MT), suggesting that the core functions of these MF myosins have been highly conserved over evolution. The structures of two DdMyo7 signature MF domains have been determined and comparison with mammalian MF structures reveals that characteristic features of MF domains are conserved. However, across millions of years of evolution conserved class-specific insertions are seen to alter the surfaces and the orientation of subdomains with respect to each other, likely resulting in new sites for binding partners. The MyTH4 domains of Myo10 and DdMyo7 bind to MT with micromolar affinity but, surprisingly, their MT binding sites are on opposite surfaces of the MyTH4 domain. The structural analysis in combination with comparison of diverse MF myosin sequences provides evidence that myosin tail domain features can be maintained without strict conservation of motifs. The results illustrate how tuning of existing features can give rise to new structures while preserving the general properties necessary for myosin tails. Thus, tinkering with the MF domain enables it to serve as a multifunctional platform for cooperative recruitment of various partners, allowing common properties such as autoinhibition of the motor and microtubule binding to arise through convergent evolution.

  12. A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP).

    Science.gov (United States)

    Heydorn, Arne; Søndergaard, Birgitte P; Ersbøll, Bjarne; Holst, Birgitte; Nielsen, Finn Cilius; Haft, Carol Renfrew; Whistler, Jennifer; Schwartz, Thue W

    2004-12-24

    Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor proteins previously proposed to be involved in post-endocytotic sorting of receptors. Of the two proteins suggested to target receptors for recycling to the cell membrane, which is the route believed to be taken by a majority of receptors, ERM (ezrin-radixin-moesin)-binding phosphoprotein 50 (EBP50) bound only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein-coupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the delta-opioid receptor, confirmed the expected nanomolar affinities for interaction with SNX1. Truncations of the NK(1) receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with N-ethylmaleimide-sensitive factor. It is concluded that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrum

  13. Rho/Rho-kinase pathway in the brainstem contributes to hypertension caused by chronic nitric oxide synthase inhibition.

    Science.gov (United States)

    Ito, Koji; Hirooka, Yoshitaka; Kishi, Takuya; Kimura, Yoshikuni; Kaibuchi, Kozo; Shimokawa, Hiroaki; Takeshita, Akira

    2004-02-01

    Central nervous system mechanisms are involved in hypertension caused by chronic inhibition of nitric oxide (NO) synthesis. Chronic inhibition of NO synthesis might also activate the Rho/Rho-kinase pathway in the vasculature. We recently demonstrated that activation of the Rho/Rho-kinase pathway in the nucleus tractus solitarii (NTS) contributes to hypertensive mechanisms in spontaneously hypertensive rats. The aim of the present study was to determine whether activation of this pathway also contributes to neurogenic hypertensive mechanisms caused by chronic NO synthesis inhibition. The NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) was administered to Wistar-Kyoto rats in their drinking water (1 mg/mL) for 2 weeks. Bilateral microinjection of Y-27632, a specific Rho-kinase inhibitor, into the NTS elicited decreases in arterial pressure, heart rate, and renal sympathetic nerve activity in control rats and L-NAME-treated rats. The magnitude of the decrease, however, was significantly greater in L-NAME-treated than in control rats. In another group of rats, the specific Rho-kinase inhibitor, Y-27632, was administered intracisternally for 2 weeks with a mini-osmotic pump from the beginning of treatment with L-NAME. Y-27632 co-treatment significantly attenuated the increase in arterial pressure. Furthermore, the expression level of membranous RhoA and phosphorylation of the target proteins of Rho-kinase, the ERM (ezrin, radixin, moesin) family members, was significantly greater in L-NAME-treated rats than in control rats. These results indicate that activation of the Rho/Rho-kinase pathway in the NTS contributes to neurogenic hypertension caused by chronic NO synthase inhibition.

  14. Binding of EBP50 to Nox organizing subunit p47phox is pivotal to cellular reactive species generation and altered vascular phenotype.

    Science.gov (United States)

    Al Ghouleh, Imad; Meijles, Daniel N; Mutchler, Stephanie; Zhang, Qiangmin; Sahoo, Sanghamitra; Gorelova, Anastasia; Henrich Amaral, Jefferson; Rodríguez, Andrés I; Mamonova, Tatyana; Song, Gyun Jee; Bisello, Alessandro; Friedman, Peter A; Cifuentes-Pagano, M Eugenia; Pagano, Patrick J

    2016-09-06

    Despite numerous reports implicating NADPH oxidases (Nox) in the pathogenesis of many diseases, precise regulation of this family of professional reactive oxygen species (ROS) producers remains unclear. A unique member of this family, Nox1 oxidase, functions as either a canonical or hybrid system using Nox organizing subunit 1 (NoxO1) or p47(phox), respectively, the latter of which is functional in vascular smooth muscle cells (VSMC). In this manuscript, we identify critical requirement of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50; aka NHERF1) for Nox1 activation and downstream responses. Superoxide (O2 (•-)) production induced by angiotensin II (AngII) was absent in mouse EBP50 KO VSMC vs. WT. Moreover, ex vivo incubation of aortas with AngII showed a significant increase in O2 (•-) in WT but not EBP50 or Nox1 nulls. Similarly, lipopolysaccharide (LPS)-induced oxidative stress was attenuated in femoral arteries from EBP50 KO vs. WT. In silico analyses confirmed by confocal microscopy, immunoprecipitation, proximity ligation assay, FRET, and gain-/loss-of-function mutagenesis revealed binding of EBP50, via its PDZ domains, to a specific motif in p47(phox) Functional studies revealed AngII-induced hypertrophy was absent in EBP50 KOs, and in VSMC overexpressing EBP50, Nox1 gene silencing abolished VSMC hypertrophy. Finally, ex vivo measurement of lumen diameter in mouse resistance arteries exhibited attenuated AngII-induced vasoconstriction in EBP50 KO vs. WT. Taken together, our data identify EBP50 as a previously unidentified regulator of Nox1 and support that it promotes Nox1 activity by binding p47(phox) This interaction is pivotal for agonist-induced smooth muscle ROS, hypertrophy, and vasoconstriction and has implications for ROS-mediated physiological and pathophysiological processes.

  15. Inversin/Nephrocystin-2 is required for fibroblast polarity and directional cell migration.

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    Iben R Veland

    Full Text Available Inversin is a ciliary protein that critically regulates developmental processes and tissue homeostasis in vertebrates, partly through the degradation of Dishevelled (Dvl proteins to coordinate Wnt signaling in planar cell polarity (PCP. Here, we investigated the role of Inversin in coordinating cell migration, which highly depends on polarity processes at the single-cell level, including the spatial and temporal organization of the cytoskeleton as well as expression and cellular localization of proteins in leading edge formation of migrating cells. Using cultures of mouse embryonic fibroblasts (MEFs derived from inv(-/- and inv(+/+ animals, we confirmed that both inv(-/- and inv(+/+ MEFs form primary cilia, and that Inversin localizes to the primary cilium in inv(+/+ MEFs. In wound healing assays, inv(-/- MEFs were severely compromised in their migratory ability and exhibited cytoskeletal rearrangements, including distorted lamellipodia formation and cilia orientation. Transcriptome analysis revealed dysregulation of Wnt signaling and of pathways regulating actin organization and focal adhesions in inv(-/- MEFs as compared to inv(+/+ MEFs. Further, Dvl-1 and Dvl-3 localized to MEF primary cilia, and β-catenin/Wnt signaling was elevated in inv(-/- MEFs, which moreover showed reduced ciliary localization of Dvl-3. Finally, inv(-/- MEFs displayed dramatically altered activity and localization of RhoA, Rac1, and Cdc42 GTPases, and aberrant expression and targeting of the Na(+/H(+ exchanger NHE1 and ezrin/radixin/moesin (ERM proteins to the edge of cells facing the wound. Phosphorylation of β-catenin at the ciliary base and formation of well-defined lamellipodia with localization and activation of ERM to the leading edge of migrating cells were restored in inv(-/- MEFs expressing Inv-GFP. Collectively, our findings point to the significance of Inversin in controlling cell migration processes, at least in part through transcriptional regulation of

  16. Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance.

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    Vicky Goler-Baron

    Full Text Available Multidrug resistance (MDR is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and

  17. Immunochemical visualization and identification of rat liver proteins adducted by 2,6-di-tert-butyl-4-methylphenol (BHT).

    Science.gov (United States)

    Reed, M; Thompson, D C

    1997-10-01

    Several alkylphenols (e.g., 2,6-di-tert-butyl-4-methylphenol, BHT) form reactive quinone methide intermediates (e.g., 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone, BHT-QM) upon oxidation by cellular enzymes. In order to pursue the role of protein alkylation in alkylphenol toxicity, we used an immunochemical approach to identify protein targets alkylated by BHT. Synthetic BHT-N-acetylcysteine (BHT-NAC) was coupled to keyhole limpet hemocyanin and used as an antigen from which polyclonal antibodies were raised in New Zealand white rabbits. Rabbit serum contained an antibody which was highly specific for BHT-NAC, as determined by competitive ELISA. The BHT antibody was used as a probe to look for the presence of BHT-protein adducts in in vitro incubations with rat liver microsomes or tissue slices and also in vivo in liver tissue from male Sprague-Dawley rats exposed to BHT. Western blotting of protein gels revealed BHT-dependent protein alkylation over a wide molecular weight range. Prominent recurrent bands were observed at approximately 34.5, 52, 64.5, 74, and 97 kDa. Detection of adducts was inhibited in microsomal incubations by cytochrome P450 inhibitors, deuterated BHT, and the omission of NADPH. Similar protein alkylation patterns were observed in rat liver microsomes exposed to synthetically prepared BHT-QM as in the enzyme-mediated incubations. In rats gavaged with up to 1000 mg/kg BHT, the amount of protein alkylation observed was maximal at 24 h postdosing and was dose-dependent. Two alkylated proteins were isolated and identified by N-terminal sequencing: a mitochondrial beta-oxidation enzyme, enoyl-CoA hydratase, and a plasma membrane/cytoskeletal linker protein from the ezrin/moesin/radixin family.

  18. VIP regulates CFTR membrane expression and function in Calu-3 cells by increasing its interaction with NHERF1 and P-ERM in a VPAC1- and PKCε-dependent manner.

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    Alshafie, Walaa; Chappe, Frederic G; Li, Mansong; Anini, Younes; Chappe, Valerie M

    2014-07-01

    Vasoactive intestinal peptide (VIP) is a topical airway gland secretagogue regulating fluid secretions, primarily by stimulating cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride secretion that contributes to the airways innate defense mechanism. We previously reported that prolonged VIP stimulation of pituitary adenylate cyclase-activating peptide receptors (VPAC1) in airway cells enhances CFTR function by increasing its membrane stability. In the present study, we identified the key effectors in the VIP signaling cascade in the human bronchial serous cell line Calu-3. Using immunocytochemistry and in situ proximity ligation assays, we found that VIP stimulation increased CFTR membrane localization by promoting its colocalization and interaction with the scaffolding protein Na(+)/H(+) exchange factor 1 (NHERF1), a PDZ protein known as a positive regulator for CFTR membrane localization. VIP stimulation also increased phosphorylation, by protein kinase Cε of the actin-binding protein complex ezrin/radixin/moesin (ERM) and its interaction with NHERF1 and CFTR complex. On the other hand, it reduced intracellular CFTR colocalization and interaction with CFTR associated ligand, another PDZ protein known to compete with NHERF1 for CFTR interaction, inducing cytoplasmic retention and lysosomal degradation. Reducing NHERF1 or ERM expression levels by specific siRNAs prevented the VIP effect on CFTR membrane stability. Furthermore, iodide efflux assays confirmed that NHERF1 and P-ERM are necessary for VIP regulation of the stability and sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions.

  19. A role of kindlin-3 in integrin αMβ2 outside-in signaling and the Syk-Vav1-Rac1/Cdc42 signaling axis.

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    Zhi-Hong Xue

    Full Text Available Integrins mediate cell-cell and cell-extracellular matrix attachments. Integrins are signaling receptors because their cytoplasmic tails are docking sites for cytoskeletal and signaling proteins. Kindlins are a family of band 4.1-ezrin-radixin-moesin-containing intracellular proteins. Apart from regulating integrin ligand-binding affinity, recent evidence suggests that kindlins are involved in integrin outside-in signaling. Kindlin-3 is expressed in platelets, hematopoietic cells and endothelial cells. In humans, loss of kindlin-3 expression accounts for the rare autosomal disease leukocyte adhesion deficiency (LAD type III that is characterized by bleeding disorders and defective recruitment of leukocytes into sites of infection. Studies have shown that the loss of kindlin-3 expression leads to poor ligand-binding properties of β1, β2 and β3 integrin subfamilies. The leukocyte-restricted β2 integrin subfamily comprises four members, namely αLβ2, αMβ2, αXβ2 and αDβ2. Integrin αMβ2 mediates leukocyte adhesion, phagocytosis, degranulation and it is involved in the maintenance of immune tolerance. Here we provide further evidence that kindlin-3 is required for integrin αMβ2-mediated cell adhesion and spreading using transfected K562 cells that expressed endogenous kindlin-3 but not β2 integrins. K562 stable cell line expressing si-RNA targeting kindlin-3, but not control-si-RNA, and transfected with constitutively activated integrin αMβ2N329S adhered and spread poorly on iC3b. We also show that kindlin-3 is required for the integrin αMβ2-Syk-Vav1 signaling axis that regulates Rac1 and Cdc42 activities. These findings reinforce a role for kindlin-3 in integrin outside-in signaling.

  20. Evidence of hybridization between Taenia saginata and Taenia asiatica.

    Science.gov (United States)

    Okamoto, Munehiro; Nakao, Minoru; Blair, David; Anantaphruti, Malinee T; Waikagul, Jitra; Ito, Akira

    2010-03-01

    There has long been a debate as to the specific status of the cestode Taenia asiatica, with some people regarding it as a distinct species and some preferring to recognize it as a strain of Taenia saginata. The balance of current opinion seems to be that T. asiatica is a distinct species. In this study we performed an allelic analysis to explore the possibility of gene exchange between these closely related taxa. In total, 38 taeniid tapeworms were collected from humans living in many localities including Kanchanaburi Province, Thailand where the two species are sympatric. A mitochondrial DNA (mtDNA)-based multiplex PCR tentatively identified those parasites as T. asiatica (n=20) and T. saginata (n=18). Phylogenetic analyses of a mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and two nuclear loci, for elongation factor-1 alpha (ef1) and ezrin-radixin-moesin (ERM)-like protein (elp), assigned all except two individual parasites to the species indicated by multiplex PCR. The two exceptional individuals, from Kanchanaburi Province, showed a discrepancy between the mtDNA and nuclear DNA phylogenies. In spite of their possession of sequences typical of the T. saginata cox1 gene, both were homozygous at the elp locus for one of the alleles found in T. asiatica. At the ef1 locus, one individual was homozygous for the allele found at high frequency in T. asiatica while the other was homozygous for the major allele in T. saginata. These findings are evidence of occasional hybridization between the two species, although the possibility of retention of ancestral polymorphism cannot be excluded.

  1. EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation.

    Science.gov (United States)

    Yao, Wenfang; Feng, Duiping; Bian, Weihua; Yang, Longyan; Li, Yang; Yang, Zhiyu; Xiong, Ying; Zheng, Junfang; Zhai, Renyou; He, Junqi

    2012-11-01

    Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.

  2. Loss of EBP50 stimulates EGFR activity to induce EMT phenotypic features in biliary cancer cells.

    Science.gov (United States)

    Clapéron, A; Guedj, N; Mergey, M; Vignjevic, D; Desbois-Mouthon, C; Boissan, M; Saubaméa, B; Paradis, V; Housset, C; Fouassier, L

    2012-03-15

    Scaffold proteins form multiprotein complexes that are central to the regulation of intracellular signaling. The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is highly expressed at the plasma membrane of normal biliary epithelial cells and binds epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with oncogenic properties. This study investigated EBP50-EGFR interplay in biliary cancer. We report that in a collection of 106 cholangiocarcinomas, EBP50 was delocalized to the cytoplasm of tumor cells in 66% of the cases. Ectopic expression of EBP50 was correlated with the presence of satellite nodules and with the expression of EGFR, which was at the plasma membrane, implying a loss of interaction with EBP50 in these cases. In vitro, loss of interaction between EBP50 and EGFR was mimicked by EBP50 depletion using a small interfering RNA approach in human biliary carcinoma cells co-expressing the two proteins at their plasma membrane, and in which interaction between EBP50 and EGFR was validated. EBP50 depletion caused an increase in EGFR expression at their surface, and a sustained activation of the receptor and of its downstream effectors (extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3) in both basal and EGF-stimulated conditions. Cells lacking EBP50 showed epithelial-to-mesenchymal transition-associated features, including reduction in E-cadherin and cytokeratin-19 expression, induction of S100A4 and of the E-cadherin transcriptional repressor, Slug, and loss of cell polarity. Accordingly, depletion of EBP50 induced the disruption of adherens junctional complexes, the development of lamellipodia structures and the subsequent acquisition of motility properties. All these phenotypic changes were prevented upon inhibition of EGFR tyrosine kinase by gefitinib. These findings indicate that loss of EBP50 at the plasma membrane in tumor cells may contribute to biliary carcinogenesis

  3. Differential effects of human L1CAM mutations on complementing guidance and synaptic defects in Drosophila melanogaster.

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    Sirisha Kudumala

    Full Text Available A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg, has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin-moesin-radixin (ERM binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis.

  4. Filopodia and membrane blebs drive efficient matrix invasion of macrophages transformed by the intracellular parasite Theileria annulata.

    Directory of Open Access Journals (Sweden)

    Min Ma

    Full Text Available Recent technical advances have broadened our understanding of processes that govern mammalian cell migration in health and disease but many of the molecular and morphological alterations that precede and accompany movement of cells - in particular in three-dimensional (3D environments - are still incompletely understood. In this manuscript, using high-resolution and time-lapse microscopy imaging approaches, we describe morphodynamic processes during rounded/amoeboid cell invasion and molecules associated with the cellular invasion structures. We used macrophages infected with the intracellular protozoan parasite Theileria annulata, which causes Tropical Theileriosis in susceptible ruminants such as domestic cattle. T. annulata transforms its host cell that, as a result, acquires many characteristics of human cancer cells including a markedly increased potential to migrate, disseminate and expand in the body of the host animal. Hence, virulence of the disease is associated with the capability of infected cells to disseminate inside the host. Using T. annulata-transformed macrophages as a model system, we described a novel mode of rounded/amoeboid macrophage migration. We show that filopodia-like membrane extensions at the leading edge lead the way and further evolve in blebbing membrane protrusions to promote progressive expansion of the matrix. Associated with focal invasion structures we detected ezrin, radixin, moesin-family proteins and their regulatory kinase MAP4K4. Furthermore, we linked Rho-kinase activity to contractile force generation, which is essential for infected cell motility. Thus, the motility mode of these parasite-transformed macrophages contrasts with those described so far in human macrophages such as the tunneling or mesenchymal modes, which require engulfment, compaction and ingestion of matrix or proteolytic matrix degradation, respectively. Together, our data reveal protrusion dynamics at the leading edge of invading

  5. A novel membrane-dependent on/off switch mechanism of talin FERM domain at sites of cell adhesion

    Institute of Scientific and Technical Information of China (English)

    Xianqiang Song; Jun Qin; Jun Yang; Jamila Hirbawi; Sheng Ye; H Dhanuja Perera; Esen Goksoy; Pallavi Dwivedi; Edward F Plow; Rongguang Zhang

    2012-01-01

    The activation of heterodimeric (α/β) integrin transmembrane receptors by cytosolic protein talin is crucial for regulating diverse cell-adhesion-dependent processes,including blood coagulation,tissue remodeling,and cancer metastasis.This process is triggered by the coincident binding of N-terminal FERM (four-point-one-protein/ezrin/radixin/moesin) domain of talin (talin-FERM) to the inner membrane surface and integrin β cytoplasmic tail,but how these binding events are spatiotemporally regulated remains obscure.Here we report the crystal structure of a dormant talin,revealing how a C-terminal talin rod segment (talin-RS) self-masks a key integrin-binding site on talin-FERM via a large interface.Unexpectedly,the structure also reveals a distinct negatively charged surface on talin-RS that electrostatically hinders the talin-FERM binding to the membrane.Such a dual inhibitory topology for talin is consistent with the biochemical and functional data,but differs significantly from a previous model.We show that upon enrichment with phosphotidylinositol-4,5-bisphosphate (PIP2) - a known talin activator,membrane strongly attracts a positively charged surface on talin-FERM and simultaneously repels the negatively charged surface on talin-RS.Such an electrostatic "pull-push" process promotes the relief of the dual inhibition of talin-FERM,which differs from the classic "steric clash" model for conventional PIP2-induced FERM domain activation.These data therefore unravel a new type of membrane-dependent FERM domain regulation and illustrate how it mediates the talin on/off switches to regulate integrin transmembrane signaling and cell adhesion.

  6. Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay

    Directory of Open Access Journals (Sweden)

    Tommy Baumann

    2013-10-01

    Full Text Available We have shown previously that the raft-associated proteins flotillin-1 and -2 are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Other proteins such as the adhesion receptor PSGL-1, the actin-membrane linker proteins ezrin/radixin/moesin (ERM and the signaling enzyme phosphatidylinositol-4-phosphate 5-kinase type Iγ90 (PIPKIγ90 also accumulate in the T-cell uropod. Using the in situ proximity ligation assay (PLA we now have investigated putative close associations of these proteins in human freshly isolated T-cells before and after chemokine addition. The PLA allows in situ subcellular localization of close proximity of endogenous proteins at single-molecule resolution in fixed cells. It allows detection also of weaker and transient complexes that would not be revealed with co-immunoprecipitation approaches. We previously provided evidence for heterodimer formation of tagged flotillin-1 and -2 in T-cells before and after chemokine addition using fluorescence resonance energy transfer (FRET. We now confirm these findings using PLA for the endogenous flotillins in fixed human T-cells. Moreover, in agreement with the literature, our PLA findings confirm a close association of endogenous PSGL-1 and ERM proteins both in resting and chemokine-activated human T-cells. In addition, we provide novel evidence using the PLA for close associations of endogenous activated ERM proteins with PIPKIγ90 and of endogenous flotillins with PSGL-1 in human T-cells, before and after chemokine addition. Our findings suggest that preformed clusters of these proteins coalesce in the uropod upon cell stimulation.

  7. Sequence Classification: 784118 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB TMB >gi|17561792|ref|NP_506340.1| FERM domain (protein 4.1-ezrin-rad...ixin-moesin) (56.9 kD) (frm-10) || http://www.ncbi.nlm.nih.gov/protein/17561792 ...

  8. Sequence Classification: 780701 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB TMB Non-TMB >gi|17507851|ref|NP_493602.1| FERM domain (protein 4.1-ezrin-rad...ixin-moesin) (frm-1) || http://www.ncbi.nlm.nih.gov/protein/17507851 ...

  9. Sequence Classification: 780698 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|17507857|ref|NP_493599.1| FERM domain (protein 4.1-ezrin-rad...ixin-moesin) (frm-1) || http://www.ncbi.nlm.nih.gov/protein/17507857 ...

  10. Sequence Classification: 780699 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|17507855|ref|NP_493600.1| FERM domain (protein 4.1-ezrin-rad...ixin-moesin) (frm-1) || http://www.ncbi.nlm.nih.gov/protein/17507855 ...

  11. Sequence Classification: 780700 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|17507853|ref|NP_493601.1| FERM domain (protein 4.1-ezrin-rad...ixin-moesin) (frm-1) || http://www.ncbi.nlm.nih.gov/protein/17507853 ...

  12. MTA-1和Ezrin在乳腺癌组织中的表达及其相关性探讨%Expression of MTA-1 and Ezrin and theirs correlation with clinical and pathology in breast cancer

    Institute of Scientific and Technical Information of China (English)

    黄俊辉; 蒋骑; 郭旭辉; 何璇; 张轶; 张曦蓓

    2009-01-01

    目的 探讨肿瘤转移相关基因MTA-1和细胞骨架连结蛋白Ezrin在乳腺癌组织中的表达及两者之间的相关性和与乳腺癌临床病理的关系.方法 分别应用分子原位杂交及免疫组织化学sP法检测80例乳腺癌组织和15例正常乳腺组织中MTA-1 mRNA、Ezrin蛋白的表达,分析两者之间的相关性及与乳腺癌患者病理类型、年龄、临床分期、淋巴结转移等参数的关系.结果 正常乳腺组织MTA-1 mR.NA(13.3%)、Ezrin蛋白(20.0%)的表达率明显低于乳腺癌组织中MTA-1 mRNA(66.2%)和Ezrin蛋白(68.7%)的表达率,P<0.05;MTA-1 mRNA与Ezrin蛋白的表达呈正相关(r=0.634,P=0.000);MTA-1 mRNA和Ezrin蛋白在乳腺癌中的表达与肿瘤临床分期和淋巴结转移状态相关,P值均<0.05;与患者年龄、肿瘤大小、病理类型无相关性,P值均>0.05.MTA-1+/Ezrin+组的无病生存率低于MTA-1-/Ezdn- 组(X2=7.610,P=0.006).结论 提示MTA-1和Ezrin在肿瘤转移中具有协同作用,高表达组患者的临床预后差与肿瘤淋巴转移有关.

  13. 4-Hydroxytamoxifen-stimulated activation of ezrin is mediated via G-protein-coupled receptor 30 and promotes human breast cancer MCF-7 cell migration%4-羟基他莫昔芬通过雌激素受体GPR30激活ezrin蛋白促进人乳腺癌MCF-7细胞迁移

    Institute of Scientific and Technical Information of China (English)

    游昕超; 王庭槐

    2014-01-01

    目的:观察4-羟基他莫昔芬(OHT)对人乳腺癌细胞株MCF-7迁移的影响并探讨其机制.方法:贴壁培养雌激素受体阳性人乳腺癌细胞株MCF-7,细胞划痕愈合实验观察OHT对MCF-7细胞迁移的影响,Western blotting检测c-Src/p-Src和ezrin/p-ezrin蛋白的表达.结果:(1)与对照组相比,单独给予MCF-7细胞雌二醇(E2)和OHT都能促进细胞迁移,OHT不能抑制E2对MCF-7细胞的促迁移效应.(2)OHT促进MCF-7细胞迁移最大效应浓度约为5 μmol/L,在6h即能观察到明显促迁移效用.(3)OHT和雌激素受体G蛋白偶联受体30 (GPR30)激动剂G1都能明显增加p-Src和p-ezrin蛋白表达.(4)分别用G15和PP2阻断GPR30和Src后,OHT激活ezrin作用都能被阻断.结论:OHT可能通过结合GPR30后激活Src蛋白,进而磷酸化激活ezrin,介导细胞骨架重构并促进MCF-7细胞迁移.

  14. p38MAPK, Rho/ROCK and PKC pathways are involved in influenza-induced cytoskeletal rearrangement and hyperpermeability in PMVEC via phosphorylating ERM.

    Science.gov (United States)

    Zhang, Chenyue; Wu, Ying; Xuan, Zinan; Zhang, Shujing; Wang, Xudan; Hao, Yu; Wu, Jun; Zhang, Shu

    2014-11-04

    Severe influenza infections are featured by acute lung injury, a syndrome of pulmonary microvascular leak. A growing number of evidences have shown that the pulmonary microvascular endothelial cells (PMVEC) are critical target of influenza virus, promoting microvascular leak. It is reported that there are multiple mechanisms by which influenza virus could elicit increased pulmonary endothelial permeability, in both direct and indirect manners. Ezrin/radixin/moesin family proteins, the linkers between plasma membrane and actin cytoskeleton, have been reported to be involved in cell adhesion, motility and may modulate endothelial permeability. Studies have also shown that ERM is phosphorylated in response to various stimuli via p38MAPK, Rho/ROCK or PKC pathways. However, it is unclear that whether influenza infection could induce ERM phosphorylation and its relocalization. In the present study, we have found that there are cytoskeletal reorganization and permeability increases in the course of influenza virus infection, accompanied by upregulated levels of p-ERM. p-ERM's aggregation along the periphery of PMVEC upon influenza virus infection was detected via confocal microscopy. Furthermore, we sought to determine the role of p38MAPK, Rho/ROCK and PKC pathways in ERM phosphorylation as well as their involvement in influenza virus-induced endothelial malfunction. The activation of p38MAPK, Rho/ROCK and PKC pathways upon influenza virus stimulation were observed, as evidenced by the evaluation of phosphorylated p38 (p-p38), phosphorylated MKK (p-MKK) in p38MAPK pathway, ROCK1 in Rho/ROCK pathway and phosphorylated PKC (p-PKC) in PKC pathway. We also showed that virus-induced ERM phosphorylation was reduced by using p38MAPK inhibitor, SB203580 (20 μM), Rho/ROCK inhibitor, Y27632 (20 μM), PKC inhibitor, LY317615 (10 μM). Additionally, influenza virus-induced F-actin reorganization and hyperpermeability were attenuated by pretreatment with SB203580, Y27632 and LY317615

  15. Evolution and origin of merlin, the product of the Neurofibromatosis type 2 (NF2 tumor-suppressor gene

    Directory of Open Access Journals (Sweden)

    Omelyanchuk Leonid V

    2005-12-01

    Full Text Available Abstract Background Merlin, the product of the Neurofibromatosis type 2 (NF2 tumor suppressor gene, belongs to the ezrin-radixin-moesin (ERM subgroup of the protein 4.1 superfamily, which links cell surface glycoproteins to the actin cytoskeleton. While merlin's functional activity has been examined in mammalian and Drosophila models, little is understood about its evolution, diversity, and overall distribution among different taxa. Results By combining bioinformatic and phylogenetic approaches, we demonstrate that merlin homologs are present across a wide range of metazoan lineages. While the phylogenetic tree shows a monophyletic origin of the ERM family, the origin of the merlin proteins is robustly separated from that of the ERM proteins. The derivation of merlin is thought to be in early metazoa. We have also observed the expansion of the ERM-like proteins within the vertebrate clade, which occurred after its separation from Urochordata (Ciona intestinalis. Amino acid sequence alignment reveals the absence of an actin-binding site in the C-terminal region of all merlin proteins from various species but the presence of a conserved internal binding site in the N-terminal domain of the merlin and ERM proteins. In addition, a more conserved pattern of amino acid residues is found in the region containing the so-called "Blue Box," although some amino acid substitutions in this region exist in the merlin sequences of worms, fish, and Ciona. Examination of sequence variability at functionally significant sites, including the serine-518 residue, the phosphorylation of which modulates merlin's intra-molecular association and function as a tumor suppressor, identifies several potentially important sites that are conserved among all merlin proteins but divergent in the ERM proteins. Secondary structure prediction reveals the presence of a conserved α-helical domain in the central to C-terminal region of the merlin proteins of various species. The

  16. Flotillins interact with PSGL-1 in neutrophils and, upon stimulation, rapidly organize into membrane domains subsequently accumulating in the uropod.

    Directory of Open Access Journals (Sweden)

    Jérémie Rossy

    -dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1. CONCLUSIONS/SIGNIFICANCE: These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1.

  17. A monoclonal antibody toolkit for C. elegans.

    Directory of Open Access Journals (Sweden)

    Gayla Hadwiger

    Full Text Available BACKGROUND: Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms. METHODOLOGY/PRINCIPAL FINDINGS: We report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1, a component of synaptic vesicles; to Rim (UNC-10, a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1, a component of centrosomes; to CENP-C (HCP-4, which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2, a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5; to the nuclear envelope protein lamin (LMN-1; to EHD1 (RME-1 a marker for recycling endosomes; to caveolin (CAV-1, a marker for caveolae; to the cytochrome P450 (CYP-33E1, a resident of the endoplasmic reticulum; to beta-1,3-glucuronyltransferase (SQV-8 that labels the Golgi; to a chaperonin (HSP-60 targeted to mitochondria; to LAMP (LMP-1, a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7 of the 26S proteasome; to dynamin (DYN-1 and to the alpha-subunit of the adaptor complex 2 (APA-2 as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1 and cadherin (HMR-1, both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1, which localized to apical membranes; to an ERBIN family protein (LET-413 which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7 which localizes to the plasma membrane at cell-cell contacts. In addition to

  18. Biological characteristics of Echinococcus f elidis%狮棘球绦虫(Echinococcus felidis)生物学特性研究进展

    Institute of Scientific and Technical Information of China (English)

    刘聪暖; 娄忠子; 李立; 范彦雷; 闫鸿斌; 贾万忠

    2014-01-01

    This review focuses on biological characteristics of Echinococcus f elidis including molecular genetic markers , species status ,host coverage ,geographical distribution ,epidemiological implications ,phylogeny ,and evolution .The molecular genetic markers are involved in mitochondrial cox1 (cytochrome C oxidase subunit 1) and nad1 (nicotinamide adenine dinucle-otide dehydrogenase subunit 1) genes ,nuclear protein-coding gene sequences such as elp (ezrin-radixin-moesin-like protein) , e f1a (elongation factor 1 alpha) ,pepck (phosphoenolpyruvate carboxykinase ) ,pold (DNA polymerase delta ) ,and ribosome RNA gene sequences such as ITS1 and 18S rRNA .The establishment of species status is based on distinctly discriminated mor-phological characteristics such as hooks on the rostrum with apparent rugae ,the special definitive host (lions) ,and divergence of DNA sequences ,etc .between E . f elidis and other Echinococcus species .In brief ,the review has provided researchers and ex-perts in the field of echinococcosis with fundamental background knowledge and guidelines for future research directions ,clinical and epidemiological investigations ,and prevention and control of echinococcosis .%本文比较了狮棘球绦虫(Echinococcus f elidis)线粒体cox1(细胞色素C氧化酶亚基1)和 nad1(烟酰胺腺嘌呤二核苷酸脱氢酶亚单位1)基因片段序列,核基因编码基因 elp(埃兹-根蛋白-膜突蛋白样蛋白)、e f1a(延伸因子1α)、pepck(磷酸烯醇式丙酮酸羧激酶)和 pold(DNA 聚合酶δ)序列,核糖体rRNA基因(ITS1和18S rRNA)序列,以及狮棘球绦虫与其它棘球绦虫之间的DNA序列的差异度,并简述了其吻钩皱褶明显的形态学特征和以独特的狮类为终末宿主的生物学特性。通过对狮棘球绦虫分子遗传标记特征、种地位的确立、种系发生、鉴定方法、宿主范围、地理分布、流行病学特征和未来研究方向的阐述,为

  19. Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins.

    Science.gov (United States)

    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.

  20. Vesicular transport of progeny parvovirus particles through ER and Golgi regulates maturation and cytolysis.

    Science.gov (United States)

    Bär, Séverine; Rommelaere, Jean; Nüesch, Jürg P F

    2013-09-01

    Progeny particles of non-enveloped lytic parvoviruses were previously shown to be actively transported to the cell periphery through vesicles in a gelsolin-dependent manner. This process involves rearrangement and destruction of actin filaments, while microtubules become protected throughout the infection. Here the focus is on the intracellular egress pathway, as well as its impact on the properties and release of progeny virions. By colocalization with cellular marker proteins and specific modulation of the pathways through over-expression of variant effector genes transduced by recombinant adeno-associated virus vectors, we show that progeny PV particles become engulfed into COPII-vesicles in the endoplasmic reticulum (ER) and are transported through the Golgi to the plasma membrane. Besides known factors like sar1, sec24, rab1, the ERM family proteins, radixin and moesin play (an) essential role(s) in the formation/loading and targeting of virus-containing COPII-vesicles. These proteins also contribute to the transport through ER and Golgi of the well described analogue of cellular proteins, the secreted Gaussia luciferase in absence of virus infection. It is therefore likely that radixin and moesin also serve for a more general function in cellular exocytosis. Finally, parvovirus egress via ER and Golgi appears to be necessary for virions to gain full infectivity through post-assembly modifications (e.g. phosphorylation). While not being absolutely required for cytolysis and progeny virus release, vesicular transport of parvoviruses through ER and Golgi significantly accelerates these processes pointing to a regulatory role of this transport pathway.

  1. Semaphorin 4A enhances lung fibrosis through activation of Akt via PlexinD1 receptor

    Indian Academy of Sciences (India)

    Hai-Ying Peng; Wei Gao; Fa-Rong Chong; Hong-Yan Liu; Ji Zhang

    2015-12-01

    Semaphorin 4A plays a regulatory role in immune function and angiogenesis. However, its specific involvement in controlling lung fibrosis, a process that is closely related to angiogenesis and inflammation is still poorly understood. In the present study, we show that treatment of Sema4A on normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including a-smooth muscle actin (-SMA), ezrin, moesin, and paxillin. We confirm that Sema4A enhances the ability of lung fibroblasts to contract collagen gel. Sema4A treatment led to resistance to apoptosis in normal lung fibroblasts. Relative to normal lung fibroblasts, fibroblasts cultured from scars of patients with the flbrotic disease Systemic Sclerosis (SSc) showed elevated Sema4A secretion, enhanced -SMA, ezrin, moesin, and paxillin expression, and high ability to induce collagen gel contraction. Using neutralizing antibody against Sema4A receptor, PlexinD1, we found that endogenous Sema4A signalling in SSc fibroblast was through PlexinD1 receptor. We then identified the signalling mechanism through which Sema4A-PlexinD1 promotes the ability of normal fibroblasts to contract a collagen gel matrix. Western blot analysis showed that Sema4A activated the Akt pathway in lung fibroblasts, and the specific inhibitor of Akt pathway, Akt inhibitor III, blocked the ability of Sema4A to promote the ability of lung fibroblasts to contract a collagen gel matrix. Thus, blocking Sema4A-PlexinD1-Akt cascades might be beneficial in reducing pulmonary fibrosis.

  2. Syntenin-1 and ezrin proteins link activated leukocyte cell adhesion molecule to the actin cytoskeleton

    NARCIS (Netherlands)

    Tudor, C.; Riet, J. te; Eich, C.; Harkes, R.; Smisdom, N.; Bouhuijzen-Wenger, J.; Ameloot, M.; Holt, M.; Kanger, J.S.; Figdor, C.G.; Cambi, A.; Subramaniam, V.

    2014-01-01

    Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both

  3. Ezrin mediates c-Myc actions in prostate cancer cell invasion

    DEFF Research Database (Denmark)

    Chuan, Yin Choy; Iglesias Gato, Diego; Fernandez-Perez, L

    2010-01-01

    The forced overexpression of c-Myc in mouse prostate and in normal human prostate epithelial cells results in tumor transformation with an invasive phenotype. How c-Myc regulates cell invasion is poorly understood. In this study, we have investigated the interplay of c-Myc and androgens in the re...

  4. Regulation of ErbB2 localization and function in breast cancer cells by ERM proteins

    Science.gov (United States)

    Asp, Nagham; Kvalvaag, Audun; Sandvig, Kirsten; Pust, Sascha

    2016-01-01

    The ERM protein family is implicated in processes such as signal transduction, protein trafficking, cell proliferation and migration. Consequently, dysregulation of ERM proteins has been described to correlate with carcinogenesis of different cancer types. However, the underlying mechanisms are poorly understood. Here, we demonstrate a novel functional interaction between ERM proteins and the ErbB2 receptor tyrosine kinase in breast cancer cells. We show that the ERM proteins ezrin and radixin are associated with ErbB2 receptors at the plasma membrane, and depletion or functional inhibition of ERM proteins destabilizes the interaction of ErbB2 with ErbB3, Hsp90 and Ebp50. Accompanied by the dissociation of this protein complex, binding of ErbB2 to the ubiquitin-ligase c-Cbl is increased, and ErbB2 becomes dephosphorylated, ubiquitinated and internalized. Furthermore, signaling via Akt- and Erk-mediated pathways is impaired upon ERM inhibition. Finally, interference with ERM functionality leads to receptor degradation and reduced cellular levels of ErbB2 and ErbB3 receptors in breast cancer cells. PMID:27029001

  5. Mst4 and Ezrin Induce Brush Borders Downstream of the Lkb1/Strad/Mo25 Polarization Complex

    NARCIS (Netherlands)

    ten Klooster, Jean Paul; Jansen, Marnix; Yuan, Jin; Oorschot, Viola; Begthel, Harry; Di Giacomo, Valeria; Colland, Frederic; de Koning, John; Maurice, Madelon M.; Hornbeck, Peter; Clevers, Hans

    2009-01-01

    The human Lkb1 kinase, encoded by the ortholog of the invertebrate Par4 polarity gene, is mutated in Peutz-Jeghers cancer syndrome. Lkb1 activity requires complex formation with the pseudokinase Strad and the adaptor protein Mo25. The complex can induce complete polarization in a single isolated int

  6. The Impact of Altered Gravity and Vibration on Endothelial Cells During a Parabolic Flight

    Directory of Open Access Journals (Sweden)

    Markus Wehland

    2013-03-01

    Full Text Available Background: Endothelial cells (EC cultured under altered gravity conditions show a cytoskeletal disorganization and differential gene expression (short-term effects, as well as apoptosis in adherently growing EC or formation of tubular 3D structures (long-term effects. Methods: Investigating short-term effects of real microgravity, we exposed EC to parabolic flight maneuvers and analysed them on both protein and transcriptional level. The effects of hypergravity and vibration were studied separately. Results: Pan-actin and tubulin proteins were elevated by vibration and down-regulated by hypergravity. β-Actin was reduced by vibration. Moesin protein was reduced by both vibration and hypergravity, ezrin potein was strongly elevated under vibration. Gene expression of ACTB, CCND1, CDC6, CDKN1A, VEGFA, FLK-1, EZR, ITBG1, OPN, CASP3, CASP8, ANXA2, and BIRC5 was reduced under vibration. With the exception of CCNA2, CCND1, MSN, RDX, OPN, BIRC5, and ACTB all investigated genes were downregulated by hypergravity. After one parabola (P CCNA2, CCND1, CDC6, CDKN1A, EZR, MSN, OPN, VEGFA, CASP3, CASP8, ANXA1, ANXA2, and BIRC5 were up-, while FLK1 was downregulated. EZR, MSN, OPN, ANXA2, and BIRC5 were upregulated after 31P. Conclusions: Genes of the cytoskeleton, angiogenesis, extracellular matrix, apoptosis, and cell cycle regulation were affected by parabolic flight maneuvers. We show that the microgravity stimulus is stronger than hypergravity/vibration.

  7. Amaranthus leucocarpus lectin recognizes a moesin-like O-glycoprotein and costimulates murine CD3-activated CD4+ T cells

    OpenAIRE

    Arenas-Del Ángel, Maria; Legorreta-Herrera, Martha; Mendoza-Hernández, Guillermo; Garfias, Yonathan; Chávez, Raul; Zenteno, Edgar; Lascurain,Ricardo

    2015-01-01

    The Galβ1,3GalNAcα1,O-Ser/Thr specific lectin from Amaranthus leucocarpus (ALL) binds a ∼70 kDa glycoprotein on murine T cell surface. We show that in the absence of antigen presenting cells, murine CD4+ T cells activated by an anti-CD3 antibody plus ALL enhanced cell proliferation similar to those cells activated via CD3/CD28 at 48 h of culture. Moreover, ALL induced the production of IL-4, IL-10, TNF-alpha, and TGF-beta in CD3-activated cells. Proteomic assay using two-dimensional electroph...

  8. Tetracycline-inducible protein expression in pancreatic cancer cells: Effects of CapG overexpression

    Institute of Scientific and Technical Information of China (English)

    Sarah Tonack; Sabina Patel; Mehdi Jalali; Taoufik Nedjadi; Rosalind E Jenkins; Christopher Goldring; John Neoptolemos; Eithne Costello

    2011-01-01

    AIM: To establish stable tetracycline-inducible pancre-atic cancer cell lines.METHODS: Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetra-cycline-controlled transactivator protein (rtTA2S-M2), under the control of either a cytomegalovirus (CMV) or a chicken β-actin promoter, and the resulting clones were characterised.RESULTS: Use of the chicken (β-actin) promoter proved superior for both the production and mainte-nance of doxycycline-inducible cell lines. The system proved versatile, enabling transient inducible expression of a variety of genes, including GST-P, CYP2E1, S100A6, and the actin capping protein, CapG. To determine the physiological utility of this system in pancreatic cancer cells, stable inducible CapG expressors were established. Overexpressed CapG was localised to the cytoplasm and the nuclear membrane, but was not observed in the nu-cleus. High CapG levels were associated with enhanced motility, but not with changes to the cell cycle, or cellu-lar proliferation. In CapG-overexpressing cells, the levels and phosphorylation status of other actin-moduating proteins (Cofilin and Ezrin/Radixin) were not altered. However, preliminary analyses suggest that the levels of other cellular proteins, such as ornithine aminotransfer-ase and enolase, are altered upon CapG induction. CONCLUSION: We have generated pancreatic-cancer derived cell lines in which gene expression is fully con-trollable.

  9. Up-regulation of hnRNP A1, Ezrin, tubulin β-2C and Annexin A1 in sentinel lymph nodes of colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate the early metastasis-associated proteins in sentinel lymph node micrometastasis (SLNMM) of colorectal cancer (CRC) through comparative proteome. METHODS: Hydrophobic protein samples were extracted from individual-matched normal lymph nodes (NLN) and SLNMM of CRC. Differentially expressed protein spots were detected by two-dimensional electrophoresis and image analysis, and subsequently identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry-mass spectro...

  10. Advance in research on structure and function of FERM domain%FERM结构域的结构与功能研究进展

    Institute of Scientific and Technical Information of China (English)

    钱玉珺; 成向明; 郭军

    2012-01-01

    FERM是一种四叶苜蓿形蛋白结构域,广泛存在于多种功能蛋白,其结构保守,能实现对多种蛋白的功能调控.FERM结构域采用分子内结合方式能屏蔽蛋白C端活性位点,实现蛋白自身抑制.通过与其他结构域的结合或被磷酸化,FERM能改变蛋白质构象,暴露其催化位点.用作连接支架时,含FERM的蛋白调节众多膜相关因子及下游信号分子,参与细胞形态构建及信号转导.本综述以ERM[埃兹蛋白(ezrin),根蛋白(radixin)与膜突蛋白(moesin)]、局部黏着斑激酶(focal adhesion kinase,FAK)、Janus蛋白酪氨酸激酶(Janus protein tyrosine kinase,JAK)、Kindlin与FERM-FA蛋白为例,阐述FERM在不同蛋白中的生物学功能.%The FERM domain is a conserved protein module found in a number of proteins that can mediate protein-protein interactions. In the canonical FERM domain containing proteins, the FERM domain interacts directly with the C-termi-nal tail domain to mask binding sites for protein interaction. Interaction with selected molecules or phosphorylation of FERM can induce confonnational changes that unmask the full-length proteins and allow its interaction with the cytoplasmic domains. Thus, the FERM domain can act as an adaptor or scaffolding unit that integrates the activities of multiple membrane-associated factors and downstream molecules, participating in cellular morphogenesis and signal transduction. In this paper, the functions of FERM in ERM, FAK, JAK, Kindlin and FERM-FA are reviewed.

  11. Localization of actin in Moloney murine leukemia virus by immunoelectron microscopy.

    Science.gov (United States)

    Nermut, M V; Wallengren, K; Pager, J

    1999-07-20

    Immunoelectron microscopy was used to detect actin in wild-type (wt) Moloney murine leukemia virus (MoMuLV) and in virus-like particles (VLP) produced by recombinant Semliki Forest virus expressing only the MoMuLV gag polyprotein. Gold immunolabeling revealed the presence of actin on the surface of delipidized VLP and delipidized wt virus particles. Statistical evaluation of the number of colloidal gold particles per VLP revealed a large range of values and a prevalence of VLP with small numbers of gold particles. Labeling for actin was lost after prolonged treatment of VLP with 1% Nonidet-P40, high-pH buffer, or gelsolin. Gold immunolabeling with antibodies to gag proteins p15 (MA) and p12 and p30 (CA) was abundant and was not affected by treatment of VLP or wt virus with 1% Nonidet or gelsolin. VLP treated with a mixture of detergent and aldehyde fixatives showed more uniform and consistent labeling for actin than without fixatives. Negative staining or heavy metal shadowing revealed a globular surface of delipidized VLP. Stereomicrographs of gold-immunolabeled VLP showed that p15gag and p12gag were associated with the globular projections. Delipidized VLP were also well labeled with antibody to p30gag, which indicated that the gag shell permitted access of antibodies to p30gag and was therefore not a closely packed structure. Labeling for actin-binding proteins moesin and ezrin was negative in both the wt virus and the VLP. The absence of Gaussian distribution of actin in the sample of VLP suggests that actin is not a structural protein and its presence in MuLV virus particles may be fortuitous. This, however, does not rule out any possible role of actin in transport, assembly, budding, or release of virus particles, events which take place in the cytoplasm or at the plasma membrane. The site of actin in VLP is discussed in relation to the present knowledge of the molecular organization of the MuLV gag shell.

  12. Induction of IFNT-Stimulated Genes by Conceptus-Derived Exosomes during the Attachment Period.

    Directory of Open Access Journals (Sweden)

    Keigo Nakamura

    Full Text Available Biochemical and/or physical communication between the conceptus and the uterine endometrium is required for conceptus implantation to the maternal endometrium, leading to placentation and the establishment of pregnancy. We previously reported that in vitro co-culture system with bovine trophoblast CT-1 cells, primary uterine endometrial epithelial cells (EECs, and uterine flushings (UFs mimics in vivo conceptus attachment process. To identify molecules in UFs responsible for this change, we first characterized protein contents of UFs from day 17 cyclic (C17 and pregnant (P17 ewes through the use of two dimensional-Polyacrylamide Gel Electrophoresis (2D-PAGE, followed by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS analysis. These analyses identified 266 proteins specific for P17 UFs, from which 172 proteins were identified as exosomal proteins. Among 172 exosomal proteins, 8 proteins that had been identified as exosomal proteins were chosen for further analysis, including macrophage-capping protein (CAPG, aldo-keto reductase family 1, member B1 protein (AKR1B1, bcl-2-like protein 15 (BCL2L15, carbonic anhydrase 2 (CA2, isocitrate dehydrogenase 2 (IDH2, eukaryotic translation elongation factor 2 (EEF2, moesin (MSN, and ezrin (EZR. CAPG and AKR1B1 were again confirmed in P15 and P17 UFs, and more importantly CAPG and AKR1B1, mRNA and protein, were found only in P15 and P17 conceptuses. Moreover, exosomes were isolated from C15, C17, P15, or P17 UFs. Only P15 and P17 exosomes, originated from the conceptus, contained interferon tau (IFNT as well as CAPG and AKR1B1, and up-regulated STAT1, STAT2, MX1, MX2, BST2, and ISG15 transcripts in EECs. These observations indicate that in addition to endometrial derived exosomes previously described, conceptus-derived exosomes are present in UFs and could function to modify endometrial response. These results suggest that exosomes secreted from conceptuses as well as endometria are involved in cell

  13. 川芎嗪对脂多糖诱导的内皮细胞损伤的保护作用%Protective effect of tetramethylpyrazine on the injury endothelial cell induced by lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    杨锡兰; 李言; 王盼; 赵士弟; 陈前芬

    2016-01-01

    Objective:To explore the effects of tetramethylpyrazine( TMP) on the levels of RhoA,Rho associated coiled coil forming protein kinase 2(ROCK2),myosin light chain(MLC) phosphorylation and ezrin-radxin-moesin(ERM) phosphorylation in the injury of human umbilical vein endothelial cells ( HUVECs ) induced by lipopolysaccharide ( LPS ) . Methods:The HUVECs were cultured in vitro,and divided into the control group,LPS group and TMP groups(0. 5,1. 0,1. 5 mg/mL). The mRNA levels of RhoA,ROCK2 and p-EZR in endothelial cells were detected by real-time fluorescence quantitative PCR,and the protein levels of RhoA,ROCK2,p-MLC and p-ERM were examined using western blotting. Results:Compared with the control group, the protein levels of RhoA, ROCK2, p-MLC and p-ERM,and the mRNA levels of RhoA,ROCK2 and p-Ezr in LPS group significantly increased(P0. 05). Compared with the LPS group,the protein levels of ROCK2,p-MLC and p-ERM,and the mRNA levels of ROCK2 and p-Ezr decreased significantly in 3 TMP groups(P0.05),ROCK2、p-MLC和p-ERM蛋白及ROCK2、p-Ezr mRNA表达均降低(P<0.05~P<0.01),且TMP 3组之间ROCK2、p-MLC和p-ERM的分子表达差异均有统计学意义(P<0.05~P<0.01)。结论:TMP对LPS诱导的HUVEC损伤的保护作用可能是通过抑制Rho/ROCK信号通路中ROCK2、p-MLC和p-ERM的表达,以减弱LPS对内皮细胞骨架的损伤。

  14. Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability.

    Science.gov (United States)

    Murchie, Ryan; Guo, Cong-Hui; Persaud, Avinash; Muise, Aleixo; Rotin, Daniela

    2014-01-14

    Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ(-/-) mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ(-/-) mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ(-/-) mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper-tyrosine-phosphorylated in the PTPσ(-/-) mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ(-/-) mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD.

  15. Androgens Regulate T47D Cells Motility and Invasion through Actin Cytoskeleton Remodeling

    Science.gov (United States)

    Montt-Guevara, Maria Magdalena; Shortrede, Jorge Eduardo; Giretti, Maria Silvia; Giannini, Andrea; Mannella, Paolo; Russo, Eleonora; Genazzani, Alessandro David; Simoncini, Tommaso

    2016-01-01

    The relationship between androgens and breast cancer is controversial. Androgens have complex effects on breast cancer progression and metastasis. Moreover, androgen receptor (AR) is expressed in approximately 70 to 90% of invasive breast carcinomas, which has prognostic relevance in basal-like cancers and in triple-negative breast cancers. Recent studies have associated the actin-binding proteins of the ezrin–radixin–moesin (ERM) family with metastasis in endocrine-sensitive cancers. We studied on T47D breast cancer cells whether androgens with different characteristics, such as testosterone (T), dihydrotestosterone (DHT), and dehydroepiandrosterone (DHEA) may regulate breast cancer cell motility and invasion through the control of actin remodeling. We demonstrate that androgens promote migration and invasion in T47D via Moesin activation. We show that T and DHEA exert their actions via the AR and estrogen receptor (ER), while the non-aromatizable androgen – DHT – only recruits AR. We further report that androgen induced significant changes in actin organization with pseudopodia along with membrane ruffles formation, and this process is mediated by Moesin. Our work identifies novel mechanisms of action of androgens on breast cancer cells. Through the modulation of Moesin, androgens alter the architecture of cytoskeleton in T47D breast cancer cell and promote cell migration and invasion. These results could help to understand the biological actions of androgens on breast cancer and, eventually, to develop new strategies for breast cancer treatment. PMID:27746764

  16. Dinitrosopiperazine-Mediated Phosphorylated-Proteins Are Involved in Nasopharyngeal Carcinoma Metastasis

    Directory of Open Access Journals (Sweden)

    Gongjun Tan

    2014-11-01

    Full Text Available N,N'-dinitrosopiperazine (DNP with organ specificity for nasopharyngeal epithelium, is involved in nasopharyngeal carcinoma (NPC metastasis, though its mechanism is unclear. To reveal the pathogenesis of DNP-induced metastasis, immunoprecipitation was used to identify DNP-mediated phosphoproteins. DNP-mediated NPC cell line (6-10B motility and invasion was confirmed. Twenty-six phosphoproteins were increased at least 1.5-fold following DNP exposure. Changes in the expression levels of selected phosphoproteins were verified by Western-blotting analysis. DNP treatment altered the phosphorylation of ezrin (threonine 567, vimentin (serine 55, stathmin (serine 25 and STAT3 (serine 727. Furthermore, it was shown that DNP-dependent metastasis is mediated in part through ezrin at threonine 567, as DNP-mediated metastasis was decreased when threonine 567 of ezrin was mutated. Strikingly, NPC metastatic tumors exhibited a higher expression of phosphorylated-ezrin at threonine 567 than the primary tumors. These findings provide novel insight into DNP-induced NPC metastasis and may contribute to a better understanding of the metastatic mechanisms of NPC tumors.

  17. Sequential Dose-Dense Doxorubicin and Ifosfamide in Advanced Soft-Tissue Sarcoma Patients in an Out-Patient-Basis Schedule

    Directory of Open Access Journals (Sweden)

    G. F. G. Almeida

    2011-01-01

    Full Text Available Aims. This phase II study explored activity/safety of front-line dose-dense chemotherapy in high-grade STS (soft tissue sarcoma patients and tested ezrin as prognostic factor. Patients and Methods. The protocol consisted of three cycles of doxorubicin (DOXO 30 mg/m2 on days 1–3 every 2 weeks, followed by three cycles of ifosfamide (IFO 2.5 g/m2 two hours a day on days 1–5 every 3 weeks, with GCSF support. Ezrin was assessed immunohistochemically. Results. Twenty patients, 13 metastatic and 7 locally advanced, were enrolled. Median age was 39 years (25–60. Median dose intensities were 42 mg/m2/week and 3.6 g/m2/week for DOXO and IFO, respectively. Grade 3/4 toxicities occurred in 18 patients. Response rate was 15% (3 of 20 by RECIST. Patients younger than 45 years with locally advanced disease and synovial histology presented longer survival. A trend towards longer survival was observed among ezrin-positive patients. Conclusions. This dose-dense schedule should not be routinely used due to its high frequency of toxic events; however, a sequential strategy with DOXO and IFO may benefit selected patients and should be further explored with lower doses. The role of ezrin as a prognostic marker should be confirmed in a larger group of patients.

  18. The chloride intracellular channel 5A stimulates podocyte Rac1, protecting against hypertension-induced glomerular injury.

    Science.gov (United States)

    Tavasoli, Mahtab; Li, Laiji; Al-Momany, Abass; Zhu, Lin-Fu; Adam, Benjamin A; Wang, Zhixiang; Ballermann, Barbara J

    2016-04-01

    Glomerular capillary hypertension elicits podocyte remodeling and is a risk factor for the progression of glomerular disease. Ezrin, which links podocalyxin to actin in podocytes, is activated through the chloride intracellular channel 5A (CLIC5A)-dependent phosphatidylinositol 4,5 bisphosphate (PI[4,5]P2) accumulation. Because Rac1 is involved in podocyte actin remodeling and can promote PI[4,5]P2 production we determined whether CLIC5A-dependent PI[4,5]P2 generation and ezrin activation are mediated by Rac1. In COS7 cells, CLIC5A expression stimulated Rac1 but not Cdc42 or Rho activity. CLIC5A also stimulated phosphorylation of the Rac1 effector Pak1 in COS7 cells and in cultured mouse podocytes. CLIC5A-induced PI[4,5]P2 accumulation and Pak1 and ezrin phosphorylation were all Rac1 dependent. In DOCA/Salt hypertension, phosphorylated Pak increased in podocytes of wild-type, but not CLIC5-deficient mice. In DOCA/salt hypertensive mice lacking CLIC5, glomerular capillary microaneurysms were more frequent and albuminuria was greater than in wild-type mice. Thus, augmented hypertension-induced glomerular capillary injury in mice lacking CLIC5 results from abrogation of Rac1-dependent Pak and ezrin activation, perhaps reducing the tensile strength of the podocyte actin cytoskeleton.

  19. EGF-induced expansion of migratory cells in the rostral migratory stream.

    Directory of Open Access Journals (Sweden)

    Olle R Lindberg

    Full Text Available The presence of neural stem cells in the adult brain is currently widely accepted and efforts are made to harness the regenerative potential of these cells. The dentate gyrus of the hippocampal formation, and the subventricular zone (SVZ of the anterior lateral ventricles, are considered the main loci of adult neurogenesis. The rostral migratory stream (RMS is the structure funneling SVZ progenitor cells through the forebrain to their final destination in the olfactory bulb. Moreover, extensive proliferation occurs in the RMS. Some evidence suggest the presence of stem cells in the RMS, but these cells are few and possibly of limited differentiation potential. We have recently demonstrated the specific expression of the cytoskeleton linker protein radixin in neuroblasts in the RMS and in oligodendrocyte progenitors throughout the brain. These cell populations are greatly altered after intracerebroventricular infusion of epidermal growth factor (EGF. In the current study we investigate the effect of EGF infusion on the rat RMS. We describe a specific increase of radixin(+/Olig2(+ cells in the RMS. Negative for NG2 and CNPase, these radixin(+/Olig2(+ cells are distinct from typical oligodendrocyte progenitors. The expanded Olig2(+ population responds rapidly to EGF and proliferates after only 24 hours along the entire RMS, suggesting local activation by EGF throughout the RMS rather than migration from the SVZ. In addition, the radixin(+/Olig2(+ progenitors assemble in chains in vivo and migrate in chains in explant cultures, suggesting that they possess migratory properties within the RMS. In summary, these results provide insight into the adaptive capacity of the RMS and point to an additional stem cell source for future brain repair strategies.

  20. MicroRNA-200c: A Novel Way to Attack Breast Cancer Metastases by Restoring the Epithelial Phenotype

    Science.gov (United States)

    2012-02-01

    factor C promotes cervical cancer metastasis via up-regulation and activation of RhoA/ROCK-2/moesin cascade. BMC Cancer 2010, 10:170. Howe et al. Breast...and CKI p16 genes. Cancer Res 1997;57:4511–6. [97] Coffey RN, Watson RW, O’Neill AJ, Mc Eleny K, Fitzpatrick JM. Androgen- mediated resistance to...attack breast cancer metastases by restoring the epithelial phenotype. PRINCIPAL INVESTIGATOR: Jennifer Richer, Ph.D

  1. Comparative actions of progesterone, medroxyprogesterone acetate, drospirenone and nestorone on breast cancer cell migration and invasion

    Directory of Open Access Journals (Sweden)

    Sitruk-Ware Regine

    2008-06-01

    Full Text Available Abstract Background Limited information is available on the effects of progestins on breast cancer progression and metastasis. Cell migration and invasion are central for these processes, and require dynamic cytoskeletal and cell membrane rearrangements for cell motility to be enacted. Methods We investigated the effects of progesterone (P, medroxyprogesterone acetate (MPA, drospirenone (DRSP and nestorone (NES alone or with 17β-estradiol (E2 on T47-D breast cancer cell migration and invasion and we linked some of these actions to the regulation of the actin-regulatory protein, moesin and to cytoskeletal remodeling. Results Breast cancer cell horizontal migration and invasion of three-dimensional matrices are enhanced by all the progestins, but differences are found in terms of potency, with MPA being the most effective and DRSP being the least. This is related to the differential ability of the progestins to activate the actin-binding protein moesin, leading to distinct effects on actin cytoskeleton remodeling and on the formation of cell membrane structures that mediate cell movement. E2 also induces actin remodeling through moesin activation. However, the addition of some progestins partially offsets the action of estradiol on cell migration and invasion of breast cancer cells. Conclusion These results imply that P, MPA, DRSP and NES alone or in combination with E2 enhance the ability of breast cancer cells to move in the surrounding environment. However, these progestins show different potencies and to some extent use distinct intracellular intermediates to drive moesin activation and actin remodeling. These findings support the concept that each progestin acts differently on breast cancer cells, which may have relevant clinical implications.

  2. Proteome analysis and tissue array for profiling protein markers associated with type B thymoma subclassification

    Institute of Scientific and Technical Information of China (English)

    SUN Qiang-ling; FANG Wen-tao; FENG Jian; ZHANG Jie; YANG Xiao-hua; GU Zhi-tao; ZHU Lei; SHA Hui-fang

    2012-01-01

    Background The prognostic relevance of World Health Organization (WHO) subtypes within type B thymomas is still controversial.Understanding of the molecular characteristics of the different histologic types of thymomas will provide meaningful information for diagnosis and therapeutic management in type B thymoma.Methods Proteins extracted from twelve type B thymoma tissue specimens (six type B1 and six type B2) were analyzed by two-dimensional electrophoresis (2-DE) coupled with MALDI-TOF-MS.Differentially expressed proteins were then assayed in sixty-nine type B thymoma tissues (including B1,B2 and B3) by tissue array analysis with immunohistochemistry staining.The relationship of their expression with clinicopathological parameters,such as tumor stage or WHO classification,was estimated by Spearman's Rank Correlation Test.Results Sixteen differentially expressed proteins between type B1 and B2 thymoma tissues were identified.The differential levels of ezrin and glutathione S-transferase pi (GSTP1) were validated using immunohistochemistry staining.A statistically significant difference was observed in the positive rate of ezrin expression between type B1 thymoma and type B3 thymoma (Z=-2.963,P <0.01).Ezrin showed a tendency to be expressed in higher classification tumors from type B1 to B3.A statistical analysis demonstrated that type B2 and B3 tumors had significantly higher positive expression of GSTP1 than the B1 group (type B2 vs.B1:Z=-2.582,P ≤0.01; type B3 vs.B1:Z=-4.012,P≤0.001).The results also showed a strong correlation between GSTP1 and WHO type staging of B1 to B3 tumors (Spearman's correlation coefficient:0.633,P≤0.001).Statistical analysis showed that there was close correlation between GSTP1 and ezrin expression with the clinical stage (Spearman's correlation coefficients,ezrin:0.481,P <0.05; GSTP1:0.484,P <0.01).Conclusions Differentially expressed proteins between type B1 and B2 thymoma tissues were analyzed by comparative proteomic

  3. A molecular switch in the scaffold NHERF1 enables misfolded CFTR to evade the peripheral quality control checkpoint.

    Science.gov (United States)

    Loureiro, Cláudia A; Matos, Ana Margarida; Dias-Alves, Ângela; Pereira, Joana F; Uliyakina, Inna; Barros, Patrícia; Amaral, Margarida D; Matos, Paulo

    2015-05-19

    The peripheral protein quality control (PPQC) checkpoint removes improperly folded proteins from the plasma membrane through a mechanism involving the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70 interacting protein). PPQC limits the efficacy of some cystic fibrosis (CF) drugs, such as VX-809, that improve trafficking to the plasma membrane of misfolded mutants of the CF transmembrane conductance regulator (CFTR), including F508del-CFTR, which retains partial functionality. We investigated the PPQC checkpoint in lung epithelial cells with F508del-CFTR that were exposed to VX-809. The conformation of the scaffold protein NHERF1 (Na(+)/H(+) exchange regulatory factor 1) determined whether the PPQC recognized "rescued" F508del-CFTR (the portion that reached the cell surface in VX-809-treated cells). Activation of the cytoskeletal regulator Rac1 promoted an interaction between the actin-binding adaptor protein ezrin and NHERF1, triggering exposure of the second PDZ domain of NHERF1, which interacted with rescued F508del-CFTR. Because binding of F508del-CFTR to the second PDZ of NHERF1 precluded the recruitment of CHIP, the coexposure of airway cells to Rac1 activator nearly tripled the efficacy of VX-809. Interference with the NHERF1-ezrin interaction prevented the increase of efficacy of VX-809 by Rac1 activation, but the actin-binding domain of ezrin was not required for the increase in efficacy. Thus, rather than mainly directing anchoring of F508del-CFTR to the actin cytoskeleton, induction of ezrin activation by Rac1 signaling triggered a conformational change in NHERF1, which was then able to bind and stabilize misfolded CFTR at the plasma membrane. These insights into the cell surface stabilization of CFTR provide new targets to improve treatment of CF.

  4. Reversible bleb formation in mast cells stimulated with antigen is Ca2+-calmodulin –dependent and bleb size is regulated by ARF6

    OpenAIRE

    Yanase, Yukki; Carvou, Nicolas; Frohman, Michael A.; Cockcroft, Shamshad

    2009-01-01

    Abstract Mast cells stimulated with antigen undergo extensive changes in their cytoskeleton. We assess here the impact of actin-modifying drugs and report that in the presence of cytochalasin D, mast cells stop membrane ruffling but instead bleb. Bleb formation is reversible following washout of the cytochalasin D and occurs in an actin polymerization-dependent manner. Bleb formation is inhibited by expression of dominant-negative ezrin-T567D. Blebbing is also inhibited by blebbist...

  5. Inhibitory effect of berberine on human skin squamous cell carcinoma A431 cells.

    Science.gov (United States)

    Li, D X; Zhang, J; Zhang, Y; Zhao, P W; Yang, L M

    2015-09-08

    Berberine (BBR) is a natural alkaloid with significant anti-tumor activity against many types of cancer cells. In this study, we investigated the molecular mechanisms employed by BBR to repress the proliferation and growth of skin squamous cell carcinoma A431 cells. Berberine was reported to inhibit the proliferation of A431 cells in a dose- and time-dependent manner and was observed to induce a series of biochemical events, including the loss of mitochondrial membrane potential, release of cytochrome-c to cytosol, induction of proteins of the Bcl-2 family and caspases, and the cleavage of poly(ADP)-ribose polymerase. This suggested its ability to induce apoptosis. The results of a wound healing test revealed that berberine inhibited the migration of A431 cells. Ezrin was transfected into A431 cells by RNA interference. The level of expression of Ezrin in the transfected A431 cells was observed to decrease with berberine treatment, which suggested that berberine might inhibit the invasion of A431 cells through Ezrin. The results of this study demonstrated that berberine could potentially inhibit proliferation, induce apoptosis, and inhibit the invasion of A431 cells.

  6. [Implant placement in the aesthetic zone: the socket-shield-technique].

    Science.gov (United States)

    Lagas, L J; Pepplinkhuizen, J J F A A; Bergé, S J; Meijer, G J

    2015-01-01

    Following the extraction of an incisor in the maxilla, resorption of the -alveolar bone always occurs, especially on the buccal side. This often indicates that in the buccocervical area, insufficient bone is present to cover the dental implant. One treatment option is to carry out a bone transplant on the buccal side prior to or during the placement of the implant. An alternative way of supporting the buccocervical gingival is to leave the buccal part of the radixin situ, the so-called socket-shield technique. The results of this treatment for 16 consecutive patients were evaluated and revealed that the socket-shield technique produces good treatment results.

  7. Assessment of response to beta-blockers by expression of βArr2 and RhoA/ROCK2 in antrum mucosa in cirrhotic patients

    DEFF Research Database (Denmark)

    Trebicka, Jonel; von Heydebrand, Matthias; Lehmann, Jennifer

    2016-01-01

    and protein expression of Ras homolog family member A (RhoA), Rho-kinase (ROCK)2, beta-arrestin2 (βArr2), endothelial nitric oxide synthase (eNOS) and the phosphorylation of downstream effectors VASP and moesin were analyzed using PCR and Western blot. Further 21 patients on NSBB were evaluated...... in cirrhosis, these expression levels might also reflect hemodynamic response to NSBB. METHODS: Biopsies from the gastric and duodenal mucosa of 25 patients with cirrhosis were collected and the hepatic venous pressure gradient (HVPG) was measured before and after an acute propranolol challenge. Transcription...

  8. Role of TGF-beta1-independent changes in protein neosynthesis, p38alphaMAPK, and cdc42 in hydrogen peroxide-induced senescence-like morphogenesis

    DEFF Research Database (Denmark)

    Chrétien, Aline; Dierick, Jean-François; Delaive, Edouard;

    2008-01-01

    The role of TGF-beta1 in hydrogen peroxide-induced senescence-like morphogenesis has been described. The aim of this work was to investigate whether TGF-beta1-independent changes in protein synthesis are involved in this morphogenesis and to study possible mechanisms occurring earlier than TGF......-beta1 overexpression. Among the multiple TGF-beta1-independent changes in protein neosynthesis, followed or not by posttranslational modifications, identified by proteomic analysis herein, those of ezrin, L-caldesmon, and HSP27 were particularly studied. Rho-GTPase cdc42 was shown to be responsible...

  9. Emodin inhibits migration and invasion of DLD-1 (PRL-3) cells via inhibition of PRL-3 phosphatase activity.

    Science.gov (United States)

    Han, Young-Min; Lee, Su-Kyung; Jeong, Dae Gwin; Ryu, Seong Eon; Han, Dong Cho; Kim, Dae Keun; Kwon, Byoung-Mog

    2012-01-01

    Anthraquinones have been reported as phosphatase inhibitors. Therefore, anthraquinone derivatives were screened to identify a potent phosphatase inhibitor against the phosphatase of regenerating liver-3 (PRL-3). Emodin strongly inhibited phosphatase activity of PRL-3 with IC(50) values of 3.5μM and blocked PRL-3-induced tumor cell migration and invasion in a dose-dependent manner. Emodin rescued the phosphorylation of ezrin, which is a known PRL-3 substrate. The results of this study reveal that emodin is a PRL-3 inhibitor and a good lead molecule for obtaining a selective PRL-3 inhibitor.

  10. Dynamic Na+-H+ Exchanger Regulatory Factor-1 Association and Dissociation Regulate Parathyroid Hormone Receptor Trafficking at Membrane Microdomains*

    Science.gov (United States)

    Ardura, Juan A.; Wang, Bin; Watkins, Simon C.; Vilardaga, Jean-Pierre; Friedman, Peter A.

    2011-01-01

    Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic PDZ (postsynaptic density 95/disc large/zona occludens) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled receptors and other membrane-delimited proteins. The parathyroid hormone receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a G protein-coupled receptor harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the receptor and induces receptor aggregation in long lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates receptor trafficking and signaling in a spatially and temporally coordinated manner. PMID:21832055

  11. Dynamic Na+-H+ exchanger regulatory factor-1 association and dissociation regulate parathyroid hormone receptor trafficking at membrane microdomains.

    Science.gov (United States)

    Ardura, Juan A; Wang, Bin; Watkins, Simon C; Vilardaga, Jean-Pierre; Friedman, Peter A

    2011-10-07

    Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic PDZ (postsynaptic density 95/disc large/zona occludens) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled receptors and other membrane-delimited proteins. The parathyroid hormone receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a G protein-coupled receptor harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the receptor and induces receptor aggregation in long lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates receptor trafficking and signaling in a spatially and temporally coordinated manner.

  12. Identification of regulators of germ layer morphogenesis using proteomics in zebrafish.

    Science.gov (United States)

    Link, Vinzenz; Carvalho, Lara; Castanon, Irinka; Stockinger, Petra; Shevchenko, Andrej; Heisenberg, Carl-Philipp

    2006-05-15

    During vertebrate gastrulation, a well-orchestrated series of morphogenetic changes leads to the formation of the three germ layers: the ectoderm, mesoderm and endoderm. The analysis of gene expression patterns during gastrulation has been central to the identification of genes involved in germ layer formation. However, many proteins are regulated on a translational or post-translational level and are thus undetectable by gene expression analysis. Therefore, we developed a 2D-gel-based comparative proteomic approach to target proteins involved in germ layer morphogenesis during zebrafish gastrulation. Proteomes of ectodermal and mesendodermal progenitor cells were compared and 35 significantly regulated proteins were identified by mass spectrometry, including several proteins with predicted functions in cytoskeletal organization. A comparison of our proteomic results with data obtained in an accompanying microarray-based gene expression analysis revealed no significant overlap, confirming the complementary nature of proteomics and transcriptomics. The regulation of ezrin2, which was identified based on a reduction in spot intensity in mesendodermal cells, was independently validated. Furthermore, we show that ezrin2 is activated by phosphorylation in mesendodermal cells and is required for proper germ layer morphogenesis. We demonstrate the feasibility of proteomics in zebrafish, concluding that proteomics is a valuable tool for analysis of early development.

  13. Solid supported membranes doped with PIP2: influence of ionic strength and pH on bilayer formation and membrane organization.

    Science.gov (United States)

    Braunger, Julia A; Kramer, Corinna; Morick, Daniela; Steinem, Claudia

    2013-11-19

    Phosphoinositides and in particular L-α-phosphatidylinositol-4,5-bisphosphate (PIP2) are key lipids controlling many cellular events and serve as receptors for a large number of intracellular proteins. To quantitatively analyze protein-PIP2 interactions in vitro in a time-resolved manner, planar membranes on solid substrates are highly desirable. Here, we describe an optimized protocol to form PIP2 containing planar solid supported membranes on silicon surfaces by vesicle spreading. Supported lipid bilayers (SLBs) were obtained by spreading POPC/PIP2 (92:8) small unilamellar vesicles onto hydrophilic silicon substrates at a low pH of 4.8. These membranes were capable of binding ezrin, resulting in large protein coverage as concluded from reflectometric interference spectroscopy and fluorescence microscopy. As deduced from fluorescence microscopy, only under low pH conditions, a homogeneously appearing distribution of fluorescently labeled PIP2 molecules in the membrane was achieved. Fluorescence recovery after photobleaching experiments revealed that PIP2 is not mobile in the bottom layer of the SLBs, while PIP2 is fully mobile in the top layer with diffusion coefficients of about 3 μm(2)/s. This diffusion coefficient was considerably reduced by a factor of about 3 if ezrin has been bound to PIP2 in the membrane.

  14. Lineage tracing quantification reveals symmetric stem cell division in Drosophila male germline stem cells.

    Science.gov (United States)

    Salzmann, Viktoria; Inaba, Mayu; Cheng, Jun; Yamashita, Yukiko M

    2013-12-01

    In the homeostatic state, adult stem cells divide either symmetrically to increase the stem cell number to compensate stem cell loss, or asymmetrically to maintain the population while producing differentiated cells. We have investigated the mode of stem cell division in the testes of Drosophila melanogaster by lineage tracing and confirm the presence of symmetric stem cell division in this system. We found that the rate of symmetric division is limited to 1-2% of total germline stem cell (GSC) divisions, but it increases with expression of a cell adhesion molecule, E-cadherin, or a regulator of the actin cytoskeleton, Moesin, which may modulate adhesiveness of germ cells to the stem cell niche. Our results indicate that the decision regarding asymmetric vs. symmetric division is a dynamically regulated process that contributes to tissue homeostasis, responding to the needs of the tissue.

  15. Molecules involved in epithelial-mesenchymal transition and epithelial-stromal interaction in phyllodes tumors: implications for histologic grade and prognosis.

    Science.gov (United States)

    Kwon, Ji Eun; Jung, Woo-Hee; Koo, Ja Seung

    2012-06-01

    The aim of this study was to investigate the expression of molecules associated with epithelial-mesenchymal transition (EMT) and epithelial-stromal interactions (ESI) and to evaluate their roles in phyllodes tumors (PTs). Tissue microarrays (TMAs) were constructed from 207 PT specimens (157 benign, 34 borderline and 16 malignant). The presence of EMT-related markers including N-cadherin, Twist, TGF-beta, HMGA2, S100A4 and Ezrin as well as ESI-related molecules such as SDF1 and CXCR4 among the TMAs was assessed immunohistochemically. Immunohistochemical results were analyzed in terms of clinicopathologic parameters. For higher grade PTs, expressions of Twist (p EMT-associated molecules, and CXCR4, an ESI-associated molecule, were increased in the stromal component of advanced grade PTs. Further, high expression of Twist in the stromal component was correlated with poorer prognoses.

  16. Rhodanine-based PRL-3 inhibitors blocked the migration and invasion of metastatic cancer cells.

    Science.gov (United States)

    Min, Garam; Lee, Su-Kyung; Kim, Hye-Nan; Han, Young-Min; Lee, Rhan-Hee; Jeong, Dae Gwin; Han, Dong Cho; Kwon, Byoung-Mog

    2013-07-01

    PRL-3, phosphatase of regenerating liver-3, plays a role in cancer progression through its involvement in invasion, migration, metastasis, and angiogenesis. We synthesized rhodanine derivatives, CG-707 and BR-1, which inhibited PRL-3 enzymatic activity with IC50 values of 0.8 μM and 1.1 μM, respectively. CG-707 and BR-1 strongly inhibited the migration and invasion of PRL-3 overexpressing colon cancer cells without exhibiting cytotoxicity. The specificity of the inhibitors on PRL-3 phosphatase activity was confirmed by the phosphorylation recovery of known PRL-3 substrates such as ezrin and cytokeratin 8. The compounds selectively inhibited PRL-3 in comparison with other phosphatases, and CG-707 regulated epithelial-to-mesenchymal transition (EMT) marker proteins. The results of the present study reveal that rhodanine is a specific PRL-3 inhibitor and a good lead molecule for obtaining a selective PRL-3 inhibitor.

  17. The brain-tumor related protein podoplanin regulates synaptic plasticity and hippocampus-dependent learning and memory.

    Science.gov (United States)

    Cicvaric, Ana; Yang, Jiaye; Krieger, Sigurd; Khan, Deeba; Kim, Eun-Jung; Dominguez-Rodriguez, Manuel; Cabatic, Maureen; Molz, Barbara; Acevedo Aguilar, Juan Pablo; Milicevic, Radoslav; Smani, Tarik; Breuss, Johannes M; Kerjaschki, Dontscho; Pollak, Daniela D; Uhrin, Pavel; Monje, Francisco J

    2016-12-01

    Podoplanin is a cell-surface glycoprotein constitutively expressed in the brain and implicated in human brain tumorigenesis. The intrinsic function of podoplanin in brain neurons remains however uncharacterized. Using an established podoplanin-knockout mouse model and electrophysiological, biochemical, and behavioral approaches, we investigated the brain neuronal role of podoplanin. Ex-vivo electrophysiology showed that podoplanin deletion impairs dentate gyrus synaptic strengthening. In vivo, podoplanin deletion selectively impaired hippocampus-dependent spatial learning and memory without affecting amygdala-dependent cued fear conditioning. In vitro, neuronal overexpression of podoplanin promoted synaptic activity and neuritic outgrowth whereas podoplanin-deficient neurons exhibited stunted outgrowth and lower levels of p-Ezrin, TrkA, and CREB in response to nerve growth factor (NGF). Surface Plasmon Resonance data further indicated a physical interaction between podoplanin and NGF. This work proposes podoplanin as a novel component of the neuronal machinery underlying neuritogenesis, synaptic plasticity, and hippocampus-dependent memory functions. The existence of a relevant cross-talk between podoplanin and the NGF/TrkA signaling pathway is also for the first time proposed here, thus providing a novel molecular complex as a target for future multidisciplinary studies of the brain function in the physiology and the pathology. Key messages Podoplanin, a protein linked to the promotion of human brain tumors, is required in vivo for proper hippocampus-dependent learning and memory functions. Deletion of podoplanin selectively impairs activity-dependent synaptic strengthening at the neurogenic dentate-gyrus and hampers neuritogenesis and phospho Ezrin, TrkA and CREB protein levels upon NGF stimulation. Surface plasmon resonance data indicates a physical interaction between podoplanin and NGF. On these grounds, a relevant cross-talk between podoplanin and NGF as well

  18. Differential molecular profiles of astrocytes in degeneration and re-innervation after sensory deafferentation of the adult rat cochlear nucleus.

    Science.gov (United States)

    Fredrich, Michaela; Zeber, Anne C; Hildebrandt, Heika; Illing, Robert-Benjamin

    2013-07-01

    Ablating the cochlea causes total sensory deafferentation of the cochlear nucleus. Over the first postoperative week, degeneration of the auditory nerve and its synaptic terminals in the cochlear nucleus temporally overlaps with its re-innervation by axon collaterals of medial olivocochlear neurons. At the same time, astrocytes increase in size and density. We investigated the time courses of the expression of ezrin, polysialic acid, matrix metalloprotease-9 and matrix metalloprotease-2 within these astrocytes during the first week following cochlear ablation. All four proteins are known to participate in degeneration, regeneration, or both, following injury of the central nervous system. In a next step, stereotaxic injections of kainic acid were made into the ventral nucleus of the trapezoid body prior to cochlear ablation to destroy the neurons that re-innervate the deafferented cochlear nucleus by axon collaterals developing growth-associated protein 43 immunoreactivity. This experimental design allowed us to distinguish between molecular processes associated with degeneration and those associated with re-innervation. Under these conditions, astrocytic growth and proliferation showed an unchanged deafferentation-induced pattern. Similarly, the distribution and amount of ezrin and matrix metalloprotease-9 in astrocytes after cochlear ablation developed in the same way as under cochlear ablation alone. In sharp contrast, the astrocytic expression of polysialic acid and matrix metalloprotease-2 normally invoked by cochlear ablation collapsed when re-innervation of the cochlear nucleus was inhibited by lesioning medial olivocochlear neurons with kainic acid. In conclusion, re-innervation, including axonal growth and synaptogenesis, seems to prompt astrocytes to recompose their molecular profile, paving the way for tissue reorganisation after nerve degeneration and loss of synaptic contacts.

  19. Correlation of microRNA-124 expression in cervical cancer tissue with cancer cell growth and invasion

    Institute of Scientific and Technical Information of China (English)

    Yi Zhu

    2016-01-01

    Objective:To study the correlation of microRNA-124 expression in cervical cancer tissue with cancer cell growth and invasion.Methods: A total of 56 cases of cervical cancer tissue samples and 60 cases of normal cervical tissue samples were selected for study, and microRNA-124 expression levels as well as protein content of proliferation, apoptosis and invasion genes in cervical tissue samples were determined.Results: The relative expression level of miR-124 in cervical cancer tissue was significantly lower than that in normal cervical tissue and the higher the FIGO staging, the lower the relative expression level of miR-124; cervical cancer tissue with different miR-124 expression was divided into group A-D according to quartile, there were differences in the protein content of cyclinD1, CDK4, CDK6, Prdx4, TNFAIP8, Piwil2, p16, p27, Caspase-3, Ezrin, CD44v6, E-cadherin andβ-catenin in cervical cancer tissue of group A, B, C and D, and the lower the relative expression level of miR-124, the higher the protein content of cyclinD1, CDK4, CDK6, Prdx4, TNFAIP8, Piwil2 as well as Ezrin and CD44v6, and the lower the protein content of p16, p27, Caspase-3 as well as E-cadherin andβ-catenin.Conclusions: microRNA-124 shows a trend of lower expression in cervical cancer tissue and is closely related to the excessive proliferation, insufficient apoptosis and invasive growth of cancer cells.

  20. Augmentation of CAR T-cell Trafficking and Antitumor Efficacy by Blocking Protein Kinase A Localization.

    Science.gov (United States)

    Newick, Kheng; O'Brien, Shaun; Sun, Jing; Kapoor, Veena; Maceyko, Steven; Lo, Albert; Puré, Ellen; Moon, Edmund; Albelda, Steven M

    2016-06-01

    Antitumor treatments based on the infusion of T cells expressing chimeric antigen receptors (CAR T cells) are still relatively ineffective for solid tumors, due to the presence of immunosuppressive mediators [such as prostaglandin E2 (PGE2) and adenosine] and poor T-cell trafficking. PGE2 and adenosine activate protein kinase A (PKA), which then inhibits T-cell receptor (TCR) activation. This inhibition process requires PKA to localize to the immune synapse via binding to the membrane protein ezrin. We generated CAR T cells that expressed a small peptide called the "regulatory subunit I anchoring disruptor" (RIAD) that inhibits the association of PKA with ezrin, thus blunting the negative effects of PKA on TCR activation. After exposure to PGE2 or adenosine in vitro, CAR-RIAD T cells showed increased TCR signaling, released more cytokines, and showed enhanced killing of tumor cells compared with CAR T cells. When injected into tumor-bearing mice, the antitumor efficacy of murine and human CAR-RIAD T cells was enhanced compared with that of CAR T cells, due to resistance to tumor-induced hypofunction and increased T-cell infiltration of established tumors. Subsequent in vitro assays showed that both mouse and human CAR-RIAD cells migrated more efficiently than CAR cells did in response to the chemokine CXCL10 and also had better adhesion to various matrices. Thus, the intracellular addition of the RIAD peptide to adoptively transferred CAR T cells augments their efficacy by increasing their effector function and by improving trafficking into tumor sites. This treatment strategy, therefore, shows potential clinical application for treating solid tumors. Cancer Immunol Res; 4(6); 541-51. ©2016 AACR.

  1. The β5/focal adhesion kinase/glycogen synthase kinase 3β integrin pathway in high-grade osteosarcoma: a protein expression profile predictive of response to neoadjuvant chemotherapy.

    Science.gov (United States)

    Le Guellec, Sophie; Moyal, Elizabeth Cohen-Jonathan; Filleron, Thomas; Delisle, Marie-Bernadette; Chevreau, Christine; Rubie, Hervé; Castex, Marie-Pierre; de Gauzy, Jerome Sales; Bonnevialle, Paul; Gomez-Brouchet, Anne

    2013-10-01

    To date, chemosensitivity to neoadjuvant chemotherapy of patients with high-grade osteosarcoma is evaluated on surgical resection by evaluation of the percentage of necrotic cells. As yet, no predictive profile of response to chemotherapy has been used in clinical practice. Because we have previously shown that the integrin pathway controls genotoxic-induced cell death and hypoxia, we hypothesized that in primary biopsies, expression of proteins involved in this pathway could be associated with sensitivity to neoadjuvant chemotherapy in high-grade osteosarcoma. We studied β1, β3, and β5 integrin expression and integrin-linked kinase, focal adhesion kinase (FAK), glycogen synthase kinase 3β (GSK3β), Rho B, angiopoietin-2, β-catenin, and ezrin expression by immunohistochemistry in 36 biopsies of osteosarcomas obtained before treatment. All patients received a chemotherapy regimen in the neoadjuvant setting. An immunoreactive score was assessed, combining the percentage of positive tumor cells and staining intensity. We evaluated the correlation of the biomarkers with response to chemotherapy, metastasis-free survival, and overall survival. A combination of 3 biomarkers (β5 integrin, FAK, and GSK3β) discriminated good and poor responders to chemotherapy, with the highest area under the curve (89.9%; 95% confidence interval, 77.4-1.00) and a diagnostic accuracy of 90.3%. Moreover, high expression of ezrin was associated with an increased risk of metastasis (hazard ratio, 3.93; 95% confidence interval, 1.19-12.9; P = .024). We report a protein expression profile in high-grade osteosarcoma associating β5 integrin, FAK, and GSK3β that significantly correlates with poor response to neoadjuvant chemotherapy. This biomarker profile could help select patients for whom an alternative protocol using inhibitors of this pathway can be proposed.

  2. Essential role for NHERF in cAMP-mediated inhibition of the Na+-HCO3- co-transporter in BSC-1 cells.

    Science.gov (United States)

    Weinman, E J; Evangelista, C M; Steplock, D; Liu, M Z; Shenolikar, S; Bernardo, A

    2001-11-01

    Prior studies have indicated a requirement for the PDZ domain-containing protein, Na(+)/H(+) Exchanger Regulatory Factor (NHERF), for protein kinase A (PKA)-mediated inhibition of the renal basolateral Na(+)-HCO(3)(-) co-transporter (NBC). The present studies explore the potential mechanisms by which NHERF transduces cAMP signals to inhibit NBC. In BSC-1 cells, cells that express NBC but lack NHERF, 8-bromo-cAMP (100 microm for 15 min) failed to inhibit transport until wild-type mNHERF-(1-355) was expressed. mNHERF-(116-355) containing PDZ II and C-terminal ezrin-binding sequences or a mutant unphosphorylated form of rabbit NHERF effectively transduced the cAMP signals that inhibited NBC. By contrast, mNHERF-(1-126) encompassing N-terminal PDZ I and mNHERF-(1-325), which lacks ezrin-binding, failed to support cAMP inhibition of NBC activity. NBC and NHERF did not associate with each other in yeast two-hybrid or co-immunoprecipitation assays, and confocal microscopy indicated distinct subcellular localization of the two proteins. NBC was phosphorylated in BSC-1 cells, but its phosphorylation was not increased by cAMP nor was immunoprecipitated NBC phosphorylated by PKA in vitro. Acute exposure of mNHERF-(1-355)-expressing BSC-1 cells to cAMP did not change cell surface expression of NBC. Although these results established an essential role for NHERF in cAMP-mediated inhibition of NBC in BSC-1 cells, they also suggest a novel mechanism for NHERF-mediated signal transduction distinct from that previously characterized from studies of other NHERF targets.

  3. RETRACTED: Downregulation of miR-204 expression correlates with poor clinical outcome of glioma patients.

    Science.gov (United States)

    Ye, Zhen-Nan; Liu, Jing-Peng; Wu, Ling-Yun; Zhang, Xiang-Sheng; Zhuang, Zong; Chen, Qiang; Lu, Yue; Liu, Ce-Gang; Zhang, Zi-Huan; Zhang, Hua-Sheng; Hou, Wen-Zhong; Hang, Chun-Hua

    2017-05-01

    Glioma is the most common type of malignant neoplasm in the central nervous system, with high incidence and mortality rate. MicroRNAs, as a class of small noncoding RNAs, play an important role in carcinogenesis and correlate with glioma diagnosis and prognosis. In this study, we investigated the microRNA-204 (miR-204) concentration in glioma tissues and its relation to the expression of ezrin and bcl-2 mRNA, as well as its potential predictive and prognostic values in glioma. The concentrations of miR-204 were significantly lower in glioma tissues than in nontumor brain tissues and also were lower in high-grade than in low-grade gliomas (World Health Organization grades III and IV versus grades I and II). The miR-204 concentration was inversely correlated with the ezrin and bcl-2 concentrations. The miR-204 concentration was classified as high or low according to the median value, and low miR-204 correlated with higher World Health Organization grade, larger tumor, and worse Karnofsky performance score. Kaplan-Meier survival analysis demonstrated that patients with low miR-204 expression had shorter progression-free survival and overall survival than patients with high miR-204 expression. In addition, univariate and multivariate analyses showed that miR-204 expression was an independent prognostic feature of overall survival and progression-free survival. In conclusion, our study indicates that miR-204 is downregulated in glioma and may be a biomarker of poor prognosis in patients with this cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Advances in molecular genetics of neurofibromatosis type 2%神经纤维瘤病Ⅱ型的分子遗传研究进展

    Institute of Scientific and Technical Information of China (English)

    李婷婷; 王超

    2010-01-01

    神经纤维瘤病Ⅱ型(neurofibromatosis type 2,NF2)是由于抑癌基因NF2突变而导致的常染色体显性遗传的多发肿瘤综合征.神经纤维瘤病Ⅱ型以神经系统肿瘤、皮肤肿瘤和晶体损害为临床特征.临床表现为双侧听神经瘤、脑膜瘤、脊髓肿瘤、周围神经肿瘤,白内障也很常见.NF2基因定位于染色体22q12.2,编码的蛋白质称为mertin(moesinezrin-radixin like protean)或schwannomin.异常的metlin影响体内的促有丝分裂途径导致肿瘤的发生.本文就其病变的分子遗传研究进展进行综述.

  5. A Critical Evaluation of Liver Pathology in Humans with Danon Disease and Experimental Correlates in a Rat Model of LAMP-2 Deficiency.

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    Wang, Lu; Wang, Jingbo; Cai, Weile; Shi, Yongquan; Zhou, Xinmin; Guo, Guanya; Guo, Changcun; Huang, Xiaofeng; Han, Zheyi; Zhang, Shuai; Ma, Shuoyi; Zhou, Xia; Fan, Daiming; Eric Gershwin, M; Han, Ying

    2017-01-26

    Danon disease is a genetic deficiency in lysosome-associated membrane protein 2 (LAMP-2), a highly glycosylated constituent of the lysosomal membrane and characterized by a cardiomyopathy, skeletal muscle myopathy, and cognitive impairment. Patients, however, often manifest hepatic abnormalities, but liver function has not been well evaluated and the syndrome is relatively uncommon. Hence, we have taken advantage of a rat that has been deleted of LAMP-2 to study the relative role of LAMP-2 on liver function. Interestingly, rats deficient in LAMP-2 develop a striking increase in serum alkaline phosphatase (ALP) and a decrease in bile flow compared with wild-type littermates. Importantly and by ultrastructural analysis, deficient rats manifest dilated canaliculi that lack microvilli with evidence of bile-containing bodies. Moreover, following bile duct ligation, LAMP-2-deficient rats develop rapid and severe evidence of advanced cholestasis, with an increase in serum bilirubin, as early as 6 h later. In wild-type control rats, multidrug resistance-associated protein 2 (Mrp2) normally concentrates at the bile canalicular membranes to secrete conjugated bilirubin into bile. However, in LAMP-2(y/-) rats, Mrp2 was detected in hepatocytes compared with other canalicular proteins including P-glycoproteins, dipeptidyl peptidase IV (CD26), and aminopeptidase (CD13). Our data further suggest that LAMP-2 interacts with the membrane cytoskeletal proteins radixin and F-actin in determining the localization of integral membrane proteins.

  6. Autosomal Recessive Nonsyndromic Hearing Impairment due to a Novel Deletion in the RDX Gene

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    Kwanghyuk Lee

    2011-01-01

    Full Text Available The RDX gene anchors cytoskeletal actin of stereocilia to hair cell transmembrane and is responsible for autosomal recessive nonsyndromic hearing impairment (ARNSHI due to DFNB24. A genome scan was performed using DNA samples from a consanguineous Pakistani family with ARNSHI. A significant maximum two-point LOD score of 4.5 (θ=0 and multipoint LOD score of 5.8 were achieved at marker D11S1998 (chr11 : 117.20 Mb. The region of homozygosity is bounded by markers D11S2000 (105.06 Mb and D11S4464 (123.13 Mb and contains the NSHI genes TECTA and RDX. Although no potentially causal variants were identified in the TECTA gene, within the RDX gene a novel deletion c.1076_1079delTTAA (p.Ile359Lysfs*6 was identified. The RDX deletion segregates with ARNSHI within the family and was not observed in 500 control chromosomes. It is predicted to cause premature truncation of radixin at the α-helical domain and to result in nonfunctional transcripts within the cochlea. RDX isoforms which encode the coiled-coil region of the α-helical domain are deemed necessary for proper function of hair cell stereocilia.

  7. Identification of Differentially Expressed Genes in Metastatic and Non-Metastatic Nasopharyngeal Carcinoma Cells by Suppression Subtractive Hybridization

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    Xu-Yu Yang

    2005-01-01

    Full Text Available Background & Objective: Nasopharyngeal carcinoma (NPC is an epithelial neoplasm with high occurrence rates in southern China. The disease often metastasizes to regional lymphnodes at a very early stage. Local recurrences and metastasis occur frequently in patients with NPC and are a leading cause of death, despite improvements on treatment modalities. The molecular mechanism underlying the metastasis of nasopharyngeal carcinoma remains poorly understood, however, and requires additional elucidation. The aim of this study was to explore possible NPC gene candidates that may play key roles in NPC metastasis. Methods: Subtractive suppression hybridization (SSH was performed to isolate differentially expressed clones between the metastatic 5-8F and non-metastatic 6-10B nasopharyngeal carcinoma cell lines. Differentially expressed clones were screened and confirmed by reverse Northern blotting. The sequences of cDNA fragments were subsequently analyzed and compared to known sequences in Genbank. Results & Discussion: The SSH library contained thousands of positive clones. Random analysis of 300 clones by PCR demonstrated that 269 clones contained inserted fragments. Reverse Northern blot confirmed that 20 out of 192 clones examined were significantly up-regulated in the 5-8F cell line. Among these 20 clones, 16 were previously identified genes (flotilin-2, ezrin, pim-3, fli-1, mel, neugrin, znf216, ASB1, raly, UBE2A, keratin6A, TMED7, EIF3S9, FTL, two ribosomal proteins RPL21 and RPL16, two were predicted genes (c9orf74 and MDS006, and two sequences shared no homology with known genes listed in GenBank and may represent novel genes. The proposed functions of the genes identified in this study include cell signal transduction, cell survival, transcription regulation, cell mobility, protein synthesis, and DNA damage repair. Flotillin-2, fli-1, pim-3 and ezrin have previously been reported to be associated with tumor metastasis and progression. The

  8. Role of Acid Sphingomyelinase-Induced Signaling in Melanoma Cells for Hematogenous Tumor Metastasis

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    Alexander Carpinteiro

    2016-01-01

    Full Text Available Background: Hematogenous metastasis of malignant tumor cells is a multistep process that requires release of tumor cells from the local tumor mass, interaction of the tumor cells with platelets in the blood, and adhesion of either the activated tumor cells or the complexes of platelets and tumor cells to the endothelial cells of the target organ. We have previously shown that the interaction of melanoma cells with platelets results in the release of acid sphingomyelinase (Asm from activated platelets. Secreted platelet-derived Asm acts on malignant tumor cells to cluster and activate integrins; such clustering and activation are necessary for tumor cell adhesion to endothelial cells and for metastasis. Methods: We examined the response of tumor cells to treatment with extracellular sphingomyelinase or co-incubation with wild-type and Asm-deficient platelets. We determined the phosphorylation and activation of several intracellular signaling molecules, in particular p38 kinase (p38K, phospholipase Cγ (PLCγ, ezrin, and extracellular signal-regulated kinases. Results: Incubation of B16F10 melanoma cells with Asm activates p38 MAP kinase (p38K, phospholipase Cγ (PLCγ, ezrin, and extracellular signal-regulated kinases. Co-incubation of B16F10 melanoma cells with wild-type or Asm-deficient platelets showed that the phosphorylation/activation of p38K is dependent on Asm. Pharmacological blockade of p38K prevents activation of β1 integrin and adhesion in vitro. Most importantly, inhibition of p38K activity in B16F10 melanoma cells prevents tumor cell adhesion and metastasis to the lung in vivo, a finding indicating the importance of p38K for metastasis. Conclusions: Asm, secreted from activated platelets after tumor cell-platelet contact, induces p38K phosphorylation in tumor cells. This in turn stimulates β1 integrin activation that is necessary for adhesion and subsequent metastasis of tumor cells. Thus, inhibition of p38K might be a novel

  9. Leucine-rich repeat kinase 2 modulates retinoic acid-induced neuronal differentiation of murine embryonic stem cells.

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    Cathrin Schulz

    Full Text Available BACKGROUND: Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2 gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture. CONCLUSION/SIGNIFICANCE: Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.

  10. Impact of the Autism-Associated Long Noncoding RNA MSNP1AS on Neuronal Architecture and Gene Expression in Human Neural Progenitor Cells

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    Jessica J. DeWitt

    2016-09-01

    Full Text Available We previously identified the long noncoding RNA (lncRNA MSNP1AS (moesin pseudogene 1, antisense as a functional element revealed by genome wide significant association with autism spectrum disorder (ASD. MSNP1AS expression was increased in the postmortem cerebral cortex of individuals with ASD and particularly in individuals with the ASD-associated genetic markers on chromosome 5p14.1. Here, we mimicked the overexpression of MSNP1AS observed in postmortem ASD cerebral cortex in human neural progenitor cell lines to determine the impact on neurite complexity and gene expression. ReNcell CX and SK-N-SH were transfected with an overexpression vector containing full-length MSNP1AS. Neuronal complexity was determined by the number and length of neuronal processes. Gene expression was determined by strand-specific RNA sequencing. MSNP1AS overexpression decreased neurite number and neurite length in both human neural progenitor cell lines. RNA sequencing revealed changes in gene expression in proteins involved in two biological processes: protein synthesis and chromatin remodeling. These data indicate that overexpression of the ASD-associated lncRNA MSNP1AS alters the number and length of neuronal processes. The mechanisms by which MSNP1AS overexpression impacts neuronal differentiation may involve protein synthesis and chromatin structure. These same biological processes are also implicated by rare mutations associated with ASD, suggesting convergent mechanisms.

  11. Force-dependent calcium signaling and its pathway of human neutrophils on P-selectin in flow.

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    Huang, Bing; Ling, Yingchen; Lin, Jiangguo; Du, Xin; Fang, Ying; Wu, Jianhua

    2017-02-01

    P-selectin engagement of P-selectin glycoprotein ligand-1 (PSGL-1) causes circulating leukocytes to roll on and adhere to the vascular surface, and mediates intracellular calcium flux, a key but unclear event for subsequent arresting firmly at and migrating into the infection or injured tissue. Using a parallel plate flow chamber technique and intracellular calcium ion detector (Fluo-4 AM), the intracellular calcium flux of firmly adhered neutrophils on immobilized P-selectin in the absence of chemokines at various wall shear stresses was investigated here in real time by fluorescence microscopy. The results demonstrated that P-selectin engagement of PSGL-1 induced the intracellular calcium flux of firmly adhered neutrophils in flow, increasing P-selectin concentration enhanced cellular calcium signaling, and, force triggered, enhanced and quickened the cytoplasmic calcium bursting of neutrophils on immobilized P-selectin. This P-selectin-induced calcium signaling should come from intracellular calcium release rather than extracellular calcium influx, and be along the mechano-chemical signal pathway involving the cytoskeleton, moesin and Spleen tyrosine kinase (Syk). These results provide a novel insight into the mechano-chemical regulation mechanism for P-selectin-induced calcium signaling of neutrophils in flow.

  12. Disruption of the Cdc42/Par6/aPKC or Dlg/Scrib/Lgl Polarity Complex Promotes Epithelial Proliferation via Overlapping Mechanisms.

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    Schimizzi, Gregory V; Maher, Meghan T; Loza, Andrew J; Longmore, Gregory D

    2016-01-01

    The establishment and maintenance of apical-basal polarity is a defining characteristic and essential feature of functioning epithelia. Apical-basal polarity (ABP) proteins are also tumor suppressors that are targeted for disruption by oncogenic viruses and are commonly mutated in human carcinomas. Disruption of these ABP proteins is an early event in cancer development that results in increased proliferation and epithelial disorganization through means not fully characterized. Using the proliferating Drosophila melanogaster wing disc epithelium, we demonstrate that disruption of the junctional vs. basal polarity complexes results in increased epithelial proliferation via distinct downstream signaling pathways. Disruption of the basal polarity complex results in JNK-dependent proliferation, while disruption of the junctional complex primarily results in p38-dependent proliferation. Surprisingly, the Rho-Rok-Myosin contractility apparatus appears to play opposite roles in the regulation of the proliferative phenotype based on which polarity complex is disrupted. In contrast, non-autonomous Tumor Necrosis Factor (TNF) signaling appears to suppress the proliferation that results from apical-basal polarity disruption, regardless of which complex is disrupted. Finally we demonstrate that disruption of the junctional polarity complex activates JNK via the Rho-Rok-Myosin contractility apparatus independent of the cortical actin regulator, Moesin.

  13. Sds22, a PP1 phosphatase regulatory subunit, regulates epithelial cell polarity and shape [Sds22 in epithelial morphology

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    Sung Hsin-Ho

    2009-02-01

    Full Text Available Abstract Background How epithelial cells adopt their particular polarised forms is poorly understood. In a screen for genes regulating epithelial morphology in Drosophila, we identified sds22, a conserved gene previously characterised in yeast. Results In the columnar epithelia of imaginal discs or follicle cells, mutation of sds22 causes contraction of cells along their apical-basal axis, resulting in a more cuboidal morphology. In addition, the mutant cells can also display altered cell polarity, forming multiple layers in follicle cells and leaving the epithelium in imaginal discs. In yeast, sds22 encodes a PP1 phosphatase regulatory subunit. Consistent with this, we show that Drosophila Sds22 binds to all four Drosophila PP1s and shares an overlapping phenotype with PP1beta9c. We also show that two previously postulated PP1 targets, Spaghetti Squash and Moesin are hyper-phosphorylated in sds22 mutants. This function is shared by the human homologue of Sds22, PPP1R7. Conclusion Sds22 is a conserved PP1 phosphatase regulatory subunit that controls cell shape and polarity.

  14. Autoimmune antigenic targets at the node of Ranvier in demyelinating disorders.

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    Stathopoulos, Panos; Alexopoulos, Harry; Dalakas, Marinos C

    2015-03-01

    Mounting evidence suggests that autoantibodies contribute to the pathogenesis of demyelination in the PNS and CNS. Rapid reversal of electrophysiological blockade after plasmapheresis or intravenous immunoglobulin treatment for acute or chronic inflammatory demyelinating polyneuropathy is more likely to result from removal or neutralization of an antibody that impairs saltatory conduction than from remyelination. Although up to 30% of patients with acute or chronic inflammatory demyelinating polyneuropathy harbour autoantibodies, specific antigens have been identified in no more than 13% of cases. To date, autoantigens identified at the node of Ranvier include neurofascin 186, gliomedin and possibly moesin in the nodal domain, and contactin-1, Caspr1 and neurofascin 155 in the paranodal domain. In some patients with multiple sclerosis, paranodal CNPase and juxtaparanodal contactin-2 trigger a humoral response. This Review explores the molecular anatomy of the node of Ranvier, focusing on proteins with extracellular domains that could serve as antigens. The clinical implications of node-specific antibody responses are addressed, and the best approaches to identify antibodies that target nodal proteins are highlighted. Also discussed are the roles of these antibodies as either secondary, disease-exacerbating responses, or as a primary effector mechanism that defines demyelination or axonal degeneration at the node, identifies disease subtypes or determines response to treatments.

  15. Proteomic analysis of proton beam irradiated human melanoma cells.

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    Sylwia Kedracka-Krok

    Full Text Available Proton beam irradiation is a form of advanced radiotherapy providing superior distributions of a low LET radiation dose relative to that of photon therapy for the treatment of cancer. Even though this clinical treatment has been developing for several decades, the proton radiobiology critical to the optimization of proton radiotherapy is far from being understood. Proteomic changes were analyzed in human melanoma cells treated with a sublethal dose (3 Gy of proton beam irradiation. The results were compared with untreated cells. Two-dimensional electrophoresis was performed with mass spectrometry to identify the proteins. At the dose of 3 Gy a minimal slowdown in proliferation rate was seen, as well as some DNA damage. After allowing time for damage repair, the proteomic analysis was performed. In total 17 protein levels were found to significantly (more than 1.5 times change: 4 downregulated and 13 upregulated. Functionally, they represent four categories: (i DNA repair and RNA regulation (VCP, MVP, STRAP, FAB-2, Lamine A/C, GAPDH, (ii cell survival and stress response (STRAP, MCM7, Annexin 7, MVP, Caprin-1, PDCD6, VCP, HSP70, (iii cell metabolism (TIM, GAPDH, VCP, and (iv cytoskeleton and motility (Moesin, Actinin 4, FAB-2, Vimentin, Annexin 7, Lamine A/C, Lamine B. A substantial decrease (2.3 x was seen in the level of vimentin, a marker of epithelial to mesenchymal transition and the metastatic properties of melanoma.

  16. Optimization of platelet concentrate quality: application of proteomic technologies to donor management.

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    Schubert, Peter; Culibrk, Brankica; Karwal, Simrath; Slichter, Sherrill J; Devine, Dana V

    2012-12-01

    Quality management of blood products is essential for blood banking. It is influenced by both processing and donor characteristics and assured by monitoring routine in vitro parameters to defined product specifications. However, these measures correlate poorly with the in vivo behavior of transfused platelets and cannot be used to select optimal donors. Since radiolabeled platelet recovery and survival studies are expensive and time consuming, there is an ongoing search for simpler measures that predict platelet transfusion outcomes. We performed a pilot study using semi-qualitative proteomics to assess changes in the platelet protein profile of donors with either acceptable or unacceptable in vivo radiolabeled autologous platelet recovery and survival measurements. Proteins changing during a 9-day storage period included cytoskeletal elements talin, vinculin and moesin as well as signal transduction proteins 14-3-3, RhoGDI and Rap1. Two of nine donations exhibited a decrease in these proteins and poor in vivo platelet recovery and survival whereas the remaining donors showed acceptable platelet recovery and survival and expected protein profiles. Analyses revealed a significant correlation between protein levels of Rap1 and RhoGDI during storage and platelet recovery and survival. This study provides for the first time preliminary data showing evidence of the utility of protein profiling to predict platelet transfusion quality. This article is part of a Special Issue entitled: Integrated omics.

  17. Cannibalism: a way to feed on metastatic tumors.

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    Fais, Stefano

    2007-12-18

    Cannibalism of tumors is an old story for pathologists, but it remained a mystery for at least one century. Recent data highlighted tumor cannibalism as a key advantage in tumor malignancy, possibly involved in resistance of tumors to the specific immune reaction. However, new data suggests also that metastatic tumor cells may use this peculiar function to feed in conditions of low nutrient supply. This makes malignant cancer cells more similar to microorganisms, rather than to normal cells undergoing malignant transformation. In cytological or histological samples of human tumors it is common to detect cells with one or many vacuoles, possibly containing cells under degradation, that push the nucleus to the periphery giving it the shape of a crescent moon. The cannibal cells may feed on sibling tumor cells, but also of the lymphocytes that should kill them. Cannibal cells eat everything without distinguishing between the feeding materials, with a mechanism that mostly differ from typical phagocytosis. Despite such phenomenon is considered mainly non-selective, a molecular framework of factors that contribute to cannibalism has been described. This machinery includes the presence of an acidic environment that allows a continuous activation of specific lytic enzymes, such as cathepsin B. Cannibalism occurs in apparently well defined structures whose main actors are big caveolar-like vacuoles and a connection between caveolin-1 and the actin cytoskeleton through the actin-linker molecule ezrin. Each of the components of the cannibal framework may represent specific tumor targets for future new strategies against cancer.

  18. Proteomics of MUC1-containing lipid rafts from plasma membranes and exosomes of human breast carcinoma cells MCF-7.

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    Staubach, Simon; Razawi, Hanieh; Hanisch, Franz-Georg

    2009-05-01

    Apically expressed human MUC1 is known to become endocytosed and either to re-enter the secretory pathway for recycling to the plasma membrane or to be exported by the cells via the formation of multi-vesicular bodies and the release of exosomes. By using recombinant fusion-tagged MUC1 as a bait protein we followed an anti-myc affinity-based approach for isolating subpopulations of lipid rafts from the plasma membranes and exosomes of MCF-7 breast cancer cells. MUC1(+) lipid rafts were not only found to contain genuine raft proteins (flotillin-1, prohibitin, G protein, annexin A2), but also raft-associated proteins linking these to the cytoskeleton (ezrin/villin-2, profilin II, HSP27, gamma-actin, beta-actin) or proteins in complexes with raft proteins, including the bait protein (HSP60, HSP70). Major overlaps were revealed for the subproteomes of plasma membranous and exosomal lipid raft preparations, indicating that MUC1 is sorted into subpopulations of rafts for its trafficking via flotillin-dependent pathways and export via exosomes.

  19. Trehalose improves cell proliferation and dehydration tolerance of human HaCaT cells

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    Lee Kyung Eun

    2015-01-01

    Full Text Available Trehalose is a disaccharide molecule that serves as a natural osmotic regulator in halophilic microorganisms and plants but not in mammals. We observed that human HaCaT cells supplied with trehalose improved cell proliferation and extended viability under dehydration. In HaCaT cells, in response to increasing concentrations of exogenous trehalose, the levels of heat shock protein (HSP 70 increased and matrix metalloproteinase (MMP 1 decreased. Proteome analysis of trehalose-treated HaCaT cells revealed remarkable increases in the levels of proteins involved in cell signaling and the cell cycle, including p21 activated kinase I, Sec I family domain protein and elongation factor G. Moreover, the proteins for cell stress resistance, tryptophan hydroxylase, serine/cysteine proteinase inhibitors and vitamin D receptors were also increased. In addition, the proteins responsible for the maintenance of the cytoskeleton and cellular structures including procollagen-lysine dioxygenase, vinculin and ezrin were increased. Proteomic data revealed that trehalose affected HaCaT cells by inducing the proteins involved in cell proliferation. These results suggest that trehalose improves the proliferation and dehydration tolerance of HaCaT cells by inducing proteins involved in cell growth and dehydration protection.

  20. The guanine nucleotide exchange factor Vav2 is a negative regulator of parathyroid hormone receptor/Gq signaling.

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    Emami-Nemini, Alexander; Gohla, Antje; Urlaub, Henning; Lohse, Martin J; Klenk, Christoph

    2012-08-01

    The parathyroid hormone receptor (PTHR) is a class B G protein-coupled receptor (GPCR) that mediates the endocrine and paracrine effects of parathyroid hormone and related peptides through the activation of phospholipase Cβ-, adenylyl cyclase-, mitogen-activated protein kinase-, and β-arrestin-initiated signaling pathways. It is currently not clear how specificity among these downstream signaling pathways is achieved. A possible mechanism involves adaptor proteins that affect receptor/effector coupling. In a proteomic screen with the PTHR C terminus, we identified vav2, a guanine nucleotide exchange factor (GEF) for Rho GTPases, as a PTHR-interacting protein. The core domains of vav2 bound to the intracellular domains of the PTHR independent of receptor activation. In addition, vav2 specifically interacted with activated Gα(q) but not with Gα(s) subunits, and it competed with PTHR for coupling to Gα(q). Consistent with its specific interaction with Gα(q), vav2 impaired G(q)-mediated inositol phosphate generation but not G(s)-mediated cAMP generation. This inhibition of G(q) signaling was specific for PTHR signaling, compared with other G(q)-coupled GPCRs. Moreover, the benefit for PTHR-mediated inositol phosphate generation in the absence of vav2 required the ezrin binding domain of Na(+)/H(+)-exchanger regulatory factor 1. Our results show that a RhoA GEF can specifically interact with a GPCR and modulate its G protein signaling specificity.

  1. Proteomic analysis of purified Newcastle disease virus particles

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    Ren Xiangpeng

    2012-05-01

    Full Text Available Abstract Background Newcastle disease virus (NDV is an enveloped RNA virus, bearing severe economic losses to the poultry industry worldwide. Previous virion proteomic studies have shown that enveloped viruses carry multiple host cellular proteins both internally and externally during their life cycle. To address whether it also occurred during NDV infection, we performed a comprehensive proteomic analysis of highly purified NDV La Sota strain particles. Results In addition to five viral structural proteins, we detected thirty cellular proteins associated with purified NDV La Sota particles. The identified cellular proteins comprised several functional categories, including cytoskeleton proteins, annexins, molecular chaperones, chromatin modifying proteins, enzymes-binding proteins, calcium-binding proteins and signal transduction-associated proteins. Among these, three host proteins have not been previously reported in virions of other virus families, including two signal transduction-associated proteins (syntenin and Ras small GTPase and one tumor-associated protein (tumor protein D52. The presence of five selected cellular proteins (i.e., β-actin, tubulin, annexin A2, heat shock protein Hsp90 and ezrin associated with the purified NDV particles was validated by Western blot or immunogold labeling assays. Conclusions The current study presented the first standard proteomic profile of NDV. The results demonstrated the incorporation of cellular proteins in NDV particles, which provides valuable information for elucidating viral infection and pathogenesis.

  2. The keratin-binding protein Albatross regulates polarization of epithelial cells.

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    Sugimoto, Masahiko; Inoko, Akihito; Shiromizu, Takashi; Nakayama, Masanori; Zou, Peng; Yonemura, Shigenobu; Hayashi, Yuko; Izawa, Ichiro; Sasoh, Mikio; Uji, Yukitaka; Kaibuchi, Kozo; Kiyono, Tohru; Inagaki, Masaki

    2008-10-06

    The keratin intermediate filament network is abundant in epithelial cells, but its function in the establishment and maintenance of cell polarity is unclear. Here, we show that Albatross complexes with Par3 to regulate formation of the apical junctional complex (AJC) and maintain lateral membrane identity. In nonpolarized epithelial cells, Albatross localizes with keratin filaments, whereas in polarized epithelial cells, Albatross is primarily localized in the vicinity of the AJC. Knockdown of Albatross in polarized cells causes a disappearance of key components of the AJC at cell-cell borders and keratin filament reorganization. Lateral proteins E-cadherin and desmoglein 2 were mislocalized even on the apical side. Although Albatross promotes localization of Par3 to the AJC, Par3 and ezrin are still retained at the apical surface in Albatross knockdown cells, which retain intact microvilli. Analysis of keratin-deficient epithelial cells revealed that keratins are required to stabilize the Albatross protein, thus promoting the formation of AJC. We propose that keratins and the keratin-binding protein Albatross are important for epithelial cell polarization.

  3. Effects of phycoerythrin from Gracilaria lemaneiformis in proliferation and apoptosis of SW480 cells.

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    Li, Peizhen; Ying, Jun; Chang, Qingli; Zhu, Wen; Yang, Guangjian; Xu, Teng; Yi, Huiguang; Pan, Ruowang; Zhang, Enyong; Zeng, Xiaofeng; Yan, Chunxia; Bao, Qiyu; Li, Shengbin

    2016-12-01

    We studied phycoerythrin (PE) in human SW480 tumor cells and the underlying molecular mechanisms of action. PE inhibited cell proliferation as evidenced by CCK-8 assay. The IC50 values of phycoerythrin were 48.2 and 27.4 µg/ml for 24 and 48 h of exposure, respectively. PE induced apoptosis and cell cycle arrest in SW480 cells as observed under electron microscopy and with flow cytometry. Apoptosis increased from 5.1 (controls) to 39.0% in 80.0 µg/ml PE-treated cells. Differences in protein expression were identified using proteomic techniques. Protein spots (1018±60 and 1010±60) were resolved in PE-treated and untreated group. Forty differential protein spots were analyzed with MALDI-TOF-MS, including GRP78 and NPM1. The expression as measured by qPCR and western blotting agreed with data from two-dimensional electrophoresis. GRP78, NPM1, MTHSP75, Ezrin and Annexin A2 were decreased and HSP60 was increased after PE treatment, indicating that PE may target multiple proteins to induce apoptosis.

  4. Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions.

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    Gez, Swetlana; Crossett, Ben; Christopherson, Richard I

    2007-09-01

    Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt's lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, alpha- and beta-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (gamma-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.

  5. ZO-1 and ZO-2 are required for extra-embryonic endoderm integrity, primitive ectoderm survival and normal cavitation in embryoid bodies derived from mouse embryonic stem cells.

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    Dominic C Y Phua

    Full Text Available The Zonula Occludens proteins ZO-1 and ZO-2 are cell-cell junction-associated adaptor proteins that are essential for the structural and regulatory functions of tight junctions in epithelial cells and their absence leads to early embryonic lethality in mouse models. Here, we use the embryoid body, an in vitro peri-implantation mouse embryogenesis model, to elucidate and dissect the roles ZO-1 and ZO-2 play in epithelial morphogenesis and de novo tight junction assembly. Through the generation of individual or combined ZO-1 and ZO-2 null embryoid bodies, we show that their dual deletion prevents tight junction formation, resulting in the disorganization and compromised barrier function of embryoid body epithelial layers. The disorganization is associated with poor microvilli development, fragmented basement membrane deposition and impaired cavity formation, all of which are key epithelial tissue morphogenetic processes. Expression of Podocalyxin, which positively regulates the formation of microvilli and the apical membrane, is repressed in embryoid bodies lacking both ZO-1 and ZO-2 and this correlates with an aberrant submembranous localization of Ezrin. The null embryoid bodies thus give an insight into how the two ZO proteins influence early mouse embryogenesis and possible mechanisms underlying the embryonic lethal phenotype.

  6. ZO-1 and ZO-2 are required for extra-embryonic endoderm integrity, primitive ectoderm survival and normal cavitation in embryoid bodies derived from mouse embryonic stem cells.

    Science.gov (United States)

    Phua, Dominic C Y; Xu, Jianliang; Ali, Safiah Mohamed; Boey, Adrian; Gounko, Natalia V; Hunziker, Walter

    2014-01-01

    The Zonula Occludens proteins ZO-1 and ZO-2 are cell-cell junction-associated adaptor proteins that are essential for the structural and regulatory functions of tight junctions in epithelial cells and their absence leads to early embryonic lethality in mouse models. Here, we use the embryoid body, an in vitro peri-implantation mouse embryogenesis model, to elucidate and dissect the roles ZO-1 and ZO-2 play in epithelial morphogenesis and de novo tight junction assembly. Through the generation of individual or combined ZO-1 and ZO-2 null embryoid bodies, we show that their dual deletion prevents tight junction formation, resulting in the disorganization and compromised barrier function of embryoid body epithelial layers. The disorganization is associated with poor microvilli development, fragmented basement membrane deposition and impaired cavity formation, all of which are key epithelial tissue morphogenetic processes. Expression of Podocalyxin, which positively regulates the formation of microvilli and the apical membrane, is repressed in embryoid bodies lacking both ZO-1 and ZO-2 and this correlates with an aberrant submembranous localization of Ezrin. The null embryoid bodies thus give an insight into how the two ZO proteins influence early mouse embryogenesis and possible mechanisms underlying the embryonic lethal phenotype.

  7. Hepatoma cell line HepG2.2.15 demonstrates distinct biological features compared with parental HepG2

    Institute of Scientific and Technical Information of China (English)

    Ran Zhao; Tian-Zhen Wang; Dan Kong; Lei Zhang; Hong-Xue Meng; Yang Jiang; Yi-Qi Wu; Zu-Xi Yu; Xiao-Ming Jin

    2011-01-01

    AIM: To investigate the biological features of hepatitis B virus (HBV)-transfected HepG2.2.15 cells. METHODS: The cell ultrastructure, cell cycle and apop-tosis, and the abilities of proliferation and invasion of HBV-transfected HepG2.2.15 and the parent HepG2 cells were examined by electron microscopy, flow cytometry, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trans-well assay. Oncogenicity of the two cell lines was compared via subcutaneous injection and orthotopic injection or implantation in nude mice, and the pathological analysis of tumor formation was performed. Two cytoskeletal proteins were detected by Western blotting.RESULTS: Compared with HepG2 cells, HepG2.2.15 cells showed organelle degeneration and filopodia disappear-ance under electron microscope. HepG2.2.15 cells pro-liferated and migrated slowly in vitro, and hardly formed tumor and lung metastasis in nude mice. Flow cytom-etry showed that the majority of HepG2.2.15 cells were arrested in G1 phase, and apoptosis was minor in both cell lines. Furthermore, the levels of cytoskeletal pro-teins F-actin and Ezrin were decreased in HepG2.2.15 cells.CONCLUSION: HepG2.2.15 cells demonstrated a low-er proliferation and invasion ability than the HepG2 cells due to HBV transfection.

  8. Autoreactivity to sweat glands and nerves in clinical scabies infection

    Directory of Open Access Journals (Sweden)

    Michael S. Howard

    2010-01-01

    Full Text Available Context: Skin changes in pregnancy can be categorized as 1 physiological/hormonal, 2 alterations in pre-existing skin diseases, or 3 represent development of new dermatoses, some of which may be pregnancy specific. Case Report: We describe a 19 years old female at 27 weeks gestation who presented with a rash on the face and breast, with intense pruritis. Hematoxylin and eosin demonstrated Scabies mites within the epidermis, with an intense perivascular infiltrate of lymphohistiocytic cells around the superficial dermal blood vessels. By direct immunofluorescence (DIF, human fibrinogen was also detected in the perivascular areas. DIF also revealed deposits of human IgG and complement C5-9/MAC deposits in the sweat glands, as well as in nerves surrounding the sweat glands subjacent to the mites. Overexpression of ezrin and junctional adhesion molecule antibodies close to the scabies infection sites were also seen. Conclusion: Given that the hallmark of clinical scabies is intense pruritus and that very limited information is available regarding the pathophysiology of this symptom, we suggest that the itching sensation may be exacerbated by nerves and eccrine sweat glands in close proximity to the sites of infection.

  9. The stromal-vascular fraction of adipose tissue contributes to major differences between subcutaneous and visceral fat depots.

    Science.gov (United States)

    Peinado, Juan R; Jimenez-Gomez, Yolanda; Pulido, Marina R; Ortega-Bellido, Maria; Diaz-Lopez, Cesar; Padillo, Francisco J; Lopez-Miranda, Jose; Vazquez-Martínez, Rafael; Malagón, María M

    2010-09-01

    Adipose tissue represents a complex tissue both in terms of its cellular composition, as it includes mature adipocytes and the various cell types comprising the stromal-vascular fraction (SVF), and in relation to the distinct biochemical, morphological and functional characteristics according to its anatomical location. Herein, we have characterized the proteomic profile of both mature adipocyte and SVF from human visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) fat depots in order to unveil differences in the expression of proteins which may underlie the distinct association of VAT and SAT to several pathologies. Specifically, 24 proteins were observed to be differentially expressed between SAT SVF versus VAT SVF from lean individuals. Immunoblotting and RT-PCR analysis confirmed the differential regulation of the nuclear envelope proteins lamin A/C, the membrane-cytoskeletal linker ezrin and the enzyme involved in retinoic acid production, aldehyde dehydrogenase 1A2, in the two fat depots. In sum, the observation that proteins with important cell functions are differentially distributed between VAT and SAT and their characterization as components of SVF or mature adipocytes pave the way for future research on the molecular basis underlying diverse adipose tissue-related pathologies such as metabolic syndrome or lipodystrophy.

  10. Purification of infectious human herpesvirus 6A virions and association of host cell proteins

    Directory of Open Access Journals (Sweden)

    Garoff Henrik

    2007-10-01

    Full Text Available Abstract Background Viruses that are incorporating host cell proteins might trigger autoimmune diseases. It is therefore of interest to identify possible host proteins associated with viruses, especially for enveloped viruses that have been suggested to play a role in autoimmune diseases, like human herpesvirus 6A (HHV-6A in multiple sclerosis (MS. Results We have established a method for rapid and morphology preserving purification of HHV-6A virions, which in combination with parallel analyses with background control material released from mock-infected cells facilitates qualitative and quantitative investigations of the protein content of HHV-6A virions. In our iodixanol gradient purified preparation, we detected high levels of viral DNA by real-time PCR and viral proteins by metabolic labelling, silver staining and western blots. In contrast, the background level of cellular contamination was low in the purified samples as demonstrated by the silver staining and metabolic labelling analyses. Western blot analyses showed that the cellular complement protein CD46, the receptor for HHV-6A, is associated with the purified and infectious virions. Also, the cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions. Conclusion Cellular proteins are associated with HHV-6A virions. The relevance of the association in disease and especially in autoimmunity will be further investigated.

  11. Evidence for elevated (LIMK2 and CFL1) and suppressed (ICAM1, EZR, MAP2K2, and NOS3) gene expressions in metabolic syndrome.

    Science.gov (United States)

    Tabur, Suzan; Oztuzcu, Serdar; Oguz, Elif; Demiryürek, Seniz; Dagli, Hasan; Alasehirli, Belgin; Ozkaya, Mesut; Demiryürek, Abdullah T

    2016-08-01

    The metabolic syndrome (MetS) is a common multicomponent condition including abdominal obesity, dyslipidemia, hypertension, and hyperglycaemia. The aim of this study was to investigate the associations of the expression of a panel of signalling genes with the MetS in a Turkish population. A total of 54 MetS patients and 42 healthy controls with similar age and sex were included to this study. mRNA from blood samples was extracted, and real-time polymerase chain reaction was performed for gene expressions using a BioMark 96.96 dynamic array system. We observed marked increases in LIM kinase 2 (LIMK2) and cofilin 1 (CFL1) gene expressions in MetS patients. However, there were significant decreases in intercellular adhesion molecules 1 (ICAM1), ezrin (EZR), mitogen-activated protein kinase kinase 2 (MAP2K2), and nitric oxide synthase 3 (NOS3) gene expressions in MetS patients. Additionally, no marked changes were noted in other 15 genes studied. This is the first study to provide evidence that activation of LIMK2/CFL1 pathway may play an important role in MetS.

  12. New insights into potential functions for the protein 4.1superfamily of proteins in kidney epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Calinisan, Venice; Gravem, Dana; Chen, Ray Ping-Hsu; Brittin,Sachi; Mohandas, Narla; Lecomte, Marie-Christine; Gascard, Philippe

    2005-06-17

    Members of the protein 4.1 family of adapter proteins are expressed in a broad panel of tissues including various epithelia where they likely play an important role in maintenance of cell architecture and polarity and in control of cell proliferation. We have recently characterized the structure and distribution of three members of the protein 4.1 family, 4.1B, 4.1R and 4.1N, in mouse kidney. We describe here binding partners for renal 4.1 proteins, identified through the screening of a rat kidney yeast two-hybrid system cDNA library. The identification of putative protein 4.1-based complexes enables us to envision potential functions for 4.1 proteins in kidney: organization of signaling complexes, response to osmotic stress, protein trafficking, and control of cell proliferation. We discuss the relevance of these protein 4.1-based interactions in kidney physio-pathology in the context of their previously identified functions in other cells and tissues. Specifically, we will focus on renal 4.1 protein interactions with beta amyloid precursor protein (beta-APP), 14-3-3 proteins, and the cell swelling-activated chloride channel pICln. We also discuss the functional relevance of another member of the protein 4.1 superfamily, ezrin, in kidney physiopathology.

  13. Evaluation of nine candidate genes in patients with normal tension glaucoma: a case control study

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    Reinthal Eva

    2009-09-01

    Full Text Available Abstract Background Normal tension glaucoma is a major subtype of glaucoma, associated with intraocular pressures that are within the statistically normal range of the population. Monogenic forms following classical inheritance patterns are rare in this glaucoma subtype. Instead, multigenic inheritance is proposed for the majority of cases. The present study tested common sequence variants in candidate genes for association with normal tension glaucoma in the German population. Methods Ninety-eight SNPs were selected to tag the common genetic variation in nine genes, namely OPTN (optineurin, RDX (radixin, SNX16 (sorting nexin 16, OPA1 (optic atrophy 1, MFN1 (mitofusin 1, MFN2 (mitofusin 2, PARL (presenilin associated, rhomboid-like, SOD2 (superoxide dismutase 2, mitochondrial and CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1. These SNPs were genotyped in 285 cases and 282 fully evaluated matched controls. Statistical analyses comprised single polymorphism association as well as haplogroup based association testing. Results Results suggested that genetic variation in five of the candidate genes (RDX, SNX16, OPA1, SOD2 and CYP1B1 is unlikely to confer major risk to develop normal tension glaucoma in the German population. In contrast, we observed a trend towards association of single SNPs in OPTN, MFN1, MFN2 and PARL. The SNPs of OPTN, MFN2 and PARL were further analysed by multimarker haplotype-based association testing. We identified a risk haplotype being more frequent in patients and a vice versa situation for the complementary protective haplotype in each of the three genes. Conclusion Common variants of OPTN, PARL, MFN1 and MFN2 should be analysed in other cohorts to confirm their involvement in normal tension glaucoma.

  14. Dynamics and molecular determinants of cytoplasmic lipid droplet clustering and dispersion.

    Directory of Open Access Journals (Sweden)

    David J Orlicky

    Full Text Available Perilipin-1 (Plin1, a prominent cytoplasmic lipid droplet (CLD binding phosphoprotein and key physiological regulator of triglyceride storage and lipolysis in adipocytes, is thought to regulate the fragmentation and dispersion of CLD that occurs in response to β-adrenergic activation of adenylate cyclase. Here we investigate the dynamics and molecular determinants of these processes using cell lines stably expressing recombinant forms of Plin1 and/or other members of the perilipin family. Plin1 and a C-terminal CLD-binding fragment of Plin1 (Plin1CT induced formation of single dense CLD clusters near the microtubule organizing center, whereas neither an N-terminal CLD-binding fragment of Plin1, nor Plin2 or Plin3 induced clustering. Clustered CLD coated by Plin1, or Plin1CT, dispersed in response to isoproterenol, or other agents that activate adenylate cyclase, in a process inhibited by the protein kinase A inhibitor, H89, and blocked by microtubule disruption. Isoproterenol-stimulated phosphorylation of CLD-associated Plin1 on serine 492 preceded their dispersion, and live cell imaging showed that cluster dispersion involved initial fragmentation of tight clusters into multiple smaller clusters, which then fragmented into well-dispersed individual CLD. siRNA knockdown of the cortical actin binding protein, moesin, induced disaggregation of tight clusters into multiple smaller clusters, and inhibited the reaggregation of dispersed CLD into tight clusters. Together these data suggest that the clustering and dispersion processes involve a complex orchestration of phosphorylation-dependent, microtubule-dependent and independent, and microfilament dependent steps.

  15. Decoding c-Myc networks of cell cycle and apoptosis regulated genes in a transgenic mouse model of papillary lung adenocarcinomas.

    Science.gov (United States)

    Ciribilli, Yari; Singh, Prashant; Spanel, Reinhard; Inga, Alberto; Borlak, Jürgen

    2015-10-13

    The c-Myc gene codes for a basic-helix-loop-helix-leucine zipper transcription factor protein and is reported to be frequently over-expressed in human cancers. Given that c-Myc plays an essential role in neoplastic transformation we wished to define its activity in lung cancer and therefore studied its targeted expression to respiratory epithelium in a transgenic mouse disease model. Using histological well-defined tumors, transcriptome analysis identified novel c-Myc responsive cell cycle and apoptosis genes that were validated as direct c-Myc targets using EMSA, Western blotting, gene reporter and ChIP assays.Through computational analyses c-Myc cooperating transcription factors emerged for repressed and up-regulated genes in cancer samples, namely Klf7, Gata3, Sox18, p53 and Elf5 and Cebpα, respectively. Conversely, at promoters of genes regulated in transgenic but non-carcinomatous lung tissue enriched binding sites for c-Myc, Hbp1, Hif1 were observed. Bioinformatic analysis of tumor transcriptomic data revealed regulatory gene networks and highlighted mortalin and moesin as master regulators while gene reporter and ChIP assays in the H1299 lung cancer cell line as well as cross-examination of published ChIP-sequence data of 7 human and 2 mouse cell lines provided strong evidence for the identified genes to be c-Myc targets. The clinical significance of findings was established by evaluating expression of orthologous proteins in human lung cancer. Taken collectively, a molecular circuit for c-Myc-dependent cellular transformation was identified and the network analysis broadened the perspective for molecularly targeted therapies.

  16. Dynamics and Molecular Determinants of Cytoplasmic Lipid Droplet Clustering and Dispersion

    Science.gov (United States)

    Stefanski, Adrianne L.; McManaman, James L.

    2013-01-01

    Perilipin-1 (Plin1), a prominent cytoplasmic lipid droplet (CLD) binding phosphoprotein and key physiological regulator of triglyceride storage and lipolysis in adipocytes, is thought to regulate the fragmentation and dispersion of CLD that occurs in response to β-adrenergic activation of adenylate cyclase. Here we investigate the dynamics and molecular determinants of these processes using cell lines stably expressing recombinant forms of Plin1 and/or other members of the perilipin family. Plin1 and a C-terminal CLD-binding fragment of Plin1 (Plin1CT) induced formation of single dense CLD clusters near the microtubule organizing center, whereas neither an N-terminal CLD-binding fragment of Plin1, nor Plin2 or Plin3 induced clustering. Clustered CLD coated by Plin1, or Plin1CT, dispersed in response to isoproterenol, or other agents that activate adenylate cyclase, in a process inhibited by the protein kinase A inhibitor, H89, and blocked by microtubule disruption. Isoproterenol-stimulated phosphorylation of CLD-associated Plin1 on serine 492 preceded their dispersion, and live cell imaging showed that cluster dispersion involved initial fragmentation of tight clusters into multiple smaller clusters, which then fragmented into well-dispersed individual CLD. siRNA knockdown of the cortical actin binding protein, moesin, induced disaggregation of tight clusters into multiple smaller clusters, and inhibited the reaggregation of dispersed CLD into tight clusters. Together these data suggest that the clustering and dispersion processes involve a complex orchestration of phosphorylation-dependent, microtubule-dependent and independent, and microfilament dependent steps. PMID:23825572

  17. Systematic identification of regulatory proteins critical for T-cell activation

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    Kolbinger Frank

    2003-09-01

    Full Text Available Abstract Background The activation of T cells, mediated by the T-cell receptor (TCR, activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. Results In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 × 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLCγ1 and SHP-1 (PTP1C as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1, transmembrane molecules (EDG1, IL-10Rα and integrin α2, cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1, and cytoskeletal molecules (moesin and vimentin. Furthermore, using truncated Lck, PLCγ1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. Conclusions This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings.

  18. [Multiplexing mapping of human cDNAs]. Final report, September 1, 1991--February 28, 1994

    Energy Technology Data Exchange (ETDEWEB)

    1994-04-01

    Using PCR with automated product analysis, 329 human brain cDNA sequences have been assigned to individual human chromosomes. Primers were designed from single-pass cDNA sequences expressed sequence tags (ESTs). Primers were used in PCR reactions with DNA from somatic cell hybrid mapping panels as templates, often with multiplexing. Many ESTs mapped match sequence database records. To evaluate of these matches, the position of the primers relative to the matching region (In), the BLAST scores and the Poisson probability values of the EST/sequence record match were determined. In cases where the gene product was stringently identified by the sequence match had already been mapped, the gene locus determined by EST was consistent with the previous position which strongly supports the validity of assigning unknown genes to human chromosomes based on the EST sequence matches. In the present cases mapping the ESTs to a chromosome can also be considered to have mapped the known gene product: rolipram-sensitive cAMP phosphodiesterase, chromosome 1; protein phosphatase 2A{beta}, chromosome 4; alpha-catenin, chromosome 5; the ELE1 oncogene, chromosome 10q11.2 or q2.1-q23; MXII protein, chromosome l0q24-qter; ribosomal protein L18a homologue, chromosome 14; ribosomal protein L3, chromosome 17; and moesin, Xp11-cen. There were also ESTs mapped that were closely related to non-human sequence records. These matches therefore can be considered to identify human counterparts of known gene products, or members of known gene families. Examples of these include membrane proteins, translation-associated proteins, structural proteins, and enzymes. These data then demonstrate that single pass sequence information is sufficient to design PCR primers useful for assigning cDNA sequences to human chromosomes. When the EST sequence matches previous sequence database records, the chromosome assignments of the EST can be used to make preliminary assignments of the human gene to a chromosome.

  19. Decoding c-Myc networks of cell cycle and apoptosis regulated genes in a transgenic mouse model of papillary lung adenocarcinomas

    Science.gov (United States)

    Ciribilli, Yari; Singh, Prashant; Spanel, Reinhard; Inga, Alberto; Borlak, Jürgen

    2015-01-01

    The c-Myc gene codes for a basic-helix-loop-helix-leucine zipper transcription factor protein and is reported to be frequently over-expressed in human cancers. Given that c-Myc plays an essential role in neoplastic transformation we wished to define its activity in lung cancer and therefore studied its targeted expression to respiratory epithelium in a transgenic mouse disease model. Using histological well-defined tumors, transcriptome analysis identified novel c-Myc responsive cell cycle and apoptosis genes that were validated as direct c-Myc targets using EMSA, Western blotting, gene reporter and ChIP assays. Through computational analyses c-Myc cooperating transcription factors emerged for repressed and up-regulated genes in cancer samples, namely Klf7, Gata3, Sox18, p53 and Elf5 and Cebpα, respectively. Conversely, at promoters of genes regulated in transgenic but non-carcinomatous lung tissue enriched binding sites for c-Myc, Hbp1, Hif1 were observed. Bioinformatic analysis of tumor transcriptomic data revealed regulatory gene networks and highlighted mortalin and moesin as master regulators while gene reporter and ChIP assays in the H1299 lung cancer cell line as well as cross-examination of published ChIP-sequence data of 7 human and 2 mouse cell lines provided strong evidence for the identified genes to be c-Myc targets. The clinical significance of findings was established by evaluating expression of orthologous proteins in human lung cancer. Taken collectively, a molecular circuit for c-Myc-dependent cellular transformation was identified and the network analysis broadened the perspective for molecularly targeted therapies. PMID:26427040

  20. Release of membrane-bound vesicles and inhibition of tumor cell adhesion by the peptide Neopetrosiamide A.

    Directory of Open Access Journals (Sweden)

    Pamela Austin

    Full Text Available BACKGROUND: Neopetrosiamide A (NeoA is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of beta1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: beta1 integrin and numerous alpha integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that beta1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane. CONCLUSIONS/SIGNIFICANCE: NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface.

  1. Expression and Significance of GRP78 in Colon cancer%GRP78蛋白在结肠癌组织中的表达与意义

    Institute of Scientific and Technical Information of China (English)

    周臣敏; 董浦江; 付小利

    2013-01-01

    Objective To study the expression and significance of glucose regulated protein 78kD(GRP78) in colon cancer.Methods Western blot was used to detect the expression level of GRP78 in 82 cases of colon cancer tissue,17 cases of benign hyperplasia tissue and 33 cases of normal mucous membrane,meanwhile,vascular endothelial growth factor(VEGF),MTA1,Ezrin was detected in colon cancer tissue.The density of blood vessels in colon cancer was detected by immunohistochemistry SABC technique.Results The expression of GRP78 in the cancer tissue was significantly higher than that of the benign hyperplasia tissue and normal colorectal membrane(P<0.05).VEGF、MTA1 、Ezrin in GRP78-higher-expression group were higher than that in other two group(P<0.05),transfer rate of tumor cells and density of blood vessels in GRP78-higher-expression group were higher than that in other two group(P<0.05).Conclusion The expression level of GRP78 shows a increase trend when cancer develop from normal tissue,benign hyperplasia tissue to carcinoma.GRP78 is involved in the development of colorectal carcinoma.The GRP78 protein is highly expressed,which may play a key role in colon cancer cell metastasis.%目的 探讨葡萄耱调节蛋白78(GRP78)在结肠癌组织中的表达及意义.方法 用蛋白免疫印迹法(Wester blot)检测82例结肠癌组织、17例结肠良性增生组织、及33例正常肠组织的GRP78及癌组织中血管内皮生长因子(VEGF)、转移相关基因1(MTA1)、Ezrin的表达情况.免疫组化SABC法测定肿瘤组织微血管密度.结果 GRP78在结肠癌组织中的表达明显高于结肠良性增生组织及正常结肠组织(P<0.05),GRP78高表达组结肠癌组织的VEGF、MTA1、Ezrin表达量高于其他两组(P<0.05),GRP78高表达组结肠癌组织肿瘤细胞转移率及血管密度明显高于其他两组(P<0.05).结论 GRP78在结肠癌组织中的表达呈增高趋势,参与了结直肠腺癌的发生过程.GRP78高表达与结肠癌细胞转移有关.

  2. Reconstruction and signal propagation analysis of the Syk signaling network in breast cancer cells

    Science.gov (United States)

    Urbach, Serge; Montcourrier, Philippe; Roy, Christian; Solassol, Jérôme; Freiss, Gilles; Radulescu, Ovidiu

    2017-01-01

    The ability to build in-depth cell signaling networks from vast experimental data is a key objective of computational biology. The spleen tyrosine kinase (Syk) protein, a well-characterized key player in immune cell signaling, was surprisingly first shown by our group to exhibit an onco-suppressive function in mammary epithelial cells and corroborated by many other studies, but the molecular mechanisms of this function remain largely unsolved. Based on existing proteomic data, we report here the generation of an interaction-based network of signaling pathways controlled by Syk in breast cancer cells. Pathway enrichment of the Syk targets previously identified by quantitative phospho-proteomics indicated that Syk is engaged in cell adhesion, motility, growth and death. Using the components and interactions of these pathways, we bootstrapped the reconstruction of a comprehensive network covering Syk signaling in breast cancer cells. To generate in silico hypotheses on Syk signaling propagation, we developed a method allowing to rank paths between Syk and its targets. We first annotated the network according to experimental datasets. We then combined shortest path computation with random walk processes to estimate the importance of individual interactions and selected biologically relevant pathways in the network. Molecular and cell biology experiments allowed to distinguish candidate mechanisms that underlie the impact of Syk on the regulation of cortactin and ezrin, both involved in actin-mediated cell adhesion and motility. The Syk network was further completed with the results of our biological validation experiments. The resulting Syk signaling sub-networks can be explored via an online visualization platform. PMID:28306714

  3. Prkci is required for a non-autonomous signal that coordinates cell polarity during cavitation.

    Science.gov (United States)

    Mah, In Kyoung; Soloff, Rachel; Izuhara, Audrey K; Lakeland, Daniel L; Wang, Charles; Mariani, Francesca V

    2016-08-01

    Polarized epithelia define boundaries, spaces, and cavities within organisms. Cavitation, a process by which multicellular hollow balls or tubes are produced, is typically associated with the formation of organized epithelia. In order for these epithelial layers to form, cells must ultimately establish a distinct apical-basal polarity. Atypical PKCs have been proposed to be required for apical-basal polarity in diverse species. Here we show that while cells null for the Prkci isozyme exhibit some polarity characteristics, they fail to properly segregate apical-basal proteins, form a coordinated ectodermal epithelium, or participate in normal cavitation. A failure to cavitate could be due to an overgrowth of interior cells or to an inability of interior cells to die. Null cells however, do not have a marked change in proliferation rate and are still capable of undergoing cell death, suggesting that alterations in these processes are not the predominant cause of the failed cavitation. Overexpression of BMP4 or EZRIN can partially rescue the phenotype possibly by promoting cell death, polarity, and differentiation. However, neither is sufficient to provide the required cues to generate a polarized epithelium and fully rescue cavitation. Interestingly, when wildtype and Prkci(-/-) ES cells are mixed together, a polarized ectodermal epithelium forms and cavitation is rescued, likely due to the ability of wildtype cells to produce non-autonomous polarity cues. We conclude that Prkci is not required for cells to respond to these cues, though it is required to produce them. Together these findings indicate that environmental cues can facilitate the formation of polarized epithelia and that cavitation requires the proper coordination of multiple basic cellular processes including proliferation, differentiation, cell death, and apical-basal polarization.

  4. Alterations of proteins in MDCK cells during acute potassium deficiency.

    Science.gov (United States)

    Peerapen, Paleerath; Ausakunpipat, Nardtaya; Chanchaem, Prangwalai; Thongboonkerd, Visith

    2016-06-01

    Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury.

  5. Boar seminal plasma exosomes: effect on sperm function and protein identification by sequencing.

    Science.gov (United States)

    Piehl, Lidia L; Fischman, M Laura; Hellman, Ulf; Cisale, Humberto; Miranda, Patricia V

    2013-04-15

    Mammalian seminal plasma contains membranous vesicles (exosomes), with a high content of cholesterol and sphingomyelin and a complex protein composition. Their physiological role is uncertain because sperm stabilization and activation effects have been reported. To analyze a putative modulatory role for semen exosomes on sperm activity in the boar, the effects of these vesicles on several sperm functional parameters were examined. Additionally, boar exosome proteins were sequenced and their incorporation into sperm was explored. Boar sperm were incubated under conditions that induce capacitation, manifested as increased tyrosine phosphorylation, cholesterol loss and greater fluidity in apical membranes, and the ability to undergo the lysophosphatidylcholine-induced acrosome reaction. After establishing this cluster of capacitation-dependent functional parameters, the effect produced by exosomes when present during or after sperm capacitation was analyzed. Exosomes inhibited the capacitation-dependent cholesterol efflux and fluidity increase in apical membranes, and the disappearance of a 14-kD phosphorylated polypeptide. In contrast, the acrosome reaction (spontaneous and lysophosphatidylcholine-induced) was not affected, and sperm binding to the oocyte zona pellucida was reduced only when vesicles were present during gamete coincubation. Liposomes with a lipid composition similar to that present in exosomes mimicked these effects, except the one on zona pellucida binding. Interaction between exosomes and sperm was confirmed by transfer of aminopeptidase activity. In addition, the major exosome protein, identified as actin, appeared to associate with sperm after coincubation. Exosome composition had a predominance for structural proteins (actin, plastin, ezrin, and condensin), enzymes, and several porcine seminal plasma-specific polypeptides (e.g., spermadhesins). Transfer of proteins from exosome to sperm and their ability to block cholesterol efflux supports a

  6. Structural basis for NHERF1 PDZ domain binding.

    Science.gov (United States)

    Mamonova, Tatyana; Kurnikova, Maria; Friedman, Peter A

    2012-04-10

    The Na(+)/H(+) exchange regulatory factor-1 (NHERF1) is a scaffolding protein that possesses two tandem PDZ domains and a carboxy-terminal ezrin-binding domain (EBD). The parathyroid hormone receptor (PTHR), type II sodium-dependent phosphate cotransporter (Npt2a), and β2-adrenergic receptor (β2-AR), through their respective carboxy-terminal PDZ-recognition motifs, individually interact with NHERF1 forming a complex with one of the PDZ domains. In the basal state, NHERF1 adopts a self-inhibited conformation, in which its carboxy-terminal PDZ ligand interacts with PDZ2. We applied molecular dynamics (MD) simulations to uncover the structural and biochemical basis for the binding selectivity of NHERF1 PDZ domains. PDZ1 uniquely forms several contacts not present in PDZ2 that further stabilize PDZ1 interactions with target ligands. The binding free energy (ΔG) of PDZ1 and PDZ2 with the carboxy-terminal, five-amino acid residues that form the PDZ-recognition motif of PTHR, Npt2a, and β2-AR was calculated and compared with the calculated ΔG for the self-association of NHERF1. The results suggest that the interaction of the PTHR, β2-adrenergic, and Npt2a involves competition between NHERF1 PDZ domains and the target proteins. The binding of PDZ2 with PTHR may also compete with the self-inhibited conformation of NHERF1, thereby contributing to the stabilization of an active NHERF1 conformation. © 2012 American Chemical Society

  7. Characterization of the murine myeloid precursor cell line MuMac-E8.

    Science.gov (United States)

    Fricke, Stephan; Pfefferkorn, Cathleen; Wolf, Doris; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

    2014-01-01

    Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies.

  8. Characterization of the murine myeloid precursor cell line MuMac-E8.

    Directory of Open Access Journals (Sweden)

    Stephan Fricke

    Full Text Available Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin, of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45, for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64, showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies.

  9. Translational implication of Kallmann syndrome-1 gene expression in hepatocellular carcinoma.

    Science.gov (United States)

    Tanaka, Yuri; Kanda, Mitsuro; Sugimoto, Hiroyuki; Shimizu, Dai; Sueoka, Satoshi; Takami, Hideki; Ezaka, Kazuhiro; Hashimoto, Ryoji; Okamura, Yukiyasu; Iwata, Naoki; Tanaka, Chie; Yamada, Suguru; Fujii, Tsutomu; Nakayama, Goro; Koike, Masahiko; Nomoto, Shuji; Fujiwara, Michitaka; Kodera, Yasuhiro

    2015-01-01

    Accumulation of epigenetic alterations causes inactivation of tumor suppressors and contributes to the initiation and progression of hepatocellular carcinoma (HCC). Identification of methylated genes is necessary to improve our understanding of the pathogenesis of HCC and develop novel biomarkers and therapeutic targets. The Kallmann syndrome-1 (KAL1) gene encodes an extracellular matrix-related protein with diverse oncological functions. However, the function of KAL1 in HCC has not been examined. We investigated the methylation status of the KAL1 promoter region in HCC cell lines, and evaluated KAL1 mRNA levels and those of genes encoding potential interacting cell adhesion factors. KAL1 mRNA expression level was heterogeneous in nine HCC cell lines, and reactivation of KAL1 mRNA expression was observed in cells with promoter hypermethylation of KAL1 gene after demethylation. In addition, KAL1 mRNA levels inversely correlated with those of ezrin in all nine HCC cell lines. KAL1 expression levels in 144 pairs of surgically-resected tissues were determined and correlated to clinicopathological parameters. KAL1 mRNA level was independent of the background liver status, whereas HCC tissues showed significantly lower KAL1 mRNA levels than corresponding noncancerous liver tissues. Downregulation of KAL1 mRNA in HCC was significantly associated with malignant phenotype characteristics, including elevated tumor markers, larger tumor size, vascular invasion, and hypermethylation of KAL1. Patients with downregulation of KAL1 were more likely to have a shorter overall survival than other patients, and multivariate analysis identified downregulation of KAL1 as an independent prognostic factor (hazard ratio 2.04, 95% confidence interval 1.11-3.90, P=0.022). Our results indicated that KAL1 may act as a putative tumor suppressor in HCC and is inactivated by promoter hypermethylation. KAL1 may serve as a biomarker of malignant phenotype of HCC.

  10. Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures

    Science.gov (United States)

    Singh, Ratnesh K.; Mallela, Ramya K.; Cornuet, Pamela K.; Reifler, Aaron N.; Chervenak, Andrew P.; West, Michael D.; Wong, Kwoon Y.; Nasonkin, Igor O.

    2015-01-01

    Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na+ and K+ currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for

  11. Metastasis-associated phosphatase PRL-2 regulates tumor cell migration and invasion.

    Science.gov (United States)

    Wang, Y; Lazo, J S

    2012-02-16

    The phosphatase of regenerating liver (PRL) family, comprising PRL-1, PRL-2 and PRL-3, is a group of prenylated phosphatases that are candidate cancer biomarkers and therapeutic targets. Although several studies have documented that altered expression of PRL-1 or PRL-3 can influence cell proliferation, migration and invasion, there is a dearth of knowledge about the biological functions of PRL-2. Thus, in the current study we have evaluated the role of PRL-2 in cell migration and invasion in human cancer cells. We found that four human lung cancer cells, including A549 cells, overexpress PRL-2 when compared with normal lung cells. PRL-2 knockdown by RNA interference markedly inhibited cell migration and invasion, and this inhibition can be restored by overexpressing the short interference RNA (siRNA)-resistant vector HA-PRL-2m. PRL-2 suppression by siRNA decreased p130Cas and vinculin expression, and decreased extracellular signal-regulated kinase (ERK) phosphorylation, while increasing the phosphorylation of ezrin on tyrosine 146. We found no significant changes in total p53, Akt and c-Src expression levels or their phosphorylation status, suggesting that PRL-2 knockdown could inhibit tumor cell migration and invasion through a Src-independent p130Cas signaling pathway. Ectopic expression of wild-type PRL-2, a catalytic inactive C101S mutant and a C-terminal CAAX deletion revealed a requirement for both the PRL-2 catalytic functionality and prenylation site. Expression of wild-type but not mutant forms of PRL-2 caused ERK phosphorylation and nuclear translocation. These results support a model in which PRL-2 promotes cell migration and invasion through an ERK-dependent signaling pathway.

  12. Reversible bleb formation in mast cells stimulated with antigen is Ca2+/calmodulin-dependent and bleb size is regulated by ARF6.

    Science.gov (United States)

    Yanase, Yuhki; Carvou, Nicolas; Frohman, Michael A; Cockcroft, Shamshad

    2009-12-14

    Mast cells stimulated with antigen undergo extensive changes in their cytoskeleton. In the present study, we assess the impact of actin-modifying drugs and report that, in the presence of cytochalasin D, mast cells stop membrane ruffling, but instead bleb. Bleb formation is reversible following washout of cytochalasin D and occurs in an actin-polymerization-dependent manner. Bleb formation is inhibited by expression of constitutively active ezrin-T567D. Blebbing is also inhibited by blebbistatin, a myosin II inhibitor, implying myosin II activation in the process. We used a selection of inhibitors and observed that myosin II activation is dependent mainly on Ca2+-calmodulin, with only a small contribution from Rho kinase. The signalling pathways stimulated by antigen include PLC (phospholipase C) and PLD (phospholipase D). Bleb formation was dependent on activation of PLC, but not PLD. Primary alcohols, used previously as a means to reduce PLD-derived phosphatidic acid, were potent inhibitors of membrane blebbing, but a more selective inhibitor of PLD, FIPI (5-fluoro-2-indolyl des-chlorohalopemide), was without effect. FIPI also did not inhibit membrane ruffling or degranulation of mast cells, indicating that inhibition by primary alcohols works through an unidentified mechanism rather than via diversion of PLD activity as assumed. We also examined the requirement for ARF6 (ADP-ribosylation factor 6) and observed that its expression led to an increase in bleb size and a further increase was observed with the dominant-active mutant, ARF6-Q67L. Since ARF6-T27N had no effect on bleb size, we conclude that ARF6 needs to be active to regulate the size of the blebs.

  13. 玄参芦头片对玄参药材整体质量的影响研究%Impact of Scrophulariae Rhizome on Overall Quality of Scrophularia ningpoensis

    Institute of Scientific and Technical Information of China (English)

    许一鸣; 吴啟南; 乐巍; 蒋征; 桑梦如; 吴达维

    2016-01-01

    A high performance liquid chromatograph (HPLC) method was established for the simultaneous determination of five components (Harpagide,Verbascoside,Angoroside-C,Harpagoside,Cinnamic acid) in Scrophulariae Rhizome and Scrophulariae Radix,in order to investigate the impact of Scrophulariae Rhizome on the overall quality of Scrophulariae Radix.The determination was performed on a Waters 2695 HPLC with a 2998 DAD detector under 210,280 nm and 330 nm.In addition,the DPPH free radical scavenging rates of Scrophulariae Radix were determined at 517 nm by Ultraviolet Spectrophotometry.Consequently,the five components reached the baseline separation and there were no significant differences in the DPPH free radical scavenging rates between the extracts of Scrophulariae Rhizome and Scrophulariae Radix.Each batch of Scrophulariae Radix generally contained Scrophulariae Rhizome,and the total contents of Harpagide and Harpagoside in Scrophulariae Rhizome was similar to those in Scrophulariae Radix.Therefore,the existence of Scrophulariae Rhizome had little impact on the overall quality of Scrophulariae Radix.%本文主要利用高效液相色谱法对玄参芦头片和根片中5种成分(哈巴苷、麦角甾苷、安格洛苷C、哈巴俄苷、肉桂酸)进行含量测定,以探讨玄参芦头片对玄参药材整体质量的影响.首先,采用高效液相色谱法,在210、280、330 nm下对样品进行检测.其次,采用紫外分光光度法,在517 nm下对样品提取液的DPPH自由基清除率进行检测.玄参中所测定的5种成分均达到基线分离,其芦头片与根片提取液的DPPH自由基清除率无显著差异.各批玄参饮片中普遍存在芦头片,实验结果表明玄参芦头片和根片中的哈巴苷和哈巴俄苷总含量差异较小,因此芦头片对玄参药材整体质量基本没有影响.

  14. Evolutionary conservation of P-selectin glycoprotein ligand-1 primary structure and function

    Directory of Open Access Journals (Sweden)

    Schapira Marc

    2007-09-01

    Full Text Available Abstract Background P-selectin glycoprotein ligand-1 (PSGL-1 plays a critical role in recruiting leukocytes in inflammatory lesions by mediating leukocyte rolling on selectins. Core-2 O-glycosylation of a N-terminal threonine and sulfation of at least one tyrosine residue of PSGL-1 are required for L- and P-selectin binding. Little information is available on the intra- and inter-species evolution of PSGL-1 primary structure. In addition, the evolutionary conservation of selectin binding site on PSGL-1 has not been previously examined in detail. Therefore, we performed multiple sequence alignment of PSGL-1 amino acid sequences of 14 mammals (human, chimpanzee, rhesus monkey, bovine, pig, rat, tree-shrew, bushbaby, mouse, bat, horse, cat, sheep and dog and examined mammalian PSGL-1 interactions with human selectins. Results A signal peptide was predicted in each sequence and a propeptide cleavage site was found in 9/14 species. PSGL-1 N-terminus is poorly conserved. However, each species exhibits at least one tyrosine sulfation site and, except in horse and dog, a T [D/E]PP [D/E] motif associated to the core-2 O-glycosylation of a N-terminal threonine. A mucin-like domain of 250–280 amino acids long was disclosed in all studied species. It lies between the conserved N-terminal O-glycosylated threonine (Thr-57 in human and the transmembrane domain, and contains a central region exhibiting a variable number of decameric repeats (DR. Interspecies and intraspecies polymorphisms were observed. Transmembrane and cytoplasmic domain sequences are well conserved. The moesin binding residues that serve as adaptor between PSGL-1 and Syk, and are involved in regulating PSGL-1-dependent rolling on P-selectin are perfectly conserved in all analyzed mammalian sequences. Despite a poor conservation of PSGL-1 N-terminal sequence, CHO cells co-expressing human glycosyltransferases and human, bovine, pig or rat PSGL-1 efficiently rolled on human L- or P

  15. Focused examination of the intestinal lamina propria yields greater molecular insight into mechanisms underlying SIV induced immune dysfunction.

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    Mahesh Mohan

    Full Text Available BACKGROUND: The Gastrointestinal (GI tract is critical to AIDS pathogenesis as it is the primary site for viral transmission and a major site of viral replication and CD4(+ T cell destruction. Consequently GI disease, a major complication of HIV/SIV infection can facilitate translocation of lumenal bacterial products causing localized/systemic immune activation leading to AIDS progression. METHODOLOGY/PRINCIPAL FINDINGS: To better understand the molecular mechanisms underlying GI disease we analyzed global gene expression profiles sequentially in the intestine of the same animals prior to and at 21 and 90d post SIV infection (PI. More importantly we maximized information gathering by examining distinct mucosal components (intraepithelial lymphocytes, lamina propria leukocytes [LPL], epithelium and fibrovascular stroma separately. The use of sequential intestinal resections combined with focused examination of distinct mucosal compartments represents novel approaches not previously attempted. Here we report data pertaining to the LPL. A significant increase (±1.7-fold in immune defense/inflammation, cell adhesion/migration, cell signaling, transcription and cell division/differentiation genes were observed at 21 and 90d PI. Genes associated with the JAK-STAT pathway (IL21, IL12R, STAT5A, IL10, SOCS1 and T-cell activation (NFATc1, CDK6, Gelsolin, Moesin were notably upregulated at 21d PI. Markedly downregulated genes at 21d PI included IL17D/IL27 and IL28B/IFNγ3 (anti-HIV/viral, activation induced cytidine deaminase (B-cell function and approximately 57 genes regulating oxidative phosphorylation, a critical metabolic shift associated with T-cell activation. The 90d transcriptome revealed further augmentation of inflammation (CXCL11, chitinase-1, JNK3, immune activation (CD38, semaphorin7A, CD109, B-cell dysfunction (CD70, intestinal microbial translocation (Lipopolysaccharide binding protein and mitochondrial antiviral signaling (NLRX1 genes

  16. The FOXP2-Driven Network in Developmental Disorders and Neurodegeneration

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    Franz Oswald

    2017-07-01

    organization, neuro-inflammation, and processing of amyloid precursor protein. Conspicuously, many links pointed to an involvement of the FOXP2-driven network in JAK/STAT signaling and the regulation of the ezrin–radixin–moesin complex. Altogether, the applied phylogenetic perspective substantiated FOXP2’s importance for nervous system development, maintenance, and functioning. However, the study also disclosed new regulatory pathways that might prove to be useful for understanding the molecular background of the aforementioned developmental disorders and neurodegenerative diseases.

  17. 鼻咽癌细胞中p53相互作用蛋白质的分离和鉴定%Separation and Identification of p53 Interacting Proteins From Human Nasopharyngeal Carcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    胡巍; 肖志强; 陈主初; 李建玲; 张鹏飞; 冯雪萍; 易红; 余艳辉; 唐新科; 刘清萍; 梁宋平

    2004-01-01

    鼻咽癌中p53基因突变罕见,但绝大部分鼻咽癌中存在p53蛋白过表达/聚集且功能失活.然而,到目前为止p53蛋白失活的机制仍然不清楚.为揭示鼻咽癌中p53蛋白功能失活的机制,采用免疫共沉淀技术分别富集鼻咽癌细胞系HNE1和HNE2的p53结合蛋白,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)对免疫沉淀复合物进行分离,从胶中切取p53结合蛋白条带,胶内酶解后进行电喷雾串联质谱(LC-ESI-MS/MS)分析,得到相应的肽序列标签(peptide sequence tags,PST),通过搜索数据库在鼻咽癌细胞系中鉴定了9个p53结合蛋白.分别是热休克蛋白70(HSP70)家族成员GRP-78和GRP-75、HSP90家族成员GRP-94、核纤层蛋白A/C(Lamin A/C)、α-actinin 4、Ezrin/Cytovillin、DNA复制准许因子/MCM3蛋白(DNA replication licensing factor/minichromosome maintenance 3 protein,MCM3)、CD98/4F2 heavy chain和蛋白激酶C(PKC).并用免疫共沉淀和蛋白质印迹分析技术对HNE1细胞蛋白条带3鉴定的p53相互作用蛋白之一HSP78进行了验证.首次在鼻咽癌细胞中鉴定了9个p53结合蛋白,为阐明鼻咽癌中p53蛋白聚集及失活的机制提供了重要依据和线索.

  18. How the NOTCH pathway contributes to the ability of osteosarcoma cells to metastasize.

    Science.gov (United States)

    Hughes, Dennis P M

    2009-01-01

    Controlling metastasis is the key to improving outcomes for osteosarcoma patients; yet our knowledge of the mechanisms regulating the metastatic process is incomplete. Clearly Fas and Ezrin are important, but other genes must play a role in promoting tumor spread. Early developmental pathways are often recapitulated in malignant tissues, and these genes are likely to be important in regulating the primitive behaviors of tumor cells, including invasion and metastasis. The Notch pathway is a highly conserved regulatory signaling network involved in many developmental processes and several cancers, at times serving as an oncogene and at others, behaving as a tumor suppressor. In normal limb development, Notch signaling maintains the apical ectodermal ridge in the developing limb bud and regulated size of bone and muscles. Here, we examine the role of Notch signaling in promoting metastasis of osteosarcoma, and the underlying regulatory processes that control Notch pathway expression and activity in the disease. We have shown that, compared to normal human osteoblasts and non-metastatic osteosarcoma cell lines, osteosarcoma cell lines with the ability to metastasize have higher levels of Notch 1, Notch 2, the Notch ligand DLL1 and the Notch-induced gene Hes1. When invasive osteosarcoma cells are treated with small molecule inhibitors of gamma-secretase, which blocks Notch activation, invasiveness is abrogated. Direct retroviral expression has shown that Hes1 expression was necessary for osteosarcoma invasiveness and accounted for the observations. In a novel orthotopic murine xenograft model of osteosarcoma pulmonary metastasis, blockade of Hes1 expression and Notch signaling eliminated spread of disease from the tibial primary tumor. In a sample of archival human osteosarcoma tumor specimens, expression of Hes1 mRNA was inversely correlated with survival (n=16 samples, p=0.04). Expression of the microRNA 34 cluster, which is known to downregulate DLL1, Notch 1 and

  19. Focused examination of the intestinal epithelium reveals transcriptional signatures consistent with disturbances in enterocyte maturation and differentiation during the course of SIV infection.

    Directory of Open Access Journals (Sweden)

    Mahesh Mohan

    Full Text Available The Gastrointestinal (GI tract plays a pivotal role in AIDS pathogenesis as it is the primary site for viral transmission, replication and CD4(+ T cell destruction. Accordingly, GI disease (enteropathy has become a well-known complication and a driver of AIDS progression. To better understand the molecular mechanisms underlying GI disease we analyzed global gene expression profiles sequentially in the intestinal epithelium of the same animals before SIV infection and at 21 and 90 days post infection (DPI. More importantly we obtained sequential excisional intestinal biopsies and examined distinct mucosal components (epithelium. intraepithelial lymphocytes, lamina propria lymphocytes, fibrovascular stroma separately. Here we report data pertaining to the epithelium. Overall genes associated with epithelial cell renewal/proliferation/differentiation, permeability and adhesion were significantly down regulated (<1.5-7 fold at 21 and 90DPI. Genes regulating focal adhesions (n = 6, gap junctions (n = 3, ErbB (n = 3 and Wnt signaling (n = 4 were markedly down at 21DPI and the number of genes in each of these groups that were down regulated doubled between 21 and 90DPI. Notable genes included FAK, ITGA6, PDGF, TGFβ3, Ezrin, FZD6, WNT10A, and TCF7L2. In addition, at 90DPI genes regulating ECM-receptor interactions (laminins and ITGB1, epithelial cell gene expression (PDX1, KLF6, polarity/tight junction formation (PARD3B&6B and histone demethylase (JMJD3 were also down regulated. In contrast, expression of NOTCH3, notch target genes (HES4, HES7 and EZH2 (histone methyltransferase were significantly increased at 90DPI. The altered expression of genes linked to Wnt signaling together with decreased expression of PDX1, PARD3B, PARD6B and SDK1 suggests marked perturbations in intestinal epithelial function and homeostasis leading to breakdown of the mucosal barrier. More importantly, the divergent expression patterns of EZH2 and JMJD3

  20. Oncogenic pathways implicated in ovarian epithelial cancer.

    Science.gov (United States)

    Nicosia, Santo V; Bai, Wenlong; Cheng, Jin Q; Coppola, Domenico; Kruk, Patricia A

    2003-08-01

    Characterization of intracellular signaling pathways should lead to a better understanding of ovarian epithelial carcinogenesis and provide an opportunity to interfere with signal transduction targets involved in ovarian tumor cell growth, survival, and progression. Challenges toward such an effort are significant because many of these signals are part of cascades within an intricate and likely redundant intracellular signaling network (Fig.1). For instance, a given signal may activate a dual intracellular pathway (ie, MEK1-MAPK and PI3K/Akt required for fibronectin-dependent activation of matrix metalloproteinase 9). A single pathway also may transduce more than one biologic or oncogenic signal (ie, PI3K signaling in epithelial and endothelial cell growth and sprouting of neovessels). Despite these challenges, evidence for therapeutic targeting of signal transduction pathways is accumulating in human cancer. For instance, the EGF-specific tyrosine kinase inhibitor ZD 1839 (Iressa) may have a beneficial therapeutic effect on ovarian epithelial cancer. Therapy of this cancer may include inhibitors of PI kinase (quercetin), ezrin and PIP kinase (genistein). The G protein-coupled family of receptors, including LPA, also is an attractive target to drugs, although their frequent pleiotropic functions may be at times toxic and lack specificity. Because of the lack of notable toxicity, PI3K/Akt pathway inhibitors such as FTIs are a promising targeted therapy of ovarian epithelial cancer. Increasing insight into the oncogenic pathways involved in ovarian epithelial cancer also is helping clinicians to understand better the phenomenon of chemoresistance in this malignancy. Oncogenic activation of gamma-synuclein promotes cell survival and provides resistance to paclitaxel, but such a resistance is partially overcome by an MEK inhibitor that suppresses ERK activity. Ovarian epithelial cancer is a complex group of neoplasms with an overall poor prognosis. Comprehension of