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Sample records for focal adhesions regulating

  1. Regulation of Cell Adhesion Strength by Peripheral Focal Adhesion Distribution

    OpenAIRE

    2011-01-01

    Cell adhesion to extracellular matrices is a tightly regulated process that involves the complex interplay between biochemical and mechanical events at the cell-adhesive interface. Previous work established the spatiotemporal contributions of adhesive components to adhesion strength and identified a nonlinear dependence on cell spreading. This study was designed to investigate the regulation of cell-adhesion strength by the size and position of focal adhesions (FA). The cell-adhesive interfac...

  2. Regulation of cell adhesion strength by peripheral focal adhesion distribution.

    Science.gov (United States)

    Elineni, Kranthi Kumar; Gallant, Nathan D

    2011-12-21

    Cell adhesion to extracellular matrices is a tightly regulated process that involves the complex interplay between biochemical and mechanical events at the cell-adhesive interface. Previous work established the spatiotemporal contributions of adhesive components to adhesion strength and identified a nonlinear dependence on cell spreading. This study was designed to investigate the regulation of cell-adhesion strength by the size and position of focal adhesions (FA). The cell-adhesive interface was engineered to direct FA assembly to the periphery of the cell-spreading area to delineate the cell-adhesive area from the cell-spreading area. It was observed that redistributing the same adhesive area over a larger cell-spreading area significantly enhanced cell-adhesion strength, but only up to a threshold area. Moreover, the size of the peripheral FAs, which was interpreted as an adhesive patch, did not directly govern the adhesion strength. Interestingly, this is in contrast to the previously reported functional role of FAs in regulating cellular traction where sizes of the peripheral FAs play a critical role. These findings demonstrate, to our knowledge for the first time, that two spatial regimes in cell-spreading area exist that uniquely govern the structure-function role of FAs in regulating cell-adhesion strength.

  3. ADAMTS-10 and -6 differentially regulate cell-cell junctions and focal adhesions

    Science.gov (United States)

    Cain, Stuart A.; Mularczyk, Ewa J.; Singh, Mukti; Massam-Wu, Teresa; Kielty, Cay M.

    2016-01-01

    ADAMTS10 and ADAMTS6 are homologous metalloproteinases with ill-defined roles. ADAMTS10 mutations cause Weill-Marchesani syndrome (WMS), implicating it in fibrillin microfibril biology since some fibrillin-1 mutations also cause WMS. However little is known about ADAMTS6 function. ADAMTS10 is resistant to furin cleavage, however we show that ADAMTS6 is effectively processed and active. Using siRNA, over-expression and mutagenesis, it was found ADAMTS6 inhibits and ADAMTS10 is required for focal adhesions, epithelial cell-cell junction formation, and microfibril deposition. Either knockdown of ADAMTS6, or disruption of its furin processing or catalytic sites restores focal adhesions, implicating its enzyme activity acts on targets in the focal adhesion complex. In ADAMTS10-depleted cultures, expression of syndecan-4 rescues focal adhesions and cell-cell junctions. Recombinant C-termini of ADAMTS10 and ADAMTS6, both of which induce focal adhesions, bind heparin and syndecan-4. However, cells overexpressing full-length ADAMTS6 lack heparan sulphate and focal adhesions, whilst depletion of ADAMTS6 induces a prominent glycocalyx. Thus ADAMTS10 and ADAMTS6 oppositely affect heparan sulphate-rich interfaces including focal adhesions. We previously showed that microfibril deposition requires fibronectin-induced focal adhesions, and cell-cell junctions in epithelial cultures. Here we reveal that ADAMTS6 causes a reduction in heparan sulphate-rich interfaces, and its expression is regulated by ADAMTS10. PMID:27779234

  4. The regulation of traction force in relation to cell shape and focal adhesions.

    Science.gov (United States)

    Rape, Andrew D; Guo, Wei-Hui; Wang, Yu-Li

    2011-03-01

    Mechanical forces provide critical inputs for proper cellular functions. The interplay between the generation of, and response to, mechanical forces regulate such cellular processes as differentiation, proliferation, and migration. We postulate that adherent cells respond to a number of physical and topographical factors, including cell size and shape, by detecting the magnitude and/or distribution of traction forces under different conditions. To address this possibility we introduce a new simple method for precise micropatterning of hydrogels, and then apply the technique to systematically investigate the relationship between cell geometry, focal adhesions, and traction forces in cells with a series of spread areas and aspect ratios. Contrary to previous findings, we find that traction force is not determined primarily by the cell spreading area but by the distance from cell center to the perimeter. This distance in turn controls traction forces by regulating the size of focal adhesions, such that constraining the size of focal adhesions by micropatterning can override the effect of geometry. We propose that the responses of traction forces to center-periphery distance, possibly through a positive feedback mechanism that regulates focal adhesions, provide the cell with the information on its own shape and size. A similar positive feedback control may allow cells to respond to a variety of physical or topographical signals via a unified mechanism. Copyright © 2010 Elsevier Ltd. All rights reserved.

  5. Regulation of brain tumor dispersal by NKCC1 through a novel role in focal adhesion regulation.

    Directory of Open Access Journals (Sweden)

    Tomas Garzon-Muvdi

    Full Text Available Glioblastoma (GB is a highly invasive and lethal brain tumor due to its universal recurrence. Although it has been suggested that the electroneutral Na(+-K(+-Cl(- cotransporter 1 (NKCC1 can play a role in glioma cell migration, the precise mechanism by which this ion transporter contributes to GB aggressiveness remains poorly understood. Here, we focused on the role of NKCC1 in the invasion of human primary glioma cells in vitro and in vivo. NKCC1 expression levels were significantly higher in GB and anaplastic astrocytoma tissues than in grade II glioma and normal cortex. Pharmacological inhibition and shRNA-mediated knockdown of NKCC1 expression led to decreased cell migration and invasion in vitro and in vivo. Surprisingly, knockdown of NKCC1 in glioma cells resulted in the formation of significantly larger focal adhesions and cell traction forces that were approximately 40% lower than control cells. Epidermal growth factor (EGF, which promotes migration of glioma cells, increased the phosphorylation of NKCC1 through a PI3K-dependant mechanism. This finding is potentially related to WNK kinases. Taken together, our findings suggest that NKCC1 modulates migration of glioma cells by two distinct mechanisms: (1 through the regulation of focal adhesion dynamics and cell contractility and (2 through regulation of cell volume through ion transport. Due to the ubiquitous expression of NKCC1 in mammalian tissues, its regulation by WNK kinases may serve as new therapeutic targets for GB aggressiveness and can be exploited by other highly invasive neoplasms.

  6. Focal Adhesion Kinase Regulates Expression of Thioredoxin-interacting Protein (TXNIP) in Cancer Cells

    OpenAIRE

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter ac...

  7. The role of focal adhesion kinase in the regulation of cellular mechanical properties

    Science.gov (United States)

    Mierke, Claudia Tanja

    2013-12-01

    The regulation of mechanical properties is necessary for cell invasion into connective tissue or intra- and extravasation through the endothelium of blood or lymph vessels. Cell invasion is important for the regulation of many healthy processes such as immune response reactions and wound healing. In addition, cell invasion plays a role in disease-related processes such as tumor metastasis and autoimmune responses. Until now the role of focal adhesion kinase (FAK) in regulating mechanical properties of cells and its impact on cell invasion efficiency is still not well known. Thus, this review focuses on mechanical properties regulated by FAK in comparison to the mechano-regulating protein vinculin. Moreover, it points out the connection between cancer cell invasion and metastasis and FAK by showing that FAK regulates cellular mechanical properties required for cellular motility. Furthermore, it sheds light on the indirect interaction of FAK with vinculin by binding to paxillin, which then impairs the binding of paxillin to vinculin. In addition, this review emphasizes whether FAK fulfills regulatory functions similar to vinculin. In particular, it discusses the differences and the similarities between FAK and vinculin in regulating the biomechanical properties of cells. Finally, this paper highlights that both focal adhesion proteins, vinculin and FAK, synergize their functions to regulate the mechanical properties of cells such as stiffness and contractile forces. Subsequently, these mechanical properties determine cellular invasiveness into tissues and provide a source sink for future drug developments to inhibit excessive cell invasion and hence, metastases formation.

  8. Angiogenin enhances cell migration by regulating stress fiber assembly and focal adhesion dynamics.

    Directory of Open Access Journals (Sweden)

    Saisai Wei

    Full Text Available Angiogenin (ANG acts on both vascular endothelial cells and cancer cells, but the underlying mechanism remains elusive. In this study, we carried out a co-immunoprecipitation assay in HeLa cells and identified 14 potential ANG-interacting proteins. Among these proteins, β-actin, α-actinin 4, and non-muscle myosin heavy chain 9 are stress fiber components and involved in cytoskeleton organization and movement, which prompted us to investigate the mechanism of action of ANG in cell migration. Upon confirmation of the interactions between ANG and the three proteins, further studies revealed that ANG co-localized with β-actin and α-actinin 4 at the leading edge of migrating cells. Down-regulation of ANG resulted in fewer but thicker stress fibers with less dynamics, which was associated with the enlargements of focal adhesions. The focal adhesion kinase activity and cell migration capacity were significantly decreased in ANG-deficient cells. Taken together, our data demonstrated that the existence of ANG in the cytoplasm optimizes stress fiber assembly and focal adhesion formation to accommodate cell migration. The finding that ANG promoted cancer cell migration might provide new clues for tumor metastasis research.

  9. Focal adhesion kinase regulates expression of thioredoxin-interacting protein (TXNIP) in cancer cells.

    Science.gov (United States)

    Ho, Baotran; Huang, Grace; Golubovskaya, Vita M

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells.

  10. Role of VASP phosphorylation for the regulation of microglia chemotaxis via the regulation of focal adhesion formation/maturation.

    Science.gov (United States)

    Lee, S; Chung, C Y

    2009-12-01

    Microglia activation and migration are known to play crucial roles for the response to brain injuries. Extracellular ADP was reported to induce microglia chemotaxis and membrane ruffles through P2Y12 receptor. In this study, we examined the role of VASP phosphorylation in ADP-induced microglia chemotaxis and membrane ruffle formation. ADP stimulation transiently increased intracellular cAMP level, VASP phosphorylation at Ser153, membrane ruffle formation, and chemotaxis. PKA inhibitor effectively inhibited VASP phosphorylation and chemotaxis, indicating that P2Y12-mediated activation of PKA and subsequent VASP phosphorylation are involved in the regulation of microglia chemotaxis. Forskolin and okadaic acid induced sustained VASP phosphorylation at a high level, causing a significant reduction of the retraction of membrane ruffles and chemotaxis. In forskolin- or okadaic acid-treated cells, phosphorylated VASP remained at the membrane cortex, and size and number of mature focal adhesions were not increased, indicating that prolonged phosphorylation of VASP could inhibit transformation of focal complexes into focal adhesions. VASP knockdown cells showed markedly reduced frequency and distance of membrane ruffling upon ADP stimulation, reinforcing the idea that VASP is required for the ruffle formation. Cells expressing GFP-VASP(S153A) also showed a significant reduction of protrusion distance during ruffle formation, but the frequency and the distance of retraction were not affected by FSK at all. This result suggests that dephosphorylation of VASP might be required for the growth of adhesion strength during membrane retraction. Our results suggest that VASP phosphorylation by PKA plays an important role in membrane ruffle formation and chemotaxis via the regulation of focal adhesion formation/maturation.

  11. Vascular Endothelial-Cadherin Regulates Cytoskeletal Tension, Cell Spreading, and Focal Adhesions by Stimulating RhoAD⃞

    Science.gov (United States)

    Nelson, Celeste M.; Pirone, Dana M.; Tan, John L.; Chen, Christopher S.

    2004-01-01

    Changes in vascular endothelial (VE)-cadherin–mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion coordinate to affect the physical and mechanical rearrangements of the endothelium, although the mechanisms for such cross talk remain undefined. Herein, we describe the regulation of focal adhesion formation and cytoskeletal tension by intercellular VE-cadherin engagement, and the molecular mechanism by which this occurs. Increasing the density of endothelial cells to increase cell-cell contact decreased focal adhesions by decreasing cell spreading. This contact inhibition of cell spreading was blocked by disrupting VE-cadherin engagement with an adenovirus encoding dominant negative VE-cadherin. When changes in cell spreading were prevented by culturing cells on a micropatterned substrate, VE-cadherin–mediated cell-cell contact paradoxically increased focal adhesion formation. We show that VE-cadherin engagement mediates each of these effects by inducing both a transient and sustained activation of RhoA. Both the increase and decrease in cell-matrix adhesion were blocked by disrupting intracellular tension and signaling through the Rho-ROCK pathway. In all, these findings demonstrate that VE-cadherin signals through RhoA and the actin cytoskeleton to cross talk with cell-matrix adhesion and thereby define a novel pathway by which cell-cell contact alters the global mechanical and functional state of cells. PMID:15075376

  12. mDia2 regulates actin and focal adhesion dynamics and organization in the lamella for efficient epithelial cell migration.

    Science.gov (United States)

    Gupton, Stephanie L; Eisenmann, Kathryn; Alberts, Arthur S; Waterman-Storer, Clare M

    2007-10-01

    Cell migration requires spatial and temporal regulation of filamentous actin (F-actin) dynamics. This regulation is achieved by distinct actin-associated proteins, which mediate polymerization, depolymerization, severing, contraction, bundling or engagement to the membrane. Mammalian Diaphanous-related (mDia) formins, which nucleate, processively elongate, and in some cases bundle actin filaments, have been extensively studied in vitro, but their function in the cell has been less well characterized. Here we study the role of mDia2 activity in the dynamic organization of F-actin in migrating epithelial cells. We find that mDia2 localizes in the lamella of migrating epithelial cells, where it is involved in the formation of a stable pool of cortical actin and in maintenance of polymerization-competent free filament barbed ends at focal adhesions. Specific inhibition of mDia2 alters focal adhesion turnover and reduces migration velocity. We suggest that the regulation of filament assembly dynamics at focal adhesions may be necessary for the formation of a stable pool of cortical lamella actin and the proper assembly and disassembly dynamics of focal adhesions, making mDia2 an important factor in epithelial cell migration.

  13. [Study on FAK regulation of migration of vascular endothelial cells depending upon focal adhesion proteins].

    Science.gov (United States)

    Gao, Min; Liu, Xiaoheng; Sun, Heng; Ren, Hongyi; Wang, Lijuan; Shen, Yang

    2013-06-01

    Tumor angiogenesis induced by vascular endothelial cells (VECs) migration is a necessary condition for tumor growth and metastasis. The purpose of this study is to investigate the effect of focal adhesion kinase (FAK) inhibitor (50nmol/mL) on the adhesion and migration of endothelial cells(ECs) and the expression of focal adhesion proteins vinculin, talin and paxillin. Scratch wound migration assay was performed to examine the effect of FAK inhibitor with 50nmol/mL on ECs migration at 0, 5, 10, 30, 60 and 120min, respectively. And immunofluorescence analysis was performed to detect the expression of F-actin in ECs treated with FAK inhibitor within 2h. Western blot was carried out to determine the effect of FAK inhibitor on expression of vinculin, talin and paxillin proteins. The results showed that the migration distance and the expression of F-actin in ECs treated with FAK inhibitor decreased significantly compared with that of the controls, and the level of vinculin showed no significant difference with increasing of treated time of FAK inhibitor. However, the talin and paxillin showed an identical decreasing tendency in 5-10min, but slowly going up in 30min and then after subsequently decreasing. The results of this study proved that blocking phosphorylation of FAK could inhibit VECs adhesion and migration by downregulating focal adhesion proteins so that it may inhibit tumor angiogenesis. This may provide a new approach for tumor therapy.

  14. MicroRNA-7 regulates glioblastoma cell invasion via targeting focal adhesion kinase expression

    Institute of Scientific and Technical Information of China (English)

    WU De-gang; WANG Xi-rui; YOU Yong-ping; LIU Ning; WANG Ying-yi; FAN Li-gang; LUO Hui; HAN Bin; SUN Li-hua; WANG Xie-feng; ZHANG Jun-xia; CAO Lei

    2011-01-01

    Background Invasion growth is the most characteristic biological phenotype of glioblastoma,but the molecular mechanism in glioma cell invasion is poorly understood.Recent data have showed that microRNA plays an essential role in tumor invasion.Our study aimed to explore the mechanism of miR-7 involved in the control of glioblastoma cell invasion.Methods Glioma cell invasion was evaluated by transwell and scratch assays after up-regulation of miR-7 using miR-7 mimics in U87 and U251 cells.Luciferase reporter assay was used to determine focal adhesion kinase (FAK) as a target of miR-7.The levels of miR-7,matrix metalloproteinases (MMP)-2 and MMP-9 mRNA were detected by PCR assay,and the levels of FAK,MMP-2,MMP-9,total and phosphorylation serine/threonine kinase (AKT),and extracellular signal-regulated kinase (ERK) 1/2 were measured by Western blotting analysis.Results Over-expression of miR-7 inhibited the invasion and migration activity of U87 and U251 cells.And up-regulation of miR-7 reduced FAK protein expression,Further,luciferase reporter assay showed that miR-7 modulated FAK expression directly by binding 3'UTR of FAK mRNA.In addition,miR-7 repressed p-ERK1/2 and p-AKT level,MMP-2 and MMP-9 expression.Finally,the inverse relationship between FAK and miR-7 expression was certificated in human glioma tissues.Conclusion To our knowledge,these data indicate for the first time that miR-7 directly regulates cell invasion by targeting FAK in glioblastoma and that miR-7 could be a potential therapeutic target for glioblastoma intervention.

  15. The emerin-binding transcription factor Lmo7 is regulated by association with p130Cas at focal adhesions

    Directory of Open Access Journals (Sweden)

    Michele A. Wozniak

    2013-08-01

    Full Text Available Loss of function mutations in the nuclear inner membrane protein, emerin, cause X-linked Emery-Dreifuss muscular dystrophy (X-EDMD. X-EDMD is characterized by contractures of major tendons, skeletal muscle weakening and wasting, and cardiac conduction system defects. The transcription factor Lmo7 regulates muscle- and heart-relevant genes and is inhibited by binding to emerin, suggesting Lmo7 misregulation contributes to EDMD disease. Lmo7 associates with cell adhesions and shuttles between the plasma membrane and nucleus, but the regulation and biological consequences of this dual localization were unknown. We report endogenous Lmo7 also associates with focal adhesions in cells, and both co-localizes and co-immunoprecipitates with p130Cas, a key signaling component of focal adhesions. Lmo7 nuclear localization and transcriptional activity increased significantly in p130Cas-null MEFs, suggesting Lmo7 is negatively regulated by p130Cas-dependent association with focal adhesions. These results support EDMD models in which Lmo7 is a downstream mediator of integrin-dependent signaling that allows tendon cells and muscles to adapt to and withstand mechanical stress.

  16. Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation.

    OpenAIRE

    1996-01-01

    It has been proposed that the focal adhesion kinase (FAK) mediates focal adhesion formation through tyrosine phosphorylation during cell adhesion. We investigated the role of FAK in focal adhesion structure and function. Loading cells with a glutathione-S-transferase fusion protein (GST-Cterm) containing the FAK focal adhesion targeting sequence, but not the kinase domain, decreased the association of endogenous FAK with focal adhesions. This displacement of endogenous FAK in both BALB/c 3T3 ...

  17. HAb18G/CD147 regulates vinculin-mediated focal adhesion and cytoskeleton organization in cultured human hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Qiang Liang

    Full Text Available Focal adhesions (FAs, integrin-mediated macromolecular complexes located at the cell membrane extracellular interface, have been shown to regulate cell adhesion and migration. Our previous studies have indicated that HAb18G/CD147 (CD147 is involved in cytoskeleton reorganization and FA formation in human hepatocellular carcinoma (HCC cells. However, the precise mechanisms underlying these processes remain unclear. In the current study, we determined that CD147 was involved in vinculin-mediated FA focal adhesion formation in HCC cells. We also found that deletion of CD147 led to reduced vinculin-mediated FA areas (P<0.0001, length/width ratios (P<0.0001, and mean intensities (P<0.0001. CD147 promoted lamellipodia formation by localizing Arp2/3 to the leading edge of the cell. Deletion of CD147 significantly reduced the fluorescence (t1/2 recovery times (22.7±3.3 s of vinculin-mediated focal adhesions (P<0.0001. In cell-spreading assays, CD147 was found to be essential for dynamic focal adhesion enlargement and disassembly. Furthermore, the current data showed that CD147 reduced tyrosine phosphorylation in vinculin-mediated focal adhesions, and enhanced the accumulation of the acidic phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2. Together, these results revealed that CD147 is involved in vinculin-mediated focal adhesion formation, which subsequently promotes cytoskeleton reorganization to facilitate invasion and migration of human HCC cells.

  18. Focal adhesion kinase regulates pathogen-killing capability and life span of neutrophils via mediating both adhesion-dependent and -independent cellular signals.

    Science.gov (United States)

    Kasorn, Anongnard; Alcaide, Pilar; Jia, Yonghui; Subramanian, Kulandayan K; Sarraj, Bara; Li, Yitang; Loison, Fabien; Hattori, Hidenori; Silberstein, Leslie E; Luscinskas, William F; Luo, Hongbo R

    2009-07-15

    Various neutrophil functions such as phagocytosis, superoxide production, and survival are regulated by integrin signaling. Despite the essential role of focal adhesion kinase (FAK) in mediating this signaling pathway, its exact function in neutrophils is ill defined. In this study, we investigated the role of FAK in neutrophils using a myeloid-specific conditional FAK knockout mouse. As reported in many other cell types, FAK is required for regulation of focal adhesion dynamics when neutrophils adhere to fibronectin or ICAM-1. Adhesion on VCAM-1-coated surfaces and chemotaxis after adhesion were not altered in FAK null neutrophils. In addition, we observed significant reduction in NADPH oxidase-mediated superoxide production and complement-mediated phagocytosis in FAK null neutrophils. As a result, these neutrophils displayed decreased pathogen killing capability both in vitro and in vivo in a mouse peritonitis model. In adherent cells, the defects associated with FAK deficiency are likely due to suppression of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) signaling and chemoattractant-elicited calcium signaling. Disruption of FAK also reduced chemoattractant-elicited superoxide production in suspended neutrophils in the absence of cell adhesion. This may be solely caused by suppression of PtdIns(3,4,5)P3 signaling in these cells, because the fMLP-elicited calcium signal was not altered. Consistent with decreased PtdIns(3,4,5)P3/Akt signaling in FAK null neutrophils, we also observed accelerated spontaneous death in these cells. Taken together, our results revealed previously unrecognized roles of FAK in neutrophil function and provided a potential therapeutic target for treatment of a variety of infectious and inflammatory diseases.

  19. Focal adhesion kinase is a phospho-regulated repressor of Rac and proliferation in human endothelial cells

    Directory of Open Access Journals (Sweden)

    Patrick W. Bryant

    2012-06-01

    Focal adhesion kinase (FAK is critically positioned to integrate signals from the extracellular matrix and cellular adhesion. It is essential for normal vascular development and has been implicated in a wide range of cellular functions including the regulation of cell proliferation, migration, differentiation, and survival. It is currently being actively targeted therapeutically using different approaches. We have used human endothelial cells as a model system to compare the effects of inhibiting FAK through several different approaches including dominant negatives, kinase inhibitors and shRNA. We find that manipulations of FAK signaling that result in inhibition of FAK 397 phosphorylation inhibit proliferation and migration. However, abolition of FAK expression using stable (shRNA or transient (siRNA approaches does not interfere with these cellular functions. The ability to regulate cell proliferation by FAK manipulation is correlated with the activation status of Rac, an essential signal for the regulation of cyclin-dependent kinase inhibitors. The knockdown of FAK, while not affecting cellular proliferation or migration, dramatically interferes with vascular morphogenesis and survival, mirroring in vivo findings. We propose a novel model of FAK signaling whereby one of the multifunctional roles of FAK as a signaling protein includes FAK as a phospho-regulated repressor of Rac activation, with important implications on interpretation of research experiments and therapeutic development.

  20. Up-regulation of Paxillin and Focal Adhesion Signaling follows Dystroglycan Complex deletions and promotes a Hypertensive State of Differentiation

    OpenAIRE

    Sen, Shamik; Tewari, Manorama; Zajac, Allison; Barton, Elisabeth; Sweeney, H. Lee; Discher, Dennis E.

    2011-01-01

    Anchorage to matrix is mediated for many cells not only by integrin-based focal adhesions but also by a parallel assembly of integral and peripheral membrane proteins known as the Dystroglycan Complex. Deficiencies in either dystrophin (mdx mice) or γ-sarcoglycan (γSG−/− mice) components of the Dystroglycan Complex lead to upregulation of numerous focal adhesion proteins, and the phosphoprotein paxillin proves to be among the most prominent. In mdx muscle, paxillin-Y31 and Y118 are both hyper...

  1. Protein kinase C, focal adhesions and the regulation of cell migration

    DEFF Research Database (Denmark)

    Fogh, Betina S; Multhaupt, Hinke A B; Couchman, John Robert

    2014-01-01

    Cell adhesion to extracellular matrix is a complex process involving protrusive activity driven by the actin cytoskeleton, engagement of specific receptors, followed by signaling and cytoskeletal organization. Thereafter, contractile and endocytic/recycling activities may facilitate migration...

  2. Defining central themes in breast cancer biology by differential proteomics: conserved regulation of cell spreading and focal adhesion kinase.

    Science.gov (United States)

    Bateman, Nicholas W; Sun, Mai; Hood, Brian L; Flint, Melanie S; Conrads, Thomas P

    2010-10-01

    Breast cancer is a highly heterogeneous disease, an observation that underscores the importance of elucidating conserved molecular characteristics, such as gene and protein expression, across breast cancer cell types toward providing a greater understanding of context-specific features central to this disease. Motivated by the goal of defining central biological themes across breast cancer cell subtypes, we conducted a global proteomic analysis of three breast cancer cell lines, MCF7, SK-BR-3, and MDA-MB-231, and compared these to a model of nontransformed mammary cells (MCF10A). Our results demonstrate modulation of proteins localized to the extracellular matrix, plasma membrane, and nucleus, along with coordinate decreases in proteins that regulate "cell spreading," a cellular event previously shown to be dysregulated in transformed cells. Protein interaction network analysis revealed the clustering of focal adhesion kinase (FAK), a fundamental regulator of cell spreading, with several proteins identified as mutually, differentially abundant across breast cancer cell lines that impact expression and activity of FAK, such as neprilysin and keratin 19. These analyses provide insights into conservation of protein expression across breast cancer cell subtypes, a subset of which warrants further investigation for their roles in the regulation of cell spreading and FAK in breast cancer.

  3. Down-regulation of integrin β1 and focal adhesion kinase in renal glomeruli under various hemodynamic conditions.

    Directory of Open Access Journals (Sweden)

    Xiaoli Yuan

    Full Text Available Given that integrin β1 is an important component of the connection to maintain glomerular structural integrity, by binding with multiple extracellular matrix proteins and mediating intracellular signaling. Focal adhesion kinase (FAK is the most essential intracellular integrator in the integrin β1-FAK signalling pathway. Here, we investigated the changes of the two molecules and visualized the possible interaction between them under various hemodynamic conditions in podocytes. Mice kidney tissues were prepared using in vivo cryotechnique (IVCT and then were stained and observed using light microscopy, confocal laser scanning microscopy and immunoelectron microscopy. The expression of these molecules were examined by western blot. Under the normal condition, integrin β1 stained continually and evenly at the membrane, and FAK was located in the cytoplasm and nuclei of the podocytes. There were significant colocalized plaques of two molecules. But under acute hypertensive and cardiac arrest conditions, integrin β1 decreased and stained intermittently. Similarly, FAK decreased and appeared uneven. Additionally, FAK translocated to the nuclei of the podocytes. As a result, the colocalization of integrin β1 and FAK reduced obviously under these conditions. Western blot assay showed a consistent result with the immunostaining. Collectively, the abnormal redistribution and decreased expressions of integrin β1 and FAK are important molecular events in regulating the functions of podocytes under abnormal hemodynamic conditions. IVCT could offer considerable advantages for morphological analysis when researching renal diseases.

  4. The Regulation of Traction Force in Relation to Cell Shape and Focal Adhesions

    OpenAIRE

    Rape, Andrew; Guo, Wei-hui; Wang, Yu-Li

    2010-01-01

    Mechanical forces provide critical inputs for proper cellular functions. The interplay between the generation of, and response to, mechanical forces regulate such cellular processes as differentiation, proliferation, and migration. We postulate that adherent cells respond to a number of physical and topographical factors, including cell size and shape, by detecting the magnitude and/or distribution of traction forces under different conditions. To address this possibility we introduce a new s...

  5. Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and alpha-Smooth Muscle Actin Expression of Retinal Muller Cells

    NARCIS (Netherlands)

    Bu, Shao-Chong; Kuijer, Roel; van der Worp, Roelofje J.; van Putten, Sander M.; Wouters, Olaf; Li, Xiao-Rong; Hooymans, Johanna M. M.; Los, Leonoor I.

    PURPOSE. The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Muller cells have been shown to be mechanosensitive; they are involved in fibrotic vitreoretinal diseases. Since fibrosis increases the

  6. Focal Adhesion-Independent Cell Migration.

    Science.gov (United States)

    Paluch, Ewa K; Aspalter, Irene M; Sixt, Michael

    2016-10-06

    Cell migration is central to a multitude of physiological processes, including embryonic development, immune surveillance, and wound healing, and deregulated migration is key to cancer dissemination. Decades of investigations have uncovered many of the molecular and physical mechanisms underlying cell migration. Together with protrusion extension and cell body retraction, adhesion to the substrate via specific focal adhesion points has long been considered an essential step in cell migration. Although this is true for cells moving on two-dimensional substrates, recent studies have demonstrated that focal adhesions are not required for cells moving in three dimensions, in which confinement is sufficient to maintain a cell in contact with its substrate. Here, we review the investigations that have led to challenging the requirement of specific adhesions for migration, discuss the physical mechanisms proposed for cell body translocation during focal adhesion-independent migration, and highlight the remaining open questions for the future.

  7. Focal adhesions and cell-matrix interactions

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1988-01-01

    Focal adhesions are areas of cell surfaces where specializations of cytoskeletal, membrane and extracellular components combine to produce stable cell-matrix interactions. The morphology of these adhesions and the components identified in them are discussed together with possible mechanisms of th...

  8. Syndecan-4 and focal adhesion function

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    2001-01-01

    Two groups have now reported the viability of mice that lack syndecan-4. These mice have wound healing/angiogenesis problems, and fibroblasts from these animals differ in adhesion and migration from normal. This is consistent with recent in vitro data indicating a need for signaling via syndecan-4...... for focal adhesion formation, and reports that overexpression of proteins that bind syndecan-4 can modify cell adhesion and migration....

  9. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Karin [Department of Medical Cell Biology, Uppsala University, Uppsala 751 23 (Sweden); Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew [HHMI, Children' s Hospital Boston, Harvard Medical School, Boston, 02115 MA (United States); Grawé, Jan [Department of Genetics and Pathology, Uppsala University, Uppsala 75185 (Sweden); McKinney-Freeman, Shannon L. [Department of Hematology, St. Jude Children' s Research Hospital, Memphis, TN 38105 (United States); Daley, George Q. [HHMI, Children' s Hospital Boston, Harvard Medical School, Boston, 02115 MA (United States); Welsh, Michael, E-mail: michael.welsh@mcb.uu.se [Department of Medical Cell Biology, Uppsala University, Uppsala 751 23 (Sweden)

    2013-07-15

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via

  10. Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and alpha-Smooth Muscle Actin Expression of Retinal Muller Cells

    NARCIS (Netherlands)

    Bu, Shao-Chong; Kuijer, Roel; van der Worp, Roelofje J.; van Putten, Sander M.; Wouters, Olaf; Li, Xiao-Rong; Hooymans, Johanna M. M.; Los, Leonoor I.

    2015-01-01

    PURPOSE. The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Muller cells have been shown to be mechanosensitive; they are involved in fibrotic vitreoretinal diseases. Since fibrosis increases the rigi

  11. Molecular Mechanisms of Mechanosensitivity in Focal Adhesions

    OpenAIRE

    2016-01-01

    Physical environment guides tissue regeneration and morphology in both health and disease. In the past three decades, several experiments illustrated that mechanical cues are captured and transduced to biochemical signals in the cellular level (mechanotransduction) mediated by cell adhesion. Cells adhere to their microenvironment through large protein assemblies known as focal adhesions that directly couple intra- and extra-cellular matrices and play a critical role in many vital cell functio...

  12. Focal adhesions and assessment of cytotoxicity

    NARCIS (Netherlands)

    van Kooten, TG; Klein, CL; Wagner, M; Kirkpatrick, CJ

    1999-01-01

    Focal adhesions are highly ordered assemblies of transmembrane receptors, extracellular matrix proteins, and a large number of cytoplasmic proteins, including structural proteins, as well as tyrosine kinases, phosphatases, and their substrates. They are now accepted as a prime component of signal tr

  13. Focal Adhesion Kinases in Adhesion Structures and Disease

    Directory of Open Access Journals (Sweden)

    Pierre P. Eleniste

    2012-01-01

    Full Text Available Cell adhesion to the extracellular matrix (ECM is essential for cell migration, proliferation, and embryonic development. Cells can contact the ECM through a wide range of matrix contact structures such as focal adhesions, podosomes, and invadopodia. Although they are different in structural design and basic function, they share common remodeling proteins such as integrins, talin, paxillin, and the tyrosine kinases FAK, Pyk2, and Src. In this paper, we compare and contrast the basic organization and role of focal adhesions, podosomes, and invadopodia in different cells. In addition, we discuss the role of the tyrosine kinases, FAK, Pyk2, and Src, which are critical for the function of the different adhesion structures. Finally, we discuss the essential role of these tyrosine kinases from the perspective of human diseases.

  14. Focal adhesion kinases in adhesion structures and disease.

    Science.gov (United States)

    Eleniste, Pierre P; Bruzzaniti, Angela

    2012-01-01

    Cell adhesion to the extracellular matrix (ECM) is essential for cell migration, proliferation, and embryonic development. Cells can contact the ECM through a wide range of matrix contact structures such as focal adhesions, podosomes, and invadopodia. Although they are different in structural design and basic function, they share common remodeling proteins such as integrins, talin, paxillin, and the tyrosine kinases FAK, Pyk2, and Src. In this paper, we compare and contrast the basic organization and role of focal adhesions, podosomes, and invadopodia in different cells. In addition, we discuss the role of the tyrosine kinases, FAK, Pyk2, and Src, which are critical for the function of the different adhesion structures. Finally, we discuss the essential role of these tyrosine kinases from the perspective of human diseases.

  15. Focal Adhesion Kinases in Adhesion Structures and Disease

    OpenAIRE

    2012-01-01

    Cell adhesion to the extracellular matrix (ECM) is essential for cell migration, proliferation, and embryonic development. Cells can contact the ECM through a wide range of matrix contact structures such as focal adhesions, podosomes, and invadopodia. Although they are different in structural design and basic function, they share common remodeling proteins such as integrins, talin, paxillin, and the tyrosine kinases FAK, Pyk2, and Src. In this paper, we compare and contrast the basic organiza...

  16. Hyperphosphorylated FAK Delocalizes from Focal Adhesions to Membrane Ruffles

    Directory of Open Access Journals (Sweden)

    Abdelkader Hamadi

    2010-01-01

    Full Text Available Cell adhesion and migration are key determinants in tumor metastasis. Adherence of tumor cell to the extracellular matrix is mediated via integrin containing focal adhesions (FAs. Binding of integrins to ECM triggers phosphorylation of two major components of FAs, focal adhesion kinase (FAK and Src, activating downstream signaling pathway which leads to FA disassembly and cell migration. In this paper, we analyze how phosphorylation of FAK regulates its trafficking at FAs in living human astrocytoma cells. Upon pervanadate-induced FAK phosphorylation, phosphorylated FAK appeared highly expressed at newly formed membrane ruffles. This effect was abolished in presence of the specific Src inhibitor PP2. Our findings demonstrate that upon phosphorylation, FAK delocalizes from FAs to membrane ruffles.

  17. Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and α-Smooth Muscle Actin Expression of Retinal Müller Cells.

    Science.gov (United States)

    Bu, Shao-Chong; Kuijer, Roel; van der Worp, Roelofje J; van Putten, Sander M; Wouters, Olaf; Li, Xiao-Rong; Hooymans, Johanna M M; Los, Leonoor I

    2015-09-01

    The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Müller cells have been shown to be mechanosensitive; they are involved in fibrotic vitreoretinal diseases. Since fibrosis increases the rigidity of the extracellular matrix, our aim was to develop an in vitro model for studying Müller cell morphology and differentiation state in relation to matrix stiffness. A spontaneously immortalized human Müller cell line (MIO-M1) was cultured on type I collagen-coated polyacrylamide gels with Young's moduli ranging from 2 to 92 kPa. Cell surface area, focal adhesion, and the expression and morphology of α-smooth muscle actin induced by transforming growth factor β (TGF-β [10 ng/mL for 48 hours]) were analyzed by immunocytology. The images were documented by using fluorescence microscopy and confocal scanning laser microscopy. MIO-M1 cells cultured on stiff substrates exhibited a significant increase in cell surface area, stress fiber, and mature focal adhesion formation. Furthermore, Müller cells treated with TGF-β1 and TGF-β2 and cultured on stiff substrates showed an increased incorporation of α-smooth muscle actin into stress fibers when compared to those grown on soft surfaces. Compliance of the surrounding matrix seems to influence the morphology and contraction of retinal Müller cells in fibrotic conditions. Development of an in vitro model simulating both the normally compliant retinal tissue and the rigid retinal fibrotic tissue helps fill the gap between the results of petri-dish cell culture with rigid surfaces and in vivo findings.

  18. Focal Adhesion Induction at the Tip of a Functionalized Nanoelectrode

    OpenAIRE

    Fuentes, Daniela E.; Bae, Chilman; Peter J Butler

    2011-01-01

    Cells dynamically interact with their physical micro-environment through the assembly of nascent focal contacts and focal adhesions. The dynamics and mechanics of these contact points are controlled by transmembrane integrins and an array of intracellular adaptor proteins. In order to study the mechanics and dynamics of focal adhesion assembly, we have developed a technique for the timed induction of a nascent focal adhesion. Bovine aortic endothelial cells were approached at the apical surfa...

  19. Endothelial paxillin and focal adhesion kinase (FAK) play a critical role in neutrophil transmigration.

    Science.gov (United States)

    Parsons, Sean A; Sharma, Ritu; Roccamatisi, Dawn L; Zhang, Hong; Petri, Björn; Kubes, Paul; Colarusso, Pina; Patel, Kamala D

    2012-02-01

    During an inflammatory response, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessel to the site of injury or infection. Although endothelial cell junctions and the cytoskeleton undergo reorganization during inflammation, little is known about another class of cellular structures, the focal adhesions. In this study, we examined several focal adhesion proteins during an inflammatory response. We found that there was selective loss of paxillin and focal adhesion kinase (FAK) from focal adhesions in proximity to transmigrating neutrophils; in contrast the levels of the focal adhesion proteins β1-integrin and vinculin were unaffected. Paxillin was lost from focal adhesions during neutrophil transmigration both under static and flow conditions. Down-regulating endothelial paxillin with siRNA blocked neutrophil transmigration while having no effect on rolling or adhesion. As paxillin dynamics are regulated partly by FAK, the role of FAK in neutrophil transmigration was examined using two complementary methods. siRNA was used to down-regulate total FAK protein while dominant-negative, kinase-deficient FAK was expressed to block FAK signaling. Disruption of the FAK protein or FAK signaling decreased neutrophil transmigration. Collectively, these findings reveal a novel role for endothelial focal adhesion proteins paxillin and FAK in regulating neutrophil transmigration.

  20. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling.

    Science.gov (United States)

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2015-01-15

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation.

  1. Cell adhesion to fibrillin-1: identification of an Arg-Gly-Asp-dependent synergy region and a heparin-binding site that regulates focal adhesion formation

    DEFF Research Database (Denmark)

    Bax, Daniel V; Mahalingam, Yashithra; Cain, Stuart;

    2007-01-01

    We have defined the molecular basis of cell adhesion to fibrillin-1, the major structural component of extracellular microfibrils that are associated with elastic fibres. Using human dermal fibroblasts, and recombinant domain swap fragments containing the Arg-Gly-Asp motif, we have demonstrated a...

  2. Focal adhesion kinase - the reversible molecular mechanosensor

    CERN Document Server

    Bell, Samuel

    2016-01-01

    Sensors are the first element of the pathways that control the response of cells to their environment. After chemical, the next most important cue is mechanical, and protein complexes that produce or enable a chemical signal in response to a mechanical stimulus are called mechanosensors. There is a sharp distinction between sensing an external force or pressure/tension applied to the cell, and sensing the mechanical stiffness of the environment. We call the first mechanosensitivity of the 1st kind, and the latter mechanosensitivity of the 2nd kind. There are two variants of protein complexes that act as mechanosensors of the 2nd kind: producing the one-off or a reversible action. The latent complex of TGF-beta is an example of the one-off action: on the release of active TGF-beta signal, the complex is discarded and needs to be replaced. In contrast, the focal adhesion kinase (FAK) in a complex with integrin is a reversible mechanosensor, which initiates the chemical signal in its active phosphorylated confor...

  3. Actin cap associated focal adhesions and their distinct role in cellular mechanosensing

    Science.gov (United States)

    Kim, Dong-Hwee; Khatau, Shyam B.; Feng, Yunfeng; Walcott, Sam; Sun, Sean X.; Longmore, Gregory D.; Wirtz, Denis

    2012-01-01

    The ability for cells to sense and adapt to different physical microenvironments plays a critical role in development, immune responses, and cancer metastasis. Here we identify a small subset of focal adhesions that terminate fibers in the actin cap, a highly ordered filamentous actin structure that is anchored to the top of the nucleus by the LINC complexes; these differ from conventional focal adhesions in morphology, subcellular organization, movements, turnover dynamics, and response to biochemical stimuli. Actin cap associated focal adhesions (ACAFAs) dominate cell mechanosensing over a wide range of matrix stiffness, an ACAFA-specific function regulated by actomyosin contractility in the actin cap, while conventional focal adhesions are restrictively involved in mechanosensing for extremely soft substrates. These results establish the perinuclear actin cap and associated ACAFAs as major mediators of cellular mechanosensing and a critical element of the physical pathway that transduce mechanical cues all the way to the nucleus. PMID:22870384

  4. Focal Adhesion Induction at the Tip of a Functionalized Nanoelectrode.

    Science.gov (United States)

    Fuentes, Daniela E; Bae, Chilman; Butler, Peter J

    2011-12-01

    Cells dynamically interact with their physical micro-environment through the assembly of nascent focal contacts and focal adhesions. The dynamics and mechanics of these contact points are controlled by transmembrane integrins and an array of intracellular adaptor proteins. In order to study the mechanics and dynamics of focal adhesion assembly, we have developed a technique for the timed induction of a nascent focal adhesion. Bovine aortic endothelial cells were approached at the apical surface by a nanoelectrode whose position was controlled with a resolution of 10s of nanometers using changes in electrode current to monitor distance from the cell surface. Since this probe was functionalized with fibronectin, a focal contact formed at the contact location. Nascent focal adhesion assembly was confirmed using time-lapse confocal fluorescent images of red fluorescent protein (RFP) - tagged talin, an adapter protein that binds to activated integrins. Binding to the cell was verified by noting a lack of change of electrode current upon retraction of the electrode. This study demonstrates that functionalized nanoelectrodes can enable precisely-timed induction and 3-D mechanical manipulation of focal adhesions and the assay of the detailed molecular kinetics of their assembly.

  5. Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation

    Science.gov (United States)

    Roa-Espitia, Ana L.; Hernández-Rendón, Eva R.; Baltiérrez-Hoyos, Rafael; Muñoz-Gotera, Rafaela J.; Cote-Vélez, Antonieta; Jiménez, Irma; González-Márquez, Humberto

    2016-01-01

    ABSTRACT Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by β1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca2+ dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton. PMID:27402964

  6. ROCK-2 is associated with focal adhesion maturation during myoblast migration.

    Science.gov (United States)

    Goetsch, K P; Snyman, C; Myburgh, K H; Niesler, C U

    2014-07-01

    Satellite cell migration is critical for skeletal muscle growth and regeneration. Controlled cell migration is dependent on the formation of mature focal adhesions between the cell and the underlying extracellular matrix (ECM). These cell-ECM interactions trigger the activation of signalling events such as the Rho/ROCK pathway. We have previously identified a specific role for ROCK-2 during myoblast migration. In this study we report that ROCK inhibition with Y-27632 increases C2C12 myoblast velocity, but at the expense of directional migration. In response to Y-27632 an increased number of smaller focal adhesions were distributed across adhesion sites that in turn were clearly larger than sites in untreated cells, suggesting a reduction in focal adhesion maturation. We also confirm ROCK-2 localisation to the focal adhesion sites in migrating myoblasts and demonstrate a change in the distribution of these ROCK-2 containing adhesions in response to Y-27632. Taken together, our observations provide further proof that ROCK-2 regulates directional myoblast migration through focal adhesion formation and maturation.

  7. Protein kinase C involvement in focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1992-01-01

    Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have...... still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form....... Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h...

  8. The nanoscale architecture of force-bearing focal adhesions.

    Science.gov (United States)

    van Hoorn, Hedde; Harkes, Rolf; Spiesz, Ewa M; Storm, Cornelis; van Noort, Danny; Ladoux, Benoit; Schmidt, Thomas

    2014-08-13

    The combination of micropillar array technology to measure cellular traction forces with super-resolution imaging allowed us to obtain cellular traction force maps and simultaneously zoom in on individual focal adhesions with single-molecule accuracy. We achieved a force detection precision of 500 pN simultaneously with a mean single-molecule localization precision of 30 nm. Key to the achievement was a two-step etching process that provided an integrated spacer next to the micropillar array that permitted stable and reproducible observation of cells on micropillars within the short working distance of a high-magnification, high numerical aperture objective. In turn, we used the technology to characterize the super-resolved structure of focal adhesions during force exertion. Live-cell imaging on MCF-7 cells demonstrated the applicability of the inverted configuration of the micropillar arrays to dynamics measurements. Forces emanated from a molecular base that was localized on top of the micropillars. What appeared as a single adhesion in conventional microscopy were in fact multiple elongated adhesions emanating from only a small fraction of the adhesion on the micropillar surface. Focal adhesions were elongated in the direction of local cellular force exertion with structural features of 100-280 nm in 3T3 Fibroblasts and MCF-7 cells. The combined measure of nanoscale architecture and force exerted shows a high level of stress accumulation at a single site of adhesion.

  9. Talin determines the nanoscale architecture of focal adhesions.

    Science.gov (United States)

    Liu, Jaron; Wang, Yilin; Goh, Wah Ing; Goh, Honzhen; Baird, Michelle A; Ruehland, Svenja; Teo, Shijia; Bate, Neil; Critchley, David R; Davidson, Michael W; Kanchanawong, Pakorn

    2015-09-01

    Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin-talin-actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.

  10. Spatiotemporal Constraints on the Force-Dependent Growth of Focal Adhesions

    Science.gov (United States)

    Stricker, Jonathan; Aratyn-Schaus, Yvonne; Oakes, Patrick W.; Gardel, Margaret L.

    2011-01-01

    Focal adhesions (FAs) are the predominant mechanism by which cells mechanically couple to and exert traction forces on their extracellular matrix (ECM). It is widely presumed that FA size is modulated by force to mediate changes in adhesion strength at different levels of cellular tension. However, previous studies seeking correlations between force and FA morphology have yielded variable and often conflicting results. Here we show that a strong correlation between adhesion size and traction force exists only during the initial stages of myosin-mediated adhesion maturation and growth. For mature adhesions, no correlation between traction stress and size is observed. Rather, the tension that is sustained at mature adhesions is more strongly influenced by proximity to the cell edge, with peripheral adhesions transmitting higher tension than adhesions near the cell center. Finally, we show that mature adhesions can withstand sixfold increases in tension without changes in size. Thus, although a strong correlation between adhesion size and mechanical tension is observed during the initial stages of myosin-mediated adhesion maturation, no correlation is observed in mature, elongated adhesions. This work places spatiotemporal constraints on the force-dependent growth of adhesions and provides insight into the mechanical regulation of cell-ECM adhesion. PMID:21689521

  11. MicroRNA-151 and its hosting gene FAK (focal adhesion kinase) regulate tumor cell migration and spreading of hepatocellular carcinoma.

    Science.gov (United States)

    Luedde, Tom

    2010-09-01

    Recurrent chromosomal aberrations are often observed in hepatocellular carcinoma (HCC), but little is known about the functional non-coding sequences, particularly microRNAs (miRNAs), at the chromosomal breakpoints in HCC. Here we show that 22 miRNAs are often amplified or deleted in HCC. MicroRNA-151 (miR-151), a frequently amplified miRNA on 8q24.3, is correlated with intrahepatic metastasis of HCC. We further show that miR-151, which is often expressed together with its host gene FAK, encoding focal adhesion kinase, significantly increases HCC cell migration and invasion in vitro and in vivo, mainly through miR-151-5p, but not through miR-151-3p. Moreover, miR-151 exerts this function by directly targeting RhoGDIA, a putative metastasis suppressor in HCC, thus leading to the activation of Rac1, Cdc42 and Rho GTPases. In addition, miR-151 can function synergistically with FAK to enhance HCC cell motility and spreading. Thus, our findings indicate that chromosome gain of miR-151 is a crucial stimulus for tumour invasion and metastasis of HCC.

  12. Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot for mechanosensitive proteins

    Science.gov (United States)

    Kirchenbüchler, David; Born, Simone; Kirchgeßner, Norbert; Houben, Sebastian; Hoffmann, Bernd; Merkel, Rudolf

    2010-05-01

    Mechanosensing is a vital prerequisite for dynamic remodeling of focal adhesions and cytoskeletal structures upon substrate deformation. For example, tissue formation, directed cell orientation or cell differentiation are regulated by such mechanosensing processes. Focal adhesions and the actin cytoskeleton are believed to be involved in these processes, but where mechanosensing molecules are located and how elastic substrate, focal adhesions and the cytoskeleton couple with each other upon substrate deformation still remains obscure. To approach these questions we have developed a sensitive method to apply defined spatially decaying deformation fields to cells cultivated on ultrasoft elastic substrates and to accurately quantify the resulting displacements of the actin cytoskeleton, focal adhesions, as well as the substrate. Displacement fields were recorded in live cell microscopy by tracking either signals from fluorescent proteins or marker particles in the substrate. As model cell type we used myofibroblasts. These cells are characterized by highly stable adhesion and force generating structures but are still able to detect mechanical signals with high sensitivity. We found a rigid connection between substrate and focal adhesions. Furthermore, stress fibers were found to be barely extendable almost over their whole lengths. Plastic deformation took place only at the very ends of actin filaments close to focal adhesions. As a result, this area became elongated without extension of existing actin filaments by polymerization. Both ends of the stress fibers were mechanically coupled with detectable plastic deformations on either site. Interestingly, traction force dependent substrate deformation fields remained mostly unaffected even when stress fiber elongations were released. These data argue for a location of mechanosensing proteins at the ends of actin stress fibers and describe, except for these domains, the whole system to be relatively rigid for tensile

  13. Cadherin-11 localizes to focal adhesions and promotes cell–substrate adhesion

    Science.gov (United States)

    Langhe, Rahul P.; Gudzenko, Tetyana; Bachmann, Michael; Becker, Sarah F.; Gonnermann, Carina; Winter, Claudia; Abbruzzese, Genevieve; Alfandari, Dominique; Kratzer, Marie-Claire; Franz, Clemens M.; Kashef, Jubin

    2016-01-01

    Cadherin receptors have a well-established role in cell–cell adhesion, cell polarization and differentiation. However, some cadherins also promote cell and tissue movement during embryonic development and tumour progression. In particular, cadherin-11 is upregulated during tumour and inflammatory cell invasion, but the mechanisms underlying cadherin-11 stimulated cell migration are still incompletely understood. Here, we show that cadherin-11 localizes to focal adhesions and promotes adhesion to fibronectin in Xenopus neural crest, a highly migratory embryonic cell population. Transfected cadherin-11 also localizes to focal adhesions in different mammalian cell lines, while endogenous cadherin-11 shows focal adhesion localization in primary human fibroblasts. In focal adhesions, cadherin-11 co-localizes with β1-integrin and paxillin and physically interacts with the fibronectin-binding proteoglycan syndecan-4. Adhesion to fibronectin mediated by cadherin-11/syndecan-4 complexes requires both the extracellular domain of syndecan-4, and the transmembrane and cytoplasmic domains of cadherin-11. These results reveal an unexpected role of a classical cadherin in cell–matrix adhesion during cell migration. PMID:26952325

  14. Structural Insight into the Mechanisms of Targeting and Signaling of Focal Adhesion Kinase

    OpenAIRE

    2002-01-01

    Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase whose focal adhesion targeting (FAT) domain interacts with other focal adhesion molecules in integrin-mediated signaling. Localization of activated FAK to focal adhesions is indispensable for its function. Here we describe a solution structure of the FAT domain bound to a peptide derived from paxillin, a FAK-binding partner. The FAT domain is composed of four helices that form a “right-turn” elongated bundle; the globular fold is ma...

  15. Bidirectional control of the inner dynamics of focal adhesions promotes cell migration

    OpenAIRE

    2009-01-01

    Focal adhesions (FA) are bidirectional mechanical biosensors that allow cells to integrate intracellular and extracellular cues. Their function is tightly regulated by changes in molecular composition and also by variation in the spatio-temporal dynamics of FA components within this structure. A closely regulated turnover of FA proteins within FA sites allows cells to respond appropriately to their environment, thereby impacting on cell shape and function. FA protein dynamics are linked to FA...

  16. Stability of focal adhesion enhanced by its inner force fluctuation

    Science.gov (United States)

    Mao, Zhi-Xiu; Chen, Xiao-Feng; Chen, Bin

    2015-08-01

    Cells actively sense and respond to mechanical signals from the extracellular matrix through focal adhesions. By representing a single focal adhesion as a cluster of slip bonds, it has been demonstrated that the cluster often became unstable under fluctuated forces. However, an unusual case was also reported, where the stability of the cluster might be substantially enhanced by a fluctuated force with a relatively low fluctuation frequency and high fluctuation amplitude. Such an observation cannot be explained by the conventional fracture theory of fatigue. Here, we intensively investigate this intriguing observation by carrying out systematic parametric studies. Our intensive simulation results indicate that stability enhancement of this kind is in fact quite robust, which can be affected by the stochastic features of a single bond and the profile of the fluctuated forces such as the average value of bond force. We then suggest that the fluctuation of traction force within a focal adhesion might enhance its stability in a certain way. Project supported by the National Natural Science Foundation of China (Grant No.*11372279).

  17. Conformational Dynamics of the Focal Adhesion Targeting Domain Control Specific Functions of Focal Adhesion Kinase in Cells

    KAUST Repository

    Kadaré, Gress

    2015-01-02

    Focal adhesion (FA) kinase (FAK) regulates cell survival and motility by transducing signals from membrane receptors. The C-terminal FA targeting (FAT) domain of FAK fulfils multiple functions, including recruitment to FAs through paxillin binding. Phosphorylation of FAT on Tyr925 facilitates FA disassembly and connects to the MAPK pathway through Grb2 association, but requires dissociation of the first helix (H1) of the four-helix bundle of FAT. We investigated the importance of H1 opening in cells by comparing the properties of FAK molecules containing wild-type or mutated FAT with impaired or facilitated H1 openings. These mutations did not alter the activation of FAK, but selectively affected its cellular functions, including self-association, Tyr925 phosphorylation, paxillin binding, and FA targeting and turnover. Phosphorylation of Tyr861, located between the kinase and FAT domains, was also enhanced by the mutation that opened the FAT bundle. Similarly phosphorylation of Ser910 by ERK in response to bombesin was increased by FAT opening. Although FAK molecules with the mutation favoring FAT opening were poorly recruited at FAs, they efficiently restored FA turnover and cell shape in FAK-deficient cells. In contrast, the mutation preventing H1 opening markedly impaired FAK function. Our data support the biological importance of conformational dynamics of the FAT domain and its functional interactions with other parts of the molecule.

  18. Focal adhesion kinase maintains, but not increases the adhesion of dental pulp cells.

    Science.gov (United States)

    Qian, Yuyan; Shao, Meiying; Zou, Wenlin; Wang, Linyan; Cheng, Ran; Hu, Tao

    2017-04-01

    Focal adhesion kinase (FAK) functions as a key enzyme in the integrin-mediated adhesion-signalling pathway. Here, we aimed to investigate the effects of FAK on adhesion of human dental pulp (HDP) cells. We transfected lentiviral vectors to silence or overexpress FAK in HDP cells ex vivo. Early cell adhesion, cell survival and focal contacts (FCs)-related proteins (FAK and paxillin) were examined. By using immunofluorescence, the formation of FCs and cytoskeleton was detected, respectively. We found that both adhesion and survival of HDP cells were suppressed by FAK inhibition. However, FAK overexpression slightly inhibited cell adhesion and exhibited no change in cell survival compared with the control. A thick rim of cytoskeleton accumulated and smaller dot-shaped FCs appeared in FAK knockdown cells. Phosphorylation of paxillin (p-paxillin) was inhibited in FAK knockdown cells, verifying that the adhesion was inhibited. Less cytoskeleton and elongated FCs were observed in FAK-overexpressed cells. However, p-paxillin had no significant difference compared with the control. In conclusion, the data suggest that FAK maintains cell adhesion, survival and cytoskeleton formation, but excessive FAK has no positive effects on these aspects.

  19. Exploring the formation of focal adhesions on patterned surfaces using super-resolution imaging.

    Science.gov (United States)

    Chien, Fan-Ching; Kuo, Chiung Wen; Yang, Zong-Han; Chueh, Di-Yen; Chen, Peilin

    2011-10-17

    The formation of focal adhesions on various sizes of fibronectin patterns, ranging from 200 μm to 250 nm, was systematically investigated by total internal reflection fluorescence microscopy and super-resolution imaging. It was found that cells adhered to and spread on these micro/nanopatterns, forming focal adhesions. On a micrometer scale the shape of the focal adhesions was elongated. However, on the nanometer scale, the shape of focal adhesions became dotlike. To further explore the distribution of focal adhesion proteins formed on surfaces, a localization-based super-resolution imaging technique was employed in order to determine the position and density of vinculin proteins. A characteristic distance of 50 nm was found between vinculin molecules in the focal adhesions, which did not depend on the size of the fibronectin nanopatterns. This distance was found to be crucial for the formation of focal adhesions. In addition, the density of vinculin at the focal adhesions formed on the nanopatterns increased as the pattern size decreased. The density of the protein was found to be 425 ± 247, 584 ± 302, and 703 ± 305 proteins μm(-2) on the 600, 400, and 250 nm fibronectin patterns respectively. Whereas 226 ± 77 proteins μm(-2) was measured for the matured focal adhesions on homogeneous fibronectin coated substrates. The increase in vinculin density implies that an increase in mechanical load was applied to the focal adhesions formed on the smaller nanopatterns.

  20. TRPM4 Is a Novel Component of the Adhesome Required for Focal Adhesion Disassembly, Migration and Contractility.

    Directory of Open Access Journals (Sweden)

    Mónica Cáceres

    Full Text Available Cellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility.

  1. FAK dimerization controls its kinase-dependent functions at focal adhesions

    KAUST Repository

    Brami-Cherrier, Karen

    2014-01-30

    Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK\\'s kinase-dependent functions-autophosphorylation of tyrosine-397-requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation. © 2014 The Authors.

  2. Assembly and mechanosensory function of focal adhesions: experiments and models.

    Science.gov (United States)

    Bershadsky, Alexander D; Ballestrem, Christoph; Carramusa, Letizia; Zilberman, Yuliya; Gilquin, Benoit; Khochbin, Saadi; Alexandrova, Antonina Y; Verkhovsky, Alexander B; Shemesh, Tom; Kozlov, Michael M

    2006-04-01

    Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex

  3. Nascent Focal Adhesions Are Responsible for the Generation of Strong Propulsive Forces in Migrating Fibroblasts

    OpenAIRE

    2001-01-01

    Fibroblast migration involves complex mechanical interactions with the underlying substrate. Although tight substrate contact at focal adhesions has been studied for decades, the role of focal adhesions in force transduction remains unclear. To address this question, we have mapped traction stress generated by fibroblasts expressing green fluorescent protein (GFP)-zyxin. Surprisingly, the overall distribution of focal adhesions only partially resembles the distribution of traction stress. In ...

  4. Stretch activates human myometrium via ERK, caldesmon and focal adhesion signaling.

    Directory of Open Access Journals (Sweden)

    Yunping Li

    Full Text Available An incomplete understanding of the molecular mechanisms responsible for myometrial activation from the quiescent pregnant state to the active contractile state during labor has hindered the development of effective therapies for preterm labor. Myometrial stretch has been implicated clinically in the initiation of labor and the etiology of preterm labor, but the molecular mechanisms involved in the human have not been determined. We investigated the mechanisms by which gestation-dependent stretch contributes to myometrial activation, by using human uterine samples from gynecologic hysterectomies and Cesarean sections. Here we demonstrate that the Ca requirement for activation of the contractile filaments in human myometrium increases with caldesmon protein content during gestation and that an increase in caldesmon phosphorylation can reverse this inhibitory effect during labor. By using phosphotyrosine screening and mass spectrometry of stretched human myometrial samples, we identify 3 stretch-activated focal adhesion proteins, FAK, p130Cas, and alpha actinin. FAK-Y397, which signals integrin engagement, is constitutively phosphorylated in term human myometrium whereas FAK-Y925, which signals downstream ERK activation, is phosphorylated during stretch. We have recently identified smooth muscle Archvillin (SmAV as an ERK regulator. A newly produced SmAV-specific antibody demonstrates gestation-specific increases in SmAV protein levels and stretch-specific increases in SmAV association with focal adhesion proteins. Thus, whereas increases in caldesmon levels suppress human myometrium contractility during pregnancy, stretch-dependent focal adhesion signaling, facilitated by the ERK activator SmAV, can contribute to myometrial activation. These results suggest that focal adhesion proteins may present new targets for drug discovery programs aimed at regulation of uterine contractility.

  5. The inner workings of stress fibers - from contractile machinery to focal adhesions and back.

    Science.gov (United States)

    Livne, Ariel; Geiger, Benjamin

    2016-04-01

    Ventral stress fibers and focal adhesions are physically coupled structures that play key roles in cellular mechanics and force sensing. The tight functional interdependence between the two is manifested not only by their apparent proximity but also by the fact that ventral stress fibers and focal adhesions are simultaneously diminished upon actomyosin relaxation, and grow when subjected to external stretching. However, whereas the apparent co-regulation of the two structures is well-documented, the underlying mechanisms remains poorly understood. In this Commentary, we discuss some of the fundamental, yet still open questions regarding ventral stress fiber structure, its force-dependent assembly, as well as its capacity to generate force. We also challenge the common approach - i.e. ventral stress fibers are variants of the well-studied striated or smooth muscle machinery - by presenting and critically discussing alternative venues. By highlighting some of the less-explored aspects of the interplay between stress fibers and focal adhesions, we hope that this Commentary will encourage further investigation in this field.

  6. Complementarity of PALM and SOFI for super-resolution live cell imaging of focal adhesions

    CERN Document Server

    Deschout, Hendrik; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

    2016-01-01

    Live cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenging task for super-resolution microscopy. We have addressed this important issue by combining photo-activated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed cell focal adhesion images, we investigated the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework was used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualized the dynamics of focal adhesions, and revealed local mean velocities around 190 nm per minute. The complementarity of PALM and SOFI was assessed in detail with a methodology that integrates a quantitative resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of m...

  7. Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

    Science.gov (United States)

    Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

    2016-12-01

    Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

  8. Why do receptor-ligand bonds in cell adhesion cluster into discrete focal-adhesion sites?

    Science.gov (United States)

    Gao, Zhiwen; Gao, Yanfei

    2016-10-01

    Cell adhesion often exhibits the clustering of the receptor-ligand bonds into discrete focal-adhesion sites near the contact edge, thus resembling a rosette shape or a contracting membrane anchored by a small number of peripheral forces. The ligands on the extracellular matrix are immobile, and the receptors in the cell plasma membrane consist of two types: high-affinity integrins (that bond to the substrate ligands and are immobile) and low-affinity integrins (that are mobile and not bonded to the ligands). Thus the adhesion energy density is proportional to the high-affinity integrin density. This paper provides a mechanistic explanation for the clustering/assembling of the receptor-ligand bonds from two main points: (1) the cellular contractile force leads to the density evolution of these two types of integrins, and results into a large high-affinity integrin density near the contact edge and (2) the front of a propagating crack into a decreasing toughness field will be unstable and wavy. From this fracture mechanics perspective, the chemomechanical equilibrium is reached when a small number of patches with large receptor-ligand bond density are anticipated to form at the cell periphery, as opposed to a uniform distribution of bonds on the entire interface. Cohesive fracture simulations show that the de-adhesion force can be significantly enhanced by this nonuniform bond density field, but the de-adhesion force anisotropy due to the substrate elastic anisotropy is significantly reduced.

  9. Regulation of G protein-linked guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and LARG by tyrosine phosphorylation: evidence of a role for focal adhesion kinase.

    Science.gov (United States)

    Chikumi, Hiroki; Fukuhara, Shigetomo; Gutkind, J Silvio

    2002-04-05

    A recently identified family of guanine nucleotide exchange factors for Rho that includes PDZ-RhoGEF, LARG, and p115RhoGEF exhibits a unique structural feature consisting in the presence of area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a structural motif by which heterotrimeric G protein alpha subunits of the Galpha(12) family can bind and regulate the activity of RhoGEFs. Hence, these newly discovered RGL domain-containing RhoGEFs provide a direct link from Galpha(12) and Galpha(13) to Rho. Recently available data suggest, however, that tyrosine kinases can regulate the ability of G protein-coupled receptors (GPCRs) to stimulate Rho, although the underlying molecular mechanisms are still unknown. Here, we found that the activation of thrombin receptors endogenously expressed in HEK-293T cells leads to a remarkable increase in the levels of GTP-bound Rho within 1 min (11-fold) and a more limited but sustained activation (4-fold) thereafter, which lasts even for several hours. Interestingly, tyrosine kinase inhibitors did not affect the early phase of Rho activation, immediately after thrombin addition, but diminished the levels of GTP-bound Rho during the delayed phase. As thrombin receptors stimulate focal adhesion kinase (FAK) potently, we explored whether this non-receptor tyrosine kinase participates in the activation of Rho by GPCRs. We obtained evidence that FAK can be activated by thrombin, Galpha(12), Galpha(13), and Galpha(q) through both Rho-dependent and Rho-independent mechanisms and that PDZ-RhoGEF and LARG can in turn be tyrosine-phosphorylated through FAK in response to thrombin, thereby enhancing the activation of Rho in vivo. These data indicate that FAK may act as a component of a positive feedback loop that results in the sustained activation of Rho by GPCRs, thus providing evidence of the existence of a novel biochemical route by which tyrosine kinases may regulate the activity of Rho through

  10. [Therapeutic effect of focal adhesion kinase gene silence on leukemia].

    Science.gov (United States)

    Xu, Lü-Hong; Fang, Jian-Pei; Weng, Wen-Jun; Xu, Hong-Gui; Zhang, Ya-Ting

    2011-06-01

    This study was aimed to investigate the effects of focal adhesion kinase (FAK) gene silence on leukemia cell growth, leukemogenesis and efficacy of chemotherapy drug. Vector containing lentiviral-FAK-shRNA was constructed and transfected into BCR/ABL-BaF3 leukemic cells, the cell growth and apoptosis were detected in vitro. The effect of FAK shRNA on leukemogenesis was studied in a murine model with leukemia. The apoptosis of leukemia cells and survival of leukemic mice treated by FAK shRNA combined with drug STI571 were monitored. The results showed that FAK gene expression was knocked down by lentiviral-FAK-shRNA. FAK gene silencing inhibited leukemia cell growth in vitro. The apoptosis test results showed that the percentages of Annexin V(+) cells in vector control group and FAK shRNA group were (3.46 ± 0.56)% and (7.3 ± 0.79)%, respectively, and the difference was statistically significant (p silence combined with drug STI571 could enhance the apoptosis of leukemia cells and prolong survival time of leukemic mice. It is concluded that FAK gene silence inhibits leukemogenesis and promotes efficacy of chemotherapy drug on leukemia cells, indicating FAK gene silence may be considered as a new therapeutic strategy for leukemia.

  11. Focal adhesion kinase overexpression and its impact on human osteosarcoma

    Science.gov (United States)

    Chen, Yong; Yang, Aizhen; Chen, Hui; Zhang, Jian; Wu, Sujia; Shi, Xin; Wang, Chen; Sun, Xiaoliang

    2015-01-01

    Focal adhesion kinase (FAK) has been implicated in tumorigenesis in various malignancies. We sought to examine the expression patterns of FAK and the activated form, phosphorylated FAK (pFAK), in human osteosarcoma and to investigate the correlation of FAK expression with clinicopathologic parameters and prognosis. In addition, the functional consequence of manipulating the FAK protein level was investigated in human osteosarcoma cell lines. Immunohistochemical staining was used to detect FAK and pFAK in pathologic archived materials from 113 patients with primary osteosarcoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognoses. The role of FAK in the cytological behavior of MG63 and 143B human osteosarcoma cell lines was studied via FAK protein knock down with siRNA. Cell proliferation, migration, invasiveness and apoptosis were assessed using the CCK8, Transwell and Annexin V/PI staining methods. Both FAK and pFAK were overexpressed in osteosarcoma. There were significant differences in overall survival between the FAK-/pFAK- and FAK+/pFAK- groups (P = 0.016), the FAK+/pFAK- and FAK+/pFAK+ groups (P = 0.012) and the FAK-/pFAK- and FAK+/pFAK+ groups (P osteosarcoma cell proliferation and apoptosis. These results collectively suggest that FAK overexpression and phosphorylation might predict more aggressive biologic behavior in osteosarcoma and may be an independent predictor of poor prognosis. PMID:26393679

  12. Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Aanei, Carmen Mariana, E-mail: caanei@yahoo.com [Laboratoire Hematologie, CHU de Saint-Etienne, 42055, Saint-Etienne (France); Department of Immunology, Gr. T. Popa University of Medicine and Pharmacy, 700115, Iasi (Romania); Eloae, Florin Zugun [Department of Immunology, Gr. T. Popa University of Medicine and Pharmacy, 700115, Iasi (Romania); Flandrin-Gresta, Pascale [Laboratoire Hematologie, CHU de Saint-Etienne, 42055, Saint-Etienne (France); CNRS UMR 5239, Universite de Lyon, 42023, Saint-Etienne (France); Tavernier, Emmanuelle [Service Hematologie Clinique, Institut de Cancerologie de la Loire, 42270, Saint-Priest-en-Jarez (France); CNRS UMR 5239, Universite de Lyon, 42023, Saint-Etienne (France); Carasevici, Eugen [Department of Immunology, Gr. T. Popa University of Medicine and Pharmacy, 700115, Iasi (Romania); Guyotat, Denis [Service Hematologie Clinique, Institut de Cancerologie de la Loire, 42270, Saint-Priest-en-Jarez (France); CNRS UMR 5239, Universite de Lyon, 42023, Saint-Etienne (France); Campos, Lydia [Laboratoire Hematologie, CHU de Saint-Etienne, 42055, Saint-Etienne (France); CNRS UMR 5239, Universite de Lyon, 42023, Saint-Etienne (France)

    2011-11-01

    Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further, because FAK is an HSP90{alpha}/{beta} client protein, these results suggest the utility of HSP90{alpha}/{beta} inhibition as a target for adjuvant therapy for myelodysplasia.

  13. Upregulation of paxillin and focal adhesion signaling follows Dystroglycan Complex deletions and promotes a hypertensive state of differentiation.

    Science.gov (United States)

    Sen, Shamik; Tewari, Manorama; Zajac, Allison; Barton, Elisabeth; Sweeney, H Lee; Discher, Dennis E

    2011-01-01

    Anchorage to matrix is mediated for many cells not only by integrin-based focal adhesions but also by a parallel assembly of integral and peripheral membrane proteins known as the Dystroglycan Complex. Deficiencies in either dystrophin (mdx mice) or γ-sarcoglycan (γSG(-/-) mice) components of the Dystroglycan Complex lead to upregulation of numerous focal adhesion proteins, and the phosphoprotein paxillin proves to be among the most prominent. In mdx muscle, paxillin-Y31 and Y118 are both hyper-phosphorylated as are key sites in focal adhesion kinase (FAK) and the stretch-stimulatable pro-survival MAPK pathway, whereas γSG(-/-) muscle exhibits more erratic hyper-phosphorylation. In cultured myotubes, cell tension generated by myosin-II appears required for localization of paxillin to adhesions while vinculin appears more stably integrated. Overexpression of wild-type (WT) paxillin has no obvious effect on focal adhesion density or the physical strength of adhesion, but WT and a Y118F mutant promote contractile sarcomere formation whereas a Y31F mutant shows no effect, implicating Y31 in striation. Self-peeling of cells as well as Atomic Force Microscopy (AFM) probing of cells with or without myosin-II inhibition indicate an increase in cell tension within paxillin-overexpressing cells. However, prednisolone, a first-line glucocorticoid for muscular dystrophies, decreases cell tension without affecting paxillin at adhesions, suggesting a non-linear relationship between paxillin and cell tension. Hypertension that results from upregulation of integrin adhesions is thus a natural and treatable outcome of Dystroglycan Complex down-regulation.

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

    Science.gov (United States)

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

    2013-12-01

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

  15. Actin cap associated focal adhesions and their distinct role in cellular mechanosensing

    OpenAIRE

    2012-01-01

    The ability for cells to sense and adapt to different physical microenvironments plays a critical role in development, immune responses, and cancer metastasis. Here we identify a small subset of focal adhesions that terminate fibers in the actin cap, a highly ordered filamentous actin structure that is anchored to the top of the nucleus by the LINC complexes; these differ from conventional focal adhesions in morphology, subcellular organization, movements, turnover dynamics, and response to b...

  16. The non-equilibrium thermodynamics and kinetics of focal adhesion dynamics.

    Directory of Open Access Journals (Sweden)

    Joseph E Olberding

    Full Text Available BACKGROUND: We consider a focal adhesion to be made up of molecular complexes, each consisting of a ligand, an integrin molecule, and associated plaque proteins. Free energy changes drive the binding and unbinding of these complexes and thereby controls the focal adhesion's dynamic modes of growth, treadmilling and resorption. PRINCIPAL FINDINGS: We have identified a competition among four thermodynamic driving forces for focal adhesion dynamics: (i the work done during the addition of a single molecular complex of a certain size, (ii the chemical free energy change associated with the addition of a molecular complex, (iii the elastic free energy change associated with deformation of focal adhesions and the cell membrane, and (iv the work done on a molecular conformational change. We have developed a theoretical treatment of focal adhesion dynamics as a nonlinear rate process governed by a classical kinetic model. We also express the rates as being driven by out-of-equilibrium thermodynamic driving forces, and modulated by kinetics. The mechanisms governed by the above four effects allow focal adhesions to exhibit a rich variety of behavior without the need to introduce special constitutive assumptions for their response. For the reaction-limited case growth, treadmilling and resorption are all predicted by a very simple chemo-mechanical model. Treadmilling requires symmetry breaking between the ends of the focal adhesion, and is achieved by driving force (i above. In contrast, depending on its numerical value (ii causes symmetric growth, resorption or is neutral, (iii causes symmetric resorption, and (iv causes symmetric growth. These findings hold for a range of conditions: temporally-constant force or stress, and for spatially-uniform and non-uniform stress distribution over the FA. The symmetric growth mode dominates for temporally-constant stress, with a reduced treadmilling regime. SIGNIFICANCE: In addition to explaining focal adhesion

  17. 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.

  18. Bacillus cereus Certhrax ADP-ribosylates vinculin to disrupt focal adhesion complexes and cell adhesion.

    Science.gov (United States)

    Simon, Nathan C; Barbieri, Joseph T

    2014-04-11

    Bacillus cereus is often associated with mild to moderate gastroenteritis; however, some recent isolates cause inhalational anthrax-like diseases and death. These potential emerging human pathogens express multiple virulence factors. B. cereus strain G9241 expresses anthrax toxin, several polysaccharide capsules, and the novel ADP-ribosyltransferase, Certhrax. In this study, we show that Certhrax ADP-ribosylates Arg-433 of vinculin, a protein that coordinates actin cytoskeleton and extracellular matrix interactions. ADP-ribosylation of vinculin disrupted focal adhesion complexes and redistributed vinculin to the cytoplasm. Exogenous vinculin rescued these phenotypes. This provides a mechanism for strain G9241 to breach host barrier defenses and promote bacterial growth and spread. Certhrax is the first bacterial toxin to add a post-translational modification to vinculin to disrupt the actin cytoskeleton.

  19. Controlled induction of focal adhesion disassembly and migration in primary fibroblasts

    DEFF Research Database (Denmark)

    Dunlevy, J R; Couchman, J R

    1993-01-01

    Fibroblast migration is an integral component of biological processes such as wound healing and embryogenesis. Previous experiments examining fibroblast locomotion from tissue explants have shown that migrating fibroblasts lack, or contain only transient, focal adhesions (focal contacts). Focal...... adhesions are specialized regions of tight cell-matrix interaction, assembled by a complex process of transmembrane signalling. Although the explant model has been used for studying several aspects of fibroblast locomotion, it is limited by the lack of control over migration, and only a small percentage...

  20. Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

    Science.gov (United States)

    Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

    2014-07-01

    Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

  1. Focal adhesion kinase modulates radial glia-dependent neuronal migration through connexin-26.

    Science.gov (United States)

    Valiente, Manuel; Ciceri, Gabriele; Rico, Beatriz; Marín, Oscar

    2011-08-10

    Focal adhesion kinase (FAK) is an intracellular kinase and scaffold protein that regulates migration in many different cellular contexts but whose function in neuronal migration remains controversial. Here, we have analyzed the function of FAK in two populations of neurons with very distinct migratory behaviors: cortical interneurons, which migrate tangentially and independently of radial glia; and pyramidal cells, which undergo glial-dependent migration. We found that FAK is dispensable for glial-independent migration but is cell-autonomously required for the normal interaction of pyramidal cells with radial glial fibers. Loss of FAK function disrupts the normal morphology of migrating pyramidal cells, delays migration, and increases the tangential dispersion of neurons arising from the same radial unit. FAK mediates this process by regulating the assembly of Connexin-26 contact points in the membrane of migrating pyramidal cells. These results indicate that FAK plays a fundamental role in the dynamic regulation of Gap-mediated adhesions during glial-guided neuronal migration in the mouse.

  2. cAMP-induced Epac-Rap activation inhibits epithelial cell migration by modulating focal adhesion and leading edge dynamics.

    Science.gov (United States)

    Lyle, Karen S; Raaijmakers, Judith H; Bruinsma, Wytse; Bos, Johannes L; de Rooij, Johan

    2008-06-01

    Epithelial cell migration is a complex process crucial for embryonic development, wound healing and tumor metastasis. It depends on alterations in cell-cell adhesion and integrin-extracellular matrix interactions and on actomyosin-driven, polarized leading edge protrusion. The small GTPase Rap is a known regulator of integrins and cadherins that has also been implicated in the regulation of actin and myosin, but a direct role in cell migration has not been investigated. Here, we report that activation of endogenous Rap by cAMP results in an inhibition of HGF- and TGFbeta-induced epithelial cell migration in several model systems, irrespective of the presence of E-cadherin adhesion. We show that Rap activation slows the dynamics of focal adhesions and inhibits polarized membrane protrusion. Importantly, forced integrin activation by antibodies does not mimic these effects of Rap on cell motility, even though it does mimic Rap effects in short-term cell adhesion assays. From these results, we conclude that Rap inhibits epithelial cell migration, by modulating focal adhesion dynamics and leading edge activity. This extends beyond the effect of integrin affinity modulation and argues for an additional function of Rap in controlling the migration machinery of epithelial cells.

  3. Inositol hexaphosphate (IP6) inhibits key events of cancer metastasis: II. Effects on integrins and focal adhesions.

    Science.gov (United States)

    Tantivejkul, Kwanchanit; Vucenik, Ivana; Shamsuddin, Abulkalam M

    2003-01-01

    We have shown that inositol hexaphosphate (IP6), a natural compound and a potent anti-cancer agent, inhibited cancer cell adhesion to the extracellular matrix (ECM) proteins, thereby leading to inhibition of cell migration and invasion. Cell adhesion to ECM is mediated by specific cell surface integrins, which transduce intracellular signals through their interaction and activation of other proteins that are recruited to the focal adhesion. We hypothesize that IP6 decreases cell adhesion by suppressing the integrin receptors and their subsequent signaling pathway. We analyzed integrin expressions of the highly invasive estrogen receptor-negative human breast cancer MDA-MB 231 cells exposed to IP6 by flow cytometry. The expression of focal adhesion proteins was investigated by immunocytochemistry and Western blotting. IP6 treatment caused a significant (P IP6 treatment. When the expression of integrins on the cell surface was assessed, there was a dramatic 82% decrease in the expression of alpha 5 beta 1 on IP6-treated cells (P IP6, was used as a control. Immunocytochemistry showed a lack of clustering of paxillin; tyrosine-phosphorylated proteins in IP6-treated cells were discontinuous and scattered around the cell periphery, whereas the patterns were more dense and localized in control cells. Consistent with these observations, focal adhesion kinase (FAK) autophosphorylation at tyrosine-397 residue was suppressed, albeit modestly, by IP6 treatment, suggesting a down-regulation in the integrin-mediated signaling pathway. The results of this study indicate that IP6-induced inhibition of cancer cell adhesion, migration and invasion may be mediated through the modulation of integrin dimerization, cell surface expression and integrin-associated signaling pathway.

  4. Theory of the mechanical response of focal adhesions to shear flow

    Science.gov (United States)

    Biton, Y. Y.; Safran, S. A.

    2010-05-01

    The response of cells to shear flow is primarily determined by the asymmetry of the external forces and moments that are sensed by each member of a focal adhesion pair connected by a contractile stress fiber. In the theory presented here, we suggest a physical model in which each member of such a pair of focal adhesions is treated as an elastic body subject to both a myosin-activated contractile force and the shear stress induced by the external flow. The elastic response of a focal adhesion complex is much faster than the active cellular processes that determine the size of the associated focal adhesions and the direction of the complex relative to the imposed flow. Therefore, the complex attains its mechanical equilibrium configuration which may change because of the cellular activity. Our theory is based on the experimental observation that focal adhesions modulate their cross-sectional area in order to attain an optimal shear. Using this assumption, our elastic model shows that such a complex can passively change its orientation to align parallel to the direction of the flow.

  5. Theory of the mechanical response of focal adhesions to shear flow

    Energy Technology Data Exchange (ETDEWEB)

    Biton, Y Y; Safran, S A, E-mail: yoav.biton@weizmann.ac.i, E-mail: sam.safran@weizmann.ac.i [Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100 (Israel)

    2010-05-19

    The response of cells to shear flow is primarily determined by the asymmetry of the external forces and moments that are sensed by each member of a focal adhesion pair connected by a contractile stress fiber. In the theory presented here, we suggest a physical model in which each member of such a pair of focal adhesions is treated as an elastic body subject to both a myosin-activated contractile force and the shear stress induced by the external flow. The elastic response of a focal adhesion complex is much faster than the active cellular processes that determine the size of the associated focal adhesions and the direction of the complex relative to the imposed flow. Therefore, the complex attains its mechanical equilibrium configuration which may change because of the cellular activity. Our theory is based on the experimental observation that focal adhesions modulate their cross-sectional area in order to attain an optimal shear. Using this assumption, our elastic model shows that such a complex can passively change its orientation to align parallel to the direction of the flow.

  6. Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1994-01-01

    Focal adhesion formation in fibroblasts results from complex transmembrane signaling processes initiated by extracellular matrix molecules. Although a role for integrins with attendant tyrosine kinases has been established, there is evidence that cell surface heparan sulfate proteoglycans (HSPGs......) are also involved with an associated role of protein kinase C. The identity of the proteoglycan has remained elusive, but we now report that syndecan 4 (ryudocan/amphiglycan) is present in focal adhesions of a number of cell types. Affinity-purified antibodies raised against a unique portion...

  7. Focal adhesive arachnoiditis of the spinal cord: Imaging diagnosis and surgical resolution

    Directory of Open Access Journals (Sweden)

    Hiroki Morisako

    2010-01-01

    Full Text Available Background: Although adhesive arachnoiditis of the spinal cord can cause progressive symptoms associated with syringomyelia or myelomalacia, its surgical resolution based on the imaging diagnosis is not well characterized. This study aims to describe the use of imaging for the diagnosis of focal adhesive arachnoiditis of the spinal cord and its surgical resolution using microsurgical arachnoidolysis. Materials and Methods: Four consecutive patients with symptomatic syringomyelia or myelomalacia caused by focal adhesive arachnoiditis underwent microsurgical arachnoidolysis. Comprehensive imaging evaluation using constructive interference in steady-state (CISS magnetic resonance imaging (MRI or myelographic MR imaging using true fast imaging with steady-state precession (TrueFISP sequences was included before surgery to determine the surgical indication. Results: In all four patients a focal adhesion was identified at the cervical or thoracic level of the spinal cord, a consequence of infection or trauma. Three patients showed modest or minor improvement in neurological function, and one patient was unchanged after surgery. The syringomyelia or myelomalacia resolved after surgery and no recurrence was noted within the follow-up period, which ranged from 5 months to 30 months. Conclusions: MRI diagnosis of focal adhesive arachnoiditis is critical to determine the surgical indication. Microsurgical arachnoidolysis appears to be a straightforward method for stabilizing the progressive symptoms, though the procedure is technically demanding.

  8. A Site-Specific Phosphorylation of the Focal Adhesion Kinase Controls the Formation of Spheroid Cell Clusters

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Gosau, Martin; Kristensen, Lars Peter

    2014-01-01

    Human dental follicle cells (DFCs) are ectomesenchymal multipotent stem cells that form spheroid cell clusters (SCCs) under serum free medium cell culture conditions (SFM). Until today, molecular mechanisms for the formation of SCCs are unknown. In this study a quantitative phosphoproteomics...... approach revealed regulated phosphorylated proteins in SCCs, which were derived from DFCs after 24 and 48 h in SFM. These regulated proteins were categorized using the Kyoto encyclopedia of genes and genomes program. Here, cellular processes and signaling pathway were identified such as the focal adhesion...

  9. A role for the juxtamembrane cytoplasm in the molecular dynamics of focal adhesions.

    Directory of Open Access Journals (Sweden)

    Haguy Wolfenson

    Full Text Available Focal adhesions (FAs are specialized membrane-associated multi-protein complexes that link the cell to the extracellular matrix and play crucial roles in cell-matrix sensing. Considerable information is available on the complex molecular composition of these sites, yet the regulation of FA dynamics is largely unknown. Based on a combination of FRAP studies in live cells, with in silico simulations and mathematical modeling, we show that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion, and a fast-diffusing cytoplasmic pool. The juxtamembrane region surrounding FAs displays a gradient of FA plaque proteins with respect to both concentration and dynamics. Based on these findings, we propose a new model for the regulation of FA dynamics in which this juxtamembrane domain acts as an intermediary layer, enabling an efficient regulation of FA formation and reorganization.

  10. Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

    Science.gov (United States)

    Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Feletti, Lely; Lasser, Theo; Radenovic, Aleksandra

    2017-02-01

    Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM-SOFI concept as a quantitative imaging framework by investigating moving focal adhesions in living cells.

  11. Redistribution of microfilament-associated proteins during the formation of focal contacts and adhesions in chick fibroblasts

    DEFF Research Database (Denmark)

    Couchman, J R; Badley, R A; Rees, D A

    1983-01-01

    fibroblast system which initially has no such adhesion specializations but then develops them sequentially over a 48 h period. Without exception, all focal contacts and focal adhesions contain both vinculin and alpha-actinin at every stage that we can detect by IRM or by double staining to reveal...... the associated microfilament bundles. Indeed the appearance of small bodies containing alpha-actinin and vinculin is shown to precede focal contact formation in our model system and such structures (not visible by IRM) are proposed to be the precursors of focal contacts and adhesions. Myosin and filamin......The roles of the microfilament-associated proteins vinculin, alpha-actinin, myosin and filamin have been studied by immunofluorescence and double fluorescence in conjunction with interference reflection microscopy (IRM), during the development of focal contacts and focal adhesions in a chick...

  12. The focal adhesion targeting domain of p130Cas confers a mechanosensing function.

    Science.gov (United States)

    Bradbury, Peta M; Turner, Kylie; Mitchell, Camilla; Griffin, Kaitlyn R; Middlemiss, Shiloh; Lau, Loretta; Dagg, Rebecca; Taran, Elena; Cooper-White, Justin; Fabry, Ben; O'Neill, Geraldine M

    2017-04-01

    Members of the Cas family of focal adhesion proteins contain a highly conserved C-terminal focal adhesion targeting (FAT) domain. To determine the role of the FAT domain in these proteins, we compared wild-type exogenous NEDD9 with a hybrid construct in which the NEDD9 FAT domain had been exchanged for the p130Cas (also known as BCAR1) FAT domain. Fluorescence recovery after photobleaching (FRAP) revealed significantly slowed exchange of the fusion protein at focal adhesions and significantly slower two-dimensional migration. No differences were detected in cell stiffness as measured using atomic force microscopy (AFM) and in cell adhesion forces measured with a magnetic tweezer device. Thus, the slowed migration was not due to changes in cell stiffness or adhesion strength. Analysis of cell migration on surfaces of increasing rigidity revealed a striking reduction of cell motility in cells expressing the p130Cas FAT domain. The p130Cas FAT domain induced rigidity-dependent phosphorylation of tyrosine residues within NEDD9. This in turn reduced post-translational cleavage of NEDD9, which we show inhibits NEDD9-induced migration. Collectively, our data therefore suggest that the p130Cas FAT domain uniquely confers a mechanosensing function. © 2017. Published by The Company of Biologists Ltd.

  13. Lateral shear forces applied to cells with single elastic micropillars to influence focal adhesion dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Heil, Patrick; Spatz, Joachim P, E-mail: spatz@mf.mpg.d [Department of New Materials and Biosystems, Max Planck Institute for Metals Research, Heisenbergstrasse 3, D-70569 Stuttgart (Germany); Department of Biophysical Chemistry, University of Heidelberg, Heisenbergstrasse 3, D-70569 Stuttgart (Germany)

    2010-05-19

    Focal adhesions (FAs) are important adhesion sites between eukaryotic cells and the extracellular matrix, their size depending on the locally applied force. To quantitatively study the mechanosensitivity of FAs, we induce their growth and disassembly by varying the distribution of intracellular stress. We present a novel method for micromanipulation of living cells to explore the dynamics of focal adhesion (FA) assembly under force. Fibroblasts are sheared laterally to their adhesion surface with single PDMS micropillars in order to apply laterally stretch or compression to focal adhesions. This allows for measuring the shear force exerted by the micropillar and correlates it with FA length and growth velocity. Furthermore, we analyze the resulting dynamics of FA molecules (paxillin) and compare intensity profiles along FAs before and after the application of external force. The responses of stretched and relaxed FAs differ fundamentally: relaxed and compressed FAs disassemble isotropically and show no length variation while stretched FAs grow unisotropically in the direction of the applied force and show protein influx only at their front.

  14. Cancer cell metastasis; perspectives from the focal adhesion

    Directory of Open Access Journals (Sweden)

    Lefteris C Zacharia

    2015-10-01

    Full Text Available In almost all cancers, most patients die from metastatic disease and not from the actual primary tumor. That is why addressing the problem of metastasis is of utmost importance for the successful treatment and improved survival of cancer patients. Metastasis is a complex process that ultimately leads to cancer cells spreading from the tumor to distant sites of the body. During this process, cancer cells tend to lose contact with the extracellular matrix (ECM and neighboring cells within the primary tumor, and are thus able to invade surrounding tissues. Hence, ECM, and the ECM-associated adhesion proteins play a critical role in the metastatic process. This review will focus on recent literature regarding interesting and novel molecules at the cell-ECM adhesion sites, namely migfilin, mitogen-inducible gene-2 (Mig-2 and Ras suppressor-1 (RSU-1, that are also critically involved in cancer cell metastasis, emphasizing on data from experiments performed in vitro in breast cancer and hepatocellular carcinoma cell lines as well as human breast cancer tissue samples.

  15. Targeting Focal Adhesion Assembly by Ethoxyfagaronine Prevents Lymphoblastic Cell Adhesion to Fibronectin

    Directory of Open Access Journals (Sweden)

    F. Ouchani

    2012-01-01

    Full Text Available Background: Leukemic cell adhesion to proteins of the bone marrow microenvironment provides signals which control morphology, motility and cell survival. We described herein the ability of ethoxyfagaronine (etxfag, a soluble synthetic derivative of fagaronine, to prevent leukemic cell adhesion to fibronectin peptide (FN/V.

  16. High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification.

    Science.gov (United States)

    Kroll, Torsten; Schmidt, David; Schwanitz, Georg; Ahmad, Mubashir; Hamann, Jana; Schlosser, Corinne; Lin, Yu-Chieh; Böhm, Konrad J; Tuckermann, Jan; Ploubidou, Aspasia

    2016-07-01

    High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging. Here, the step-by-step development of an HCA assay protocol and an HCS bioinformatics analysis pipeline are described. The protocol's power is demonstrated by application to focal adhesion (FA) detection, quantitative analysis of multiple FA features, and functional annotation of signaling pathways regulating FA size, using primary data of a published RNAi screen. The assay and the underlying strategy are aimed at researchers performing microscopy-based quantitative analysis of subcellular features, on a small scale or in large HCS experiments. © 2016 by John Wiley & Sons, Inc.

  17. Apigenin Attenuates Melanoma Cell Migration by Inducing Anoikis through Integrin and Focal Adhesion Kinase Inhibition.

    Science.gov (United States)

    Hasnat, Md Abul; Pervin, Mehnaz; Lim, Ji Hong; Lim, Beong Ou

    2015-11-27

    Apigenin, a nonmutagenic flavonoid, has been found to have antitumor properties and is therefore particularly relevant for the development of chemotherapeutic agents for cancers. In this study, time- and dose-dependent cell viability and cytotoxicity were assessed to determine the effects of apigenin on A2058 and A375 melanoma cells. Melanoma cells were pretreated with different concentrations of apigenin and analyzed for morphological changes, anoikis induction, cell migration, and levels of proteins associated with apoptosis. Apigenin reduced integrin protein levels and inhibited the phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK1/2), which induce anoikis in human cutaneous melanoma cells. Apigenin exhibited dose-dependent inhibition of melanoma cell migration, unlike untreated controls. Furthermore, apigenin treatment increased apoptotic factors such as caspase-3 and cleaved poly(ADP-ribose) polymerase in a dose-dependent manner, demonstrating the metastasis of melanoma cells. Our results provide a new insight into the mechanisms by which apigenin prevents melanoma metastasis by sensitizing anoikis induced by the loss of integrin proteins in the FAK/ERK1/2 signaling pathway. These findings elucidate the related mechanisms and suggest the potential of apigenin in developing clinical treatment strategies against malignant melanoma.

  18. Targeting Focal Adhesion Kinase Suppresses the Malignant Phenotype in Rhabdomyosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Alicia M. Waters

    2016-08-01

    Full Text Available Despite the tremendous advances in the treatment of childhood solid tumors, rhabdomyosarcoma (RMS continues to provide a therapeutic challenge. Children with metastatic or relapsed disease have a disease-free survival rate under 30%. Focal adhesion kinase (FAK is a nonreceptor tyrosine kinase that is important in many facets of tumorigenesis. Signaling pathways both upstream and downstream to FAK have been found to be important in sarcoma tumorigenesis, leading us to hypothesize that FAK would be present in RMS and would impact cellular survival. In the current study, we showed that FAK was present and phosphorylated in pediatric alveolar and embryonal RMS tumor specimens and cell lines. We also examined the effects of FAK inhibition upon two RMS cell lines utilizing parallel approaches including RNAi and small molecule inhibitors. FAK inhibition resulted in decreased cellular survival, invasion, and migration and increased apoptosis. Furthermore, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse RMS xenograft model. The findings from this study will help to further our understanding of the regulation of tumorigenesis in RMS and may provide desperately needed novel therapeutic strategies for these difficult-to-treat tumors.

  19. Copper deficiency induced emphysema is associated with focal adhesion kinase inactivation.

    Directory of Open Access Journals (Sweden)

    Shiro Mizuno

    Full Text Available BACKGROUND: Copper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1α dependent vascular endothelial growth factor (VEGF expression, and is also required for the activity of lysyl oxidase (LOX to effect matrix protein cross-linking. Cell detachment from the extracellular matrix can induce apoptosis (anoikis via inactivation of focal adhesion kinase (FAK. METHODOLOGY: To examine the molecular mechanisms whereby copper depletion causes the destruction of the normal alveolar architecture via anoikis, Male Sprague-Dawley rats were fed a copper deficient diet for 6 weeks while being treated with the copper chelator, tetrathiomolybdate. Other groups of rats were treated with the inhibitor of auto-phosphorylation of FAK, 1,2,4,5-benzenetetraamine tetrahydrochloride (1,2,4,5-BT or FAK small interfering RNA (siRNA. PRINCIPAL FINDINGS: Copper depletion caused emphysematous changes, decreased HIF-1α activity, and downregulated VEGF expression in the rat lungs. Cleaved caspase-3, caspase-8 and Bcl-2 interacting mediator of cell death (Bim expression was increased, and the phosphorylation of FAK was decreased in copper depleted rat lungs. Administration of 1,2,4,5-BT and FAK siRNA caused emphysematous lung destruction associated with increased expression of cleaved capase-3, caspase-8 and Bim. CONCLUSIONS: These data indicate that copper-dependent mechanisms contribute to the pathogenesis of emphysema, which may be associated with decreased HIF-1α and FAK activity in the lung.

  20. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions

    KAUST Repository

    Walkiewicz, Katarzyna Wiktoria

    2015-06-17

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein ‘nanomachines’ to become activated in a site-specific manner.

  1. Regulative mechanisms of chondrocyte adhesion

    DEFF Research Database (Denmark)

    Schmal, Hagen; Mehlhorn, Alexander T; Fehrenbach, Miriam

    2006-01-01

    Interaction between chondrocytes and extracellular matrix is considered a key factor in the generation of grafts for matrix-associated chondrocyte transplantation. Therefore, our objective was to study the influence of differentiation status on cellular attachment. Adhesion of chondrocytes to col...

  2. Talin contains a C-terminal calpain2 cleavage site important in focal adhesion dynamics.

    Directory of Open Access Journals (Sweden)

    Neil Bate

    Full Text Available Talin is a large (∼2540 residues dimeric adaptor protein that associates with the integrin family of cell adhesion molecules in cell-extracellular matrix junctions (focal adhesions; FAs, where it both activates integrins and couples them to the actin cytoskeleton. Calpain2-mediated cleavage of talin between the head and rod domains has previously been shown to be important in FA turnover. Here we identify an additional calpain2-cleavage site that removes the dimerisation domain from the C-terminus of the talin rod, and show that an E2492G mutation inhibits calpain cleavage at this site in vitro, and increases the steady state levels of talin1 in vivo. Expression of a GFP-tagged talin1 E2492G mutant in CHO.K1 cells inhibited FA turnover and the persistence of cell protrusion just as effectively as a L432G mutation that inhibits calpain cleavage between the talin head and rod domains. Moreover, incorporation of both mutations into a single talin molecule had an additive effect clearly demonstrating that calpain cleavage at both the N- and C-terminal regions of talin contribute to the regulation of FA dynamics. However, the N-terminal site was more sensitive to calpain cleavage suggesting that lower levels of calpain are required to liberate the talin head and rod fragments than are needed to clip off the C-terminal dimerisation domain. The talin head and rod liberated by calpain2 cleavage have recently been shown to play roles in an integrin activation cycle important in FA turnover and in FAK-dependent cell cycle progression respectively. The half-life of the talin head is tightly regulated by ubiquitination and we suggest that removal of the C-terminal dimerisation domain from the talin rod may provide a mechanism both for terminating the signalling function of the talin rod and indeed for inactivating full-length talin thereby promoting FA turnover at the rear of the cell.

  3. Vimentin contributes to epithelial-mesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation.

    Science.gov (United States)

    Liu, Ching-Yi; Lin, Hsi-Hui; Tang, Ming-Jer; Wang, Yang-Kao

    2015-06-30

    Modulations of cytoskeletal organization and focal adhesion turnover correlate to tumorigenesis and epithelial-mesenchymal transition (EMT), the latter process accompanied by the loss of epithelial markers and the gain of mesenchymal markers (e.g., vimentin). Clinical microarray results demonstrated that increased levels of vimentin mRNA after chemotherapy correlated to a poor prognosis of breast cancer patients. We hypothesized that vimentin mediated the reorganization of cytoskeletons to maintain the mechanical integrity in EMT cancer cells. By using knockdown strategy, the results showed reduced cell proliferation, impaired wound healing, loss of directional migration, and increased large membrane extension in MDA-MB 231 cells. Vimentin depletion also induced reorganization of cytoskeletons and reduced focal adhesions, which resulted in impaired mechanical strength because of reduced cell stiffness and contractile force. In addition, overexpressing vimentin in MCF7 cells increased cell stiffness, elevated cell motility and directional migration, reoriented microtubule polarity, and increased EMT phenotypes due to the increased β1-integrin and the loss of junction protein E-cadherin. The EMT-related transcription factor slug was also mediated by vimentin. The current study demonstrated that vimentin serves as a regulator to maintain intracellular mechanical homeostasis by mediating cytoskeleton architecture and the balance of cell force generation in EMT cancer cells.

  4. Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer*

    Science.gov (United States)

    Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S.; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2015-01-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. PMID:26330541

  5. Gangliosides regulate tumor cell adhesion to collagen.

    Science.gov (United States)

    Kazarian, Tamara; Jabbar, Adnan A; Wen, Fei-Qui; Patel, Dharmesh A; Valentino, Leonard A

    2003-01-01

    The ability of tumor cells to adhere to extracellular matrix proteins is critical for migration and invasion. The factors that regulate tumor cell adhesion are poorly characterized. Gangliosides promote platelet adhesion and may also play a role in the adhesion of other cell types. We hypothesized that pharmacological depletion of membrane gangliosides from adherent cells would abrogate adhesion to collagen and promote migration and invasion. To test these hypotheses, LA-N1 neuroblastoma cells, which avidly adhere to collagen and are rich with membrane gangliosides (43.69 nmol/10(8) cells), were cultured in the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-HCl. Endogenous gangliosides were reduced by 98% (0.76 nmol/10(8) cells) and adhesion to collagen decreased by 67%. There were no changes in cell morphology, viability, proliferation rate or apoptosis. Pre-incubation of ganglioside-depleted cells in conditioned medium from control cells restored adhesion to collagen (0.45 +/- 0.002), comparable to that of control cells (0.49 +/- 0.035). Similarly, pre-incubation of ganglioside-depleted cells with purified GD2 completely restored adhesion in a concentration-dependent manner. When LA-N1 cells were cultured with retinoic acid, a biological response modifier known to increase endogenous gangliosides, adhesion to collagen increased. Next, we questioned whether changes in adhesion would be reflected as changes in migration and invasion. Cells depleted of endogenous cellular gangliosides migrated more than control cells. Finally, control cells replete with their endogenous gangliosides demonstrated less invasive potential than control cells. The data demonstrate that endogenous tumor gangliosides increase neuroblastoma cell adhesion to collagen and reduce migration and invasion in vitro.

  6. A site-specific phosphorylation of the focal adhesion kinase controls the formation of spheroid cell clusters.

    Science.gov (United States)

    Beck, Hans Christian; Gosau, Martin; Kristensen, Lars Peter; Morsczeck, Christian

    2014-07-01

    Human dental follicle cells (DFCs) are ectomesenchymal multipotent stem cells that form spheroid cell clusters (SCCs) under serum free medium cell culture conditions (SFM). Until today, molecular mechanisms for the formation of SCCs are unknown. In this study a quantitative phosphoproteomics approach revealed regulated phosphorylated proteins in SCCs, which were derived from DFCs after 24 and 48 h in SFM. These regulated proteins were categorized using the Kyoto encyclopedia of genes and genomes program. Here, cellular processes and signaling pathway were identified such as the focal adhesion kinase (FAK) signaling pathway. In addition to the phosphoproteomics approach we showed that a specific phosphorylation of FAK (Y397) was required for the formation of SCCs. In conclusion, this study disclosed the phosphoproteome of SCCs for the first time and showed that the FAK signaling pathway is required for the formation of SCCs.

  7. Organization and post-transcriptional processing of focal adhesion kinase gene

    Directory of Open Access Journals (Sweden)

    Enslen Hervé

    2006-08-01

    Full Text Available Abstract Background Focal adhesion kinase (FAK is a non-receptor tyrosine kinase critical for processes ranging from embryo development to cancer progression. Although isoforms with specific molecular and functional properties have been characterized in rodents and chicken, the organization of FAK gene throughout phylogeny and its potential to generate multiple isoforms are not well understood. Here, we study the phylogeny of FAK, the organization of its gene, and its post-transcriptional processing in rodents and human. Results A single orthologue of FAK and the related PYK2 was found in non-vertebrate species. Gene duplication probably occurred in deuterostomes after the echinoderma embranchment, leading to the evolution of PYK2 with distinct properties. The amino acid sequence of FAK and PYK2 is conserved in their functional domains but not in their linker regions, with the absence of autophosphorylation site in C. elegans. Comparison of mouse and human FAK genes revealed the existence of multiple combinations of conserved and non-conserved 5'-untranslated exons in FAK transcripts suggesting a complex regulation of their expression. Four alternatively spliced coding exons (13, 14, 16, and 31, previously described in rodents, are highly conserved in vertebrates. Cis-regulatory elements known to regulate alternative splicing were found in conserved alternative exons of FAK or in the flanking introns. In contrast, other reported human variant exons were restricted to Homo sapiens, and, in some cases, other primates. Several of these non-conserved exons may correspond to transposable elements. The inclusion of conserved alternative exons was examined by RT-PCR in mouse and human brain during development. Inclusion of exons 14 and 16 peaked at the end of embryonic life, whereas inclusion of exon 13 increased steadily until adulthood. Study of various tissues showed that inclusion of these exons also occurred, independently from each other, in a

  8. Differential regulation of adhesion complex turnover by ROCK1 and ROCK2.

    Directory of Open Access Journals (Sweden)

    Frances E Lock

    Full Text Available BACKGROUND: ROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration. METHODOLOGY/PRINCIPAL FINDINGS: In this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibits signalling via focal adhesion kinase resulting in a failure of immature adhesion complexes to form mature stable focal adhesions. In contrast, loss of ROCK2 expression results in a significant reduction in adhesion complex turnover leading to formation of large, stable focal adhesions. Interestingly, loss of either ROCK1 or ROCK2 expression significantly impairs cell migration indicating both ROCK isoforms are required for normal keratinocyte migration. CONCLUSIONS: ROCK1 and ROCK2 have distinct and separate roles in adhesion complex assembly and turnover in human epidermal keratinocytes.

  9. Nanoengineered surfaces for focal adhesion guidance trigger mesenchymal stem cell self-organization and tenogenesis.

    Science.gov (United States)

    Iannone, Maria; Ventre, Maurizio; Formisano, Lucia; Casalino, Laura; Patriarca, Eduardo J; Netti, Paolo A

    2015-03-11

    The initial conditions for morphogenesis trigger a cascade of events that ultimately dictate structure and functions of tissues and organs. Here we report that surface nanopatterning can control the initial assembly of focal adhesions, hence guiding human mesenchymal stem cells (hMSCs) through the process of self-organization and differentiation. This process self-sustains, leading to the development of macroscopic tissues with molecular profiles and microarchitecture reminiscent of embryonic tendons. Therefore, material surfaces can be in principle engineered to set off the hMSC program toward tissuegenesis in a deterministic manner by providing adequate sets of initial environmental conditions.

  10. Focal adhesion kinase modulates activation of NF-κB by flow in endothelial cells

    OpenAIRE

    Petzold, Tobias; Orr, A. Wayne; Hahn, Cornelia; Jhaveri, Krishna A.; Parsons, J Thomas; Schwartz, Martin Alexander

    2009-01-01

    Atherogenesis involves activation of NF-κB in endothelial cells by fluid shear stress. Because this pathway involves integrins, we investigated the involvement of focal adhesion kinase (FAK). We found that FAK was not required for flow-stimulated translocation of the p65 NF-κB subunit to the nucleus but was essential for phosphorylation of p65 on serine 536 and induction of ICAM-1, an NF-κB-dependent gene. NF-κB activation by TNF-α or hydrogen peroxide was FAK independent. Events upstream of ...

  11. Silencing of focal adhesion kinase by tumor direct injection of small interfering RNA decreases in vivo tumor growth.

    Science.gov (United States)

    Tsutsumi, Kae; Yamaura, Takeshi; Nakajima, Motowo; Honda, Toshiyuki; Kasaoka, Tatsuhiko

    2009-07-01

    Focal adhesion kinase (FAK) is shown to be frequently correlated with malignancy of the tumor and poor prognosis of the diseases.Because FAK resides immediately downstream of the interaction of cell surface adhesion molecules and extracellular matricies, it is considered to be critical to regulate several cellular processes including growth, differentiation, adhesion, motility and apoptosis. However, the studies on the role of FAK related to cell proliferation have been limited even in vitro. Here, in order to validate the role of FAK in in vivo tumor formation and proliferation, we employed direct intratumoral injection of short hairpin RNA (shRNA) targeting FAK with cationic liposome. Using shRNAs targeting FAK selected from the constructed shRNA library for FAK and by optimization of in vivo delivery conditions, we demonstrated different patterns of the association of FAK inhibition with in vivo tumor formation/proliferation inhibition in two models, PC3M heterotopic xenograft and 4T1 orthotopic syngraft models. These observations indicated that the roles of FAK in tumorigenesis are different among the tumor species. In addition, we showed that ERK is the critical MAP kinase in the signaling pathway down stream of FAK in in vivo proliferation of 4T1 tumor cells.

  12. The role of focal adhesion complexes in fibroblast mechanotransduction during scar formation.

    Science.gov (United States)

    Rustad, Kristine C; Wong, Victor W; Gurtner, Geoffrey C

    2013-10-01

    Historically, great efforts have been made to elucidate the biochemical pathways that direct the complex process of wound healing; however only recently has there been recognition of the importance that mechanical signals play in the process of tissue repair and scar formation. The body's physiologic response to injury involves a dynamic interplay between mechanical forces and biochemical cues which directs a cascade of signals leading ultimately to the formation of fibrotic scar. Fibroblasts are a highly mechanosensitive cell type and are also largely responsible for the generation of the fibrotic matrix during scar formation and are thus a critical player in the process of mechanotransduction during tissue repair. Mechanotransduction is initiated at the interface between the cell membrane and the extracellular matrix where mechanical signals are first translated into a biochemical response. Focal adhesions are dynamic multi-protein complexes through which the extracellular matrix links to the intracellular cytoskeleton. These focal adhesion complexes play an integral role in the propagation of this initial mechanical cue into an extensive network of biochemical signals leading to widespread downstream effects including the influx of inflammatory cells, stimulation of angiogenesis, keratinocyte migration, fibroblast proliferation and collagen synthesis. Increasing evidence has demonstrated the importance of the biomechanical milieu in healing wounds and suggests that an integrated approach to the discovery of targets to decrease scar formation may prove more clinically efficacious than previous purely biochemical strategies.

  13. Myxococcus xanthus gliding motors are elastically coupled to the substrate as predicted by the focal adhesion model of gliding motility

    CERN Document Server

    Balagam, Rajesh; Czerwinski, Fabian; Sun, Mingzhai; Kaplan, Heidi B; Shaevitz, Joshua W; Igoshin, Oleg A

    2014-01-01

    Myxococcus xanthus is a model organism for studying bacterial social behaviors due to its ability to form complex multi-cellular structures. Knowledge of M. xanthus surface gliding motility and the mechanisms that coordinate it are critically important to our understanding of collective cell behaviors. Although the mechanism of gliding motility is still under investigation, recent experiments suggest that there are two possible mechanisms underlying force production for cell motility: the focal adhesion mechanism and the helical rotor mechanism which differ in the biophysics of the cell-substrate interactions. Whereas the focal adhesion model predicts an elastic coupling, the helical rotor model predicts a viscous coupling. Using a combination of computational modeling, imaging, and force microscopy, we find evidence for elastic coupling in support of the focal adhesion model. Using a biophysical model of the M. xanthus cell, we investigated how the mechanical interactions between cells are affected by intera...

  14. Alveolar Type II Cells Escape Stress Failure Caused by Tonic Stretch through Transient Focal Adhesion Disassembly

    Directory of Open Access Journals (Sweden)

    Xiao-Yang Liu, Xiao-Fei Chen, Yan-Hong Ren, Qing-Yuan Zhan, Chen Wang, Chun Yang

    2011-01-01

    Full Text Available Mechanical ventilation-induced excessive stretch of alveoli is reported to induce cellular stress failure and subsequent lung injury, and is therefore an injurious factor to the lung. Avoiding cellular stress failure is crucial to ventilator-induced lung injury (VILI treatment. In the present study, primary rat alveolar type II (ATII cells were isolated to evaluate their viability and the mechanism of their survival under tonic stretch. By the annexin V/ PI staining and flow cytometry assay, we demonstrated that tonic stretch-induced cell death is an immediate injury of mechanical stress. In addition, immunofluorescence and immunoblots assay showed that the cells experienced an expansion-contraction-reexpansion process, accompanied by partial focal adhesion (FA disassembly during contraction. Manipulation of integrin adherent affinity by altering bivalent cation levels in the culture medium and applying an integrin neutralizing antibody showed that facilitated adhesion affinity promoted cell death under tonic stretch, while lower level of adhesion protected the cells from stretch-induced stress failure. Finally, a simplified numerical model was established to reveal that adequate disassembly of FAs reduced the forces transmitting throughout the cell. Taken together, these results indicate that ATII cells escape stress failure caused by tonic stretch via active cell morphological remodeling, during which cells transiently disassemble FAs to unload mechanical forces.

  15. The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co-localize at focal adhesions.

    OpenAIRE

    1995-01-01

    Focal adhesions are sites of cell-extracellular matrix interactions that function in anchoring stress fibers to the plasma membrane and in adhesion-mediated signal transduction. Both focal adhesion structure and signaling ability involve protein tyrosine phosphorylation. LAR is a broadly expressed transmembrane protein tyrosine phosphatase comprised of a cell adhesion-like ectodomain and two intracellular protein tyrosine phosphatase domains. We have identified a novel cytoplasmic 160 kDa pho...

  16. PI3K{gamma} activation by CXCL12 regulates tumor cell adhesion and invasion

    Energy Technology Data Exchange (ETDEWEB)

    Monterrubio, Maria; Mellado, Mario; Carrera, Ana C. [Department of Immunology and Oncology, Centro Nacional de Biotecnologia/CSIC, Campus de Cantoblanco, E-28049 Madrid (Spain); Rodriguez-Frade, Jose Miguel, E-mail: jmrfrade@cnb.csic.es [Department of Immunology and Oncology, Centro Nacional de Biotecnologia/CSIC, Campus de Cantoblanco, E-28049 Madrid (Spain)

    2009-10-16

    Tumor dissemination is a complex process, in which certain steps resemble those in leukocyte homing. Specific chemokine/chemokine receptor pairs have important roles in both processes. CXCL12/CXCR4 is the most commonly expressed chemokine/chemokine receptor pair in human cancers, in which it regulates cell adhesion, extravasation, metastatic colonization, angiogenesis, and proliferation. All of these processes require activation of signaling pathways that include G proteins, phosphatidylinositol-3 kinase (PI3K), JAK kinases, Rho GTPases, and focal adhesion-associated proteins. We analyzed these pathways in a human melanoma cell line in response to CXCL12 stimulation, and found that PI3K{gamma} regulates tumor cell adhesion through mechanisms different from those involved in cell invasion. Our data indicate that, following CXCR4 activation after CXCL12 binding, the invasion and adhesion processes are regulated differently by distinct downstream events in these signaling cascades.

  17. Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer.

    Science.gov (United States)

    Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2015-11-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Xiangyang [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanchang University, Nanchang, Jiangxi 330006 (China); State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047 (China); Wang, Yao [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Liu, Chengmei [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047 (China); Lu, Quqin [Department of Biostatistics and Epidemiology, School of Public Health, Nanchang University, Nanchang, Jiangxi 330006 (China); Liu, Tao [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Chen, Guoan [Department of Hematology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006 (China); Rao, Hai [Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, TX 78229 (United States); Luo, Shiwen, E-mail: shiwenluo@ncu.edu.cn [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China)

    2014-08-01

    Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer. - Highlights: • HSP90β protects FAK from degradation by the ubiquitin-proteasome pathway. • Inhibition of HSP90β or FAK attenuates tumorigenesis of breast cancer cells. • Genetic repression of HSP90β or FAK inhibits tumor cell migration and proliferation. • Inhibition of HSP90β or FAK interferes cell invasion and cytoskeleton.

  19. A novel type 3 secretion system effector, YspI of Yersinia enterocolitica, induces cell paralysis by reducing total focal adhesion kinase.

    Science.gov (United States)

    LeGrand, Karen; Matsumoto, Hiroyuki; Young, Glenn M

    2015-05-01

    Some of the world's most important diseases are caused by bacterial pathogens that deliver toxic effector proteins directly into eukaryotic cells using type III secretion systems. The myriad of pathological outcomes caused by these pathogens is determined, in part, by the manipulation of host cell physiology due to the specific activities of individual effectors among the unique suite each pathogen employs. YspI was found to be an effector, delivered by Yersinia enterocolitica Biovar 1B, that inhibits host cell motility. The action of YspI comes about through its specific interaction with focal adhesion kinase, FAK, which is a fulcrum of focal adhesion complexes for controlling cellular motility. The interaction was defined by a specific domain of YspI that bound to the FAK kinase domain. Further examination revealed that YspI-FAK interaction leads to a reduction of FAK steady-state levels without altering its phosphorylation state. This collection of observations and results showed YspI displays unique functionality by targeting the key regulator of focal adhesion complexes to inhibit cellular movement.

  20. Biphasic function of focal adhesion kinase in endothelial tube formation induced by fibril-forming collagens.

    Science.gov (United States)

    Nakamura, Junko; Shigematsu, Satoshi; Yamauchi, Keishi; Takeda, Teiji; Yamazaki, Masanori; Kakizawa, Tomoko; Hashizume, Kiyoshi

    2008-10-03

    Migration and tube formation of endothelial cells are important in angiogenesis and require a coordinated response to the extra-cellular matrix (ECM) and growth factor. Since focal adhesion kinase (FAK) integrates signals from both ECM and growth factor, we investigated its role in angiogenesis. Type I and II collagens are fibril-forming collagens and stimulate human umbilical vein endothelial cells (HUVECs) to form tube structure. Although knockdown of FAK restrained cell motility and resulted in inhibition of tube formation, FAK degradation and tube formation occurred simultaneously after incubation with fibril-forming collagens. The compensation for the FAK degradation by a calpain inhibitor or transient over-expression of FAK resulted in disturbance of tube formation. These phenomena are specific to fibril-forming collagens and mediated via alpha2beta1 integrin. In conclusion, our data indicate that FAK is functioning in cell migration, but fibril-forming collagen-induced FAK degradation is necessary for endothelial tube formation.

  1. Composite resin's adhesive resistance to dentin: influence of Er:YAG laser focal distance variation.

    Science.gov (United States)

    Corona, Silmara Aparecida Milori; Atoui, Juliana Abdallah; Chimello, Daniela Thomazatti; Borsatto, Maria Cristina; Pecora, Jesus Djalma; Dibb, Regina Guenka Palma

    2005-04-01

    The aim of this study was to analyze in vitro the influence of Er:YAG laser focal distance variation on tensile bond strength of a composite resin to dentin. Although there are several studies using the Er:YAG laser for dentin treatment, there is a lack of available literature related to the Er:YAG laser focal distance variation. Sixty vestibular and lingual dentin surfaces from extracted human third molars, kept in a 0.4% azide sodium solution, were ground and assigned to six groups. The control group was conditioned with 35% phosphoric acid (CA). In the lased groups, the dentin surface treatment was performed by irradiation with Er:YAG laser (80 mJ/2 Hz), varying the focal distance (11, 12, 14, 16, and 17 mm), followed by acid etching. The Single Bond/Filtek Z250 (3M) resinous system was used for the specimen manufacture. The tensile bond strength tests were performed in a Universal Testing Machine with 50 kgf load cell and 0.5 mm/min cross head speed. The averages in MPa were: CA: 18.03 (+/-2.09); 11 mm; 9.92 (+/-3.34); 12 mm: 9.49 (+/-2.29); 14 mm: 10.99 (+/-3.45); 16 mm: 10.56 (+/-1.93); and 17 mm: 17.05 (+/-2.31). It was concluded that the application of Er:YAG laser in a defocused mode (17 mm) associated with acid etching was similar to the treatment of acid solely. Er:YAG laser irradiation in a focused (12 mm) and a defocused (11, 14, and 16 mm) mode coupled with acid conditioning produced the lowest values of adhesion.

  2. STROBE-compliant integrin through focal adhesion involve in cancer stem cell and multidrug resistance of ovarian cancer

    Science.gov (United States)

    Wei, Luwei; Yin, Fuqiang; Zhang, Wei; Li, Li

    2017-01-01

    Abstract Cancer stem cells (CSCs) are considered to be the root of carcinoma relapse and drug resistance in ovarian cancer. Hunting for the potential CSC genes and explain their functions would be a feasible strategy to meet the challenge of the drug resistance in ovarian cancer. In this study, we performed bioinformatic approaches such as biochip data extraction and pathway enrichment analyses to elucidate the mechanism of the CSC genes in regulation of drug resistance. Potential key genes, integrins, were identified to be related to CSC in addition to their associations with drug resistance and prognosis in ovarian cancer. A total of 36 ovarian CSC genes involved in regulation of drug resistance were summarized, and potential drug resistance-related CSC genes were identified based on 3 independent microarrays retrieved from the Gene Expression Omnibus (GEO) Profiles. Pathway enrichment of CSC genes associated with drug resistance in ovarian cancer indicated that focal adhesion signaling might play important roles in CSC genes-mediated drug resistance. Integrins are members of the adhesion molecules family, and integrin subunit alpha 1, integrin subunit alpha 5, and integrin subunit alpha 6 (ITGA6) were identified as central CSC genes and their expression in side population cells, cisplatin-resistant SKOV3 (SKOV3/DDP2) cells, and cisplatin-resistant A2780 (A2780/DDP) cells were dysregulated as measured by real-time quantitative polymerase chain reaction. The high expression of ITGA6 in 287 ovarian cancer patients of TCGA cohort was significantly associated with poorer progression-free survival. This study provide the basis for further understanding of CSC genes in regulation of drug resistance in ovarian cancer, and integrins could be a potential biomarker for prognosis of ovarian cancer. PMID:28328815

  3. MVL-PLA2, a snake venom phospholipase A2, inhibits angiogenesis through an increase in microtubule dynamics and disorganization of focal adhesions.

    Directory of Open Access Journals (Sweden)

    Amine Bazaa

    Full Text Available Integrins are essential protagonists of the complex multi-step process of angiogenesis that has now become a major target for the development of anticancer therapies. We recently reported and characterized that MVL-PLA2, a novel phospholipase A2 from Macrovipera lebetina venom, exhibited anti-integrin activity. In this study, we show that MVL-PLA2 also displays potent anti-angiogenic properties. This phospholipase A2 inhibited adhesion and migration of human microvascular-endothelial cells (HMEC-1 in a dose-dependent manner without being cytotoxic. Using Matrigel and chick chorioallantoic membrane assays, we demonstrated that MVL-PLA2, as well as its catalytically inactivated form, significantly inhibited angiogenesis both in vitro and in vivo. We have also found that the actin cytoskeleton and the distribution of alphav beta3 integrin, a critical regulator of angiogenesis and a major component of focal adhesions, were disturbed after MVL-PLA2 treatment. In order to further investigate the mechanism of action of this protein on endothelial cells, we analyzed the dynamic instability behavior of microtubules in living endothelial cells. Interestingly, we showed that MVL-PLA2 significantly increased microtubule dynamicity in HMEC-1 cells by 40%. We propose that the enhancement of microtubule dynamics may explain the alterations in the formation of focal adhesions, leading to inhibition of cell adhesion and migration.

  4. MUC16 contributes to the metastasis of pancreatic ductal adenocarcinoma through focal adhesion mediated signaling mechanism

    Science.gov (United States)

    Chugh, Seema; Rachagani, Satyanarayana; Lakshmanan, Imayavaramban; Gupta, Suprit; Seshacharyulu, Parthasarathy; Smith, Lynette M.; Ponnusamy, Moorthy P.; Batra, Surinder K.

    2016-01-01

    MUC16, a heavily glycosylated type-I transmembrane mucin is overexpressed in several cancers including pancreatic ductal adenocarcinoma (PDAC). Previously, we have shown that MUC16 is significantly overexpressed in human PDAC tissues. However, the functional consequences and its role in PDAC is poorly understood. Here, we show that MUC16 knockdown decreases PDAC cell proliferation, colony formation and migration in vitro. Also, MUC16 knockdown decreases the tumor formation and metastasis in orthotopic xenograft mouse model. Mechanistically, immunoprecipitation and immunofluorescence analyses confirms MUC16 interaction with galectin-3 and mesothelin in PDAC cells. Adhesion assay displayed decreased cell attachment of MUC16 knockdown cells with recombinant galectin-1 and galectin-3 protein. Further, CRISPR/Cas9-mediated MUC16 knockout cells show decreased tumor-associated carbohydrate antigens (T and Tn) in PDAC cells. Importantly, carbohydrate antigens were decreased in the region that corresponds to MUC16 and suggests for the decreased MUC16-galectin interactions. Co-immunoprecipitation also revealed a novel interaction between MUC16 and FAK in PDAC cells. Interestingly, we observed decreased expression of mesenchymal and increased expression of epithelial markers in MUC16-silenced cells. Additionally, MUC16 loss showed a decreased FAK-mediated Akt and ERK/MAPK activation. Altogether, these findings suggest that MUC16-focal adhesion signaling may play a critical role in facilitating PDAC growth and metastasis. PMID:27382435

  5. Integrin-Associated Focal Adhesion Kinase Protects Human Embryonic Stem Cells from Apoptosis, Detachment, and Differentiation

    Directory of Open Access Journals (Sweden)

    Loriana Vitillo

    2016-08-01

    Full Text Available Human embryonic stem cells (hESCs can be maintained in a fully defined niche on extracellular matrix substrates, to which they attach through integrin receptors. However, the underlying integrin signaling mechanisms, and their contribution to hESC behavior, are largely unknown. Here, we show that focal adhesion kinase (FAK transduces integrin activation and supports hESC survival, substrate adhesion, and maintenance of the undifferentiated state. After inhibiting FAK kinase activity we show that hESCs undergo cell detachment-dependent apoptosis or differentiation. We also report deactivation of FAK downstream targets, AKT and MDM2, and upregulation of p53, all key players in hESC regulatory networks. Loss of integrin activity or FAK also induces cell aggregation, revealing a role in the cell-cell interactions of hESCs. This study provides insight into the integrin signaling cascade activated in hESCs and reveals in FAK a key player in the maintenance of hESC survival and undifferentiated state.

  6. PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

    Science.gov (United States)

    Prolactin-Induced Tyrosine Phosphorylation, Activation and ReceptorAssociation of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells. Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental ProtectionAgency, MD-72, Research Triangle Park, NC 27711, and

  7. Focal adhesion kinase is required for intestinal regeneration and tumorigenesis downstream of Wnt/c-Myc signaling

    NARCIS (Netherlands)

    Ashton, G.H.; Morton, J.P.; Myant, K.; Phesse, T.J.; Ridgway, R.A.; Marsh, V.; Wilkins, J.A.; Athineos, D.; Muncan, V.; Kemp, R.; Neufeld, K.; Clevers, H.; Brunton, V.; Winton, D.J.; Wang, X.; Sears, R.; Clarke, A.R.; Frame, M.C.; Sansom, O.J.

    2010-01-01

    The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration followin

  8. Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation.

    NARCIS (Netherlands)

    Miltenburg, van M.H.; Nimwegen, van M.J.; Tijdens, R.B.; Lalai, R.A.; Kuiper, R.; Klarenbeek, S.; Schouten, P.C.; Vries, de A.; Jonkers, J.M.M..; Water, van de B.

    2014-01-01

    BACKGROUND Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneo

  9. alpha2-Adrenoceptor stimulation promotes actin polymerization and focal adhesion in 3T3F442A and BFC-1beta preadipocytes.

    Science.gov (United States)

    Bétuing, S; Daviaud, D; Valet, P; Bouloumié, A; Lafontan, M; Saulnier-Blache, J S

    1996-12-01

    We previously demonstrated that in white fat cell precursors alpha2-adrenoceptor stimulation lead to the phosphorylation of p44 and p42 mitogen-activated protein kinases and an increase in cell number. Regulation of cell adhesion and cell cytoskeleton plays a crucial role in the control of cell growth by various growth factors. Here, we report that in mouse 3T3F442A preadipocytes expressing 2500 fmol/mg protein of the human alpha2C10-adrenoceptor (alpha2AF2 cells), alpha2-adrenergic stimulation rapidly restored the spreading of cells previously retracted by serum withdrawal. This effect was pertussis toxin sensitive and was blocked by pretreatment of the cells with dihydrocytochalasin B (a blocker of actin polymerization), genistein (a tyrosine kinase inhibitor), or agents that increase cell cAMP content. Spreading was accompanied by cell membrane ruffling, formation of lamelipodia and filipodia, appearance of focal adhesion plaques, and induction of actin stress fibers. alpha2-Adrenergic stimulation also lead to a rapid Gi- and actin-dependent tyrosine phosphorylation of the pp125 focal adhesion kinase (FAK) as well as of the p42 and p44 mitogen-activated protein kinases. alpha2-Adrenergic-dependent spreading and FAK and mitogen-activated protein kinase phosphorylation were also observed in 3T3F442A preadipocytes permanently expressing 20 fmol/mg protein of the human alpha2C10-adrenoceptor (alpha2AF3 cells) as well as in BFC-1beta preadipocytes, which constitutively express 25 fmol/mg protein of mouse alpha2A-adrenoceptors. In BFC-1beta preadipocytes, alpha2-adrenergic-dependent spreading and pp125FAK phosphorylation were counteracted by beta-adrenergic stimulation. Our results suggest that alpha2-adrenergic control of actin polymerization and focal adhesion assembly could play a crucial role in the regulation of preadipocyte growth by the sympathetic nervous system.

  10. Translucent titanium coating altered the composition of focal adhesions and promoted migration of osteoblast-like MG-63 cells on glass.

    Science.gov (United States)

    Ho, Yi; Kok, Sang-Heng; Wang, Juo-Song; Lin, Li-Deh

    2014-04-01

    "TiGlass" was designed and was known to promote initial adhesion and increase migration of rat calvarial osteoblats. In this article, migration study and a series of epifluorescence microscopic studies were conducted to find out the composition of focal adhesion on titanium surface. The translucent titanium surface was applied in random migration analysis and immunofluorescence cell staining. In the immunofluorescent double staining, phosphorylated focal adhesion kinase was tested with vinculin. Various integrin subunits were then tested with vinculin to study the composition of activated focal adhesions. Integrin subunit α5 and αV were tested against β3; integrin subunits α5, αV, β3, and αVβ3 were tested with F-actin, respectively. The MG-63 cells began migration earlier and migrated faster on "TiGlass." Immunofluorescent double staining revealed that all focal adhesion kinase in the focal adhesions were activated on both the surfaces. The osteoblast was inferred to made adhesion to titanium and glass through integrins. The focal adhesions on glass were found to be composed of integrin subunits αV and β3. However, on "TiGlass," integrin subunits α5 might have supplemented the adhesion to titanium. Results from double staining of integrin subunits α5, αV, β3, and αVβ3 with F-actin also supported integrin subunits α5 might have involved in adhesion of titanium.

  11. Flexible nanopillars to regulate cell adhesion and movement

    Science.gov (United States)

    Chien, Fan-Ching; Dai, Yang-Hong; Kuo, Chiung Wen; Chen, Peilin

    2016-11-01

    Flexible polymer nanopillar substrates were used to systematically demonstrate cell alignment and migration guided by the directional formation of focal adhesions. The polymer nanopillar substrates were constructed to various height specifications to provide an extensive variation of flexibility; a rectangular arrangement created spatial confinement between adjacent nanopillars, providing less spacing in the horizontal and vertical directions. Three polymer nanopillar substrates with the diameter of 400 nm and the heights of 400, 800, and 1200 nm were fabricated. Super-resolution localization imaging and protein pair-distance analysis of vinculin proteins revealed that Chinese hamster ovary (CHO) cells formed mature focal adhesions on 1200 nm high nanopillar substrates by bending adjacent nanopillars to link dot-like adhesions. The spacing confinement of the adjacent nanopillars enhanced the orthogonal directionality of the formation tendency of the mature focal adhesions. The directional formation of the mature focal adhesions also facilitated the organization of actin filaments in the horizontal and vertical directions. Moreover, 78% of the CHO cells were aligned in these two directions, in conformity with the flexibility and nanotopographical cues of the nanopillars. Biased cell migration was observed on the 1200 nm high nanopillar substrates.

  12. Laminin α2-mediated focal adhesion kinase activation triggers Alport glomerular pathogenesis.

    Directory of Open Access Journals (Sweden)

    Duane Delimont

    Full Text Available It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.

  13. Laminin α2-mediated focal adhesion kinase activation triggers Alport glomerular pathogenesis.

    Science.gov (United States)

    Delimont, Duane; Dufek, Brianna M; Meehan, Daniel T; Zallocchi, Marisa; Gratton, Michael Anne; Phillips, Grady; Cosgrove, Dominic

    2014-01-01

    It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.

  14. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes.

    Science.gov (United States)

    Bellance, Catherine; Khan, Junaid A; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-05-01

    Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(-) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl-amino)-phenyl-17β-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes.

  15. Diamagnetic levitation causes changes in the morphology, cytoskeleton, and focal adhesion proteins expression in osteocytes.

    Science.gov (United States)

    Qian, A R; Wang, L; Gao, X; Zhang, W; Hu, L F; Han, J; Li, J B; Di, S M; Shang, Peng

    2012-01-01

    Diamagnetic levitation technology is a novel simulated weightless technique and has recently been applied in life-science research. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels, namely, μg (diamagnetic levitation), 1g, and 2g for diamagnetic materials. In this study, the effects of LG-HMF on the activity, morphology, and cytoskeleton (actin filament, microtubules, and vimentin intermediate filaments) in osteocyte - like cell line MLO-Y4 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods, hematoxylin-eosin (HE) staining, and laser scanning confocal microscopy (LSCM), respectively. The changes induced by LG-HMF in distribution and expression of focal adhesion (FA) proteins, including vinculin, paxillin, and talin in MLO-Y4 were determined by LSCM and Western blotting. The results showed that LG-HMF produced by superconducting magnet had no lethal effects on MLO-Y4. Compared to control, diamagnetic levitation (μg) affected MLO-Y4 morphology, nucleus size, cytoskeleton architecture, and FA proteins distribution and expression. The study indicates that osteocytes are sensitive to altered gravity and FA proteins (vinculin, paxillin, and talin) may be involved in osteocyte mechanosensation. The diamagnetic levitation may be a novel ground-based space-gravity simulator and can be used for biological experiment at cellular level. © 2011 IEEE

  16. Focal adhesion kinase is involved in the migration of human osteosarcoma cells

    Science.gov (United States)

    FENG, SITAN; SHI, XIN; REN, KE; WU, SUJIA; SUN, XIAOLIANG

    2015-01-01

    The aim of the present study was to analyze the expression of focal adhesion kinase (FAK) in osteosarcoma (OS) cell lines with different migration abilities in order to determine the role of FAK in migration. A number of different 143B subclone cell lines (A1, A2, A3, A4 and A5) were obtained by a limiting dilution method, and the expression of FAK was detected using western blot analysis. The role of FAK in the migration of OS cells was investigated using small interfering RNA (siRNA), and the ratio of the number of lamellipodia was compared by immunofluorescence staining. The A2 and A3 OS 143B subclone cell lines demonstrated a stronger migration ability and exhibited higher FAK expression compared with the A1 cell line (P<0.05). Following transfection with FAK-siRNA, the migration ability of the A3 cells was significantly decreased (P<0.05), and the ratio of the number of lamellipodia formed was reduced from 35 to 11% (P<0.05). In conclusion, the level of FAK expression was higher in the cell lines with a stronger migration ability. FAK affects the migration ability of OS cells by suppressing the formation of lamellipodia. PMID:26137126

  17. GFAP isoforms control intermediate filament network dynamics, cell morphology, and focal adhesions.

    Science.gov (United States)

    Moeton, Martina; Stassen, Oscar M J A; Sluijs, Jacqueline A; van der Meer, Vincent W N; Kluivers, Liselot J; van Hoorn, Hedde; Schmidt, Thomas; Reits, Eric A J; van Strien, Miriam E; Hol, Elly M

    2016-11-01

    Glial fibrillary acidic protein (GFAP) is the characteristic intermediate filament (IF) protein in astrocytes. Expression of its main isoforms, GFAPα and GFAPδ, varies in astrocytes and astrocytoma implying a potential regulatory role in astrocyte physiology and pathology. An IF-network is a dynamic structure and has been functionally linked to cell motility, proliferation, and morphology. There is a constant exchange of IF-proteins with the network. To study differences in the dynamic properties of GFAPα and GFAPδ, we performed fluorescence recovery after photobleaching experiments on astrocytoma cells with fluorescently tagged GFAPs. Here, we show for the first time that the exchange of GFP-GFAPδ was significantly slower than the exchange of GFP-GFAPα with the IF-network. Furthermore, a collapsed IF-network, induced by GFAPδ expression, led to a further decrease in fluorescence recovery of both GFP-GFAPα and GFP-GFAPδ. This altered IF-network also changed cell morphology and the focal adhesion size, but did not alter cell migration or proliferation. Our study provides further insight into the modulation of the dynamic properties and functional consequences of the IF-network composition.

  18. Force Fluctuations within Focal Adhesions Mediate ECM-Rigidity Sensing to Guide Directed Cell Migration

    Science.gov (United States)

    Plotnikov, Sergey V.; Pasapera, Ana M.; Sabass, Benedikt; Waterman, Clare M.

    2013-01-01

    Summary Cell migration toward areas of higher extracellular matrix (ECM) rigidity via a process called “durotaxis” is thought to contribute to development, immune response, and cancer metastasis. To understand how cells sample ECM rigidity to guide durotaxis, we characterized cell-generated forces on the nanoscale within single mature integrin-based focal adhesions (FAs). We found that individual FAs act autonomously, exhibiting either stable or dynamically fluctuating (“tugging”) traction. We show that a FAK/phosphopaxillin/vinculin pathway is essential for high FA traction and to enable tugging FA traction over a broad range of ECM rigidities. We show that tugging FA traction is dispensable for FA maturation, chemotaxis, and haptotaxis but is critical to direct cell migration toward rigid ECM. We conclude that individual FAs dynamically sample rigidity by applying fluctuating pulling forces to the ECM to act as sensors to guide durotaxis, and that FAK/phosphopaxillin/vinculin signaling defines the rigidity range over which this dynamic sensing process operates. PMID:23260139

  19. Two distinct actin networks mediate traction oscillations to confer mechanosensitivity of focal adhesions

    Science.gov (United States)

    Wu, Zhanghan; Plotnikov, Sergey; Waterman, Clare; Liu, Jian

    Cells sense the mechanical stiffness of their extracellular matrix (ECM) by exerting traction force through focal adhesions (FAs), which are integrin-based protein assemblies. Strikingly, FA-mediated traction forces oscillate in time and space and govern durotaxis - the tendency of most cell types to migrate toward stiffer ECM. The underlying mechanism of this intriguing oscillation of FA traction force is unknown. Combing theory and experiment, we develop a model of FA growth, which integrates coordinated contributions of a branched actin network and stress fibers in the process. We show that retrograde flux of branched actin network contributes to a traction peak near the FA distal tip and that stress fiber-mediated actomyosin Contractility generates a second traction peak near the FA center. Formin-mediated stress fiber elongation negatively feeds back with actomyosin Contractility, resulting in the central traction peak oscillation. This underpins observed spatio-temporal patterns of the FA traction, and broadens the ECM stiffness range, over which FAs could accurately adapt with traction force generation. Our findings shed light on the fundamental mechanism of FA mechanosensing and hence durotaxis.

  20. Focal Adhesion Kinase-mediated Phosphorylation of Beclin1 Protein Suppresses Cardiomyocyte Autophagy and Initiates Hypertrophic Growth*♦

    Science.gov (United States)

    Cheng, Zhaokang; Zhu, Qiang; Dee, Rachel; Opheim, Zachary; Mack, Christopher P.; Cyr, Douglas M.; Taylor, Joan M.

    2017-01-01

    Autophagy is an evolutionarily conserved intracellular degradation/recycling system that is essential for cellular homeostasis but is dysregulated in a number of diseases, including myocardial hypertrophy. Although it is clear that limiting or accelerating autophagic flux can result in pathological cardiac remodeling, the physiological signaling pathways that fine-tune cardiac autophagy are poorly understood. Herein, we demonstrated that stimulation of cardiomyocytes with phenylephrine (PE), a well known hypertrophic agonist, suppresses autophagy and that activation of focal adhesion kinase (FAK) is necessary for PE-stimulated autophagy suppression and subsequent initiation of hypertrophic growth. Mechanistically, we showed that FAK phosphorylates Beclin1, a core autophagy protein, on Tyr-233 and that this post-translational modification limits Beclin1 association with Atg14L and reduces Beclin1-dependent autophagosome formation. Remarkably, although ectopic expression of wild-type Beclin1 promoted cardiomyocyte atrophy, expression of a Y233E phosphomimetic variant of Beclin1 failed to affect cardiomyocyte size. Moreover, genetic depletion of Beclin1 attenuated PE-mediated/FAK-dependent initiation of myocyte hypertrophy in vivo. Collectively, these findings identify FAK as a novel negative regulator of Beclin1-mediated autophagy and indicate that this pathway can facilitate the promotion of compensatory hypertrophic growth. This novel mechanism to limit Beclin1 activity has important implications for treating a variety of pathologies associated with altered autophagic flux. PMID:27994061

  1. Homozygous mutation of focal adhesion kinase in embryonic stem cell derived neurons: normal electrophysiological and morphological properties in vitro

    Directory of Open Access Journals (Sweden)

    Komiyama NH

    2006-06-01

    Full Text Available Abstract Background Genetically manipulated embryonic stem (ES cell derived neurons (ESNs provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro. Here we report a study of focal adhesion kinase (FAK, which has been implicated in synapse formation and regulation of ion channels, using the ESN system to circumvent the embryonic lethality of homozygous FAK mutant mice. Results Mouse ES cells carrying homozygous null mutations (FAK-/- were generated and differentiated in vitro into neurons. FAK-/- ESNs extended axons and dendrites and formed morphologically and electrophysiologically intact synapses. A detailed study of NMDA receptor gated currents and voltage sensitive calcium currents revealed no difference in their magnitude, or modulation by tyrosine kinases. Conclusion FAK does not have an obligatory role in neuronal differentiation, synapse formation or the expression of NMDA receptor or voltage-gated calcium currents under the conditions used in this study. The use of genetically modified ESNs has great potential for rapidly and effectively examining the consequences of neuronal gene manipulation and is complementary to mouse studies.

  2. Vascular growth responses to chronic arterial occlusion are unaffected by myeloid specific focal adhesion kinase (FAK) deletion

    Science.gov (United States)

    Heuslein, Joshua L.; Murrell, Kelsey P.; Leiphart, Ryan J.; Llewellyn, Ryan A.; Meisner, Joshua K.; Price, Richard J.

    2016-05-01

    Arteriogenesis, or the lumenal expansion of pre-existing arterioles in the presence of an upstream occlusion, is a fundamental vascular growth response. Though alterations in shear stress stimulate arteriogenesis, the migration of monocytes into the perivascular space surrounding collateral arteries and their differentiation into macrophages is critical for this vascular growth response to occur. Focal adhesion kinase’s (FAK) role in regulating cell migration has recently been expanded to primary macrophages. We therefore investigated the effect of the myeloid-specific conditional deletion of FAK on vascular remodeling in the mouse femoral arterial ligation (FAL) model. Using laser Doppler perfusion imaging, whole mount imaging of vascular casted gracilis muscles, and immunostaining for CD31 in gastrocnemius muscles cross-sections, we found that there were no statistical differences in perfusion recovery, arteriogenesis, or angiogenesis 28 days after FAL. We therefore sought to determine FAK expression in different myeloid cell populations. We found that FAK is expressed at equally low levels in Ly6Chi and Ly6Clo blood monocytes, however expression is increased over 2-fold in bone marrow derived macrophages. Ultimately, these results suggest that FAK is not required for monocyte migration to the perivascular space and that vascular remodeling following arterial occlusion occurs independently of myeloid specific FAK.

  3. Inhibition of Focal Adhesion Kinase (FAK) Leads to Abrogation of the Malignant Phenotype in Aggressive Pediatric Renal Malignancies

    Science.gov (United States)

    Megison, Michael L.; Gillory, Lauren A.; Stewart, Jerry E.; Nabers, Hugh C.; Mrozcek-Musulman, Elizabeth; Beierle, Elizabeth A.

    2014-01-01

    Despite the tremendous advances in the treatment of childhood kidney tumors, there remain subsets of pediatric renal tumors that continue to pose a therapeutic challenge, mainly malignant rhabdoid kidney tumors and non-osseous renal Ewing sarcoma. Children with advanced, metastatic or relapsed disease have a disease-free survival rate under 30%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult renal cellular carcinoma, leading us to hypothesize that FAK would be present in pediatric kidney tumors and would impact their cellular survival. In the current study, we showed that FAK was present and phosphorylated in pediatric kidney tumor specimens. We also examined the effects of FAK inhibition upon G401 and SK-NEP-1 cell lines utilizing a number of parallel approaches to block FAK including RNAi and small molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion and migration, and increased apoptosis. Further, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse SK-NEP-1 xenograft model. The findings from this study will help to further our understanding of the regulation of tumorigenesis in rare pediatric renal tumors, and may provide desperately needed novel therapeutic strategies and targets for these rare, but difficult to treat, malignancies. PMID:24464916

  4. Loss of keratinocyte focal adhesion kinase stimulates dermal proteolysis through upregulation of MMP9 in wound healing.

    Science.gov (United States)

    Wong, Victor W; Garg, Ravi K; Sorkin, Michael; Rustad, Kristine C; Akaishi, Satoshi; Levi, Kemal; Nelson, Emily R; Tran, Misha; Rennert, Robert; Liu, Wei; Longaker, Michael T; Dauskardt, Reinhold H; Gurtner, Geoffrey C

    2014-12-01

    To investigate how epithelial mechanotransduction pathways impact wound repair. Mechanical forces are increasingly recognized to influence tissue repair, but their role in chronic wound pathophysiology remains unknown. Studies have shown that chronic wounds exhibit high levels of matrix metalloproteinase 9 (MMP9), a key proteolytic enzyme that regulates wound remodeling. We hypothesized that epithelial mechanosensory pathways regulated by keratinocyte-specific focal adhesion kinase (FAK) control dermal remodeling via MMP9. A standard wound model was applied to keratinocyte-specific FAK knockout (KO) and control mice. Rates of wound healing were measured and tissue was obtained for histologic and molecular analyses. Transcriptional and immunoblot assays were used to assess the activation of FAK, intracellular kinases, and MMP9 in vitro. A cell suspension model was designed to validate the importance of FAK mechanosensing, p38, and MMP9 secretion in human cells. Biomechanical testing was utilized to evaluate matrix tensile properties in FAK KO and control wounds. Wound healing in FAK KO mice was significantly delayed compared with controls (closure at 15 days compared with 20 days, P = 0.0003). FAK KO wounds demonstrated decreased dermal thickness and collagen density. FAK KO keratinocytes exhibited overactive p38 and MMP9 signaling in vitro, findings recapitulated in human keratinocytes via the deactivation of FAK in the cell suspension model. Functionally, FAK KO wounds were significantly weaker and more brittle than control wounds, results consistent with the histologic and molecular analyses. Keratinocyte FAK is highly responsive to mechanical cues and may play a critical role in matrix remodeling via regulation of p38 and MMP9. These findings suggest that aberrant epithelial mechanosensory pathways may contribute to pathologic dermal proteolysis and wound chronicity.

  5. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation

    DEFF Research Database (Denmark)

    Grossi, Alberto; Karlsson, Anders H; Lawson, Moira Ann

    2008-01-01

    reorganization due to the activity of the ubiquitous proteolytic enzymes, calpains, has been reported. Whether there is a link between stretch- or load-induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have...... demonstrated that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m-calpain relocates into focal adhesion complexes and is followed by a breakdown...... of specific focal adhesion proteins previously identified as substrates for this enzyme. We show that stimulation also leads to an increase in calpain activity in these cells. These data support the pivotal role for m-calpain in the control of muscle precursor cell differentiation and thus strengthen the idea...

  6. Activation of focal adhesion kinase enhances the adhesion of Fusarium solani to human corneal epithelial cells via the tyrosine-specific protein kinase signaling pathway.

    Science.gov (United States)

    Pan, Xiaojing; Wang, Ye; Zhou, Qingjun; Chen, Peng; Xu, Yuanyuan; Chen, Hao; Xie, Lixin

    2011-03-05

    To determine the role of the integrin-FAK signaling pathway triggered by the adherence of F. solani to human corneal epithelial cells (HCECs). After pretreatment with/without genistein, HCECs were incubated with F. solani spores at different times (0-24 h). Cell adhesion assays were performed by optical microscopy. Changes of the ultrastructure were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The expression of F-actin and Paxillin (PAX) were detected by immunofluorescence and western blotting to detect the expression of these key proteins with/without genistein treatment. Cell adhesion assays showed that the number of adhered spores began to rise at 6 h after incubation and peaked at 8 h. SEM and TEM showed that the HCECs exhibited a marked morphological alteration induced by the attachment and entry of the spores. The expression of PAX increased, while the expression of F-actin decreased by stimulation with F. solani. The interaction of F. solani with HCECs causes actin rearrangement in HCECs. Genistein strongly inhibited FAK phosphorylation and the activation of the downstream protein (PAX). F. solani-induced enhancement of cell adhesion ability was inhibited along with the inhibition of FAK phosphorylation. Our results suggest that the integrin-FAK signaling pathway is involved in the control of F. solani adhesion to HCECs and that the activation of focal adhesion kinase enhances the adhesion of human corneal epithelial cells to F. solani via the tyrosine-specific protein kinase signaling pathway.

  7. The oncogenic epidermal growth factor receptor variant Xiphophorus melanoma receptor kinase induces motility in melanocytes by modulation of focal adhesions.

    Science.gov (United States)

    Meierjohann, Svenja; Wende, Elisabeth; Kraiss, Anita; Wellbrock, Claudia; Schartl, Manfred

    2006-03-15

    One of the most prominent features of malignant melanoma is the fast generation of metastasizing cells, resulting in the poor prognosis of patients with this tumor type. For this process, cells must gain the ability to migrate. The oncogenic receptor Xmrk (Xiphophorus melanoma receptor kinase) from the Xiphophorus melanoma system is a mutationally activated version of the epidermal growth factor receptor that induces the malignant transformation of pigment cells. Here, we show that the activation of Xmrk leads to a clear increase of pigment cell motility in a fyn-dependent manner. Stimulation of Xmrk induces its interaction with the focal adhesion kinase (FAK) and the interaction of active, receptor-bound fyn with FAK. This results in changes in FAK activity and induces the modulation of stress fibers and focal adhesions. Overexpression of dominant-negative FAK shows that the activity of innate FAK and a receptor-induced focal adhesion turnover are a prerequisite for pigment cell migration. Our findings show that in our system, Xmrk is sufficient for the induction of pigment cell motility and underlines a role of the src family protein tyrosine kinase fyn in melanoma development and progression.

  8. Orientation-specific responses to sustained uniaxial stretching in focal adhesion growth and turnover

    Science.gov (United States)

    Chen, Yun; Pasapera, Ana M.; Koretsky, Alan P.; Waterman, Clare M.

    2013-01-01

    Cells are mechanosensitive to extracellular matrix (ECM) deformation, which can be caused by muscle contraction or changes in hydrostatic pressure. Focal adhesions (FAs) mediate the linkage between the cell and the ECM and initiate mechanically stimulated signaling events. We developed a stretching apparatus in which cells grown on fibronectin-coated elastic substrates can be stretched and imaged live to study how FAs dynamically respond to ECM deformation. Human bone osteosarcoma epithelial cell line U2OS was transfected with GFP-paxillin as an FA marker and subjected to sustained uniaxial stretching. Two responses at different timescales were observed: rapid FA growth within seconds after stretching, and delayed FA disassembly and loss of cell polarity that occurred over tens of minutes. Rapid FA growth occurred in all cells; however, delayed responses to stretch occurred in an orientation-specific manner, specifically in cells with their long axes perpendicular to the stretching direction, but not in cells with their long axes parallel to stretch. Pharmacological treatments demonstrated that FA kinase (FAK) promotes but Src inhibits rapid FA growth, whereas FAK, Src, and calpain 2 all contribute to delayed FA disassembly and loss of polarity in cells perpendicular to stretching. Immunostaining for phospho-FAK after stretching revealed that FAK activation was maximal at 5 s after stretching, specifically in FAs oriented perpendicular to stretch. We hypothesize that orientation-specific activation of strain/stress-sensitive proteins in FAs upstream to FAK and Src promote orientation-specific responses in FA growth and disassembly that mediate polarity rearrangement in response to sustained stretch. PMID:23754369

  9. Two Distinct Actin Networks Mediate Traction Oscillations to Confer Focal Adhesion Mechanosensing.

    Science.gov (United States)

    Wu, Zhanghan; Plotnikov, Sergey V; Moalim, Abdiwahab Y; Waterman, Clare M; Liu, Jian

    2017-02-28

    Focal adhesions (FAs) are integrin-based transmembrane assemblies that connect a cell to its extracellular matrix (ECM). They are mechanosensors through which cells exert actin cytoskeleton-mediated traction forces to sense the ECM stiffness. Interestingly, FAs themselves are dynamic structures that adapt their growth in response to mechanical force. It is unclear how the cell manages the plasticity of the FA structure and the associated traction force to accurately sense ECM stiffness. Strikingly, FA traction forces oscillate in time and space, and govern the cell mechanosensing of ECM stiffness. However, precisely how and why the FA traction oscillates is unknown. We developed a model of FA growth that integrates the contributions of the branched actin network and stress fibers (SFs). Using the model in combination with experimental tests, we show that the retrograde flux of the branched actin network promotes the proximal growth of the FA and contributes to a traction peak near the FA's distal tip. The resulting traction gradient within the growing FA favors SF formation near the FA's proximal end. The SF-mediated actomyosin contractility further stabilizes the FA and generates a second traction peak near the center of the FA. Formin-mediated SF elongation negatively feeds back with actomyosin contractility, resulting in central traction peak oscillation. This underpins the observed FA traction oscillation and, importantly, broadens the ECM stiffness range over which FAs can accurately adapt to traction force generation. Actin cytoskeleton-mediated FA growth and maturation thus culminate with FA traction oscillation to drive efficient FA mechanosensing. Published by Elsevier Inc.

  10. Doxycycline inhibits leukemic cell migration via inhibition of matrix metalloproteinases and phosphorylation of focal adhesion kinase.

    Science.gov (United States)

    Wang, Chunhuai; Xiang, Ru; Zhang, Xiangzhong; Chen, Yunxian

    2015-09-01

    Doxycycline, a tetracycline-based antibiotic, has been reported to attenuate melanoma cell migration through inhibiting the focal adhesion kinase (FAK) signaling pathway. However, it remains to be elucidated whether doxycycline exerts this effect on leukemia cell migration. The present study aimed to examine the role of doxycycline in leukemia cell migration. The invasion capacities of the human leukemia cell lines KG1a (acute myelogenous leukemia) and K562 (chronic myelogenous leukemia) were evaluated using Matrigel® matrix‑coated Transwell® chamber assays; leukemic cell lines treated with doxycycline (1 µg/ml) or anti‑β1‑integrin antibodies were added to the upper chamber, while untreated cells were included as controls. Reverse transcription quantitative polymerase chain reaction was performed in order to further understand the influence of doxycycline treatment on the expression of FAK and gelatinases in the KG1a and K562 leukemic cell lines. In addition, FAK protein expression and phosphorylation were determined using western blot analysis in order to investigate the mechanism by which doxycycline inhibited leukemic cell migration. The results revealed that doxycycline treatment significantly attenuated the migration of KG1a and K562 cells, which was demonstrated to be associated with inhibition of the expression and phosphorylation of FAK. In addition, doxycycline treatment inhibited matrix metalloproteinase (MMP)‑2 and MMP‑9 expression. Furthermore, incubation with blocking anti‑β1‑integrin antibodies had an analogous inhibitory effect on leukemic cell migration to that of doxycycline. In conclusion, the results of the present study suggested that doxycycline attenuated leukemic cell migration through inhibiting the FAK signaling pathway. Therefore, doxycycline may have potential for use as a novel strategy for the treatment of leukemia.

  11. Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

    Science.gov (United States)

    Dormond, Olivier; Ponsonnet, Lionel; Hasmim, Meriem; Foletti, Alessandro; Rüegg, Curzio

    2004-07-01

    Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

  12. Progesterone promotes focal adhesion formation and migration in breast cancer cells through induction of protease-activated receptor-1.

    Science.gov (United States)

    Diaz, Jorge; Aranda, Evelyn; Henriquez, Soledad; Quezada, Marisol; Espinoza, Estefanía; Bravo, Maria Loreto; Oliva, Bárbara; Lange, Soledad; Villalon, Manuel; Jones, Marius; Brosens, Jan J; Kato, Sumie; Cuello, Mauricio A; Knutson, Todd P; Lange, Carol A; Leyton, Lisette; Owen, Gareth I

    2012-08-01

    Progesterone and progestins have been demonstrated to enhance breast cancer cell migration, although the mechanisms are still not fully understood. The protease-activated receptors (PARs) are a family of membrane receptors that are activated by serine proteases in the blood coagulation cascade. PAR1 (F2R) has been reported to be involved in cancer cell migration and overexpressed in breast cancer. We herein demonstrate that PAR1 mRNA and protein are upregulated by progesterone treatment of the breast cancer cell lines ZR-75 and T47D. This regulation is dependent on the progesterone receptor (PR) but does not require PR phosphorylation at serine 294 or the PR proline-rich region mPRO. The increase in PAR1 mRNA was transient, being present at 3  h and returning to basal levels at 18  h. The addition of a PAR1-activating peptide (aPAR1) to cells treated with progesterone resulted in an increase in focal adhesion (FA) formation as measured by the cellular levels of phosphorylated FA kinase. The combined but not individual treatment of progesterone and aPAR1 also markedly increased stress fiber formation and the migratory capacity of breast cancer cells. In agreement with in vitro findings, data mining from the Oncomine platform revealed that PAR1 expression was significantly upregulated in PR-positive breast tumors. Our observation that PAR1 expression and signal transduction are modulated by progesterone provides new insight into how the progestin component in hormone therapies increases the risk of breast cancer in postmenopausal women.

  13. Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain.

    Science.gov (United States)

    Kintner, C

    1992-04-17

    Differential adhesion between embryonic cells has been proposed to be mediated by a family of closely related glycoproteins called the cadherins. The cadherins mediate adhesion in part through an interaction between the cadherin cytoplasmic domain and intracellular proteins, called the catenins. To determine whether these interactions could regulate cadherin function in embryos, a form of N-cadherin was generated that lacks an extracellular domain. Expression of this mutant in Xenopus embryos causes a dramatic inhibition of cell adhesion. Analysis of the mutant phenotype shows that at least two regions of the N-cadherin cytoplasmic domain can inhibit adhesion and that the mutant cadherin can inhibit catenin binding to E-cadherin. These results suggest that cadherin-mediated adhesion can be regulated by cytoplasmic interactions and that this regulation may contribute to morphogenesis when emerging tissues coexpress several cadherin types.

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

    Science.gov (United States)

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

    2015-10-01

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

  15. Regulation of cadherin-mediated adhesion in morphogenesis.

    Science.gov (United States)

    Gumbiner, Barry M

    2005-08-01

    Cadherin cell-adhesion proteins mediate many facets of tissue morphogenesis. The dynamic regulation of cadherins in response to various extracellular signals controls cell sorting, cell rearrangements and cell movements. Cadherins are regulated at the cell surface by an inside-out signalling mechanism that is analogous to the integrins in platelets and leukocytes. Signal-transduction pathways impinge on the catenins (cytoplasmic cadherin-associated proteins), which transduce changes across the membrane to alter the state of the cadherin adhesive bond.

  16. Effect of bioactive extruded PLA/HA composite films on focal adhesion formation of preosteoblastic cells.

    Science.gov (United States)

    Persson, Maria; Lorite, Gabriela S; Kokkonen, Hanna E; Cho, Sung-Woo; Lehenkari, Petri P; Skrifvars, Mikael; Tuukkanen, Juha

    2014-09-01

    The quality of the initial cell attachment to a biomaterial will influence any further cell function, including spreading, proliferation, differentiation and viability. Cell attachment is influenced by the material's ability to adsorb proteins, which is related to the surface chemistry and topography of the material. In this study, we incorporated hydroxyapatite (HA) particles into a poly(lactic acid) (PLA) composite and evaluated the surface structure and the effects of HA density on the initial cell attachment in vitro of murine calvarial preosteoblasts (MC3T3-EI). Scanning electron microscopy (SEM), atomic force microscopy (AFM) and infrared spectroscopy (FTIR) showed that the HA particles were successfully incorporated into the PLA matrix and located at the surface which is of importance in order to maintain the bioactive effect of the HA particles. SEM and AFM investigation revealed that the HA density (particles/area) as well as surface roughness increased with HA loading concentration (i.e. 5, 10, 15 and 20wt%), which promoted protein adsorption. Furthermore, the presence of HA on the surface enhanced cell spreading, increased the formation of actin stress fibers and significantly improved the expression of vinculin in MC3T3-E1 cells which is a key player in the regulation of cell adhesion. These results suggest the potential utility of PLA/HA composites as biomaterials for use as a bone substitute material and in tissue engineering applications.

  17. Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase C alpha (PKC alpha) increases focal adhesion localization of PKC alpha

    DEFF Research Database (Denmark)

    Lim, Ssang-Taek; Longley, Robert L; Couchman, John R

    2003-01-01

    Syndecan-4 is a transmembrane heparan sulfate proteoglycan that acts as a coreceptor with integrins in focal adhesion formation. The central region of syndecan-4 cytoplasmic domain (4V; LGKKPIYKK) binds phosphatidylinositol 4,5-bisphosphate, and together they regulate protein kinase C alpha (PKC...... alpha) activity. Syndecan 4V peptide directly potentiates PKC alpha activity, leading to "superactivation" of the enzyme, apparently through an interaction with its catalytic domain. We now have performed yeast two-hybrid and in vitro binding assays to determine the interaction sites between 4V and PKC...... alpha. Full-length PKC alpha weakly interacted with 4V by yeast two-hybrid assays, but PKC alpha constructs that lack the pseudosubstrate region or constructs of the whole catalytic domain interacted more strongly. A mutated 4V sequence (4V(YF): LGKKPIFKK) did not interact with PKC alpha, indicating...

  18. Cellular adhesome screen identifies critical modulators of focal adhesion dynamics, cellular traction forces and cell migration behaviour.

    Science.gov (United States)

    Fokkelman, Michiel; Balcıoğlu, Hayri E; Klip, Janna E; Yan, Kuan; Verbeek, Fons J; Danen, Erik H J; van de Water, Bob

    2016-08-17

    Cancer cells migrate from the primary tumour into surrounding tissue in order to form metastasis. Cell migration is a highly complex process, which requires continuous remodelling and re-organization of the cytoskeleton and cell-matrix adhesions. Here, we aimed to identify genes controlling aspects of tumour cell migration, including the dynamic organization of cell-matrix adhesions and cellular traction forces. In a siRNA screen targeting most cell adhesion-related genes we identified 200+ genes that regulate size and/or dynamics of cell-matrix adhesions in MCF7 breast cancer cells. In a subsequent secondary screen, the 64 most effective genes were evaluated for growth factor-induced cell migration and validated by tertiary RNAi pool deconvolution experiments. Four validated hits showed significantly enlarged adhesions accompanied by reduced cell migration upon siRNA-mediated knockdown. Furthermore, loss of PPP1R12B, HIPK3 or RAC2 caused cells to exert higher traction forces, as determined by traction force microscopy with elastomeric micropillar post arrays, and led to considerably reduced force turnover. Altogether, we identified genes that co-regulate cell-matrix adhesion dynamics and traction force turnover, thereby modulating overall motility behaviour.

  19. Resveratrol and Estradiol Exert Disparate Effects on Cell Migration, Cell Surface Actin Structures, and Focal Adhesion Assembly in MDA-MB-231 Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Nicolas G. Azios

    2005-02-01

    Full Text Available Resveratrol, a grape polyphenol, is thought to be a cancer preventive, yet its effects on metastatic breast cancer are relatively unknown. Since cancer cell invasion is dependent on cell migration, the chemotactic response of MDA-MB-231 metastatic human breast cancer cells to resveratrol, estradiol (E2, or epidermal growth factor (EGF was investigated. Resveratrol decreased while E2 and EGF increased directed cell migration. Resveratrol may inhibit cell migration by altering the cytoskeleton. Resveratrol induced a rapid global array of filopodia and decreased focal adhesions and focal adhesion kinase (FAK activity. E2 or EGF treatment did not affect filopodia extension but increased lamellipodia and associated focal adhesions that are integral for cell migration. Combined resveratrol and E2 treatment resulted in a filopodia and focal adhesion response similar to resveratrol alone. Combined resveratrol and EGF resulted in a lamellipodia and focal adhesion response similar to EGF alone. E2 and to a lesser extent resveratrol increased EGFR activity. The cytoskeletal changes and EGFR activity in response to E2 were blocked by EGFR1 inhibitor indicating that E2 may increase cell migration via crosstalk with EGFR signaling. These data suggest a promotional role for E2 in breast cancer cell migration but an antiestrogenic, preventative role for resveratrol.

  20. Glycogen synthase kinase 3β dictates podocyte motility and focal adhesion turnover by modulating paxillin activity: implications for the protective effect of low-dose lithium in podocytopathy.

    Science.gov (United States)

    Xu, Weiwei; Ge, Yan; Liu, Zhihong; Gong, Rujun

    2014-10-01

    Aberrant focal adhesion turnover is centrally involved in podocyte actin cytoskeleton disorganization and foot process effacement. The structural and dynamic integrity of focal adhesions is orchestrated by multiple cell signaling molecules, including glycogen synthase kinase 3β (GSK3β), a multitasking kinase lately identified as a mediator of kidney injury. However, the role of GSK3β in podocytopathy remains obscure. In doxorubicin (Adriamycin)-injured podocytes, lithium, a GSK3β inhibitor and neuroprotective mood stabilizer, obliterated the accelerated focal adhesion turnover, rectified podocyte hypermotility, and restored actin cytoskeleton integrity. Mechanistically, lithium counteracted the doxorubicin-elicited GSK3β overactivity and the hyperphosphorylation and overactivation of paxillin, a focal adhesion-associated adaptor protein. Moreover, forced expression of a dominant negative kinase dead mutant of GSK3β highly mimicked, whereas ectopic expression of a constitutively active GSK3β mutant abolished, the effect of lithium in doxorubicin-injured podocytes, suggesting that the effect of lithium is mediated, at least in part, through inhibition of GSK3β. Furthermore, paxillin interacted with GSK3β and served as its substrate. In mice with doxorubicin nephropathy, a single low dose of lithium ameliorated proteinuria and glomerulosclerosis. Consistently, lithium therapy abrogated GSK3β overactivity, blunted paxillin hyperphosphorylation, and reinstated actin cytoskeleton integrity in glomeruli associated with an early attenuation of podocyte foot process effacement. Thus, GSK3β-modulated focal adhesion dynamics might serve as a novel therapeutic target for podocytopathy.

  1. Fluid-flow-induced mesenchymal stem cell migration: role of focal adhesion kinase and RhoA kinase sensors.

    Science.gov (United States)

    Riehl, Brandon D; Lee, Jeong Soon; Ha, Ligyeom; Lim, Jung Yul

    2015-03-01

    The study of mesenchymal stem cell (MSC) migration under flow conditions with investigation of the underlying molecular mechanism could lead to a better understanding and outcome in stem-cell-based cell therapy and regenerative medicine. We used peer-reviewed open source software to develop methods for efficiently and accurately tracking, measuring and processing cell migration as well as morphology. Using these tools, we investigated MSC migration under flow-induced shear and tested the molecular mechanism with stable knockdown of focal adhesion kinase (FAK) and RhoA kinase (ROCK). Under steady flow, MSCs migrated following the flow direction in a shear stress magnitude-dependent manner, as assessed by root mean square displacement and mean square displacement, motility coefficient and confinement ratio. Silencing FAK in MSCs suppressed morphology adaptation capability and reduced cellular motility for both static and flow conditions. Interestingly, ROCK silencing significantly increased migration tendency especially under flow. Blocking ROCK, which is known to reduce cytoskeletal tension, may lower the resistance to skeletal remodelling during the flow-induced migration. Our data thus propose a potentially differential role of focal adhesion and cytoskeletal tension signalling elements in MSC migration under flow shear.

  2. Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocyte activation by the 29-kDa amino-terminal fibronectin fragment.

    Science.gov (United States)

    Gemba, Takefumi; Valbracht, Jean; Alsalameh, Saifeddin; Lotz, Martin

    2002-01-11

    The 29-kDa amino-terminal fibronectin fragment (FN-f) has a potent chondrolytic effect and is thought to be involved in cartilage degradation in arthritis. However, little is known about signal transduction pathways that are activated by FN-f. Here we demonstrated that FN-f induced nitric oxide (NO) production from human articular chondrocytes. Expression of inducible nitric-oxide synthase (iNOS) mRNA and NO production were observed at 6 and 48 h after FN-f treatment, respectively. Interleukin-1beta (IL-1beta) mRNA up-regulation was stimulated by FN-f in human chondrocytes. To address the possibility that FN-f-induced NO release is mediated by IL-1beta production, the effect of IL-1 receptor antagonist (IL-1ra) was determined. IL-1ra partially inhibited FN-f-induced NO release although it almost completely inhibited IL-1beta-induced NO release. Tyrosine phosphorylation of focal adhesion kinase was induced transiently by FN-f treatment. Blocking antibodies to alpha(5) or beta(1) integrin and Arg-Gly-Asp-containing peptides did not inhibit FN-f-induced NO production. PP2, a Src family kinase inhibitor, or cytochalasin D, which selectively disrupts the network of actin filaments, inhibited both FAK phosphorylation and NO production induced by FN-f, but the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect. Analysis of mitogen-activated protein kinases (MAPK) showed activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 MAPK. High concentrations of SB203580, which inhibit both JNK and p38 MAPK, and PD98059 a selective inhibitor of MEK1/2 that blocks ERK activation, inhibited FN-f induced NO production. These data suggest that focal adhesion kinase and MAPK mediate FN-f induced activation of human articular chondrocytes.

  3. Therapeutic effects of tyroservatide on metastasis of lung cancer and its mechanism affecting integrin–focal adhesion kinase signal transduction

    Science.gov (United States)

    Huang, Yu-ting; Zhao, Lan; Fu, Zheng; Zhao, Meng; Song, Xiao-meng; Jia, Jing; Wang, Song; Li, Jin-ping; Zhu, Zhi-feng; Lin, Gang; Lu, Rong; Yao, Zhi

    2016-01-01

    Tyroservatide (YSV) can inhibit the growth and metastasis of mouse lung cancer significantly. This study investigated the therapeutic effects of tripeptide YSV on metastasis of human lung cancer cells and explored its possible mechanism that affects integrin–focal adhesion kinase (FAK) signal transduction in tumor cells. YSV significantly inhibited the adhesion and the invasion of highly metastatic human lung cancer cell lines 95D, A549, and NCI-H1299. In addition, YSV significantly inhibited phosphorylation of FAK Tyr397 and FAK Tyr576/577 in the 95D, A549, and NCI-H1299 human lung cancer cells in vitro. And the mRNA level and protein expression of FAK in these human lung cancer cells decreased at the same time. YSV also significantly inhibited mRNA and protein levels of integrin β1 and integrin β3 in the 95D, A549, and NCI-H1299 human lung cancer cells. Our research showed that YSV inhibited adhesion and invasion of human lung cancer cells and exhibited therapeutic effects on metastasis of lung cancer. PMID:27041993

  4. The human papillomavirus E6 oncogene represses a cell adhesion pathway and disrupts focal adhesion through degradation of TAp63β upon transformation.

    Directory of Open Access Journals (Sweden)

    Youcef Ben Khalifa

    2011-09-01

    Full Text Available Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63β isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63β is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and β. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63β although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63β in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63β is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63β therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63β in anchorage independent growth that might represent a new critical

  5. Focal nodular hyperplasia of the liver: composition of the extracellular matrix and expression of cell-cell and cell-matrix adhesion molecules.

    Science.gov (United States)

    Scoazec, J Y; Flejou, J F; D'Errico, A; Couvelard, A; Kozyraki, R; Fiorentino, M; Grigioni, W F; Feldmann, G

    1995-10-01

    We studied by immunohistochemistry 25 cases of focal nodular hyperplasia (FNH) to evaluate the composition of the extracellular matrix and the expression and distribution of endothelial cell-cell adhesion molecules and integrin receptors. The extracellular matrix of FNH retained the overall organization of that of normal liver. The matrix of central scars resembled that of portal tracts. The main difference was the presence of large vitronectin deposits, which might indicate the existence of local hemodynamic disturbances. The matrix lining the sinusoid-like vessels running in the hyperplastic parenchyma retained characteristic features of the normal perisinusoidal matrix, such as the presence of tenascin. In the zone surrounding the central scars, it contained large amounts of laminin, von Willebrand factor, and thrombospondin, suggesting the development of perisinusoidal fibrosis. Laminin deposition was accompanied by the induction of cell-cell adhesion molecules on adjacent endothelial cells and by the up-regulation of specific integrin receptors on both hepatocytes and sinusoidal endothelial cells. In conclusion, our study: (1) reinforces the hypothesis that FNH is merely a hyperplastic response of liver parenchyma to local vascular abnormalities, and (2) shows that the lesions of perisinusoidal fibrosis associated with FNH are accompanied by the induction of integrin receptors on hepatocytes and sinusoidal endothelial cells.

  6. Regulation of embryonic cell adhesion by the prion protein.

    Directory of Open Access Journals (Sweden)

    Edward Málaga-Trillo

    2009-03-01

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

  7. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    Directory of Open Access Journals (Sweden)

    Donejko M

    2017-03-01

    Full Text Available Magdalena Donejko,1 Edyta Rysiak,2 Elżbieta Galicka,1 Robert Terlikowski,3 Edyta Katarzyna Głażewska,1 Andrzej Przylipiak1 1Department of Esthetic Medicine, 2Department of Medicinal Chemistry, Faculty of Pharmacy, 3Department of Health Restoration, Medical University of Białystok, Białystok, Poland Aim: The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK, and the influence of HA on those processes. Materials and methods: Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results: Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion: This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. Keywords: apoptosis, skin fibroblast, focal adhesion kinase, hyaluronic acid, ethanol

  8. Heterogeneous response of traction force at focal adhesions of vascular smooth muscle cells subjected to macroscopic stretch on a micropillar substrate.

    Science.gov (United States)

    Nagayama, Kazuaki; Adachi, Akifumi; Matsumoto, Takeo

    2011-10-13

    Traction force generated at focal adhesions (FAs) of cells plays an essential role in regulating cellular functions. However, little is known about how the traction force at each FA changes during cell stretching. Here we investigated dynamic changes in traction force at FAs during macroscopic stretching of porcine aortic smooth muscle cells (SMCs) cultured on elastic micropillar substrates. SMCs were cultured on polydimethylsiloxane (PDMS)-based substrates with a micropillar array, and stretched approximately in the direction of their major axis and then released by stretching and relaxing the substrates. This stretch-release cycle was repeated twice with cell strain rates of 0.3%/15s up to a 3% strain, and the deflection of the PDMS micropillars was measured simultaneously to obtain the traction force at each FA F, total force in the cell's major axis direction F(all), and whole-cell strain ε(cell). Traction forces of SMCs during stretching varied widely with location: their changes at some pillars synchronized well with the applied strain ε(cell), but others did not synchronized. Whole-cell stiffness estimated as the slope of the loading limb of the F(all)-ε(cell) curves was ∼10nN/%, which was the same order of magnitude of the reported stiffness of cultured SMCs obtained in a tensile test. Interestingly, F(all) at a zero-strain state (pretension at the whole-cell level) actively increased in some cells following the loading/unloading process, as did whole-cell stiffness. Such a change did not occur in cultured SMCs in the tensile test in which cells were held with a pair of micropipettes coated with nonspecific adhesive. These results indicate that SMCs showed a myogenic response when stretched through their multiple FAs, but not through nonspecific adhesions on their membrane. SMCs may behave differently depending on the sites through which they are stretched. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Focal Adhesion Kinase-Dependent Role of the Soluble Form of Neurotensin Receptor-3/Sortilin in Colorectal Cancer Cell Dissociation

    Directory of Open Access Journals (Sweden)

    Sophie Béraud-Dufour

    2016-11-01

    Full Text Available The aim of the present review is to unravel the mechanisms of action of the soluble form of the neurotensin (NT receptor-3 (NTSR3, also called Sortilin, in numerous physiopathological processes including cancer development, cardiovascular diseases and depression. Sortilin/NTSR3 is a transmembrane protein thought to exert multiple functions both intracellularly and at the level of the plasma membrane. The Sortilin/NTSR3 extracellular domain is released by shedding from all the cells expressing the protein. Although the existence of the soluble form of Sortilin/NTSR3 (sSortilin/NTSR3 has been evidenced for more than 10 years, the studies focusing on the role of this soluble protein at the mechanistic level remain rare. Numerous cancer cells, including colonic cancer cells, express the receptor family of neurotensin (NT, and particularly Sortilin/NTSR3. This review aims to summarize the functional role of sSortilin/NTSR3 characterized in the colonic cancer cell line HT29. This includes mechanisms involving signaling cascades through focal adhesion kinase (FAK, a key pathway leading to the weakening of cell–cell and cell–extracellular matrix adhesions, a series of events which could be responsible for cancer metastasis. Finally, some future approaches targeting the release of sNTSR3 through the inhibition of matrix metalloproteases (MMPs are suggested.

  10. Focal Adhesion Kinase-Dependent Role of the Soluble Form of Neurotensin Receptor-3/Sortilin in Colorectal Cancer Cell Dissociation

    Science.gov (United States)

    Béraud-Dufour, Sophie; Devader, Christelle; Massa, Fabienne; Roulot, Morgane; Coppola, Thierry; Mazella, Jean

    2016-01-01

    The aim of the present review is to unravel the mechanisms of action of the soluble form of the neurotensin (NT) receptor-3 (NTSR3), also called Sortilin, in numerous physiopathological processes including cancer development, cardiovascular diseases and depression. Sortilin/NTSR3 is a transmembrane protein thought to exert multiple functions both intracellularly and at the level of the plasma membrane. The Sortilin/NTSR3 extracellular domain is released by shedding from all the cells expressing the protein. Although the existence of the soluble form of Sortilin/NTSR3 (sSortilin/NTSR3) has been evidenced for more than 10 years, the studies focusing on the role of this soluble protein at the mechanistic level remain rare. Numerous cancer cells, including colonic cancer cells, express the receptor family of neurotensin (NT), and particularly Sortilin/NTSR3. This review aims to summarize the functional role of sSortilin/NTSR3 characterized in the colonic cancer cell line HT29. This includes mechanisms involving signaling cascades through focal adhesion kinase (FAK), a key pathway leading to the weakening of cell–cell and cell–extracellular matrix adhesions, a series of events which could be responsible for cancer metastasis. Finally, some future approaches targeting the release of sNTSR3 through the inhibition of matrix metalloproteases (MMPs) are suggested. PMID:27834811

  11. Hydrogen peroxide regulates cell adhesion through the redox sensor RPSA.

    Science.gov (United States)

    Vilas-Boas, Filipe; Bagulho, Ana; Tenente, Rita; Teixeira, Vitor H; Martins, Gabriel; da Costa, Gonçalo; Jerónimo, Ana; Cordeiro, Carlos; Machuqueiro, Miguel; Real, Carla

    2016-01-01

    To become metastatic, a tumor cell must acquire new adhesion properties that allow migration into the surrounding connective tissue, transmigration across endothelial cells to reach the blood stream and, at the site of metastasis, adhesion to endothelial cells and transmigration to colonize a new tissue. Hydrogen peroxide (H2O2) is a redox signaling molecule produced in tumor cell microenvironment with high relevance for tumor development. However, the molecular mechanisms regulated by H2O2 in tumor cells are still poorly known. The identification of H2O2-target proteins in tumor cells and the understanding of their role in tumor cell adhesion are essential for the development of novel redox-based therapies for cancer. In this paper, we identified Ribosomal Protein SA (RPSA) as a target of H2O2 and showed that RPSA in the oxidized state accumulates in clusters that contain specific adhesion molecules. Furthermore, we showed that RPSA oxidation improves cell adhesion efficiency to laminin in vitro and promotes cell extravasation in vivo. Our results unravel a new mechanism for H2O2-dependent modulation of cell adhesion properties and identify RPSA as the H2O2 sensor in this process. This work indicates that high levels of RPSA expression might confer a selective advantage to tumor cells in an oxidative environment.

  12. E-cadherin-dependent stimulation of traction force at focal adhesions via the Src and PI3K signaling pathways.

    Science.gov (United States)

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

    2012-07-18

    The interplay between cadherin- and integrin-dependent signals controls cell behavior, but the precise mechanisms that regulate the strength of adhesion to the extracellular matrix remains poorly understood. We deposited cells expressing a defined repertoire of cadherins and integrins on fibronectin (FN)-coated polyacrylamide gels (FN-PAG) and on FN-coated pillars used as a micro-force sensor array (μFSA), and analyzed the functional relationship between these adhesion receptors to determine how it regulates cell traction force. We found that cadherin-mediated adhesion stimulated cell spreading on FN-PAG, and this was modulated by the substrate stiffness. We compared S180 cells with cells stably expressing different cadherins on μFSA and found that traction forces were stronger in cells expressing cadherins than in parental cells. E-cadherin-mediated contact and mechanical coupling between cells are required for this increase in cell-FN traction force, which was not observed in isolated cells, and required Src and PI3K activities. Traction forces were stronger in cells expressing type I cadherins than in cells expressing type II cadherins, which correlates with our previous observation of a higher intercellular adhesion strength developed by type I compared with type II cadherins. Our results reveal one of the mechanisms whereby molecular cross talk between cadherins and integrins upregulates traction forces at cell-FN adhesion sites, and thus provide additional insight into the molecular control of cell behavior. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. Structural and functional assessment of the interaction between focal adhesion kinase and sarcomeric myosin

    OpenAIRE

    Aline Mara dos Santos

    2011-01-01

    Resumo: A tirosino-quinase de adesão focal (FAK) tem papel crítico na mediação da migração, sobrevivência e proliferação celular. Estudos anteriores de nosso laboratório demonstraram que a FAK é ativada pelo estresse mecânico em miócitos cardíacos e que ela se coimunoprecipita com a miosina sarcomérica. No presente trabalho foi demonstrado que o domínio FERM da FAK medeia à interação com a miosina sarcomérica, sendo que esta interação leva a inibição da autofosforilação da FAK, enquanto que a...

  14. Focal DNA copy number changes in neuroblastoma target MYCN regulated genes.

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    Candy Kumps

    Full Text Available Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17~92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17~92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17~92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1 target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2 serve as a resource for identifying new molecular targets for treatment.

  15. PRL-3 engages the focal adhesion pathway in triple-negative breast cancer cells to alter actin structure and substrate adhesion properties critical for cell migration and invasion.

    Science.gov (United States)

    Gari, Hamid H; DeGala, Gregory D; Ray, Rahul; Lucia, M Scott; Lambert, James R

    2016-10-01

    Triple-negative breast cancers (TNBCs) are among the most aggressive cancers characterized by a high propensity to invade, metastasize and relapse. We previously reported that the TNBC-specific inhibitor, AMPI-109, significantly impairs the ability of TNBC cells to migrate and invade by reducing levels of the metastasis-promoting phosphatase, PRL-3. Here, we examined the mechanisms by which AMPI-109 and loss of PRL-3 impede cell migration and invasion. AMPI-109 treatment or knock down of PRL-3 expression were associated with deactivation of Src and ERK signaling and concomitant downregulation of RhoA and Rac1/2/3 GTPase protein levels. These cellular changes led to rearranged filamentous actin networks necessary for cell migration and invasion. Conversely, overexpression of PRL-3 promoted TNBC cell invasion by upregulating matrix metalloproteinase 10, which resulted in increased TNBC cell adherence to, and degradation of, the major basement membrane component laminin. Our data demonstrate that PRL-3 engages the focal adhesion pathway in TNBC cells as a key mechanism for promoting TNBC cell migration and invasion. Collectively, these data suggest that blocking PRL-3 activity may be an effective method for reducing the metastatic potential of TNBC cells.

  16. Epac Activation Regulates Human Mesenchymal Stem Cells Migration and Adhesion.

    Science.gov (United States)

    Yu, Jiao-Le; Deng, Ruixia; Chung, Sookja K; Chan, Godfrey Chi-Fung

    2016-04-01

    How to enhance the homing of human mesenchymal stem cells (hMSCs) to the target tissues remains a clinical challenge nowadays. To overcome this barrier, the mechanism responsible for the hMSCs migration and engraftment has to be defined. Currently, the exact mechanism involved in migration and adhesion of hMSCs remains unknown. Exchange protein directly activated by cAMP (Epac), a novel protein discovered in cAMP signaling pathway, may have a potential role in regulating cells adhesion and migration by triggering the downstream Rap family signaling cascades. However, the exact role of Epac in cells homing is elusive. Our study evaluated the role of Epac in the homing of hMSCs. We confirmed that hMSCs expressed functional Epac and its activation enhanced the migration and adhesion of hMSCs significantly. The Epac activation was further found to be contributed directly to the chemotactic responses induced by stromal cell derived factor-1 (SDF-1) which is a known chemokine in regulating hMSCs homing. These findings suggested Epac is connected to the SDF-1 signaling cascades. In conclusion, our study revealed that Epac plays a role in hMSCs homing by promoting adhesion and migration. Appropriate manipulation of Epac may enhance the homing of hMSCs and facilitate their future clinical applications.

  17. Migration of periodontal ligament fibroblasts on nanometric topographical patterns: influence of filopodia and focal adhesions on contact guidance.

    Directory of Open Access Journals (Sweden)

    Douglas W Hamilton

    Full Text Available Considered to be the "holy grail" of dentistry, regeneration of the periodontal ligament in humans remains a major clinical problem. Removal of bacterial biofilms is commonly achieved using EDTA gels or lasers. One side effect of these treatment regimens is the etching of nanotopographies on the surface of the tooth. However, the response of periodontal ligament fibroblasts to such features has received very little attention. Using laser interference lithography, we fabricated precisely defined topographies with continuous or discontinuous nanogrooves to assess the adhesion, spreading and migration of PDL fibroblasts. PDL fibroblasts adhered to and spread on all tested surfaces, with initial spreading and focal adhesion formation slower on discontinuous nanogrooves. Cells had a significantly smaller planar area on both continuous and discontinuous nanogrooves in comparison with cells on non-patterned controls. At 24 h post seeding, cells on both types of nanogrooves were highly elongated parallel to the groove long axis. Time-lapse video microscopy revealed that PDL fibroblast movement was guided on both types of grooves, but migration velocity was not significantly different from cells cultured on non-patterned controls. Analysis of filopodia formation using time-lapse video microscopy and labeling of vinculin and F-actin revealed that on nanogrooves, filopodia were highly aligned at both ends of the cell, but with increasing time filopodia and membrane protrusions developed at the side of the cell perpendicular to the cell long axis. We conclude that periodontal ligament fibroblasts are sensitive to nanotopographical depths of 85-100 µm, which could be utilized in regeneration of the periodontal ligament.

  18. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    Science.gov (United States)

    Donejko, Magdalena; Rysiak, Edyta; Galicka, Elżbieta; Terlikowski, Robert; Głażewska, Edyta Katarzyna; Przylipiak, Andrzej

    2017-01-01

    Aim The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes. Materials and methods Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. PMID:28293103

  19. Three-dimensional cell body shape dictates the onset of traction force generation and growth of focal adhesions.

    Science.gov (United States)

    Fouchard, Jonathan; Bimbard, Célian; Bufi, Nathalie; Durand-Smet, Pauline; Proag, Amsha; Richert, Alain; Cardoso, Olivier; Asnacios, Atef

    2014-09-09

    Cell shape affects proliferation and differentiation, which are processes known to depend on integrin-based focal adhesion (FA) signaling. Because shape results from force balance and FAs are mechanosensitive complexes transmitting tension from the cell structure to its mechanical environment, we investigated the interplay between 3D cell shape, traction forces generated through the cell body, and FA growth during early spreading. Combining measurements of cell-scale normal traction forces with FA monitoring, we show that the cell body contact angle controls the onset of force generation and, subsequently, the initiation of FA growth at the leading edge of the lamella. This suggests that, when the cell body switches from convex to concave, tension in the apical cortex is transmitted to the lamella where force-sensitive FAs start to grow. Along this line, increasing the stiffness resisting cell body contraction led to a decrease of the lag time between force generation and FA growth, indicating mechanical continuity of the cell structure and force transmission from the cell body to the leading edge. Remarkably, the overall normal force per unit area of FA increased with stiffness, and its values were similar to those reported for local tangential forces acting on individual FAs. These results reveal how the 3D cell shape feeds back on its internal organization and how it may control cell fate through FA-based signaling.

  20. Focal adhesion kinase is involved in type III group B streptococcal invasion of human brain microvascular endothelial cells.

    Science.gov (United States)

    Shin, Sooan; Paul-Satyaseela, Maneesh; Maneesh, Paul-Satyaseela; Lee, Jong-Seok; Romer, Lewis H; Kim, Kwang Sik

    2006-01-01

    Group B streptococcus (GBS), the leading cause of neonatal meningitis, has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. GBS invasion of HBMEC has been shown to require the host cell actin cytoskeleton rearrangements. The present study examined the mechanisms underlying actin cytoskeleton rearrangements that are involved in type III GBS invasion of HBMEC. We showed that type III GBS invasion was inhibited by genistein, a general tyrosine kinase inhibitor (mean 54% invasion decrease at 100 microM), and LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor (mean 70% invasion decrease at 50 microM), but not by PP2, an inhibitor of the Src family tyrosine kinases. We subsequently showed that the focal adhesion kinase (FAK) was the one of the host proteins tyrosine phosphorylated by type III GBS. Over-expression of a dominant negative form of the FAK C-terminal domain significantly decreased type III GBS invasion of HBMEC (mean 51% invasion decrease). In addition, we showed that FAK phosphorylation correlated with its association of paxillin, an adapter protein of actin filament, and PI3-kinase subunit p85. This is the first demonstration that FAK phosphorylation and its association with paxillin and PI3 kinase play a key role in type III GBS invasion of HBMEC.

  1. Exploring the interaction between human focal adhesion kinase and inhibitors: a molecular dynamic simulation and free energy calculations.

    Science.gov (United States)

    Zhan, Jiu-Yu; Zhang, Ji-Long; Wang, Yan; Li, Ye; Zhang, Hong-Xing; Zheng, Qing-Chuan

    2016-11-01

    Focal adhesion kinase is an important target for the treatment of many kinds of cancers. Inhibitors of FAK are proposed to be the anticancer agents for multiple tumors. The interaction characteristic between FAK and its inhibitors is crucial to develop new inhibitors. In the present article, we used Molecular Dynamic (MD) simulation method to explore the characteristic of interaction between FAK and three inhibitors (PHM16, TAE226, and ligand3). The MD simulation results together with MM-GB/SA calculations show that the combinations are enthalpy-driven process. Cys502 and Asp564 are both essential residues due to the hydrogen bond interactions with inhibitors, which was in good agreement with experimental data. Glu500 can form a non-classical hydrogen bond with each inhibitor. Arg426 can form electrostatic interactions with PHM16 and ligand3, while weaker with TAE226. The electronic static potential was employed, and we found that the ortho-position methoxy of TAE226 has a weaker negative charge than the meta-position one in PHM16 or ligand3. Ile428, Val436, Ala452, Val484, Leu501, Glu505, Glu506, Leu553, Gly563 Leu567, Ser568 are all crucial residues in hydrophobic interactions. The key residues in this work will be available for further inhibitor design of FAK and also give assistance to further research of cancer.

  2. Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions

    Energy Technology Data Exchange (ETDEWEB)

    Sakwe, Amos M., E-mail: asakwe@mmc.edu [Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208 (United States); Koumangoye, Rainelli; Guillory, Bobby [Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208 (United States); Ochieng, Josiah [Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208 (United States); Center for Aids Health Disparity Research, Meharry Medical College, Nashville, TN 37208 (United States); Department of Cancer Biology, Vanderbilt University, Nashville, TN (United States)

    2011-04-01

    The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell-cell cohesion, cell adhesion/spreading onto collagen type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell-cell and/or cell-ECM contacts and anchorage-independent cell proliferation.

  3. Focal adhesion kinase activation is required for TNF-α-induced production of matrix metalloproteinase-2 and proinflammatory cytokines in cultured human periodontal ligament fibroblasts.

    Science.gov (United States)

    Zhang, Peng; Li, Ya-jing; Guo, Liu-yun; Wang, Guo-fang; Lu, Ke; Yue, Er-li

    2015-08-01

    Since focal adhesion kinase (FAK) was proposed as a mediator of the inflammatory response, we have investigated the role of this molecule in the release of inflammatory cytokines by cultured human periodontal ligament fibroblasts (HPDLFs), cells that are thought to be important in the patient's response to periodontal infection. Human periodontal ligament fibroblasts were stimulated by tumor necrosis factor alpha (TNF-α) and its effects on interleukin (IL)-6 and IL-8 release were measured by ELISA. Expression of matrix metalloproteinase 2 (MMP-2) protein was analysed by western blotting. The levels of IL6, IL8, and MMP2 mRNA were evaluated by real-time PCR. Tumor necrosis factor alpha dose-dependently induced the phosphorylation of FAK, whereas small interfering FAK (siFAK) inhibited TNF-α-induced FAK phosphorylation. Tumor necrosis factor alpha also stimulated the production of IL-6, IL-8, and MMP-2 in a dose-dependent manner. Knockdown of FAK significantly suppressed TNF-α-induced expression of IL6 and IL8 mRNA and release of IL-6 and IL-8 protein in HPDLFs. Similarly, MMP-2 down-regulation was significantly prevented by siFAK. Our results strongly suggest that knockdown of FAK can decrease the production of TNF-α-induced IL-6, IL-8, and MMP-2 in HPDLFs. These effects may help in understanding the mechanisms that control expression of inflammatory cytokines in the pathogenesis of periodontitis.

  4. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK and Mitogen-Activated Protein Kinases (MAP Kinases Signaling Pathway in Keratinocytes

    Directory of Open Access Journals (Sweden)

    Yun-Hee Choi

    2015-11-01

    Full Text Available Mycosporine-like amino acids (MAAs are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS. In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH, Mycosporine-glycine (M-Gly, and Porphyra (P334 were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK, extracellular signal-regulated kinases (ERK, and c-Jun N-terminal kinases (JNK. These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.

  5. Dynamics of Actin Stress Fibers and Focal Adhesions during Slow Migration in Swiss 3T3 Fibroblasts: Intracellular Mechanism of Cell Turning

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    Michiko Sugawara

    2016-01-01

    Full Text Available To understand the mechanism regulating the spontaneous change in polarity that leads to cell turning, we quantitatively analyzed the dynamics of focal adhesions (FAs coupling with the self-assembling actin cytoskeletal structure in Swiss 3T3 fibroblasts. Fluorescent images were acquired from cells expressing GFP-actin and RFP-zyxin by laser confocal microscopy. On the basis of the maximum area, duration, and relocation distance of FAs extracted from the RFP-zyxin images, the cells could be divided into 3 regions: the front region, intermediate lateral region, and rear region. In the intermediate lateral region, FAs appeared close to the leading edge and were stabilized gradually as its area increased. Simultaneously, bundled actin stress fibers (SFs were observed vertically from the positions of these FAs, and they connected to the other SFs parallel to the leading edge. Finally, these connecting SFs fused to form a single SF with matured FAs at both ends. This change in SF organization with cell retraction in the first cycle of migration followed by a newly formed protrusion in the next cycle is assumed to lead to cell turning in migrating Swiss 3T3 fibroblasts.

  6. Hepatic stellate cells induce hepatocellular carcinoma cell resistance to sorafenib through the laminin-332/α3 integrin axis recovery of focal adhesion kinase ubiquitination.

    Science.gov (United States)

    Azzariti, Amalia; Mancarella, Serena; Porcelli, Letizia; Quatrale, Anna Elisa; Caligiuri, Alessandra; Lupo, Luigi; Dituri, Francesco; Giannelli, Gianluigi

    2016-12-01

    In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin (Ln)-332, produced by hepatic stellate cells (HSCs). Ln-332 is the ligand of α3β1 and α6β4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln-332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln-332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC-CM or Ln-332 inhibited sorafenib's effectiveness in HCC cells expressing both α3β1 and α6β4. Inhibiting α3 but not α6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln-332 and HSC-CM. Hep3B cells expressing α6β4 but lacking the α3 integrin were insensitive to Ln-332 and HSC-CM protective effects. Hep3B α3-positive, but not wild-type and scramble transfected, cells acquired protection by sorafenib when plated on Ln-332-CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal-regulated kinases 1/2, whereas Ln-332 and HSC-CM partially restored the pathways. Silencing FAK, but not extracellular signal-regulated kinases 1/2, abrogated the protection induced by Ln-332 and HSC-CM, suggesting a specific role for FAK. Sorafenib down-regulated total FAK, inducing its proteasomal degradation, while Ln-332 and HSC-CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization.

  7. Mechanical Stimulation of C2C12 Cells Increases m-Calpain Expression and Activity, Focal Adhesion Plaque Degradation and Cell Fusion

    DEFF Research Database (Denmark)

    Grossi, Alberto; Lawson, Moira Ann; Karlsson, Anders H

    Abstract Mechanical Stimulation of C2C12 Cells Increases m-calpain Expression and Activity, Focal Adhesion Plaque Degradation and Cell Fusion A. Grossi, A. H. Karlsson, M. A. Lawson; Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark...... to the activity of ubiquitous proteolytic enzymes known as calpains has been reported. Whether there is a link between stretch- or load induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have demonstrated...... that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m-calpain relocates into focal adhesion complexes and is followed by a breakdown of specific...

  8. Suppression of β3-integrin in mice triggers a neuropilin-1-dependent change in focal adhesion remodelling that can be targeted to block pathological angiogenesis

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    Tim S. Ellison

    2015-09-01

    Full Text Available Anti-angiogenic treatments against αvβ3-integrin fail to block tumour growth in the long term, which suggests that the tumour vasculature escapes from angiogenesis inhibition through αvβ3-integrin-independent mechanisms. Here, we show that suppression of β3-integrin in mice leads to the activation of a neuropilin-1 (NRP1-dependent cell migration pathway in endothelial cells via a mechanism that depends on NRP1's mobilisation away from mature focal adhesions following VEGF-stimulation. The simultaneous genetic targeting of both molecules significantly impairs paxillin-1 activation and focal adhesion remodelling in endothelial cells, and therefore inhibits tumour angiogenesis and the growth of already established tumours. These findings provide a firm foundation for testing drugs against these molecules in combination to treat patients with advanced cancers.

  9. PCTK3/CDK18 regulates cell migration and adhesion by negatively modulating FAK activity

    Science.gov (United States)

    Matsuda, Shinya; Kawamoto, Kohei; Miyamoto, Kenji; Tsuji, Akihiko; Yuasa, Keizo

    2017-01-01

    PCTAIRE kinase 3 (PCTK3) is a member of the cyclin dependent kinase family, but its physiological function remains unknown. We previously reported that PCTK3-knockdown HEK293T cells showed actin accumulation at the leading edge, suggesting that PCTK3 is involved in the regulation of actin reorganization. In this study, we investigated the physiological function and downstream signal transduction molecules of PCTK3. PCTK3 knockdown in HEK293T cells increased cell motility and RhoA/Rho-associated kinase activity as compared with control cells. We also found that phosphorylation at residue Tyr-397 in focal adhesion kinase (FAK) was increased in PCTK3-knockdown cells. FAK phosphorylation at Tyr-397 was increased in response to fibronectin stimulation, whereas its phosphorylation was suppressed by PCTK3. In addition, excessive expression of PCTK3 led to the formation of filopodia during the early stages of cell adhesion in HeLa cells. These results indicate that PCTK3 controls actin cytoskeleton dynamics by negatively regulating the FAK/Rho signaling pathway. PMID:28361970

  10. Regulation of cell-cell adhesion by Rap1.

    Science.gov (United States)

    Fujita, Yasuyuki; Hogan, Catherine; Braga, Vania M M

    2006-01-01

    Rap1 has been implicated in the regulation of morphogenesis and cell-cell contacts in vivo (Asha et al., 1999; Hariharan et al., 1991; Knox and Brown, 2002) and in vitro (Hogan et al., 2004; Price et al., 2004). Among cell-cell adhesion molecules regulated by Rap1 is cadherin, a calcium-dependent adhesive receptor. Assembly of cadherin-mediated cell-cell contacts triggers Rap1 activation, and Rap function is necessary for the stability of cadherins at junctions (Hogan et al., 2004; Price et al., 2004). Here we describe assays to access the effects of Rap1 on cadherin-dependent adhesion in epithelia, in particular the method used for Rap1 localization, activation, and function modulation by microinjection. We focus on controls and culture conditions to determine the specificity of the phenotype with respect to cadherin receptors. This is important, because different receptors that accumulate at sites of cell-cell contacts are also able to activate Rap1 (Fukuyama et al., 2005; Mandell et al., 2005).

  11. Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

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    Ohannessian Arthur

    2004-05-01

    Full Text Available Abstract Background Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK. Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK. Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC lines. Methods Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. Results In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. Conclusions We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

  12. Mammalian adenylyl cyclase-associated protein 1 (CAP1) regulates cofilin function, the actin cytoskeleton, and cell adhesion.

    Science.gov (United States)

    Zhang, Haitao; Ghai, Pooja; Wu, Huhehasi; Wang, Changhui; Field, Jeffrey; Zhou, Guo-Lei

    2013-07-19

    CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

  13. Cell invasion by Neisseria meningitidis requires a functional interplay between the focal adhesion kinase, Src and cortactin.

    Directory of Open Access Journals (Sweden)

    Heiko Slanina

    Full Text Available Entry of Neisseria meningitidis (the meningococcus into human brain microvascular endothelial cells (HBMEC is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK, which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells.

  14. A TRP Channel in the Lysosome Regulates Large Particle Phagocytosis via Focal Exocytosis

    OpenAIRE

    2013-01-01

    Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here we identified Mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers, and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML...

  15. LNK (SH2B3) is a key regulator of integrin signaling in endothelial cells and targets α-parvin to control cell adhesion and migration

    Science.gov (United States)

    Devallière, Julie; Chatelais, Mathias; Fitau, Juliette; Gérard, Nathalie; Hulin, Philippe; Velazquez, Laura; Turner, Christopher E.; Charreau, Béatrice

    2012-01-01

    Focal adhesion (FA) formation and disassembly play an essential role in adherence and migration of endothelial cells. These processes are highly regulated and involve various signaling molecules that are not yet completely identified. Lnk [Src homology 2-B3 (SH2B3)] belongs to a family of SH2-containing proteins with important adaptor functions. In this study, we showed that Lnk distribution follows that of vinculin, localizing Lnk in FAs. Inhibition of Lnk by RNA interference resulted in decreased spreading, whereas sustained expression dramatically increases the number of focal and cell-matrix adhesions. We demonstrated that Lnk expression impairs FA turnover and cell migration and regulates β1-integrin-mediated signaling via Akt and GSK3β phosphorylation. Moreover, the α-parvin protein was identified as one of the molecular targets of Lnk responsible for impaired FA dynamics and cell migration. Finally, we established the ILK protein as a new molecular partner for Lnk and proposed a model in which Lnk regulates α-parvin expression through its interaction with ILK. Collectively, our results underline the adaptor Lnk as a novel and effective key regulator of integrin-mediated signaling controlling endothelial cell adhesion and migration.—Devallière, J., Chatelais, M., Fitau, J., Gérard, N., Hulin, P., Velazquez, L., Turner, C. E. Charreau, B. LNK (SH2B3) is a key regulator of integrin signaling in endothelial cells and targets α-parvin to control cell adhesion and migration. PMID:22441983

  16. The relative roles of collagen adhesive receptor DDR2 activation and matrix stiffness on the downregulation of focal adhesion kinase in vascular smooth muscle cells.

    Science.gov (United States)

    Bhadriraju, Kiran; Chung, Koo-Hyun; Spurlin, Tighe A; Haynes, Ross J; Elliott, John T; Plant, Anne L

    2009-12-01

    Cells within tissues derive mechanical anchorage and specific molecular signals from the insoluble extracellular matrix (ECM) that surrounds them. Understanding the role of different cues that extracellular matrices provide cells is critical for controlling and predicting cell response to scaffolding materials. Using an engineered extracellular matrix of Type I collagen we examined how the stiffness, supramolecular structure, and glycosylation of collagen matrices influence the protein levels of cellular FAK and the activation of myosin II. Our results show that (1) cellular FAK is downregulated on collagen fibrils, but not on a non-fibrillar monolayer of collagen, (2) the downregulation of FAK is independent of the stiffness of the collagen fibrils, and (3) FAK levels are correlated with levels of tyrosine phosphorylation of the collagen adhesion receptor DDR2. Further, siRNA depletion of DDR2 blocks FAK downregulation. Our results suggest that the collagen receptor DDR2 is involved in the regulation of FAK levels in vSMC adhered to Type I collagen matrices, and that regulation of FAK levels in these cells appears to be independent of matrix stiffness.

  17. The Sal-like 4 - integrin α6β1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cells.

    Science.gov (United States)

    Itou, Junji; Tanaka, Sunao; Li, Wenzhao; Iida, Atsuo; Sehara-Fujisawa, Atsuko; Sato, Fumiaki; Toi, Masakazu

    2017-01-01

    During metastasis, cancer cell migration is enhanced. However, the mechanisms underlying this process remain elusive. Here, we addressed this issue by functionally analyzing the transcription factor Sal-like 4 (SALL4) in basal-like breast cancer cells. Loss-of-function studies of SALL4 showed that this transcription factor is required for the spindle-shaped morphology and the enhanced migration of cancer cells. SALL4 also up-regulated integrin gene expression. The impaired cell migration observed in SALL4 knockdown cells was restored by overexpression of integrin α6 and β1. In addition, we clarified that integrin α6 and β1 formed a heterodimer. At the molecular level, loss of the SALL4 - integrin α6β1 network lost focal adhesion dynamics, which impairs cell migration. Over-activation of Rho is known to inhibit focal adhesion dynamics. We observed that SALL4 knockdown cells exhibited over-activation of Rho. Aberrant Rho activation was suppressed by integrin α6β1 expression, and pharmacological inhibition of Rho activity restored cell migration in SALL4 knockdown cells. These results indicated that the SALL4 - integrin α6β1 network promotes cell migration via modulation of Rho activity. Moreover, our zebrafish metastasis assays demonstrated that this gene network enhances cell migration in vivo. Our findings identify a potential new therapeutic target for the prevention of metastasis, and provide an improved understanding of cancer cell migration. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Focal adhesion kinase antisense oligodeoxynucleotides inhibit human pulmonary artery smooth muscle cells proliferation and promote human pulmonary artery smooth muscle cells apoptosis

    Institute of Scientific and Technical Information of China (English)

    LIN Chun-long; ZHANG Zhen-xiang; XU Yong-jian; NI Wang; CHEN Shi-xin

    2005-01-01

    Background Pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling. At present, the mechanisms related to proliferation of PASMCs are not clear. Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase. Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells. Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs). We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein.Methods Cultured HPASMCs stimulated by fibronectin (40 μg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense-FAK respectively. Expression of FAK, Jun NH2-terminal kinase (JNK), cyclin-dependent kinase 2 (CDK 2) and caspase-3 proteins were detected by immunoprecipitation and Western blots. Cell cycle and cell apoptosis were analysed by flow cytometry. In addition, cytoplasmic FAK expression was detected by immunocytochemical staining.Results When compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense-FAK ODNs group and increased in sense-FAK ODNs group significantly. Caspase-3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs. When compared with mismatch sense ODNs group, the proportion of cells at G1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly. In contrast, compared with mismatch sense ODNs group, the proportion of cells at G1 phase was increased significantly in antisense-FAK ODNs group. The level of cell apoptosis in antisense-FAK group

  19. Activin Receptor Signaling Regulates Prostatic Epithelial Cell Adhesion and Viability

    Directory of Open Access Journals (Sweden)

    Derek P. Simon

    2009-04-01

    Full Text Available Mutational changes coupled with endocrine, paracrine, and/or autocrine signals regulate cell division during carcinogenesis. The hormone signals remain undefined, although the absolute requirement in vitro for fetal serum indicates the necessity for a fetal serum factor(s in cell proliferation. Using prostatic cancer cell (PCC lines as a model of cancer cell proliferation, we have identified the fetal serum component activin A and its signaling through the activin receptor type II (ActRII, as necessary, although not sufficient, for PCC proliferation. Activin A induced Smad2 phosphorylation and PCC proliferation, but only in the presence of fetal bovine serum (FBS. Conversely, activin A antibodies and inhibin A suppressed FBS-induced PCC proliferation confirming activin A as one of multiple serum components required for PCC proliferation. Basic fibroblast growth factor was subsequently shown to synergize activin A-induced PCC proliferation. Inhibition of ActRII signaling using a blocking antibody or antisense-P decreased mature ActRII expression, Smad2 phosphorylation, and the apparent viability of PCCs and neuroblastoma cells grown in FBS. Suppression of ActRII signaling in PCC and neuroblastoma cells did not induce apoptosis as indicated by the ratio of active/inactive caspase 3 but did correlate with increased cell detachment and ADAM-15 expression, a disintegrin whose expression is strongly correlated with prostatic metastasis. These findings indicate that ActRII signaling is required for PCC and neuroblastoma cell viability, with ActRII mediating cell fate via the regulation of cell adhesion. That ActRII signaling governs both cell viability and cell adhesion has important implications for developing therapeutic strategies to regulate cancer growth and metastasis.

  20. Interleukin-2 induces beta2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

    DEFF Research Database (Denmark)

    Brockdorff, J; Kanner, S B; Nielsen, M

    1998-01-01

    B-tyrosine phosphorylation in beta2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125(FAK). In conclusion, our data indicate that IL-2 induces beta2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB.......beta2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of beta2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4(+) T cell lines obtained from healthy donors...... experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in beta2-integrin-positive but not in beta2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fak...

  1. CADM1 controls actin cytoskeleton assembly and regulates extracellular matrix adhesion in human mast cells.

    Directory of Open Access Journals (Sweden)

    Elena P Moiseeva

    Full Text Available CADM1 is a major receptor for the adhesion of mast cells (MCs to fibroblasts, human airway smooth muscle cells (HASMCs and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM. Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion.

  2. Interleukin-2 induces beta2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

    DEFF Research Database (Denmark)

    Brockdorff, J; Kanner, S B; Nielsen, M;

    1998-01-01

    experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in beta2-integrin-positive but not in beta2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fakB......-tyrosine phosphorylation in beta2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125(FAK). In conclusion, our data indicate that IL-2 induces beta2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB....... and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in beta2 integrin (CD18)-positive but not in beta2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation...

  3. A novel role for Lsc/p115 RhoGEF and LARG in regulating RhoA activity downstream of adhesion to fibronectin.

    Science.gov (United States)

    Dubash, Adi D; Wennerberg, Krister; García-Mata, Rafael; Menold, Marisa M; Arthur, William T; Burridge, Keith

    2007-11-15

    Adhesion of cells to extracellular matrix proteins such as fibronectin initiates signaling cascades that affect cell morphology, migration and survival. Some of these signaling pathways involve the Rho family of GTPases, such as Cdc42, Rac1 and RhoA, which play a key role in regulating the organization of the cytoskeleton. Although significant advances have been made in understanding how Rho proteins control cytoskeletal architecture, less is known about the signals controlling activation of the GTPases themselves. The focus of this study was to determine which guanine nucleotide exchange factor(s) are responsible for activation of RhoA downstream of adhesion to fibronectin. Using an affinity pulldown assay for activated exchange factors, we show that the RhoA-specific exchange factors Lsc/p115 RhoGEF and LARG are activated when cells are plated onto fibronectin, but not other exchange factors such as Ect2 or Dbl. Knockdown of Lsc and LARG together significantly decreases RhoA activation and formation of stress fibers and focal adhesions downstream of fibronectin adhesion. Similarly, overexpression of a catalytically inactive mutant of Lsc/p115 RhoGEF inhibits RhoA activity and formation of stress fibers and focal adhesions on fibronectin. These data establish a previously uncharacterized role for the exchange factors Lsc/p115 RhoGEF and LARG in linking fibronectin signals to downstream RhoA activation.

  4. CEACAM6 cross-linking induces caveolin-1-dependent, Src-mediated focal adhesion kinase phosphorylation in BxPC3 pancreatic adenocarcinoma cells.

    Science.gov (United States)

    Duxbury, Mark S; Ito, Hiromichi; Ashley, Stanley W; Whang, Edward E

    2004-05-28

    Despite lacking transmembrane or intracellular domains, glycosylphosphatidylinositol-anchored proteins can modulate intracellular signaling events, in many cases through aggregation within membrane "lipid raft" microdomains. CEACAM6 is a glycosylphosphatidylinositol-linked cell surface protein of importance in the anchorage-independent survival and metastasis of pancreatic adenocarcinoma cells. We examined the effects of antibody-mediated cross-linking of CEACAM6 on intracellular signaling events and anchorage-independent survival of the CEACAM6-overexpressing pancreatic ductal adenocarcinoma cell line, BxPC3. CEACAM6 cross-linking increased c-Src activation and induced tyrosine phosphorylation of p125(FAK) focal adhesion kinase. Focal adhesion kinase phosphorylation was dependent on c-Src kinase activation, for which caveolin-1 was required. CEACAM6 cross-linking induced a significant increase in cellular resistance to anoikis. These observations represent the first characterization of the mechanism through which this important cell surface oncoprotein influences intracellular signaling events and hence malignant cellular behavior.

  5. Tbx1 regulates oral epithelial adhesion and palatal development

    Science.gov (United States)

    Funato, Noriko; Nakamura, Masataka; Richardson, James A.; Srivastava, Deepak; Yanagisawa, Hiromi

    2012-01-01

    Cleft palate, the most frequent congenital craniofacial birth defect, is a multifactorial condition induced by the interaction of genetic and environmental factors. In addition to complete cleft palate, a large number of human cases involve soft palate cleft and submucosal cleft palate. However, the etiology of these forms of cleft palate has not been well understood. T-box transcriptional factor (Tbx) family of transcriptional factors has distinct roles in a wide range of embryonic differentiation or response pathways. Here, we show that genetic disruption of Tbx1, a major candidate gene for the human congenital disorder 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome), led to abnormal epithelial adhesion between the palate and mandible in mouse, resulting in various forms of cleft palate similar to human conditions. We found that hyperproliferative epithelium failed to undergo complete differentiation in Tbx1-null mice (Tbx1−/−). Inactivation of Tbx1 specifically in the keratinocyte lineage (Tbx1KCKO) resulted in an incomplete cleft palate confined to the anterior region of the palate. Interestingly, Tbx1 overexpression resulted in decreased cell growth and promoted cell-cycle arrest in MCF7 epithelial cells. These findings suggest that Tbx1 regulates the balance between proliferation and differentiation of keratinocytes and is essential for palatal fusion and oral mucosal differentiation. The impaired adhesion separation of the oral epithelium together with compromised palatal mesenchymal growth is an underlying cause for various forms of cleft palate phenotypes in Tbx1−/− mice. Our present study reveals new pathogenesis of incomplete and submucous cleft palate during mammalian palatogenesis. PMID:22371266

  6. Spatial organization of adhesion: force-dependent regulation and function in tissue morphogenesis.

    Science.gov (United States)

    Papusheva, Ekaterina; Heisenberg, Carl-Philipp

    2010-08-18

    Integrin- and cadherin-mediated adhesion is central for cell and tissue morphogenesis, allowing cells and tissues to change shape without loosing integrity. Studies predominantly in cell culture showed that mechanosensation through adhesion structures is achieved by force-mediated modulation of their molecular composition. The specific molecular composition of adhesion sites in turn determines their signalling activity and dynamic reorganization. Here, we will review how adhesion sites respond to mecanical stimuli, and how spatially and temporally regulated signalling from different adhesion sites controls cell migration and tissue morphogenesis.

  7. Down-regulation of Bcl-2 in rat substantia nigra after focal cerebral ischemia.

    Science.gov (United States)

    Arango-Dávila, Cesar A; Cardona-Gomez, Gloria P; Gallego-Gomez, Juan C; Garcia-Segura, Luis M; Pimienta, Hernán J

    2004-06-28

    After occlusion of the middle cerebral artery in rats, a robust neuronal loss occurs in the ipsilateral substantia nigra reticulata. In this study we have assessed whether degeneration of the substantia nigra is accompanied by changes in the expression of the anti-apoptotic protein Bcl-2. Neuronal loss was assessed by neuronal nuclei (NeuN) immunoreactivity. A significant decrease of Bcl-2 expression was observed in the substantia nigra 12, 24 and 72 h after middle cerebral artery occlusion. These results suggest that the secondary neuronal loss in the substantia nigra could be related with the modification of proteins regulating programmed cell death. Exo-focal cell death may explain the appearance of neuropsychiatric symptoms that are not correlated with the primary site of lesion.

  8. A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; McCarthy, J B; Furcht, L T

    1993-01-01

    Cell adhesion to extracellular matrix molecules such as fibronectin involves complex transmembrane signaling processes. Attachment and spreading of primary fibroblasts can be promoted by interactions of cell surface integrins with RGD-containing fragments of fibronectin, but the further process o...

  9. cAMP-induced Epac-Rap activation inhibits epithelial cell migration by modulating focal adhesion and leading edge dynamics

    NARCIS (Netherlands)

    Lyle, Karen S.; Raaijmakers, J.H.; Bruinsma, Wytse; Bos, Johannes L.; Rooij, J. de

    2008-01-01

    Epithelial cell migration is a complex process crucial for embryonic development, wound healing and tumor metastasis. It depends on alterations in cell–cell adhesion and integrin–extracellular matrix interactions and on actomyosin-driven, polarized leading edge protrusion. The small GTPase Rap is a

  10. Mild hypothermia effects on intercellular adhesion molecule-1 and serum interleukin-6 expression in brain tissues of a rat focal ischemia model

    Institute of Scientific and Technical Information of China (English)

    Shengqi Fu; Lei Yang; Shuling Zhang; Shilong Sun; Xingai Mao

    2008-01-01

    BACKGROUND: Previous studies have confirmed the neuroprotective effect of mild hypothermia on ischemic brain injury.OBJECTIVE: To investigate the effects of mild hypothermia on intercellular adhesion molecule-1 expression and serum interleukin-6 levels in ischemic brain tissues of focal brain ischemia rats, and to explore the neuroprotective effects of mild hypothermia on ischemic brain injury.DESIGN, TIME AND SETTING: A randomized, controlled, neurobiological experiment was performed at the Central Laboratory, First Affiliated Hospital, Xinxiang Medical College, China from February to July 2006.MATERIALS: Thirty healthy, adult, Sprague Dawley rats were used to establish middle cerebral artery occlusion models using the suture method. The immunohistochemistry (streptavidin-biotin-peroxidase complex method) kit was purchased from Boster, China. Interleukin-6 radioimmunoassay was supplied by Institute of Radioimmunity, Technology Development Center, General Hospital of Chinese PLA. METHODS: The rats were equally and randomly assigned into mild hypothermia and control groups, and middle cerebral artery occlusion models were established. The rectal temperature was maintained at (37 ± 0.5)℃ in the control group. In the mild hypothermia group, the rectal temperature was maintained at (33±1)℃.MAIN OUTCOME MEASURES: At 12 hours after model establishment, the ischemic brain hemispheres were coronally sliced at the level of the optic chiasm. The number of intercellular adhesion molecule- 1 -positive vessels per high-power field was observed with an optical microscope. Serum interleukin-6 levels were measured by radioimmunoassay.RESULTS: Compared with the control group, intercellular adhesion molecule-I and serum interleukin-6 expressions were significantly decreased in ischemic brain tissues of the mild hypothermia group (P < 0.01).CONCLUSION: Mild hypothermia exhibits a neuroprotective effect by reducing serum interleukin-6 and intercellular adhesion molecule- 1

  11. Osthole Suppresses the Migratory Ability of Human Glioblastoma Multiforme Cells via Inhibition of Focal Adhesion Kinase-Mediated Matrix Metalloproteinase-13 Expression

    Directory of Open Access Journals (Sweden)

    Cheng-Fang Tsai

    2014-03-01

    Full Text Available Glioblastoma multiforme (GBM is the most common type of primary and malignant tumor occurring in the adult central nervous system. GBM often invades surrounding regions of the brain during its early stages, making successful treatment difficult. Osthole, an active constituent isolated from the dried C. monnieri fruit, has been shown to suppress tumor migration and invasion. However, the effects of osthole in human GBM are largely unknown. Focal adhesion kinase (FAK is important for the metastasis of cancer cells. Results from this study show that osthole can not only induce cell death but also inhibit phosphorylation of FAK in human GBM cells. Results from this study show that incubating GBM cells with osthole reduces matrix metalloproteinase (MMP-13 expression and cell motility, as assessed by cell transwell and wound healing assays. This study also provides evidence supporting the potential of osthole in reducing FAK activation, MMP-13 expression, and cell motility in human GBM cells.

  12. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation and cell differentiation

    DEFF Research Database (Denmark)

    Grossi, Alberto; Lawson, Moira Ann

    Abstract Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation and cell differentiation. A. Grossi, M. A. Lawson; Department of Food Science, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark The process of muscle...... documented and has been shown to affect transcription of specific gene sequences, protein synthesis, the immune system and increase in Ca2+ influx. The past 10 years has seen a dramatic increase in the understanding of how proteolytic enzymes such as calpains can affect the growth of muscle. In vivo studies...... have shown that m-calpain is necessary for myoblast fusion leading to the formation of muscle fibers and that inhibition of this enzyme restricts myotube formation. Whether there is a link between stretchor load induced signaling and m-calpain expression and activation is not known. Using a magnetic...

  13. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation and cell differentiation

    DEFF Research Database (Denmark)

    Grossi, Alberto; Lawson, Moira Ann

    Abstract Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation and cell differentiation. A. Grossi, M. A. Lawson; Department of Food Science, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark The process of muscle...... development and growth is a complex sequence of events whereby muscle cells respond to a number of stimuli in order to form organised muscle tissue. Increase in muscle mass is greatly influenced by the rate of skeletal muscle protein synthesis and degradation, processes that can be altered by mechanical...... forces. Stretch- or load-induced signaling is now beginning to be understood as a factor which affects the mass and phenotype of muscles as well as the expression of a number of proteins within muscle cells. Use of magnetic field to produce mechanical forces to stimulate cell populations has been well...

  14. Impact of extracellular matrix derived from osteoarthritis subchondral bone osteoblasts on osteocytes: role of integrinβ1 and focal adhesion kinase signaling cues.

    Science.gov (United States)

    Prasadam, Indira; Farnaghi, Saba; Feng, Jian Q; Gu, Wenyi; Perry, Samuel; Crawford, Ross; Xiao, Yin

    2013-10-09

    Our recent study indicated that subchondral bone pathogenesis in osteoarthritis (OA) is associated with osteocyte morphology and phenotypic abnormalities. However, the mechanism underlying this abnormality needs to be identified. In this study we investigated the effect of extracellular matrix (ECM) produced from normal and OA bone on osteocytic cells function. De-cellularized matrices, resembling the bone provisional ECM secreted from primary human subchondral bone osteoblasts (SBOs) of normal and OA patients were used as a model to study the effect on osteocytic cells. Osteocytic cells (MLOY4 osteocyte cell line) cultured on normal and OA derived ECMs were analyzed by confocal microscopy, scanning electron microscopy (SEM), cell attachment assays, zymography, apoptosis assays, qRT-PCR and western blotting. The role of integrinβ1 and focal adhesion kinase (FAK) signaling pathways during these interactions were monitored using appropriate blocking antibodies. The ECM produced by OA SBOs contained less mineral content, showed altered organization of matrix proteins and matrix structure compared with the matrices produced by normal SBOs. Culture of osteocytic cells on these defective OA ECM resulted in a decrease of integrinβ1 expression and the de-activation of FAK cell signaling pathway, which subsequently affected the initial osteocytic cell's attachment and functions including morphological abnormalities of cytoskeletal structures, focal adhesions, increased apoptosis, altered osteocyte specific gene expression and increased Matrix metalloproteinases (MMP-2) and -9 expression. This study provides new insights in understanding how altered OA bone matrix can lead to the abnormal osteocyte phenotypic changes, which is typical in OA pathogenesis.

  15. Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration

    Institute of Scientific and Technical Information of China (English)

    Takuya Watanabe; Masumi Tsuda; Yoshinori Makino; Tassos Konstantinou; Hiroshi Nishihara; Tokifumi Majima; Akio Minami; Stephan M Feller; Shinya Tanaka

    2009-01-01

    Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gab1 without extraceilular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of Crkll demonstrated that the SH2 domain, but not the SH3(N) or the regulatory Y221 residue of Crkll, is critical for the induction of Gabl-Y307 phosphorylation. SH2 mutation of Crkll also decreased the interaction with Gab1. In GST pull-down assay, Crk-SH2 bound to wild-type Gabl, whereas Crk-SH3(N) interacted with the Gabl mutant, which lacks the clus-tered tyrosine region (residues 242-410). Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The GabI-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxiilin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory-lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

  16. Research progress on focal adhesion kinase in malignant tumors%FAK 在恶性肿瘤中的研究进展

    Institute of Scientific and Technical Information of China (English)

    赵美娜(综述); 陈公琰(审校)

    2016-01-01

    Focal adhesion kinase( FAK) ,a cytoplasmic non-receptor protein tyrosine kinase,serves as both a molecular scaffold and a mediator participating in multiple signal transduction pathways.FAK is involved in tumor cell survival,proliferation,migration and metastasis.Recent studies have shown that FAK is expressed in many tumor cells.Currently,FAK has been regarded as a potential target for cancer therapy.This review is to summarize the relationship between FAK and tumor progression.%黏着斑激酶( Focal adhesion kinase,FAK)是细胞内重要的骨架蛋白,属于一种非受体型酪氨酸蛋白激酶,也是多种信号通路的关键性分子。在肿瘤发生、发展、迁移以及侵袭的各个阶段FAK都具有重要作用。近年来,人们对FAK的研究越来越多,综合国内外研究资料表明,FAK在许多肿瘤组织中表达增高,提示FAK可能与肿瘤的发生发展密切相关,可能是肿瘤治疗的潜在靶点。该综述将对FAK分子结构及功能特点,与肿瘤的关系进行系统阐述。

  17. Focal uptake at the rotator interval or inferior capsule of shoulder on {sup 18}F-FDG PET/CT is associated with adhesive capsulitis

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Radhika [Universiti Kebangsaan Malaysia Medical Centre, Department of Diagnostic Imaging, Kuala Lumpur (Malaysia); Engle, Mitchell Philip [The University of Texas M.D. Anderson Cancer Center, Department of Pain Medicine, Houston, TX (United States); Garg, Naveen [The University of Texas M.D. Anderson Cancer Center, Department of Diagnostic Imaging, Houston, TX (United States); Wei, Wei; Amini, Behrang [The University of Texas M.D. Anderson Cancer Center, Department of Biostatistics, Houston, TX (United States)

    2017-04-15

    To determine if focal increased uptake at the rotator interval (RI) and/or inferior capsule (IC) on{sup 18}F-FDG PET/CT (''positive PET'') predicts the presence of adhesive capsulitis (AC). Three populations were retrospectively examined. Group 1 included 1,137 consecutive {sup 18}F-FDG PET/CT studies and was used to determine the prevalence of focal uptake at the RI or IC. Group 2 included 361 cases from a 10-year period with {sup 18}F-FDG PET/CT and MRI of shoulder performed within 45 days of each other and was used to enrich the study group. Group 3 included 109 randomly selected patients from the same time frame as groups 1 and 2 and was used to generate the control group. The study group consisted of 15 cases from the three groups, which had positive PET findings. PET/CT images were assessed in consensus by two musculoskeletal radiologists. The reference standard for a diagnosis of AC was clinical and was made by review of the medical record by a pain medicine physician. The prevalence of focal activity at either the RI or IC (''positive PET'') was 0.53%. Nine patients had a clinical diagnosis of AC and 15 patients had a positive PET. The sensitivity and specificity of PET for detection of AC was 56% and 87%, respectively. PET/CT had a positive likelihood ratio for AC of 6.3 (95% CI: 2.8-14.6). Increased uptake at the RI or IC on PET/CT confers a moderate increase in the likelihood of AC. (orig.)

  18. Cell adhesion molecules regulate contractile ring-independent cytokinesis in Dictyostelium discoideum

    Institute of Scientific and Technical Information of China (English)

    Akira Nagasaki; Masamitsu Kanada; Taro QP Uyeda

    2009-01-01

    To investigate the roles of substrate adhesion in cytokinesis, we established cell lines lacking paxiUin (PAXB) or vinculin (VINA), and those expressing the respective GFP fusion proteins in Dictyostelium discoideum. As in mammalian cells, GFP-PAXB and GFP-VINA formed focal adhesion-like complexes on the cell bottom, paxB cells in suspension grew normally, but on substrates, often failed to divide after regression of the furrow. The efficient cytokinesis of paxB cells in suspension is not because of shear forces to assist abscission, as they divided normally in static suspension culture as well. Double knockout strains lacking mhcA, which codes for myosin I1, and paxB or vinA displayed more severe cytokinetic defects than each single knockout strain. In mitotic wild-type cells, GFP-PAXB was diffusely distributed on the basal membrane, but was strikingly condensed along the polar edges in mitotic mhcA cells. These results are consistent with our idea that Dictyostelium displays two forms of cytokinesis, one that is contractile ringdependent and adhesion-independent, and the other that is contractile ring-independent and adhesion-dependent, and that the latter requires PAXB and VINA. Furthermore, that paxB cells fail to divide normally in the presence of substrate adhesion suggests that this adhesion molecule may play additional signaling roles.

  19. Focal adhesion kinase regulates collagen I-induced airway smooth muscle phenotype switching

    NARCIS (Netherlands)

    Dekkers, Bart G J; Spanjer, Anita I R; van der Schuyt, Robert D; Kuik, Willem Jan; Zaagsma, Johan; Meurs, Herman

    2013-01-01

    Increased extracellular matrix (ECM) deposition and airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma. Recently, we demonstrated that the ECM protein collagen I, which is increased surrounding asthmatic ASM, induces a proliferative, hypocontractile ASM phenotype.

  20. HPK1 associates with SKAP-HOM to negatively regulate Rap1-mediated B-lymphocyte adhesion.

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    Sebastian Königsberger

    Full Text Available BACKGROUND: Hematopoietic progenitor kinase 1 (HPK1 is a Ste20-related serine/threonine kinase activated by a range of environmental stimuli including genotoxic stress, growth factors, inflammatory cytokines and antigen receptor triggering. Being inducibly recruited to membrane-proximal signalling scaffolds to regulate NFAT, AP-1 and NFkappaB-mediated gene transcription in T-cells, the function of HPK1 in B-cells to date remains rather ill-defined. METHODOLOGY/PRINCIPAL FINDINGS: By using two loss of function models, we show that HPK1 displays a novel function in regulating B-cell integrin activity. Wehi 231 lymphoma cells lacking HPK1 after shRNA mediated knockdown exhibit increased basic activation levels of Ras-related protein 1 (Rap1, accompanied by a severe lymphocyte function-associated antigen-1 (LFA-1 dependent homotypic aggregation and increased adhesion to intercellular adhesion molecule 1 (ICAM-1. The observed phenotype of enhanced integrin activity is caused downstream of Src, by a signalling module independent of PI3K and PLC, involving HPK1, SKAP55 homologue (SKAP-HOM and Rap1-GTP-interacting adaptor molecule (RIAM. This alters actin dynamics and renders focal adhesion kinase (FAK constitutively phosphorylated. Bone marrow and splenic B-cell development of HPK1(-/- mice are largely unaffected, except age-related tendencies for increased splenic cellularity and BCR downregulation. In addition, naïve splenic knockout B-cells appear hyperresponsive to a range of stimuli applied ex vivo as recently demonstrated by others for T-cells. CONCLUSIONS/SIGNIFICANCE: We therefore conclude that HPK1 exhibits a dual function in B-cells by negatively regulating integrin activity and controlling cellular activation, which makes it an interesting candidate to study in pathological settings like autoimmunity and cancer.

  1. The Small GTPase Rap1b: A Bidirectional Regulator of Platelet Adhesion Receptors

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    Gianni Francesco Guidetti

    2012-01-01

    Full Text Available Integrins and other families of cell adhesion receptors are responsible for platelet adhesion and aggregation, which are essential steps for physiological haemostasis, as well as for the development of thrombosis. The modulation of platelet adhesive properties is the result of a complex pattern of inside-out and outside-in signaling pathways, in which the members of the Rap family of small GTPases are bidirectionally involved. This paper focuses on the regulation of the main Rap GTPase expressed in circulating platelets, Rap1b, downstream of adhesion receptors, and summarizes the most recent achievements in the investigation of the function of this protein as regulator of platelet adhesion and thrombus formation.

  2. Patterning of cell assemblies regulated by adhesion receptors of the cadherin superfamily.

    OpenAIRE

    2000-01-01

    During morphogenesis, cell-cell association patterns are dynamically altered. We are interested in how cell adhesion molecules can regulate the patterning of cellular assemblies. Cadherins, a group of cell-cell adhesion receptors, are crucial for the organized assembly of many cell types, but they also regulate dynamic aspects of cell association. For example, during neural crest emigration from the neural tube, the cadherin subtypes expressed by crest cells are switched from one subtype to a...

  3. PRL-3/PTP4A3 phosphatase regulates integrin β1 in adhesion structures during migration of human ocular melanoma cells.

    Science.gov (United States)

    Foy, Malika; Anézo, Océane; Saule, Simon; Planque, Nathalie

    2017-03-08

    In a previous transcriptomic analysis of 63 ocular melanomas of the uvea, we found that expression of the PRL-3/PTP4A3 gene, encoding a phosphatase that is anchored to the plasma membrane, was associated with the risk of metastasis, and a poor prognosis. We also showed that PRL-3 overexpression in OCM-1 ocular melanoma cells significantly increased cell migration in vitro and invasiveness in vivo, suggesting a direct role for PRL-3 in the metastatic spreading of uveal melanoma. Here, we aimed to identify PRL-3 substrates at the plasma membrane involved in adhesion to the extracellular matrix. We focused on integrin β1, which is the most highly expressed integrin in our cohort of uveal melanomas. We show that preventing PRL-3 anchorage to the plasma membrane i) abolishes PRL-3-induced migration in OCM-1 cells, ii) specifically enhances the spreading of OCM-1 cells overexpressing PRL-3, and iii) favors the maturation of large focal adhesions (FAs) containing integrin β1 on collagen I. Knockdown experiments confirmed integrin β1 involvement in PRL3-induced migration. We identified interactions between PRL-3 and integrin β1, as well as with FAK P-Y397, an auto-activated form of Focal Adhesion Kinase found in FAs. We also show that integrin β1 may be dephosphorylated by PRL-3 in its intracytoplasmic S/T region, an important motif for integrin-mediated cell adhesion. Finally, we observed that PRL-3 regulated the clustering of integrin β1 in FAs on collagen I but not on fibronectin. This work identifies PRL-3 as a new regulator of cell adhesion structures to the extracellular matrix, and further supports PRL-3 as a key actor of metastasis in uveal melanoma, of which molecular mechanisms are still poorly understood.

  4. A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis.

    Science.gov (United States)

    Samie, Mohammad; Wang, Xiang; Zhang, Xiaoli; Goschka, Andrew; Li, Xinran; Cheng, Xiping; Gregg, Evan; Azar, Marlene; Zhuo, Yue; Garrity, Abigail G; Gao, Qiong; Slaugenhaupt, Susan; Pickel, Jim; Zolov, Sergey N; Weisman, Lois S; Lenk, Guy M; Titus, Steve; Bryant-Genevier, Marthe; Southall, Noel; Juan, Marugan; Ferrer, Marc; Xu, Haoxing

    2013-09-16

    Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML1 overexpression and TRPML1 agonists facilitate both lysosomal exocytosis and particle uptake. Using time-lapse confocal imaging and direct patch clamping of phagosomal membranes, we found that particle binding induces lysosomal PI(3,5)P2 elevation to trigger TRPML1-mediated lysosomal Ca2+ release specifically at the site of uptake, rapidly delivering TRPML1-resident lysosomal membranes to nascent phagosomes via lysosomal exocytosis. Thus phagocytic ingestion of large particles activates a phosphoinositide- and Ca2+-dependent exocytosis pathway to provide membranes necessary for pseudopod extension, leading to clearance of senescent and apoptotic cells in vivo.

  5. Integrin-linked kinase regulates oligodendrocyte cytoskeleton, growth cone, and adhesion dynamics.

    Science.gov (United States)

    Michalski, John-Paul; Cummings, Sarah E; O'Meara, Ryan W; Kothary, Rashmi

    2016-02-01

    Integrin-linked kinase (ILK), a focal adhesion protein, brokers the link between cytoskeleton, cell membrane, and extracellular environment. Here, we demonstrate a role for ILK in laminin-2-mediated adhesion in primary murine oligodendrocytes (OLs) - with ILK loss leading to severe defects in process branching and outgrowth. These defects were partially recovered when the ILK-depleted OLs were instead grown on the non-integrin-activating substrate poly-l-lysine. Intriguingly, ILK loss on the neutral poly-l-lysine substrate led to swelling at the tips of OL processes, which we identified as enlarged growth cones. Employing the bloated ILK-depleted growth cones as template, we demonstrate the appearance of distinct cytoskeletal domains within OL growth cones bearing classic neuronal growth cone architecture. Further, microtubule organization was severely perturbed following ILK loss, with centripetal microtubule looping and failure to bundle occurring in a laminin-2-independent manner. Together, our work highlights differences in specific aspects of OL biology as driven by laminin-2-dependent or independent ILK governed mechanisms. We also reinforce the idea of OLs as growth cone bearing cells and describe the neuronal-like cytoskeleton therein. Finally, we demonstrate a role for ILK in OL growth cone maturation through microtubule regulation, the loss of which translates to decreased process length and myelin production capacity. We describe herein how different substrates fundamentally alter the oligodendrocyte's response to loss of integrin-linked kinase (ILK). On laminin-2 (Ln-2), ILK-depleted oligodendrocytes appear stunted and malformed, while on the non-integrin-activating substrate PLL branching and membrane formation are restored. We also reinforce the idea of oligodendrocytes as growth cone-bearing cells, detailing the growth cone's cytoskeletal architecture. Strikingly, loss of ILK on poly-l-lysine leads to growth cone swelling, the structure's size and

  6. Bidirectional remodeling of β1-integrin adhesions during chemotropic regulation of nerve growth

    Directory of Open Access Journals (Sweden)

    Carlstrom Lucas P

    2011-11-01

    Full Text Available Abstract Background Chemotropic factors in the extracellular microenvironment guide nerve growth by acting on the growth cone located at the tip of extending axons. Growth cone extension requires the coordination of cytoskeleton-dependent membrane protrusion and dynamic adhesion to the extracellular matrix, yet how chemotropic factors regulate these events remains an outstanding question. We demonstrated previously that the inhibitory factor myelin-associated glycoprotein (MAG triggers endocytic removal of the adhesion receptor β1-integrin from the growth cone surface membrane to negatively remodel substrate adhesions during chemorepulsion. Here, we tested how a neurotrophin might affect integrin adhesions. Results We report that brain-derived neurotropic factor (BDNF positively regulates the formation of substrate adhesions in axonal growth cones during stimulated outgrowth and prevents removal of β1-integrin adhesions by MAG. Treatment of Xenopus spinal neurons with BDNF rapidly triggered β1-integrin clustering and induced the dynamic formation of nascent vinculin-containing adhesion complexes in the growth cone periphery. Both the formation of nascent β1-integrin adhesions and the stimulation of axon extension by BDNF required cytoplasmic calcium ion signaling and integrin activation at the cell surface. Exposure to MAG decreased the number of β1-integrin adhesions in the growth cone during inhibition of axon extension. In contrast, the BDNF-induced adhesions were resistant to negative remodeling by MAG, correlating with the ability of BDNF pretreatment to counteract MAG-inhibition of axon extension. Pre-exposure to MAG prevented the BDNF-induced formation of β1-integrin adhesions and blocked the stimulation of axon extension by BDNF. Conclusions Altogether, these findings demonstrate the neurotrophin-dependent formation of integrin-based adhesions in the growth cone and reveal how a positive regulator of substrate adhesions can block

  7. Endoglin regulates mural cell adhesion in the circulatory system.

    Science.gov (United States)

    Rossi, Elisa; Smadja, David M; Boscolo, Elisa; Langa, Carmen; Arevalo, Miguel A; Pericacho, Miguel; Gamella-Pozuelo, Luis; Kauskot, Alexandre; Botella, Luisa M; Gaussem, Pascale; Bischoff, Joyce; Lopez-Novoa, José M; Bernabeu, Carmelo

    2016-04-01

    The circulatory system is walled off by different cell types, including vascular mural cells and podocytes. The interaction and interplay between endothelial cells (ECs) and mural cells, such as vascular smooth muscle cells or pericytes, play a pivotal role in vascular biology. Endoglin is an RGD-containing counter-receptor for β1 integrins and is highly expressed by ECs during angiogenesis. We find that the adhesion between vascular ECs and mural cells is enhanced by integrin activators and inhibited upon suppression of membrane endoglin or β1-integrin, as well as by addition of soluble endoglin (SolEng), anti-integrin α5β1 antibody or an RGD peptide. Analysis of different endoglin mutants, allowed the mapping of the endoglin RGD motif as involved in the adhesion process. In Eng (+/-) mice, a model for hereditary hemorrhagic telangectasia type 1, endoglin haploinsufficiency induces a pericyte-dependent increase in vascular permeability. Also, transgenic mice overexpressing SolEng, an animal model for preeclampsia, show podocyturia, suggesting that SolEng is responsible for podocytes detachment from glomerular capillaries. These results suggest a critical role for endoglin in integrin-mediated adhesion of mural cells and provide a better understanding on the mechanisms of vessel maturation in normal physiology as well as in pathologies such as preeclampsia or hereditary hemorrhagic telangiectasia.

  8. Ligation of the adhesion-GPCR EMR2 regulates human neutrophil function.

    Science.gov (United States)

    Yona, Simon; Lin, Hsi-Hsien; Dri, Pietro; Davies, John Q; Hayhoe, Richard P G; Lewis, Sion M; Heinsbroek, Sigrid E M; Brown, K Alun; Perretti, Mauro; Hamann, Jörg; Treacher, David F; Gordon, Siamon; Stacey, Martin

    2008-03-01

    At present, approximately 150 different members of the adhesion-G protein-coupled receptor (GPCR) family have been identified in metazoans. Surprisingly, very little is known about their function, although they all possess large extracellular domains coupled to a seven-transmembrane domain, suggesting a potential role in cell adhesion and signaling. Here, we demonstrate how the human-restricted adhesion-GPCR, EMR2 (epidermal growth factor-like module-containing mucin-like hormone receptor), regulates neutrophil responses by potentiating the effects of a number of proinflammatory mediators and show that the transmembrane region is critical for adhesion-GPCR function. Using an anti-EMR2 antibody, ligation of EMR2 increases neutrophil adhesion and migration, and augments superoxide production and proteolytic enzyme degranulation. On neutrophil activation, EMR2 is rapidly translocated to membrane ruffles and the leading edge of the cell. Further supporting the role in neutrophil activation, EMR2 expression on circulating neutrophils is significantly increased in patients with systemic inflammation. These data illustrate a definitive function for a human adhesion-GPCR within the innate immune system and suggest an important role in potentiating the inflammatory response. Ligation of the adhesion-GPCR EMR2 regulates human neutrophil function.

  9. Correlation between {sup 99m}Tc-(V)-DMSA uptake and constitutive level of phosphorylated focal adhesion kinase in an in vitro model of cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Denoyer, Delphine; Perek, Nathalie; Jeune, Nathalie Le; Dubois, Francis [University of Saint-Etienne, Department of Biophysics and Radiopharmaceuticals, ' ' Cell Survival and Adhesion' ' Research Group, Jacques Lisfranc Faculty of Medicine, Saint-Etienne (France); Cornillon, Jerome [University of Saint-Etienne, Department of Haematology, ' ' Cell Survival and Adhesion' ' Research Group, Jacques Lisfranc Faculty of Medicine, Saint-Etienne (France)

    2005-07-01

    Although a number of prognostic indicators have been developed, it is still difficult to predict the biological behaviour of all cancer types. {sup 99m}Tc-(V)-DMSA (V DMSA) uptake and focal adhesion kinase (FAK) expression and activation level could be potential agents for this purpose. We hypothesised the existence of a correlation between V DMSA, whose uptake is linked to phosphate ions, essential compounds for tumour growth and cell proliferation, and the adhesion protein FAK, whose elevated expression and level of constitutive activation are implicated in cancer progression. The aim of this study was to assess the relationship between V DMSA incorporation rate and FAK expression and activation by phosphorylation on tyrosine 397 residue. We determined V DMSA uptake in six different cancer cell lines and we measured FAK expression and activation by using Western Blotting analysis. Correlations with factors known to be associated with poor prognosis, such as invasive potential, resistance to chemotherapy and proliferation rate, were also investigated. The cell lines exhibited different V DMSA incorporation rates. In addition, these cells showed the same FAK expression, but various degrees of activation. A correlation was observed between V DMSA uptake and level of FAK phosphorylation and between V DMSA or constitutive FAK activation and proliferation rate. However, no correlation was shown between these parameters and the other factors tested, i.e. invasive potential and anticancer drug resistance. The results of this in vitro study clearly demonstrate that phosphorylation of FAK, proliferation rate and V DMSA uptake are closely related. Because proliferation and a high level of constitutive FAK activation are linked to cancer progression, it can be assumed that in vivo V DMSA uptake reflects tumour aggressiveness. (orig.)

  10. Role of Protease-Activated Receptor 2 in Regulating Focal Segmental Glomerulosclerosis

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    Yongjun Wang

    2017-02-01

    Full Text Available Background /Aims: The underlying mechanisms leading to focal segmental glomerulosclerosis (FSGS are lacking. In this report, we examined the role of protease-activated receptors (PARs subtype PAR2 and its downstream signals in regulating the pathophysiological process of FSGS. Methods: Nephropathy was induced by intravenous injections of adriamycin (ADR in rats to study FSGS. Western Blot analysis and ELISA were employed to determine the protein expression levels of PAR2 and its downstream signal pathways as well as the levels of PICs. Results: In ADR rats, expression of PAR2, PKCε and PKA was amplified and this was accompanied with increases of pro-inflammatory cytokines (PICs including IL-1β, IL-6 and TNF-α. Inhibition of PAR2 signal by systemic administration of FSLLRY-NH2 (FSL attenuated amplification of PICs. Notably, FSL further influenced key molecular mediators during development of FSGS. i.e., it specifically restored the impaired nephrin and attenuated the exaggerated transforming growth factor beta 1 (TGF-β1, caspase-9 and desmin thereby improving worsened renal functions and glomerular injury. Consistent with this, in cultured podocytes FSL also largely restored downregulation of nephrin and attenuated amplifications of caspase-9 and desmin induced by TGF-β1. Conclusions: Results of this study suggest that PAR2 plays an important role in mediating renal injury induced by glomerulosclerosis. Inhibition of PAR2 signal pathway has a protective effect on FSGS mainly via PIC and TGF-β1 mechanisms. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of FSGS observed in patients.

  11. Cadherin-mediated adhesion regulates posterior body formation

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    Fasanmi Oluwafoyinsa

    2007-11-01

    Full Text Available Abstract Background The anterior-posterior axis of the vertebrate embryo undergoes a dramatic elongation during early development. Convergence and extension of the mesoderm, occurring during gastrulation, initiates the narrowing and lengthening of the embryo. However the lengthening of the axis continues during post-gastrula stages in the tailbud region, and is thought to involve convergent extension movements as well as other cell behaviors specific to posterior regions. Results We demonstrate here, using a semi-dominant N-cadherin allele, that members of the classical cadherin subfamily of cell-cell adhesion molecules are required for tailbud elongation in the zebrafish. In vivo imaging of cell behaviors suggests that the extension of posterior axial mesodermal cells is impaired in embryos that carry the semi-dominant N-cadherin allele. This defect most likely results from a general loss of cell-cell adhesion in the tailbud region. Consistent with these observations, N-cadherin is expressed throughout the tailbud during post-gastrulation stages. In addition, we show that N-cadherin interacts synergistically with vang-like 2, a member of the non-canonical Wnt signaling/planar cell polarity pathway, to mediate tail morphogenesis. Conclusion We provide the first evidence here that N-cadherin and other members of the classical cadherin subfamily function in parallel with the planar cell polarity pathway to shape the posterior axis during post-gastrulation stages. These findings further highlight the central role that adhesion molecules play in the cellular rearrangements that drive morphogenesis in vertebrates and identify classical cadherins as major contributors to tail development.

  12. Antitumor effect of focal adhesion kinase inhibitor PF562271 against human osteosarcoma in vitro and in vivo.

    Science.gov (United States)

    Hu, Chuanzhen; Chen, Xu; Wen, Junxiang; Gong, Liangzhi; Liu, Zhuochao; Wang, Jun; Liang, Jing; Hu, Fangqiong; Zhou, Qi; Wei, Li; Shen, Yuhui; Zhang, Weibin

    2017-07-01

    Focal adhesion kinase (FAK) overexpression is related to invasive and metastatic properties in different kinds of cancers. Target therapy by inhibiting FAK has achieved promising effect in some cancer treatments, but its effect in human osteosarcoma has not been well studied. In the present study, we analyzed the antitumor efficacy of PF562271, an FAK inhibitor, against osteosarcoma in vitro and in vivo. Phosphorylated FAK (Y397) was highly expressed in primary human osteosarcoma tumor samples and was associated with osteosarcoma prognosis and lung metastasis. PF562271 greatly suppressed proliferation and colony formation in human osteosarcoma cell lines. In addition, treatment of osteosarcoma cell lines with PF562271 induced apoptosis and downregulated the activity of the protein kinase B/mammalian target of rapamycin pathway. PF562271 also impaired the tube formation ability of endothelial cells in vitro. Finally, oral treatment with PF562271 in mice dramatically reduced tumor volume, weight, and angiogenesis of osteosarcoma xenografts in vivo. These results indicate that FAK inhibitor PF562271 can potentially be effectively used for the treatment of osteosarcoma. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  13. A small physiological electric field mediated responses of extravillous trophoblasts derived from HTR8/SVneo cells: involvement of activation of focal adhesion kinase signaling.

    Directory of Open Access Journals (Sweden)

    Juan Zhang

    Full Text Available Moderate invasion of trophoblast cells into endometrium is essential for the placental development and normal pregnancy. Electric field (EF-induced effects on cellular behaviors have been observed in many cell types. This study was to investigate the effect of physiological direct current EF (dc EF on cellular responses such as elongation, orientation and motility of trophoblast cells. Immortalized first trimester extravillous trophoblast cells (HTR-8/SVneo were exposed to the dc EF at physiological magnitude. Cell images were recorded and analyzed by image analyzer. Cell lysates were used to detect protein expression by Western blot. Cultured in the dc EFs the cells showed elongation, orientation and enhanced migration rate compared with non-EF stimulated cells at field strengths of 100 mV/mm to 200 mV/mm. EF exposure increased focal adhesion kinase (FAK phosphorylation in a time-dependent manner and increased expression levels of MMP-2. Pharmacological inhibition of FAK impaired the EF-induced responses including motility and abrogated the elevation of MMP-2 expression. However, the expression levels of integrins like integrin α1, α5, αV and β1 were not affected by EF stimulation. Our results demonstrate the importance of FAK activation in migration/motility of trophobalst cells driven by EFs. In addition, it raises the feasibility of using applied EFs to promote placentation through effects on trophoblast cells.

  14. A ZIP6-ZIP10 heteromer controls NCAM1 phosphorylation and integration into focal adhesion complexes during epithelial-to-mesenchymal transition

    Science.gov (United States)

    Brethour, Dylan; Mehrabian, Mohadeseh; Williams, Declan; Wang, Xinzhu; Ghodrati, Farinaz; Ehsani, Sepehr; Rubie, Elizabeth A.; Woodgett, James R.; Sevalle, Jean; Xi, Zhengrui; Rogaeva, Ekaterina; Schmitt-Ulms, Gerold

    2017-01-01

    The prion protein (PrP) evolved from the subbranch of ZIP metal ion transporters comprising ZIPs 5, 6 and 10, raising the prospect that the study of these ZIPs may reveal insights relevant for understanding the function of PrP. Building on data which suggested PrP and ZIP6 are critical during epithelial-to-mesenchymal transition (EMT), we investigated ZIP6 in an EMT paradigm using ZIP6 knockout cells, mass spectrometry and bioinformatic methods. Reminiscent of PrP, ZIP6 levels are five-fold upregulated during EMT and the protein forms a complex with NCAM1. ZIP6 also interacts with ZIP10 and the two ZIP transporters exhibit interdependency during their expression. ZIP6 contributes to the integration of NCAM1 in focal adhesion complexes but, unlike cells lacking PrP, ZIP6 deficiency does not abolish polysialylation of NCAM1. Instead, ZIP6 mediates phosphorylation of NCAM1 on a cluster of cytosolic acceptor sites. Substrate consensus motif features and in vitro phosphorylation data point toward GSK3 as the kinase responsible, and interface mapping experiments identified histidine-rich cytoplasmic loops within the ZIP6/ZIP10 heteromer as a novel scaffold for GSK3 binding. Our data suggests that PrP and ZIP6 inherited the ability to interact with NCAM1 from their common ZIP ancestors but have since diverged to control distinct posttranslational modifications of NCAM1. PMID:28098160

  15. Cytomegalovirus Destruction of Focal Adhesions Revealed in a High-Throughput Western Blot Analysis of Cellular Protein Expression† ▿

    OpenAIRE

    Stanton, Richard James; McSharry, Brian Patrick; Rickards, Carole Ruth; Wang, Edward Chung Yern; Tomasec, Peter; Wilkinson, Gavin William Grahame

    2007-01-01

    Human cytomegalovirus (HCMV) systematically manages the expression of cellular functions, rather than exerting the global shutoff of host cell protein synthesis commonly observed with other herpesviruses during the lytic cycle. While microarray technology has provided remarkable insights into viral control of the cellular transcriptome, HCMV is known to encode multiple mechanisms for posttranscriptional and posttranslation regulation of cellular gene expression. High-throughput Western blotti...

  16. The focal adhesion-associated proteins DOCK5 and GIT2 comprise a rheostat in control of epithelial invasion

    DEFF Research Database (Denmark)

    Frank, S R; Köllmann, C P; van Lidth de Jeude, J F;

    2017-01-01

    DOCK proteins are guanine nucleotide exchange factors for Rac and Cdc42 GTPases. DOCK1 is the founding member of the family and acts downstream of integrins via the canonical Crk-p130Cas complex to activate Rac GTPases in numerous contexts. In contrast, DOCK5, which possesses the greatest...... with these cells. Collectively, our work identifies DOCK5 as a key regulator of epithelial invasion and metastasis, and demonstrates that suppression of DOCK5 by GIT2 represents a previously unappreciated mechanism for coordination of Rho and Rac GTPases.Oncogene advance online publication, 26 September 2016; doi...

  17. NDRG2 inhibits hepatocellular carcinoma adhesion, migration and invasion by regulating CD24 expression

    Directory of Open Access Journals (Sweden)

    Tao Yurong

    2011-06-01

    Full Text Available Abstract Background The prognosis of most hepatocellular carcinoma (HCC patients is poor due to the high metastatic rate of the disease. Understanding the molecular mechanisms underlying HCC metastasis is extremely urgent. The role of CD24 and NDRG2 (N-myc downstream-regulated gene 2, a candidate tumor suppressor gene, has not yet been explored in HCC. Methods The mRNA and protein expression of CD24 and NDRG2 was analyzed in MHCC97H, Huh7 and L-02 cells. Changes in cell adhesion, migration and invasion were detected by up- or down-regulating NDRG2 by adenovirus or siRNA. The expression pattern of NDRG2 and CD24 in HCC tissues and the relationship between NDRG2 and HCC clinical features was analyzed by immunohistochemical and western blotting analysis. Results NDRG2 expression was negatively correlated with malignancy in HCC. NDRG2 exerted anti-tumor activity by regulating CD24, a molecule that mediates cell-cell interaction, tumor proliferation and adhesion. NDRG2 up-regulation decreased CD24 expression and cell adhesion, migration and invasion. By contrast, NDRG2 down-regulation enhanced CD24 expression and cell adhesion, migration and invasion. Immunohistochemical analysis of 50 human HCC clinical specimens showed a strong correlation between NDRG2 down-regulation and CD24 overexpression (P = 0.04. In addition, increased frequency of NDRG2 down-regulation was observed in patients with elevated AFP serum level (P = 0.006, late TNM stage (P = 0.009, poor differentiation grade (P = 0.002, tumor invasion (P = 0.004 and recurrence (P = 0.024. Conclusions Our findings indicate that NDRG2 and CD24 regulate HCC adhesion, migration and invasion. The expression level of NDRG2 is closely related to the clinical features of HCC. Thus, NDRG2 plays an important physiological role in HCC metastasis.

  18. Mutations in X-linked PORCN, a putative regulator of Wnt signaling, cause focal dermal hypoplasia

    Science.gov (United States)

    Focal dermal hypoplasia is an X-linked dominant disorder characterized by patchy hypoplastic skin and digital, ocular, and dental malformations. We used array comparative genomic hybridization to identify a 219-kb deletion in Xp11.23 in two affected females. We sequenced genes in this region and fou...

  19. Targeted inhibition of Focal Adhesion Kinase Attenuates Cardiac Fibrosis and Preserves Heart Function in Adverse Cardiac Remodeling

    Science.gov (United States)

    Zhang, Jie; Fan, Guangpu; Zhao, Hui; Wang, Zhiwei; Li, Fei; Zhang, Peide; Zhang, Jing; Wang, Xu; Wang, Wei

    2017-01-01

    Cardiac fibrosis in post-myocardial infarction (MI), seen in both infarcted and non-infarcted myocardium, is beneficial to the recovery of heart function. But progressively pathological fibrosis impairs ventricular function and leads to poor prognosis. FAK has recently received attention as a potential mediator of fibrosis, our previous study reported that pharmacological inhibition of FAK can attenuate cardiac fibrosis in post MI models. However, the long-term effects on cardiac function and adverse cardiac remodelling were not clearly investigated. In this study, we tried to determine the preliminary mechanisms in regulating CF transformation to myofibroblasts and ECM synthesis relevant to the development of adverse cardiac remolding in vivo and in vitro. Our study provides even more evidence that FAK is directly related to the activation of CF in hypoxia condition in a dose-dependent and time-dependent manner. Pharmacological inhibition of FAK significantly reduces myofibroblast differentiation; our in vivo data demonstrated that a FAK inhibitor significantly decreases fibrotic score, and preserves partial left ventricular function. Both PI3K/AKT signalling and ERK1/2 are necessary for hypoxia-induced CF differentiation and ECM synthesis; this process also involves lysyl oxidase (LOX). These findings suggest that pharmacological inhibition of FAK may become an effective therapeutic strategy against adverse fibrosis. PMID:28225063

  20. The direct effect of Focal Adhesion Kinase (FAK, dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

    Directory of Open Access Journals (Sweden)

    Zhang Li

    2009-08-01

    Full Text Available Abstract Background Focal adhesion kinase (FAK is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis. Methods To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD, and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors. Results Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p Conclusion Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal

  1. Quorum Sensing Regulation of Adhesion in Serratia Marcescens MG1 is surface dependent

    DEFF Research Database (Denmark)

    Labbate, M.; Zhu, H.; Thung, L.;

    2007-01-01

    Serratia marcescens is an opportunistic pathogen and a major cause of ocular infections. In previous studies of S. marcescens MG1, we showed that biofilm maturation and sloughing were regulated by N-acyl homoserine lactone (AHL)-based quorum sensing (QS). Because of the importance of adhesion...

  2. Synopsis and meta-analysis of genetic association studies in osteoporosis for the focal adhesion family genes: the CUMAGAS-OSTEOporosis information system

    Directory of Open Access Journals (Sweden)

    Kastanis Alkibiadis

    2011-01-01

    Full Text Available Abstract Background Focal adhesion (FA family genes have been studied as candidate genes for osteoporosis, but the results of genetic association studies (GASs are controversial. To clarify these data, a systematic assessment of GASs for FA genes in osteoporosis was conducted. Methods We developed Cumulative Meta-Analysis of GAS-OSTEOporosis (CUMAGAS-OSTEOporosis, a web-based information system that allows the retrieval, analysis and meta-analysis (for allele contrast, recessive, dominant, additive and codominant models of data from GASs on osteoporosis with the capability of update. GASs were identified by searching the PubMed and HuGE PubLit databases. Results Data from 72 studies involving 13 variants of 6 genes were analyzed and catalogued in CUMAGAS-OSTEOporosis. Twenty-two studies produced significant associations with osteoporosis risk under any genetic model. All studies were underpowered (1 (COL1A1 G2046T (all genetic models, COL1A1 G-1997T (allele contrast and dominant model and integrin β-chain β3 (ITGB3 T176C (recessive and additive models. In COL1A1 G2046T, subgroup analysis has shown significant associations for Caucasians, adults, females, males and postmenopausal women. A differential magnitude of effect in large versus small studies (that is, indication of publication bias was detected for the variant COL1A1 G2046T. Conclusion There is evidence of an implication of FA family genes in osteoporosis. CUMAGAS-OSTEOporosis could be a useful tool for current genomic epidemiology research in the field of osteoporosis.

  3. A novel orally available inhibitor of focal adhesion signaling increases survival in a xenograft model of diffuse large B-cell lymphoma with central nervous system involvement.

    Science.gov (United States)

    Bosch, Rosa; Moreno, María José; Dieguez-Gonzalez, Rebeca; Céspedes, María Virtudes; Gallardo, Alberto; Trias, Manuel; Grañena, Albert; Sierra, Jorge; Casanova, Isolda; Mangues, Ramon

    2013-08-01

    Central nervous system dissemination is a relatively uncommon but almost always fatal complication in diffuse large B-cell lymphoma patients. Optimal therapy for central nervous involvement in this malignancy has not been established. In this paper, we aimed to evaluate the therapeutic effect of E7123, a celecoxib derivative that inhibits focal adhesion signaling, in a novel xenograft model of diffuse large B-cell lymphoma with central nervous system involvement. Cells obtained after disaggregation of HT subcutaneous tumors (HT-SC cells) were intravenously injected in NOD/SCID mice. These mice received oral vehicle or 75 mg/kg of E7123 daily until they were euthanized for weight loss or signs of sickness. The antitumor effect of E7123 was validated in an independent experiment using a bioluminescent mouse model. Intravenously injected HT-SC cells showed higher take rate and higher central nervous system tropism (associated with increased expression of β1-integrin and p130Cas proteins) than HT cells. The oral administration of E7123 significantly increased survival time in 2 independent experiments using mice injected with unmodified or bioluminescent HT-SC cells. We have developed a new xenograft model of diffuse large B-cell lymphoma with central nervous system involvement that can be used in the pre-clinical evaluation of new drugs for this malignancy. E7123 is a new, well-tolerated and orally available therapeutic agent that merits further investigation since it may improve current management of diffuse large B-cell lymphoma patients with central nervous system involvement.

  4. Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

    Science.gov (United States)

    Fang, Jung-Da; Lee, Sheau-Ling

    2014-07-01

    Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

  5. A phase I study of VS-6063, a second-generation focal adhesion kinase inhibitor, in patients with advanced solid tumors.

    Science.gov (United States)

    Jones, Suzanne F; Siu, Lillian L; Bendell, Johanna C; Cleary, James M; Razak, Albiruni R A; Infante, Jeffrey R; Pandya, Shuchi S; Bedard, Philippe L; Pierce, Kristen J; Houk, Brett; Roberts, W Gregory; Shreeve, S Martin; Shapiro, Geoffrey I

    2015-10-01

    VS-6063 (also known as defactinib or PF-04554878) is a second-generation inhibitor of focal adhesion kinase (FAK) and proline-rich tyrosine kinase-2 (Pyk2). This phase I dose-escalation study was conducted in patients with advanced solid malignancies. Using a traditional 3 + 3 design, VS-6063 was administered orally twice daily (b.i.d.) in 21-day cycles to cohorts of three to six patients. In cycle 1, a lead-in dose was administered to assess single-dose pharmacokinetics; steady-state pharmacokinetics was assessed after 15 days of continuous dosing. Dose escalation was performed in the fasted state, and repeated in two additional cohorts in the fed state. Forty-six patients were treated across nine dose levels (12.5-750 mg b.i.d.). Dose-limiting toxicities, comprising headache (n = 1), fatigue (n = 1) and unconjugated hyperbilirubinemia (n = 3), occurred at the 300- or 425-mg b.i.d. dose level and were reversible. Frequent adverse events included nausea (37 %), fatigue (33 %), vomiting (28 %), diarrhea (22 %) and headache (22 %). A maximum-tolerated dose was not defined. Dose escalation was stopped at the 750-mg b.i.d. dose due to decreased serum exposure in the 500- and 750-mg versus 300- and 425-mg groups. Food delayed the time to peak serum concentration without affecting serum drug exposure. No radiographic responses were reported. Disease stabilization at ~12 weeks occurred in six of 37 (16 %) patients receiving doses ≥100 mg b.i.d. VS-6063 has an acceptable safety profile. Treatment-related adverse events were mild to moderate, and reversible. The recommended phase II fasting dose of VS-6063 is 425 mg b.i.d.

  6. Genetic regulation of the intercellular adhesion locus in staphylococci

    Directory of Open Access Journals (Sweden)

    David R Cue

    2012-03-01

    Full Text Available The formation of biofilms by Staphylococcus aureus and Staphylococcus epidermidis is an important aspect of many staphylococcal infections, most notably endocarditis, osteomyelitis and infections associated with indwelling medical devices. The major constituents of S. aureus biofilms are polysaccharides, such as poly N-acetyl glucosamine (PIA/PNAG, cell surface and secreted bacterial proteins, and extracellular DNA. The exact composition of biofilms often varies considerably between different strains of staphylococci and between different sites of infection by the same strain. PIA/PNAG is synthesized by the products of 4 genes, icaADBC, that are encoded in a single operon. A fifth gene, icaR, is a negative regulator of icaADBC. Expression of icaADBC is tightly regulated, but can often be induced in vitro by growing staphylococci in the presence of high salt, high glucose or ethanol. Regulation of icaADBC is complex and numerous regulatory factors have been implicated in control of icaADBC. Many of these are well known global transcriptional regulatory factors like SarA and sigmaB, whereas other regulators, such as IcaR, seem to affect expression of relatively few genes. Here, we will attempt to summarize how various regulatory factors affect the production of PIA/PNAG in staphylococci.

  7. The integrin-ligand interaction regulates adhesion and migration through a molecular clutch.

    Directory of Open Access Journals (Sweden)

    Lingfeng Chen

    Full Text Available Adhesive and migratory behavior can be cell type, integrin, and substrate dependent. We have compared integrin and substrate differences using three integrin receptors: α5β1, α6β1, and αLβ2 expressed in a common cell type, CHO.B2 cells, which lack integrin α subunits, as well as in different cell types that express one or more of these integrins. We find that CHO.B2 cells expressing either α6β1 or αLβ2 integrins migrate and protrude faster and are more directionally persistent on laminin or ICAM-1, respectively, than CHO.B2 cells expressing α5β1 on fibronectin. Despite rapid adhesion maturation and the presence of large adhesions in both the α6β1- and αLβ2-expressing cells, they display robust tyrosine phosphorylation. In addition, whereas myosin II regulates adhesion maturation and turnover, protrusion rates, and polarity in cells migrating on fibronectin, surprisingly, it does not have comparable effects in cells expressing α6β1 or αLβ2. This apparent difference in the integration of myosin II activity, adhesion, and migration arises from alterations in the ligand-integrin-actin linkage (molecular clutch. The elongated adhesions in the protrusions of the α6β1-expressing cells on laminin or the αLβ2-expressing cells on ICAM-1 display a novel, rapid retrograde flux of integrin; this was largely absent in the large adhesions in protrusions of α5β1-expressing cells on fibronectin. Furthermore, the force these adhesions exert on the substrate in protrusive regions is reduced compared to similar regions in α5-expressing cells, and the adhesion strength is reduced. This suggests that intracellular forces are not efficiently transferred from actomyosin to the substratum due to altered adhesion strength, that is, avidity, affinity, or the ligand-integrin-actin interaction. Finally, we show that the migration of fast migrating leukocytes on fibronectin or ICAM-1 is also largely independent of myosin II; however, their

  8. Patterning of cell assemblies regulated by adhesion receptors of the cadherin superfamily.

    Science.gov (United States)

    Takeichi, M; Nakagawa, S; Aono, S; Usui, T; Uemura, T

    2000-07-29

    During morphogenesis, cell-cell association patterns are dynamically altered. We are interested in how cell adhesion molecules can regulate the patterning of cellular assemblies. Cadherins, a group of cell-cell adhesion receptors, are crucial for the organized assembly of many cell types, but they also regulate dynamic aspects of cell association. For example, during neural crest emigration from the neural tube, the cadherin subtypes expressed by crest cells are switched from one subtype to another. Artificial perturbation of this switch results in blocking of their escape from the neural tube. Intracellular modulations of cadherin activity also seem to play a role in regulation of cell adhesion. We identified p120ctn as a regulator of cadherin function in carcinoma cells. With such regulators, cells may make a choice as to whether they should maintain stable cell contacts or disrupt their association. Finally, we found another type of cadherin-mediated cell patterning: Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity in Drosophila imaginal discs. Thus, the cadherin superfamily receptors control the patterning of cell assemblies through a variety of mechanisms.

  9. Differential Regulation of Adhesion Complex Turnover by ROCK1 and ROCK2

    OpenAIRE

    Lock, Frances E.; Katie R Ryan; Poulter, Natalie S.; Maddy Parsons; Hotchin, Neil A

    2012-01-01

    BACKGROUND: ROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration. METHODOLOGY/PRINCIPAL FINDINGS: In this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibi...

  10. Msh homeobox genes regulate cadherin-mediated cell adhesion and cell-cell sorting.

    Science.gov (United States)

    Lincecum, J M; Fannon, A; Song, K; Wang, Y; Sassoon, D A

    1998-07-01

    Msx-1 and Msx-2 are two closely related homeobox genes expressed in cephalic neural crest tooth buds, the optic cup endocardial cushions, and the developing limb [Hill and Davidson, 1991; Monaghan et al., 1991; Robert et al., 1991]. These sites correspond to regions of active cell segregation and proliferation under the influence of epithelial-mesenchymal cell interactions [Brown et al., 1993; Davidson et al., 1991], suggesting that Msx-1 and Msx-2 regulate cell-cell interactions. We have investigated the potential relationship between expression of the Msh homeobox genes (Msx-1 and Msx-2) and cadherin-mediated cell adhesion and cell sorting. We report that cell lines stably expressing Msx-1 or Msx-2 differentially sort on the basis of Msh gene expression. We demonstrate in vitro that initial cell aggregation involves calcium-dependent adhesion molecules (cadherins) and that Msh genes regulate cadherin-mediated adhesion. These results support the hypothesis that Msh genes play a role in the regulation of cell-cell adhesion and provide a link between the genetic phenomena of homeobox gene expression and cellular events involved in morphogenesis, including cell sorting and proliferation.

  11. Siah regulation of Pard3A controls neuronal cell adhesion during germinal zone exit.

    Science.gov (United States)

    Famulski, Jakub K; Trivedi, Niraj; Howell, Danielle; Yang, Yuan; Tong, Yiai; Gilbertson, Richard; Solecki, David J

    2010-12-24

    The brain's circuitry is established by directed migration and synaptogenesis of neurons during development. Although neurons mature and migrate in specific patterns, little is known about how neurons exit their germinal zone niche. We found that cerebellar granule neuron germinal zone exit is regulated by proteasomal degradation of Pard3A by the Seven in Absentia homolog (Siah) E3 ubiquitin ligase. Pard3A gain of function and Siah loss of function induce precocious radial migration. Time-lapse imaging using a probe to measure neuronal cell contact reveals that Pard3A promotes adhesive interactions needed for germinal zone exit by recruiting the epithelial tight junction adhesion molecule C to the neuronal cell surface. Our findings define a Siah-Pard3A signaling pathway that controls adhesion-dependent exit of neuronal progenitors or immature neurons from a germinal zone niche.

  12. Preparation and regulating cell adhesion of anion-exchangeable layered double hydroxide micropatterned arrays.

    Science.gov (United States)

    Yao, Feng; Hu, Hao; Xu, Sailong; Huo, Ruijie; Zhao, Zhiping; Zhang, Fazhi; Xu, Fujian

    2015-02-25

    We describe a reliable preparation of MgAl-layered double hydroxide (MgAl-LDH) micropatterned arrays on gold substrate by combining SO3(-)-terminated self-assembly monolayer and photolithography. The synthesis route is readily extended to prepare LDH arrays on the SO3(-)-terminated polymer-bonded glass substrate amenable for cell imaging. The anion-exchangeable MgAl-LDH micropattern can act both as bioadhesive region for selective cell adhesion and as nanocarrier for drug molecules to regulate cell behaviors. Quantitative analysis of cell adhesion shows that selective HepG2 cell adhesion and spreading are promoted by the micropatterned MgAl-LDH, and also suppressed by methotrexate drug released from the LDH interlayer galleries.

  13. Down-regulation of vimentin expression inhibits carcinoma cell migration and adhesion.

    Science.gov (United States)

    McInroy, Lorna; Määttä, Arto

    2007-08-17

    Vimentin is a type III Intermediate filament protein that is expressed frequently in epithelial carcinomas correlating with invasiveness and poor prognosis. We have analysed migration and adhesion to collagenous matrix of a panel of carcinoma cell lines. In vitro invasiveness was highest in vimentin-positive SW480 colon cancer and MDA-MB-231 breast cancer cells and the role of vimentin in these cell lines was investigated by RNA interference. Down-regulation of vimentin expression resulted in impaired migration in both scratch-wound experiments and in invasion assays through cell culture inserts coated with collagen gel. Compromised migration was observed in both cell lines, whereas cell attachment assays revealed impaired adhesion to fibrillar collagen in MDA-MB-231 cells while the adhesion of vimentin-ablated SW480 cells, that express both vimentin and keratin intermediate filaments was not affected. In conclusion, ablation of vimentin expression inhibits migration and invasion of colon and breast cancer cell lines.

  14. A dual role for Sonic hedgehog in regulating adhesion and differentiation of neuroepithelial cells.

    Science.gov (United States)

    Jarov, Artem; Williams, Kevin P; Ling, Leona E; Koteliansky, Victor E; Duband, Jean-Loup; Fournier-Thibault, Claire

    2003-09-15

    In vertebrates, the nervous system arises from a flat sheet of epithelial cells, the neural plate, that gradually transforms into a hollow neural tube. This process, called neurulation, involves sequential changes in cellular interactions that are precisely coordinated both spatially and temporally by the combined actions of morphogens. To gain further insight into the molecular events regulating cell adhesion during neurulation, we investigated whether the adhesive and migratory capacities of neuroepithelial cells might be modulated by Sonic hedgehog (Shh), a signaling molecule involved in the control of cell differentiation in the ventral neural tube. When deposited onto extracellular matrix components in vitro, neural plates explanted from avian embryos at early neurulation readily dispersed into monolayers of spread cells, thereby revealing their intrinsic ability to migrate. In the presence of Shh added in solution to the culture medium, the explants still exhibited the same propensity to disperse. In contrast, when Shh was immobilized to the substrate or produced by neuroepithelial cells themselves after transfection, neural plate explants failed to disperse and instead formed compact structures. Changes in the adhesive capacities of neuroepithelial cells caused by Shh could be accounted for by inactivation of surface beta1-integrins combined with an increase in N-cadherin-mediated cell adhesion. Furthermore, immobilized Shh promoted differentiation of neuroepithelial cells into motor neurons and floor plate cells with the same potency as soluble Shh. However, the effect of Shh on the neuroepithelial cell adhesion was discernible and apparently independent from its differentiation effect and was not mediated by the signaling cascade elicited by the Patched-Smoothened receptor and involving the Gli transcription factors. Thus, our experiments indicate that Shh is able to control sequentially adhesion and differentiation of neuroepithelial cells through

  15. ALCAM/CD166 adhesive function is regulated by the tetraspanin CD9.

    Science.gov (United States)

    Gilsanz, Alvaro; Sánchez-Martín, Lorena; Gutiérrez-López, María Dolores; Ovalle, Susana; Machado-Pineda, Yesenia; Reyes, Raquel; Swart, Guido W; Figdor, Carl G; Lafuente, Esther M; Cabañas, Carlos

    2013-02-01

    ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM-ALCAM) or heterophilic (ALCAM-CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenomena, with particular relevance in leukocyte extravasation, stabilization of the immunological synapse, T cell activation and proliferation and tumor growth and metastasis. Although the functional implications of ALCAM in these processes is well established, the mechanisms regulating its adhesive capacity remain obscure. Using confocal microscopy colocalization, and biochemical and functional analyses, we found that ALCAM directly associates with the tetraspanin CD9 on the leukocyte surface in protein complexes that also include the metalloproteinase ADAM17/TACE. The functional relevance of these interactions is evidenced by the CD9-induced upregulation of both homophilic and heterophilic ALCAM interactions, as reflected by increased ALCAM-mediated cell adhesion and T cell migration, activation and proliferation. The enhancement of ALCAM function induced by CD9 is mediated by a dual mechanism involving (1) augmented clustering of ALCAM molecules, and (2) upregulation of ALCAM surface expression due to inhibition of ADAM17 sheddase activity.

  16. Neuropeptide-induced androgen independence in prostate cancer cells: roles of nonreceptor tyrosine kinases Etk/Bmx, Src, and focal adhesion kinase.

    Science.gov (United States)

    Lee, L F; Guan, J; Qiu, Y; Kung, H J

    2001-12-01

    The bombesin/gastrin-releasing peptide (GRP) family of neuropeptides has been implicated in various in vitro and in vivo models of human malignancies including prostate cancers. It was previously shown that bombesin and/or neurotensin (NT) acts as a survival and migratory factor(s) for androgen-independent prostate cancers. However, a role in the transition from an androgen-dependent to -refractory state has not been addressed. In this study, we investigate the biological effects and signal pathways of bombesin and NT on LNCaP, a prostate cancer cell line which requires androgen for growth. We show that both neurotrophic factors can induce LNCaP growth in the absence of androgen. Concurrent transactivation of reporter genes driven by the prostate-specific antigen promoter or a promoter carrying an androgen-responsive element (ARE) indicate that growth stimulation is accompanied by androgen receptor (AR) activation. Furthermore, neurotrophic factor-induced gene activation was also present in PC3 cells transfected with the AR but not in the parental line which lacks the AR. Given that bombesin does not directly bind to the AR and is known to engage a G-protein-coupled receptor, we investigated downstream signaling events that could possibly interact with the AR pathway. We found that three nonreceptor tyrosine kinases, focal adhesion kinase (FAK), Src, and Etk/BMX play important parts in this process. Etk/Bmx activation requires FAK and Src and is critical for neurotrophic factor-induced growth, as LNCaP cells transfected with a dominant-negative Etk/BMX fail to respond to bombesin. Etk's activation requires FAK, Src, but not phosphatidylinositol 3-kinase. Likewise, bombesin-induced AR activation is inhibited by the dominant-negative mutant of either Src or FAK. Thus, in addition to defining a new G-protein pathway, this report makes the following points regarding prostate cancer. (i) Neurotrophic factors can activate the AR, thus circumventing the normal growth

  17. Focal Adhesion Kinase Inhibitors in Combination with Erlotinib Demonstrate Enhanced Anti-Tumor Activity in Non-Small Cell Lung Cancer.

    Directory of Open Access Journals (Sweden)

    Grant A Howe

    Full Text Available Blockade of epidermal growth factor receptor (EGFR activity has been a primary therapeutic target for non-small cell lung cancers (NSCLC. As patients with wild-type EGFR have demonstrated only modest benefit from EGFR tyrosine kinase inhibitors (TKIs, there is a need for additional therapeutic approaches in patients with wild-type EGFR. As a key component of downstream integrin signalling and known receptor cross-talk with EGFR, we hypothesized that targeting focal adhesion kinase (FAK activity, which has also been shown to correlate with aggressive stage in NSCLC, would lead to enhanced activity of EGFR TKIs. As such, EGFR TKI-resistant NSCLC cells (A549, H1299, H1975 were treated with the EGFR TKI erlotinib and FAK inhibitors (PF-573,228 or PF-562,271 both as single agents and in combination. We determined cell viability, apoptosis and 3-dimensional growth in vitro and assessed tumor growth in vivo. Treatment of EGFR TKI-resistant NSCLC cells with FAK inhibitor alone effectively inhibited cell viability in all cell lines tested; however, its use in combination with the EGFR TKI erlotinib was more effective at reducing cell viability than either treatment alone when tested in both 2- and 3-dimensional assays in vitro, with enhanced benefit seen in A549 cells. This increased efficacy may be due in part to the observed inhibition of Akt phosphorylation when the drugs were used in combination, where again A549 cells demonstrated the most inhibition following treatment with the drug combination. Combining erlotinib with FAK inhibitor was also potent in vivo as evidenced by reduced tumor growth in the A549 mouse xenograft model. We further ascertained that the enhanced sensitivity was irrespective of the LKB1 mutational status. In summary, we demonstrate the effectiveness of combining erlotinib and FAK inhibitors for use in known EGFR wild-type, EGFR TKI resistant cells, with the potential that a subset of cell types, which includes A549, could be

  18. Cellular prion protein is required for neuritogenesis: fine-tuning of multiple signaling pathways involved in focal adhesions and actin cytoskeleton dynamics

    Directory of Open Access Journals (Sweden)

    Alleaume-Butaux A

    2013-07-01

    Full Text Available Aurélie Alleaume-Butaux,1,2 Caroline Dakowski,1,2 Mathéa Pietri,1,2 Sophie Mouillet-Richard,1,2 Jean-Marie Launay,3,4 Odile Kellermann,1,2 Benoit Schneider1,2 1INSERM, UMR-S 747, 2Paris Descartes University, Sorbonne Paris Cité, UMR-S 747, 3Public Hospital of Paris, Department of Biochemistry, INSERM UMR-S 942, Lariboisière Hospital, Paris, France; 4Pharma Research Department, Hoffmann La Roche Ltd, Basel, Switzerland Abstract: Neuritogenesis is a dynamic phenomenon associated with neuronal differentiation that allows a rather spherical neuronal stem cell to develop dendrites and axon, a prerequisite for the integration and transmission of signals. The acquisition of neuronal polarity occurs in three steps: (1 neurite sprouting, which consists of the formation of buds emerging from the postmitotic neuronal soma; (2 neurite outgrowth, which represents the conversion of buds into neurites, their elongation and evolution into axon or dendrites; and (3 the stability and plasticity of neuronal polarity. In neuronal stem cells, remodeling and activation of focal adhesions (FAs associated with deep modifications of the actin cytoskeleton is a prerequisite for neurite sprouting and subsequent neurite outgrowth. A multiple set of growth factors and interactors located in the extracellular matrix and the plasma membrane orchestrate neuritogenesis by acting on intracellular signaling effectors, notably small G proteins such as RhoA, Rac, and Cdc42, which are involved in actin turnover and the dynamics of FAs. The cellular prion protein (PrPC, a glycosylphosphatidylinositol (GPI-anchored membrane protein mainly known for its role in a group of fatal neurodegenerative diseases, has emerged as a central player in neuritogenesis. Here, we review the contribution of PrPC to neuronal polarization and detail the current knowledge on the signaling pathways fine-tuned by PrPC to promote neurite sprouting, outgrowth, and maintenance. We emphasize that Pr

  19. Endothelial Cell Junctional Adhesion Molecules: Role and Regulation of Expression in Inflammation.

    Science.gov (United States)

    Reglero-Real, Natalia; Colom, Bartomeu; Bodkin, Jennifer Victoria; Nourshargh, Sussan

    2016-10-01

    Endothelial cells line the lumen of all blood vessels and play a critical role in maintaining the barrier function of the vasculature. Sealing of the vessel wall between adjacent endothelial cells is facilitated by interactions involving junctionally expressed transmembrane proteins, including tight junctional molecules, such as members of the junctional adhesion molecule family, components of adherence junctions, such as VE-Cadherin, and other molecules, such as platelet endothelial cell adhesion molecule. Of importance, a growing body of evidence indicates that the expression of these molecules is regulated in a spatiotemporal manner during inflammation: responses that have significant implications for the barrier function of blood vessels against blood-borne macromolecules and transmigrating leukocytes. This review summarizes key aspects of our current understanding of the dynamics and mechanisms that regulate the expression of endothelial cells junctional molecules during inflammation and discusses the associated functional implications of such events in acute and chronic scenarios. © 2016 American Heart Association, Inc.

  20. Endocytosis regulates cell soma translocation and the distribution of adhesion proteins in migrating neurons.

    Directory of Open Access Journals (Sweden)

    Jennifer C Shieh

    Full Text Available Newborn neurons migrate from their birthplace to their final location to form a properly functioning nervous system. During these movements, young neurons must attach and subsequently detach from their substrate to facilitate migration, but little is known about the mechanisms cells use to release their attachments. We show that the machinery for clathrin-mediated endocytosis is positioned to regulate the distribution of adhesion proteins in a subcellular region just proximal to the neuronal cell body. Inhibiting clathrin or dynamin function impedes the movement of migrating neurons both in vitro and in vivo. Inhibiting dynamin function in vitro shifts the distribution of adhesion proteins to the rear of the cell. These results suggest that endocytosis may play a critical role in regulating substrate detachment to enable cell body translocation in migrating neurons.

  1. Tissue organization by cadherin adhesion molecules: dynamic molecular and cellular mechanisms of morphogenetic regulation

    Science.gov (United States)

    Niessen, Carien M.; Leckband, Deborah; Yap, Alpha S.

    2013-01-01

    This review addresses the cellular and molecular mechanisms of cadherin-based tissue morphogenesis. Tissue physiology is profoundly influenced by the distinctive organizations of cells in organs and tissues. In metazoa, adhesion receptors of the classical cadherin family play important roles in establishing and maintaining such tissue organization. Indeed, it is apparent that cadherins participate in a range of morphogenetic events that range from support of tissue integrity to dynamic cellular rearrangements. A comprehensive understanding of cadherin-based morphogenesis must then define the molecular and cellular mechanisms that support these distinct cadherin biologies. Here we focus on four key mechanistic elements: the molecular basis for adhesion through cadherin ectodomains; the regulation of cadherin expression at the cell surface; cooperation between cadherins and the actin cytoskeleton; and regulation by cell signaling. We discuss current progress and outline issues for further research in these fields. PMID:21527735

  2. Microtubule-dependent modulation of adhesion complex composition.

    Science.gov (United States)

    Ng, Daniel H J; Humphries, Jonathan D; Byron, Adam; Millon-Frémillon, Angélique; Humphries, Martin J

    2014-01-01

    The microtubule network regulates the turnover of integrin-containing adhesion complexes to stimulate cell migration. Disruption of the microtubule network results in an enlargement of adhesion complex size due to increased RhoA-stimulated actomyosin contractility, and inhibition of adhesion complex turnover; however, the microtubule-dependent changes in adhesion complex composition have not been studied in a global, unbiased manner. Here we used label-free quantitative mass spectrometry-based proteomics to determine adhesion complex changes that occur upon microtubule disruption with nocodazole. Nocodazole-treated cells displayed an increased abundance of the majority of known adhesion complex components, but no change in the levels of the fibronectin-binding α5β1 integrin. Immunofluorescence analyses confirmed these findings, but revealed a change in localisation of adhesion complex components. Specifically, in untreated cells, α5-integrin co-localised with vinculin at peripherally located focal adhesions and with tensin at centrally located fibrillar adhesions. In nocodazole-treated cells, however, α5-integrin was found in both peripherally located and centrally located adhesion complexes that contained both vinculin and tensin, suggesting a switch in the maturation state of adhesion complexes to favour focal adhesions. Moreover, the switch to focal adhesions was confirmed to be force-dependent as inhibition of cell contractility with the Rho-associated protein kinase inhibitor, Y-27632, prevented the nocodazole-induced conversion. These results highlight a complex interplay between the microtubule cytoskeleton, adhesion complex maturation state and intracellular contractile force, and provide a resource for future adhesion signaling studies. The proteomics data have been deposited in the ProteomeXchange with identifier PXD001183.

  3. Regulation of promyogenic signal transduction by cell-cell contact and adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Robert S., E-mail: Robert.Krauss@mssm.edu [Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY 10029 (United States)

    2010-11-01

    Skeletal myoblast differentiation involves acquisition of the muscle-specific transcriptional program and morphological changes, including fusion into multinucleated myofibers. Differentiation is regulated by extracellular signaling cues, including cell-cell contact and adhesion. Cadherin and Ig adhesion receptors have been implicated in distinct but overlapping stages of myogenesis. N-cadherin signals through the Ig receptor Cdo to activate p38 MAP kinase, while the Ig receptor neogenin signals to activate FAK; both processes promote muscle-specific gene expression and myoblast fusion. M-cadherin activates Rac1 to enhance fusion. Specific Ig receptors (Kirre and Sns) are essential for myoblast fusion in Drosophila, also signaling through Rac, and vertebrate orthologs of Kirre and Sns have partially conserved function. Mice lacking specific cytoplasmic signaling factors activated by multiple receptors (e.g., Rac1) have strong muscle phenotypes in vivo. In contrast, mice lacking individual adhesion receptors that lie upstream of these factors have modest phenotypes. Redundancy among receptors may account for this. Many of the mammalian Ig receptors and cadherins associate with each other, and multivalent interactions within these complexes may require removal of multiple components to reveal dramatic defects in vivo. Nevertheless, it is possible that the murine adhesion receptors rate-limiting in vivo have not yet been identified or fully assessed.

  4. A dynamic cell adhesion surface regulates tissue architecture in growth plate cartilage.

    Science.gov (United States)

    Romereim, Sarah M; Conoan, Nicholas H; Chen, Baojiang; Dudley, Andrew T

    2014-05-01

    The architecture and morphogenetic properties of tissues are founded in the tissue-specific regulation of cell behaviors. In endochondral bones, the growth plate cartilage promotes bone elongation via regulated chondrocyte maturation within an ordered, three-dimensional cell array. A key event in the process that generates this cell array is the transformation of disordered resting chondrocytes into clonal columns of discoid proliferative cells aligned with the primary growth vector. Previous analysis showed that column-forming chondrocytes display planar cell divisions, and the resulting daughter cells rearrange by ∼90° to align with the lengthening column. However, these previous studies provided limited information about the mechanisms underlying this dynamic process. Here we present new mechanistic insights generated by application of a novel time-lapse confocal microscopy method along with immunofluorescence and electron microscopy. We show that, during cell division, daughter chondrocytes establish a cell-cell adhesion surface enriched in cadherins and β-catenin. Rearrangement into columns occurs concomitant with expansion of this adhesion surface in a process more similar to cell spreading than to migration. Column formation requires cell-cell adhesion, as reducing cadherin binding via chelation of extracellular calcium inhibits chondrocyte rearrangement. Importantly, physical indicators of cell polarity, such as cell body alignment, are not prerequisites for oriented cell behavior. Our results support a model in which regulation of adhesive surface dynamics and cortical tension by extrinsic signaling modifies the thermodynamic landscape to promote organization of daughter cells in the context of the three-dimensional growth plate tissue.

  5. Loss of miR-203 regulates cell shape and matrix adhesion through ROBO1/Rac/FAK in response to stiffness.

    Science.gov (United States)

    Le, Lily Thao-Nhi; Cazares, Oscar; Mouw, Janna K; Chatterjee, Sharmila; Macias, Hector; Moran, Angel; Ramos, Jillian; Keely, Patricia J; Weaver, Valerie M; Hinck, Lindsay

    2016-03-14

    Breast tumor progression is accompanied by changes in the surrounding extracellular matrix (ECM) that increase stiffness of the microenvironment. Mammary epithelial cells engage regulatory pathways that permit dynamic responses to mechanical cues from the ECM. Here, we identify a SLIT2/ROBO1 signaling circuit as a key regulatory mechanism by which cells sense and respond to ECM stiffness to preserve tensional homeostasis. We observed that Robo1 ablation in the developing mammary gland compromised actin stress fiber assembly and inhibited cell contractility to perturb tissue morphogenesis, whereas SLIT2 treatment stimulated Rac and increased focal adhesion kinase activity to enhance cell tension by maintaining cell shape and matrix adhesion. Further investigation revealed that a stiff ECM increased Robo1 levels by down-regulating miR-203. Consistently, patients whose tumor expressed a low miR-203/high Robo1 expression pattern exhibited a better overall survival prognosis. These studies show that cells subjected to stiffened environments up-regulate Robo1 as a protective mechanism that maintains cell shape and facilitates ECM adherence.

  6. Loss of miR-203 regulates cell shape and matrix adhesion through ROBO1/Rac/FAK in response to stiffness

    Science.gov (United States)

    Le, Lily Thao-Nhi; Cazares, Oscar; Mouw, Janna K.; Chatterjee, Sharmila; Macias, Hector; Moran, Angel; Ramos, Jillian; Keely, Patricia J.; Weaver, Valerie M.

    2016-01-01

    Breast tumor progression is accompanied by changes in the surrounding extracellular matrix (ECM) that increase stiffness of the microenvironment. Mammary epithelial cells engage regulatory pathways that permit dynamic responses to mechanical cues from the ECM. Here, we identify a SLIT2/ROBO1 signaling circuit as a key regulatory mechanism by which cells sense and respond to ECM stiffness to preserve tensional homeostasis. We observed that Robo1 ablation in the developing mammary gland compromised actin stress fiber assembly and inhibited cell contractility to perturb tissue morphogenesis, whereas SLIT2 treatment stimulated Rac and increased focal adhesion kinase activity to enhance cell tension by maintaining cell shape and matrix adhesion. Further investigation revealed that a stiff ECM increased Robo1 levels by down-regulating miR-203. Consistently, patients whose tumor expressed a low miR-203/high Robo1 expression pattern exhibited a better overall survival prognosis. These studies show that cells subjected to stiffened environments up-regulate Robo1 as a protective mechanism that maintains cell shape and facilitates ECM adherence. PMID:26975850

  7. Regulation of human cerebro-microvascular endothelial baso-lateral adhesion and barrier function by S1P through dual involvement of S1P1 and S1P2 receptors.

    Science.gov (United States)

    Wiltshire, Rachael; Nelson, Vicky; Kho, Dan Ting; Angel, Catherine E; O'Carroll, Simon J; Graham, E Scott

    2016-01-27

    Herein we show that S1P rapidly and acutely reduces the focal adhesion strength and barrier tightness of brain endothelial cells. xCELLigence biosensor technology was used to measure focal adhesion, which was reduced by S1P acutely and this response was mediated through both S1P1 and S1P2 receptors. S1P increased secretion of several pro-inflammatory mediators from brain endothelial cells. However, the magnitude of this response was small in comparison to that mediated by TNFα or IL-1β. Furthermore, S1P did not significantly increase cell-surface expression of any key cell adhesion molecules involved in leukocyte recruitment, included ICAM-1 and VCAM-1. Finally, we reveal that S1P acutely and dynamically regulates microvascular endothelial barrier tightness in a manner consistent with regulated rapid opening followed by closing and strengthening of the barrier. We hypothesise that the role of the S1P receptors in this process is not to cause barrier dysfunction, but is related to controlled opening of the endothelial junctions. This was revealed using real-time measurement of barrier integrity using ECIS ZΘ TEER technology and endothelial viability using xCELLigence technology. Finally, we show that these responses do not occur simply though the pharmacology of a single S1P receptor but involves coordinated action of S1P1 and S1P2 receptors.

  8. 局部黏着斑激酶作为肿瘤治疗靶点的研究进展%Focal adhesion kinase, a novel target for cancer therapy

    Institute of Scientific and Technical Information of China (English)

    张文静; 黄启来; 华子春

    2011-01-01

    局部黏着斑激酶(focal adhesion kinase,FAK)是一种非受体型酪氨酸蛋白激酶,是细胞内重要的骨架蛋白与多种信号通路的关键分子.FAK在肿瘤发生、发展、迁移和侵袭的各个阶段都具有重要作用,FAK己经被当作潜在的肿瘤治疗靶点来研究.该综述将对FAK与肿瘤的关系以及FAK作为肿瘤治疗靶点的研究进展进行探讨.%Focal adhesion kinase (FAK), a cytoplasmic non-receptor protein tyrosine kinase, serves as both a molecular scaffold and a mediator participating in multiple signal transduction pathways. FAK is involved in tumor cell survival, proliferation, migration and metastasis. Currently, FAK has been regarded as a potential target for cancer therapy. This review is to summarize the relationship between FAK and tumor progression, and to discuss the strategies targeting FAK for cancer treatment.

  9. Focal adhesion kinase (FAK) mediates the induction of pro-oncogenic and fibrogenic phenotypes in hepatitis C virus (HCV)-infected cells.

    Science.gov (United States)

    Alisi, Anna; Arciello, Mario; Petrini, Stefania; Conti, Beatrice; Missale, Gabriele; Balsano, Clara

    2012-01-01

    Hepatitis C Virus (HCV) infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC). The pivotal role of hepatic stellate cells (HCSs) and extracellular matrix (ECM) in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP). These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in HSCs.

  10. MiR-126 and miR-126* regulate shear-resistant firm leukocyte adhesion to human brain endothelium

    Science.gov (United States)

    Cerutti, Camilla; Edwards, Laura J.; de Vries, Helga E.; Sharrack, Basil; Male, David K.; Romero, Ignacio A.

    2017-01-01

    Leukocyte adhesion to brain endothelial cells, the blood-brain barrier main component, is a critical step in the pathogenesis of neuroinflammatory diseases such as multiple sclerosis (MS). Leukocyte adhesion is mediated mainly by selectins, cell adhesion molecules and chemokines induced by pro-inflammatory cytokines such as TNFα and IFNγ, but the regulation of this process is not fully clear. This study investigated the regulation of firm leukocyte adhesion to human brain endothelium by two different brain endothelial microRNAs (miRs), miR-126 and miR-126*, that are downregulated by TNFα and IFNγ in a human brain endothelial cell line, hCMEC/D3. Using a leukocyte adhesion in vitro assay under shear forces mimicking blood flow, we observed that reduction of endothelial miR-126 and miR-126* enhanced firm monocyte and T cell adhesion to hCMEC/D3 cells, whereas their increased expression partially prevented THP1, Jurkat and primary MS patient-derived PBMC firm adhesion. Furthermore, we observed that miR-126* and miR-126 downregulation increased E-selectin and VCAM1, respectively, while miR-126 overexpression reduced VCAM1 and CCL2 expression by hCMEC/D3 cells, suggesting that these miRs regulate leukocyte adhesion by modulating the expression of adhesion-associated endothelial mRNA targets. Hence, human brain endothelial miR-126 and miR-126* could be used as a therapeutic tool to reduce leukocyte adhesion and thus reduce neuroinflammation. PMID:28358058

  11. Interleukin-18-induced cell adhesion molecule expression is associated with feedback regulation by PPAR-γ and NF-κB in Apo E-/- mice.

    Science.gov (United States)

    Bhat, Owais Mohammad; Uday Kumar, P; Harishankar, N; Ravichandaran, L; Bhatia, A; Dhawan, Veena

    2017-02-07

    Focal recruitment of monocytes and lymphocytes is one of the earliest detectable cellular responses in atherosclerotic lesion formation. Endothelium may regulate leukocyte recruitment by expressing specific adhesion molecules. Interleukin-18 is a proinflammatory cytokine that plays an important role in vascular pathologies. The present study highlights the modulation of adhesion molecules and PPAR-γ by IL-18 and proposes a novel feedback mechanism by which PPAR-γ may regulate IL-18 expression. Three groups of normal chow diet-fed, male Apo E-/- mice, aged 12 weeks (n = 6/group) were employed: Gp I, phosphate-buffered saline (PBS) (2 mo): Gp II, recombinant IL-18 (rIL-18) (1 mo) followed by PBS (1 mo); Gp III, rIL-18 (1 mo) followed by pyrrolidine dithiocarbamate (PDTC) (1 mo). Significantly augmented mRNA expression of ICAM-1 (~5.7-fold), VCAM-1 (~3.6-fold), and NF-κB (~7-fold) was observed in Gp II mice as compared to Gp I, whereas PPAR-γ expression was not altered. PDTC treatment caused a significant downregulation of ICAM-1 (~4.2-fold), VCAM-1(~2-fold), and NF-κB (~4.5-fold) and upregulation of PPAR-γ expression (~5-fold) in Gp III mice. A similar trend was observed in protein expression. In vivo imaging results demonstrated a marked increase in probe (CF750 dye conjugated to VCAM-1 antibody) fluorescence intensity for VCAM-1 expression in Gp II mice, whereas it was moderately decreased in Gp III. PPAR-γ was found to significantly downregulate both IL-18 levels and IL-18-induced adhesion molecules. The underlying mechanism was found to be via inhibition of NF-κB activity by PDTC, thereby leading to decreased adherence of monocytes to the activated endothelial cells and a step to halt the progression and development of atherosclerotic lesions.

  12. PDE8 regulates rapid Teff cell adhesion and proliferation independent of ICER.

    Directory of Open Access Journals (Sweden)

    Amanda G Vang

    Full Text Available BACKGROUND: Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs is a prerequisite for effector T (Teff cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions. METHODOLOGY/PRINCIPAL FINDINGS: We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem-/- mice lacking the inducible cAMP early repressor (ICER. Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection. CONCLUSION/SIGNIFICANCE: Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.

  13. MICA Expression Is Regulated by Cell Adhesion and Contact in a FAK/Src-Dependent Manner

    Science.gov (United States)

    Moncayo, Gerald; Lin, Da; McCarthy, Michael T.; Watson, Aleksandra A.; O’Callaghan, Christopher A.

    2017-01-01

    MICA is a major ligand for the NKG2D immune receptor, which plays a key role in activating natural killer (NK) cells and cytotoxic T cells. We analyzed NKG2D ligand expression on a range of cell types and could demonstrate that MICA expression levels were closely linked to cellular growth mode. While the expression of other NKG2D ligands was largely independent of cell growth mode, MICA expression was mainly found on cells cultured as adherent cells. In addition, MICA surface expression was reduced through increase in cell–cell contact or loss of cell–matrix adherence. Furthermore, we found that the reduction in MICA expression was modulated by focal adhesion kinase (FAK)/Src signaling and associated with increased susceptibility to NK cell-mediated killing. While the mechanisms of tumor immune evasion are not fully understood, the reduction of MICA expression following loss of attachment poises a potential way by which metastasizing tumor cells avoid immune detection. The role of FAK/Src in this process indicates a potential therapeutic approach to modulate MICA expression and immune recognition of tumor cells during metastasis. PMID:28154561

  14. Cell Adhesion Regulates Expression of the Androgen Receptor and Coregulators in Different Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sheng Li

    2007-02-01

    Full Text Available Prostate cancer cells adhere to a tumor basement membrane, while secretoryepithelial cells reside in a suprabasal cell compartment. Since tumor cells are derived fromsuprabasal epithelial cells, they experience de-novo substratum adhesion in the context ofoncogenesis. We therefore analyzed whether cell-matrix adhesion could affect the proteinexpression and activity of the AR. In this study, AR protein expression declined uponsuspension of BPH-1-AR cells, but not in PC-3-AR cells shown by Western blot. In a timecourse study, BPH-1 cell lost AR expression within 6 hours, and the synthetic androgen,R1881 reduced the loss of AR expression. We further explored the mechanism of AR loss insuspended BPH-1 cells. BPH-1-AR cells underwent apoptosis (anoikis when suspended for2 - 5 hours. Suspension did not induce significant apoptosis or decreasing of AR expressionin PC-3 cells. Inhibition of apoptosis in suspended BPH-1-AR cells, either by expression ofBcl-2 or Bcl-xl or by treatment with Z-VAD, a caspase inhibitor, prevented loss of ARprotein. In contrast, the calpain protease inhibitor , ALLN, accelerated the loss of AR proteinexpression. Additionally, cell-matrix adhesion changed the expression of coregulators of ARin the mRNA level of prostate cancer cells. Our results demonstrate that AR proteinexpression was reduced through activation of cell death pathways, and thus indirectly through cell suspension in BPH-AR cells. The activity of AR can also be regulated by adhesion in PC-3-AR and LNCaP cells through affecting the coregulators level.

  15. Prednisone inhibits the focal adhesion kinase/receptor activator of NF-κB ligand/mitogen-activated protein kinase signaling pathway in rats with adriamycin-induced nephropathy.

    Science.gov (United States)

    Ye, Minyuan; Zheng, Jing; Chen, Xiaoying; Chen, Xuelan; Wu, Xinhong; Lin, Xiuqin; Liu, Yafang

    2015-11-01

    The aim of the present study was to investigate the mechanisms underlying the effects of prednisone on adriamycin-induced nephritic rat kidney damage via the focal adhesion kinase (FAK)/receptor activator of nuclear factor-κB ligand (RANKL)/mitogen‑activated protein kinase (MAPK) signaling pathway. An adriamycin‑induced nephritic rat model was established to investigate these mechanisms. A total of 30 healthy male Sprague‑Dawley rats were randomly assigned to the normal, model or prednisone group. Samples of urine were collected over the course of 24 h at days 7, 14, and 28, and renal cortex tissue samples were harvested at days 14, and 28 following nephritic rat model establishment. The total urinary protein content was measured by biuret colorimetry. Pathological changes in the kidney tissue samples were observed using an electron microscope. The mRNA expressions levels of FAK, RANKL, p38, extracellular signal‑regulated kinase (ERK), c‑Jun N‑terminal kinase (JNK), and nephrin were then quantified by reverse transcription‑quantitative polymerase chain reaction. In addition, the protein expressions levels of FAK, RANKL, p38, ERK, JNK, phosphorylated (p)‑FAK, p‑ERK, and p‑JNK were quantified by western blotting. As compared with the normal group, the protein expression levels of FAK, RANKL, p-FAK, p38 and p-ERK in the model group were increased. In the prednisone group, the protein expression levels of p-ERK decreased, as compared with the normal group. In the prednisone group, the urinary protein levels, the protein expression levels of FAK, RANKL, p38, p-FAK, p-p38 and the mRNA expression levels of FAK, p38, RANKL, ERK, JNK decreased, as compared with the model group. In the prednisone group, the mRNA and protein expression levels of nephrin and the serum expression levels of RANKL increased, the serum expression levels of osteoprotegerin (OPG) were decreased, as compared with the model group. No significant changes in the protein expression

  16. Inhibition on Apoptosis Induced by Elevated Hydrostatic Pressure in Retinal Ganglion Cell-5 via Laminin Upregulating β1-integrin/Focal Adhesion Kinase/Protein Kinase B Signaling Pathway

    Institute of Scientific and Technical Information of China (English)

    Yi Li; Yan-Ming Chen; Ming-Ming Sun; Xiao-Dan Guo; Ya-Chen Wang; Zhong-Zhi Zhang

    2016-01-01

    Background:Glaucoma is a progressive optic neuropathy characterized by degeneration of neurons due to loss of retinal ganglion cells (RGCs).High intraocular pressure (HIOP),the main risk factor,causes the optic nerve damage.However,the precise mechanism of HIOP-induced RGC death is not yet completely understood.This study was conducted to determine apoptosis of RGC-5 cells induced by elevated hydrostatic pressures,explore whether laminin is associated with apoptosis under pressure,whether laminin can protect RGCs from apoptosis and affirm the mechanism that regulates the process of RGCs survival.Methods:RGC-5 cells were exposed to 0,20,40,and 60 mmHg in a pressurized incubator for 6,12,and 24 h,respectively.The effect of elevated hydrostatic pressure on RGC-5 cells was measured by Annexin V-fluorescein isothiocyanate/propidium iodide staining,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,and Western blotting of cleaved caspase-3 protein.Location and expression oflaminin were detected by immunofluorescence.The expression of β 1-integrin,phosphorylation of focal adhesion kinase (FAK) and protein kinase B (PKB,or AKT) were investigated with real-time polymerase chain reaction and Western blotting analysis.Results:Elevated hydrostatic pressure induced apoptosis in cultured RGC-5 cells.Pressure with 40 mmHg for 24 h induced a maximum apoptosis.Laminin was declined in RGC-5 cells after exposing to 40 mmHg for 24 h.After pretreating with laminin,RGC-5 cells survived from elevated pressure.Furthermore,β1-integrin and phosphorylation of FAK and AKT were increased compared to 40 mmHg group.Conclusions:The data show apoptosis tendency of RGC-5 cells with elevated hydrostatic pressure.Laminin can protect RGC-5 cells against high pressure via β 1-integrin/FAK/AKT signaling pathway.These results suggest that the decreased laminin of RGC-5 cells might be responsible for apoptosis induced by elevated hydrostatic pressure,and laminin or activating β1

  17. Focal adhesion kinase (FAK mediates the induction of pro-oncogenic and fibrogenic phenotypes in hepatitis C virus (HCV-infected cells.

    Directory of Open Access Journals (Sweden)

    Anna Alisi

    Full Text Available Hepatitis C Virus (HCV infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC. The pivotal role of hepatic stellate cells (HCSs and extracellular matrix (ECM in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP. These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in

  18. Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer

    Directory of Open Access Journals (Sweden)

    Chen Hairu

    2010-10-01

    Full Text Available Abstract Background Activated leukocyte cell adhesion molecule (ALCAM is implicated in the prognosis of multiple cancers with low level expression associated with metastasis and early death in breast cancer. Despite this significance, mechanisms that regulate ALCAM gene expression and ALCAM's role in adhesion of pre-metastatic circulating tumor cells have not been defined. We studied ALCAM expression in 20 tumor cell lines by real-time PCR, western blot and immunochemistry. Epigenetic alterations of the ALCAM promoter were assessed using methylation-specific PCR and bisulfite sequencing. ALCAM's role in adhesion of tumor cells to the vascular wall was studied in isolated perfused lungs. Results A common site for transcription initiation of the ALCAM gene was identified and the ALCAM promoter sequenced. The promoter contains multiple cis-active elements including a functional p65 NF-κB motif, and it harbors an extensive array of CpG residues highly methylated exclusively in ALCAM-negative tumor cells. These CpG residues were modestly demethylated after 5-aza-2-deoxycytidine treatment. Restoration of high-level ALCAM expression using an ALCAM cDNA increased clustering of MDA-MB-435 tumor cells perfused through the pulmonary vasculature of ventilated rat lungs. Anti-ALCAM antibodies reduced the number of intravascular tumor cell clusters. Conclusion Our data suggests that loss of ALCAM expression, due in part to DNA methylation of extensive segments of the promoter, significantly impairs the ability of circulating tumor cells to adhere to each other, and may therefore promote metastasis. These findings offer insight into the mechanisms for down-regulation of ALCAM gene expression in tumor cells, and for the positive prognostic value of high-level ALCAM in breast cancer.

  19. β-Catenin-regulated myeloid cell adhesion and migration determine wound healing.

    Science.gov (United States)

    Amini-Nik, Saeid; Cambridge, Elizabeth; Yu, Winston; Guo, Anne; Whetstone, Heather; Nadesan, Puviindran; Poon, Raymond; Hinz, Boris; Alman, Benjamin A

    2014-06-01

    A β-catenin/T cell factor-dependent transcriptional program is critical during cutaneous wound repair for the regulation of scar size; however, the relative contribution of β-catenin activity and function in specific cell types in the granulation tissue during the healing process is unknown. Here, cell lineage tracing revealed that cells in which β-catenin is transcriptionally active express a gene profile that is characteristic of the myeloid lineage. Mice harboring a macrophage-specific deletion of the gene encoding β-catenin exhibited insufficient skin wound healing due to macrophage-specific defects in migration, adhesion to fibroblasts, and ability to produce TGF-β1. In irradiated mice, only macrophages expressing β-catenin were able to rescue wound-healing deficiency. Evaluation of scar tissue collected from patients with hypertrophic and normal scars revealed a correlation between the number of macrophages within the wound, β-catenin levels, and cellularity. Our data indicate that β-catenin regulates myeloid cell motility and adhesion and that β-catenin-mediated macrophage motility contributes to the number of mesenchymal cells and ultimate scar size following cutaneous injury.

  20. Fibroblast growth factor-regulated palmitoylation of the neural cell adhesion molecule determines neuronal morphogenesis.

    Science.gov (United States)

    Ponimaskin, Evgeni; Dityateva, Galina; Ruonala, Mika O; Fukata, Masaki; Fukata, Yuko; Kobe, Fritz; Wouters, Fred S; Delling, Markus; Bredt, David S; Schachner, Melitta; Dityatev, Alexander

    2008-09-03

    During development of the nervous system, short- and long-range signals cooperate to promote axonal growth, guidance, and target innervation. Particularly, a short-range signal transducer, the neural cell adhesion molecule (NCAM), stimulates neurite outgrowth via mechanisms that require posttranslational modification of NCAM and signaling via receptors to a long-range messenger, the fibroblast growth factor (FGF). In the present study we further characterized a mechanism which regulates the functional interplay between NCAM and FGF receptor(s). We show that activation of FGF receptor(s) by FGF2 leads to palmitoylation of the two major transmembrane NCAM isoforms, NCAM140 and NCAM180, translocation of NCAM to GM1 ganglioside-containing lipid rafts, and stimulation of neurite outgrowth of hippocampal neurons. Ablation of NCAM, mutation of NCAM140 or NCAM180 palmitoylation sites, or pharmacological suppression of NCAM signaling inhibited FGF2-stimulated neurite outgrowth. Of the 23 members of the aspartate-histidine-histidine-cysteine (DHHC) domain containing proteins, DHHC-7 most strongly stimulated palmitoylation of NCAM, and enzyme activity was enhanced by FGF2. Thus, our study uncovers a molecular mechanism by which a growth factor regulates neuronal morphogenesis via activation of palmitoylation, which in turn modifies subcellular location and thus signaling via an adhesion molecule.

  1. Regulator of G protein signaling 20 enhances cancer cell aggregation, migration, invasion and adhesion.

    Science.gov (United States)

    Yang, Lei; Lee, Maggie M K; Leung, Manton M H; Wong, Yung H

    2016-11-01

    Several RGS (regulator of G protein signaling) proteins are known to be upregulated in a variety of tumors but their roles in modulating tumorigenesis remain undefined. Since the expression of RGS20 is elevated in metastatic melanoma and breast tumors, we examined the effects of RGS20 overexpression and knockdown on the cell mobility and adhesive properties of different human cancer cell lines, including cervical cancer HeLa, breast adenocarcinoma MDA-MB-231, and non-small cell lung carcinoma H1299 and A549 cells. Expression of RGS20 enhanced cell aggregation, migration, invasion and adhesion as determined by hanging drop aggregation, wound healing, transwell chamber migration and invasion assays. Conversely, shRNA-mediated knockdown of endogenous RGS20 impaired these responses. In addition, RGS20 elevated the expression of vimentin (a mesenchymal cell marker) but down-regulated the expression of E-cadherin, two indicators commonly associated with metastasis. These results suggest that the expression of RGS20 may promote metastasis of tumor cells.

  2. Role of dystrophins and utrophins in platelet adhesion process.

    Science.gov (United States)

    Cerecedo, Doris; Mondragón, Ricardo; Cisneros, Bulmaro; Martínez-Pérez, Francisco; Martínez-Rojas, Dalila; Rendón, Alvaro

    2006-07-01

    Platelets are crucial at the site of vascular injury, adhering to the sub-endothelial matrix through receptors on their surface, leading to cell activation and aggregation to form a haemostatic plug. Platelets display focal adhesions as well as stress fibres to contract and facilitate expulsion of growth and pro-coagulant factors contained in the granules and to constrict the clot. The interaction of F-actin with different actin-binding proteins determines the properties and composition of the focal adhesions. Recently, we demonstrated the presence of dystrophin-associated protein complex corresponding to short dystrophin isoforms (Dp71d and Dp71) and the uthophin gene family (Up400 and Up71), which promote shape change, adhesion, aggregation, and granule centralisation. To elucidate participation of both complexes during the platelet adhesion process, their potential association with integrin beta-1 fraction and the focal adhesion system (alpha-actinin, vinculin and talin) was evaluated by immunofluorescence and immunoprecipitation assays. It was shown that the short dystrophin-associated protein complex participated in stress fibre assembly and in centralisation of cytoplasmic granules, while the utrophin-associated protein complex assembled and regulated focal adhesions. The simultaneous presence of dystrophin and utrophin complexes indicates complementary structural and signalling mechanisms to the actin network, improving the platelet haemostatic role.

  3. Integrins stimulate E-cadherin-mediated intercellular adhesion by regulating Src-kinase activation and actomyosin contractility.

    Science.gov (United States)

    Martinez-Rico, Clara; Pincet, Frederic; Thiery, Jean-Paul; Dufour, Sylvie

    2010-03-01

    Cadherins and integrins are major adhesion molecules regulating cell-cell and cell-matrix interactions. In vitro and in vivo studies have demonstrated the existence of crosstalk between integrins and cadherins in cell adhesion and motility. We used a dual pipette assay to measure the force required to separate E-cadherin-producing cell doublets and to investigate the role of integrin in regulating the strength of intercellular adhesion. A greater force was required to separate cell doublets bound to fibronectin or vitronectin-coated beads than for doublets bound to polylysine-coated beads. This effect depended on cell spreading and the duration of stimulation. Cells expressing type II cadherin-7 also responded to fibronectin stimulation to produce a higher intercellular adhesion. Establishment of cadherin-mediated adhesion needed ROCK, MLCK and myosin ATPase II activity. The regulation of intercellular adhesion strength by integrin stimulation required activation of Src family kinases, ROCK and actomyosin contractility. These findings highlight the importance and mechanisms of molecular crosstalk between cadherins and integrins in the control of cell plasticity during histogenesis and morphogenesis.

  4. Pathogenesis of Focal Segmental Glomerulosclerosis

    Directory of Open Access Journals (Sweden)

    Beom Jin Lim

    2016-11-01

    Full Text Available Focal segmental glomerulosclerosis (FSGS is characterized by focal and segmental obliteration of glomerular capillary tufts with increased matrix. FSGS is classified as collapsing, tip, cellular, perihilar and not otherwise specified variants according to the location and character of the sclerotic lesion. Primary or idiopathic FSGS is considered to be related to podocyte injury, and the pathogenesis of podocyte injury has been actively investigated. Several circulating factors affecting podocyte permeability barrier have been proposed, but not proven to cause FSGS. FSGS may also be caused by genetic alterations. These genes are mainly those regulating slit diaphragm structure, actin cytoskeleton of podocytes, and foot process structure. The mode of inheritance and age of onset are different according to the gene involved. Recently, the role of parietal epithelial cells (PECs has been highlighted. Podocytes and PECs have common mesenchymal progenitors, therefore, PECs could be a source of podocyte repopulation after podocyte injury. Activated PECs migrate along adhesion to the glomerular tuft and may also contribute to the progression of sclerosis. Markers of activated PECs, including CD44, could be used to distinguish FSGS from minimal change disease. The pathogenesis of FSGS is very complex; however, understanding basic mechanisms of podocyte injury is important not only for basic research, but also for daily diagnostic pathology practice.

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

    Science.gov (United States)

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

    2014-01-01

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

  6. Pancreatic cancer cell glycosylation regulates cell adhesion and invasion through the modulation of α2β1 integrin and E-cadherin function.

    Directory of Open Access Journals (Sweden)

    Sònia Bassagañas

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

  7. The Neuroplastin adhesion molecules: key regulators of neuronal plasticity and synaptic function.

    Science.gov (United States)

    Beesley, Philip W; Herrera-Molina, Rodrigo; Smalla, Karl-Heinz; Seidenbecher, Constanze

    2014-11-01

    The Neuroplastins Np65 and Np55 are neuronal and synapse-enriched immunoglobulin superfamily molecules that play important roles in a number of key neuronal and synaptic functions including, for Np65, cell adhesion. In this review we focus on the physiological roles of the Neuroplastins in promoting neurite outgrowth, regulating the structure and function of both inhibitory and excitatory synapses in brain, and in neuronal and synaptic plasticity. We discuss the underlying molecular and cellular mechanisms by which the Neuroplastins exert their physiological effects and how these are dependent upon the structural features of Np65 and Np55, which enable them to bind to a diverse range of protein partners. In turn this enables the Neuroplastins to interact with a number of key neuronal signalling cascades. These include: binding to and activation of the fibroblast growth factor receptor; Np65 trans-homophilic binding leading to activation of p38 MAPK and internalization of glutamate (GluR1) receptor subunits; acting as accessory proteins for monocarboxylate transporters, thus affecting neuronal energy supply, and binding to GABAA α1, 2 and 5 subunits, thus regulating the composition and localization of GABAA receptors. An emerging theme is the role of the Neuroplastins in regulating the trafficking and subcellular localization of specific binding partners. We also discuss the involvement of Neuroplastins in a number of pathophysiological conditions, including ischaemia, schizophrenia and breast cancer and the role of a single nucleotide polymorphism in the human Neuroplastin (NPTN) gene locus in impairment of cortical development and cognitive functions. Neuroplastins are neuronal cell adhesion molecules, which induce neurite outgrowth and play important roles in synaptic maturation and plasticity. This review summarizes the functional implications of Neuroplastins for correct synaptic membrane protein localization, neuronal energy supply, expression of LTP and LTD

  8. Anterior gradient protein-2 is a regulator of cellular adhesion in prostate cancer.

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    Diptiman Chanda

    Full Text Available Anterior Gradient Protein (AGR-2 is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis.

  9. Human Bone Marrow Mesenchymal Stem Cells Regulate Biased DNA Segregation in Response to Cell Adhesion Asymmetry

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    Delphine Freida

    2013-11-01

    Full Text Available Biased DNA segregation is a mitotic event in which the chromatids carrying the original template DNA strands and those carrying the template copies are not segregated randomly into the two daughter cells. Biased segregation has been observed in several cell types, but not in human mesenchymal stem cells (hMSCs, and the factors affecting this bias have yet to be identified. Here, we have investigated cell adhesion geometries as a potential parameter by plating hMSCs from healthy donors on fibronectin-coated micropatterns. On symmetric micropatterns, the segregation of sister chromatids to the daughter cells appeared random. In contrast, on asymmetric micropatterns, the segregation was biased. This sensitivity to asymmetric extracellular cues was reproducible in cells from all donors but was not observed in human skin-derived fibroblasts or in a fibroblastic cell line used as controls. We conclude that the asymmetry of cell adhesion is a major factor in the regulation of biased DNA segregation in hMSCs.

  10. Amigo Adhesion Protein Regulates Development of Neural Circuits in Zebrafish Brain*

    Science.gov (United States)

    Zhao, Xiang; Kuja-Panula, Juha; Sundvik, Maria; Chen, Yu-Chia; Aho, Vilma; Peltola, Marjaana A.; Porkka-Heiskanen, Tarja; Panula, Pertti; Rauvala, Heikki

    2014-01-01

    The Amigo protein family consists of three transmembrane proteins characterized by six leucine-rich repeat domains and one immunoglobulin-like domain in their extracellular moieties. Previous in vitro studies have suggested a role as homophilic adhesion molecules in brain neurons, but the in vivo functions remain unknown. Here we have cloned all three zebrafish amigos and show that amigo1 is the predominant family member expressed during nervous system development in zebrafish. Knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. A similar defect in fiber tract development is caused by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. Analysis of differentiated neural circuits reveals defects in the catecholaminergic system. At the behavioral level, the disturbed formation of neural circuitry is reflected in enhanced locomotor activity and in the inability of the larvae to perform normal escape responses. We suggest that Amigo1 is essential for the development of neural circuits of zebrafish, where its mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion. PMID:24904058

  11. Adhesive activity of Lu glycoproteins is regulated by interaction with spectrin

    Energy Technology Data Exchange (ETDEWEB)

    An, Xiuli; Gauthier, Emilie; Zhang, Xihui; Guo, Xinhua; Anstee, David; Mohandas, Narla; Anne Chasis, Joel

    2008-03-18

    The Lutheran (Lu) and Lu(v13) blood group glycoproteins function as receptors for extracellular matrix laminins. Lu and Lu(v13) are linked to the erythrocyte cytoskeleton through a direct interaction with spectrin. However, neither the molecular basis of the interaction nor its functional consequences have previously been delineated. In the present study, we defined the binding motifs of Lu and Lu(v13) on spectrin and identified a functional role for this interaction. We found that the cytoplasmic domains of both Lu and Lu(v13) bound to repeat 4 of the spectrin chain. The interaction of full-length spectrin dimer to Lu and Lu(v13) was inhibited by repeat 4 of {alpha}-spectrin. Further, resealing of this repeat peptide into erythrocytes led to weakened Lu-cytoskeleton interaction as demonstrated by increased detergent extractability of Lu. Importantly, disruption of the Lu-spectrin linkage was accompanied by enhanced cell adhesion to laminin. We conclude that the interaction of the Lu cytoplasmic tail with the cytoskeleton regulates its adhesive receptor function.

  12. Regulation of the elastic modulus of polyurethane microarrays and its influence on gecko-inspired dry adhesion

    Science.gov (United States)

    Li, Ming; Zhao, Aiwu; Jiang, Rui; Wang, Dapeng; Li, Da; Guo, Hongyan; Tao, Wenyu; Gan, Zibao; Zhang, Maofeng

    2011-02-01

    We studied the influence of the elastic modulus on the gecko-inspired dry adhesion by regulating the elastic modulus of bulk polyurethane combined with changing the size of microarrays. Segmented polyurethane (PU) was utilized to fabricate micro arrays by the porous polydimethyl siloxane (PDMS) membrane molding method. The properties of the micro arrays, such as the elastic modulus and adhesion, were investigated by Triboindenter. The study demonstrates that bulk surfaces show the highest elastic modulus, with similar values at around 175 MPa and decreasing the arrays radius causes a significant decrease in E, down to 0.62 MPa. The corresponding adhesion experiments show that decrease of the elastic modulus can enhance the adhesion which is consistent with the recent theoretical models.

  13. Green Tea Epigallocatechin Gallate Exhibits Anticancer Effect in Human Pancreatic Carcinoma Cells via the Inhibition of Both Focal Adhesion Kinase and Insulin-Like Growth Factor-I Receptor

    Directory of Open Access Journals (Sweden)

    Hoang Anh Vu

    2010-01-01

    Full Text Available The exact molecular mechanism by which epigallocatechin gallate (EGCG suppresses human pancreatic cancer cell proliferation is unclear. We show here that EGCG-treated pancreatic cancer cells AsPC-1 and BxPC-3 decrease cell adhesion ability on micro-pattern dots, accompanied by dephosphorylations of both focal adhesion kinase (FAK and insulin-like growth factor-1 receptor (IGF-1R whereas retained the activations of mitogen-activated protein kinase and mammalian target of rapamycin. The growth of AsPC-1 and BxPC-3 cells can be significantly suppressed by EGCG treatment alone in a dose-dependent manner. At a dose of 100 μM which completely abolishes activations of FAK and IGF-1R, EGCG suppresses more than 50% of cell proliferation without evidence of apoptosis analyzed by PARP cleavage. Finally, the MEK1/2 inhibitor U0126 enhances growth-suppressive effect of EGCG. Our data suggests that blocking FAK and IGF-1R by EGCG could prove valuable for targeted therapy, which can be used in combination with other therapies, for pancreatic cancer.

  14. Rho-kinase regulates adhesive and mechanical mechanisms of pulmonary recruitment of neutrophils in abdominal sepsis.

    Science.gov (United States)

    Palani, Karzan; Rahman, Milladur; Hasan, Zirak; Zhang, Su; Qi, Zhongquan; Jeppsson, Bengt; Thorlacius, Henrik

    2012-05-05

    We hypothesized that Rho-kinase signaling plays a role in mechanical and adhesive mechanisms of neutrophil accumulation in lung. Male C57BL/6 mice were treated with the Rho-kinase inhibitor Y-27632 prior to cecal ligation and puncture (CLP). Lung levels of myeloperoxidase (MPO) and histological tissue damage were determined 6h and 24h after CLP. Expression of Mac-1 and F-actin formation in neutrophils were quantified by using flow cytometry 6h after CLP. Mac-1 expression and F-actin formation were also determined in isolated neutrophils up to 3h after stimulation with CXCL2. Labeled and activated neutrophils co-incubated with Y-27632, an anti-Mac-1 antibody and cytochalasin B were adoptively transferred to CLP mice. Y-27632 reduced the CLP-induced pulmonary injury and MPO activity as well as Mac-1 on neutrophils. Neutrophil F-actin formation peaked at 6h and returned to baseline levels 24h after CLP induction. Rho-kinase inhibition decreased CLP-provoked F-actin formation in neutrophils. CXCL2 rapidly increased Mac-1 expression and F-actin formation in neutrophils. Co-incubation with Y-27632 abolished CXCL2-induced Mac-1 up-regulation and formation of F-actin in neutrophils. Notably, co-incubation with cytochalasin B inhibited formation of F-actin but did not reduce Mac-1 expression on activated neutrophils. Adoptive transfer experiments revealed that co-incubation of neutrophils with the anti-Mac-1 antibody or cytochalasin B significantly decreased pulmonary accumulation of neutrophils in septic mice. Our data show that targeting Rho-kinase effectively reduces neutrophil recruitment and tissue damage in abdominal sepsis. Moreover, these findings demonstrate that Rho-kinase-dependent neutrophil accumulation in septic lung injury is regulated by both adhesive and mechanical mechanisms.

  15. CsrA regulates Helicobacter pylori J99 motility and adhesion by controlling flagella formation.

    Science.gov (United States)

    Kao, Cheng-Yen; Sheu, Bor-Shyang; Wu, Jiunn-Jong

    2014-12-01

    Motility mediated by the flagella of Helicobacter pylori has been shown to be required for normal colonization and is thought to be important for the bacteria to move toward the gastric mucus in niches adjacent to the epithelium. Barnard et al. showed that CsrA appears to be necessary for full motility and the ability to infect mice, but its mechanism of regulation is still unclear. Motility and cell adhesion ability were determined in wild-type, csrA mutant, and revertant J99 strains. The bacterial shape and flagellar structure were evaluated by transmission electron microscopy. The expression of two major flagellins, flaA/flaB, and the alternative sigma factor rpoN (σ(54)) were determined by real-time quantitative RT-PCR and Western blot. The csrA mutant showed loss of motility and lower adhesion ability compared with the wild-type and revertant J99 strains. The csrA mutant was not flagellated. Transcription of flaA and flaB mRNA decreased to only 40% and 16%, respectively, in the csrA mutant compared with the wild-type J99 (p = .006 and <.0001, respectively), and Western blot analysis showed dramatically reduced FlaA/FlaB proteins in a csrA mutant. The disruption of csrA also decreased expression of rpoN to 48% in the csrA mutant, but the degradation rate of rpoN mRNA was not changed. These results suggest that CsrA regulates H. pylori J99 flagella formation and thereby affects bacterial motility. © 2014 John Wiley & Sons Ltd.

  16. PERP regulates enamel formation via effects on cell-cell adhesion and gene expression.

    Science.gov (United States)

    Jheon, Andrew H; Mostowfi, Pasha; Snead, Malcolm L; Ihrie, Rebecca A; Sone, Eli; Pramparo, Tiziano; Attardi, Laura D; Klein, Ophir D

    2011-03-01

    Little is known about the role of cell-cell adhesion in the development of mineralized tissues. Here we report that PERP, a tetraspan membrane protein essential for epithelial integrity, regulates enamel formation. PERP is necessary for proper cell attachment and gene expression during tooth development, and its expression is controlled by P63, a master regulator of stratified epithelial development. During enamel formation, PERP is localized to the interface between the enamel-producing ameloblasts and the stratum intermedium (SI), a layer of cells subjacent to the ameloblasts. Perp-null mice display dramatic enamel defects, which are caused, in part, by the detachment of ameloblasts from the SI. Microarray analysis comparing gene expression in teeth of wild-type and Perp-null mice identified several differentially expressed genes during enamel formation. Analysis of these genes in ameloblast-derived LS8 cells upon knockdown of PERP confirmed the role for PERP in the regulation of gene expression. Together, our data show that PERP is necessary for the integrity of the ameloblast-SI interface and that a lack of Perp causes downregulation of genes that are required for proper enamel formation.

  17. ALCAM/CD166 adhesive function is regulated by the tetraspanin CD9

    NARCIS (Netherlands)

    Gilsanz, A.; Sanchez-Martin, L.; Gutierrez-Lopez, M.D.; Ovalle, S.; Machado-Pineda, Y.; Reyes, R.; Swart, G.W.; Figdor, C.G.; Lafuente, E.M.; Cabanas, C.

    2013-01-01

    ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM-ALCAM) or heterophilic (ALCAM-CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenom

  18. Regulation of Adhesion and Migration by the Rsu1- and PINCH1-mediated Inhibition of Focal Adhesion Formation and Actin Polymerization

    Science.gov (United States)

    2012-06-08

    signaling: new phosphodiesterases and new functions. Current opinion in cell biology. 12:174- 179. Sonoda, Y., T. Ozawa , K.D. Aldape, D.F. Deen, M.S...Cancer research. 61:6674- 6678. 72 Sonoda, Y., T. Ozawa , Y. Hirose, K.D. Aldape, M. McMahon, M.S. Berger, and R.O. Pieper. 2001b. Formation

  19. B-cell receptor-associated protein 31 regulates human embryonic stem cell adhesion, stemness, and survival via control of epithelial cell adhesion molecule.

    Science.gov (United States)

    Kim, Won-Tae; Seo Choi, Hong; Min Lee, Hyun; Jang, Young-Joo; Ryu, Chun Jeih

    2014-10-01

    B-Cell receptor-associated protein 31 (BAP31) regulates the export of secreted membrane proteins from the endoplasmic reticulum (ER) to the downstream secretory pathway. Previously, we generated a monoclonal antibody 297-D4 against the surface molecule on undifferentiated human embryonic stem cells (hESCs). Here, we found that 297-D4 antigen was localized to pluripotent hESCs and downregulated during early differentiation of hESCs and identified that the antigen target of 297-D4 was BAP31 on the hESC-surface. To investigate the functional role of BAP31 in hESCs, BAP31 expression was knocked down by small interfering RNA. BAP31 depletion impaired hESC self-renewal and pluripotency and drove hESC differentiation into multicell lineages. BAP31 depletion hindered hESC proliferation by arresting cell cycle at G0/G1 phase and inducing caspase-independent cell death. Interestingly, BAP31 depletion reduced hESC adhesion to extracellular matrix (ECM). Analysis of cell surface molecules showed decreased expression of epithelial cell adhesion molecule (EpCAM) in BAP31-depleted hESCs, while ectopic expression of BAP31 elevated the expression of EpCAM. EpCAM depletion also reduced hESC adhesion to ECM, arrested cell cycle at G0/G1 phase and induced cell death, producing similar effects to those of BAP31 depletion. BAP31 and EpCAM were physically associated and colocalized at the ER and cell surface. Both BAP31 and EpCAM depletion decreased cyclin D1 and E expression and suppressed PI3K/Akt signaling, suggesting that BAP31 regulates hESC stemness and survival via control of EpCAM expression. These findings provide, for the first time, mechanistic insights into how BAP31 regulates hESC stemness and survival via control of EpCAM expression.

  20. Three fim genes required for the regulation of length and mediation of adhesion of Escherichia coli type 1 fimbriae

    DEFF Research Database (Denmark)

    Klemm, P; Christiansen, Gunna

    1987-01-01

    Three novel fim genes of Escherichia coli, fimF, fimG and fimH, were characterized. These genes were not necessary for the production of fimbriae but were shown to be involved in the adhesive property and longitudinal regulation of these structures. Complementation experiments indicated that both...

  1. The reported clinical utility of taurine in ischemic disorders may reflect a down-regulation of neutrophil activation and adhesion.

    Science.gov (United States)

    McCarty, M F

    1999-10-01

    The first publications regarding clinical use of taurine were Italian reports claiming therapeutic efficacy in angina, intermittent claudication and symptomatic cerebral arteriosclerosis. A down-regulation of neutrophil activation and endothelial adhesion might plausibly account for these observations. Endothelial platelet-activating factor (PAF) is a crucial stimulus to neutrophil adhesion and activation, whereas endothelial nitric oxide (NO) suppresses PAF production and acts in various other ways to antagonize binding and activation of neutrophils. Hypochlorous acid (HOCl), a neutrophil product which avidly oxidizes many sulfhydryl-dependent proteins, can be expected to inhibit NO synthase while up-regulating PAF generation; thus, a vicious circle can be postulated whereby HOCl released by marginating neutrophils acts on capillary or venular endothelium to promote further neutrophil adhesion and activation. Taurine is the natural detoxicant of HOCl, and thus has the potential to intervene in this vicious circle, promoting a less adhesive endothelium and restraining excessive neutrophil activation. Agents which inhibit the action of PAF on neutrophils, such as ginkgolides and pentoxifylline, have documented utility in ischemic disorders and presumably would complement the efficacy of taurine in this regard. Fish oil, which inhibits endothelial expression of various adhesion factors and probably PAF as well, and which suppresses neutrophil leukotriene production, may likewise be useful in ischemia. These agents may additionally constitute a non-toxic strategy for treating inflammatory disorders in which activated neutrophils play a prominent pathogenic role. Double-blind studies to confirm the efficacy of taurine in symptomatic chronic ischemia are needed.

  2. Peritoneal Adhesion and Angiogenesis in Ovarian Carcinoma Are Inversely Regulated by Hyaluronan: The Role of Gonadotropins

    Directory of Open Access Journals (Sweden)

    Yael Chagit Tzuman

    2010-01-01

    Full Text Available Ovarian carcinoma is the leading cause of death among gynecologic cancers. Although transformation of the outer ovarian epithelium was linked with ovulation, the disease is significantly more prevalent and severe in postmenopausal women. We postulated that menopause could augment ovarian cancer progression through the effects of gonadotropins on multifocal seeding to the mesothelial layer lining the peritoneum. This seeding is mediated by integrins as well as by CD44 interaction with hyaluronan (HA. Here, we report the effect of gonadotropins on HA synthesis and degradation and on peritoneal adhesion. A significant concentration- and time-dependent induction in expression levels of HA synthases (HASs and hyaluronidases (Hyals was observed in vitro on stimulation of human epithelial ovarian carcinoma cells by gonadotropins. Hormonal regulation of HA-mediated adhesion was manifested in vivo as well, by fluorescence microscopy of stained MLS multicellular tumor spheroids. The number of spheroids adhered to the mesothelium of ovariectomized CD-1 nude mice 9.5 hours after intraperitoneal insertion was significantly higher than in nonovariectomized mice. Inhibition of HA synthesis by 6-diazo-5-oxo-1-norleucine (DON both in spheroids and ovariectomized mice significantly reduced the number of adhered spheroids. Thus, the change in the hormonal environment during menopause assists in HA-dependent adherence of ovarian cancer spheroids onto the peritoneum. However, HA is antiangiogenic and it can significantly suppress tumor progression. Accordingly, angiogenesis of the adhered spheroids was significantly elevated in DON-treated tumors. These results can explain the selective pressure that can lead to simultaneously increased tumor expression of both HASs and Hyals.

  3. Upregulated expression of Nogo-A and NgR in an experimental model of focal microgyria regulates the migration, proliferation and self-renewal of subventricular zone neural progenitors

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Sixun; Shu, Haifeng; Yang, Tao; Huang, Haidong [Department of Neurosurgery, General Hospital of the People' s Liberation Army Chengdu Military Region, Chengdu, Sichuan, 610083 (China); Li, Song [Department of Neurosurgery, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Zhao, Ziyi [Central Laboratory, Teaching Hospital of Chengdu University of Traditional Chinese Medicine, 610075 (China); Kuang, Yongqin, E-mail: kuangyongqin@163.com [Department of Neurosurgery, General Hospital of the People' s Liberation Army Chengdu Military Region, Chengdu, Sichuan, 610083 (China)

    2016-04-29

    Nogo-A and its receptor (NgR) were first described as myelin-associated inhibitors of neuronal regeneration in response to injury. In recent years, knowledge about the important role of the Nogo-A protein in several neuronal pathologies has grown considerably. Here, we employed a neonatal cortex freeze-lesion (NFL) model in neonatal rats and measured the expression of Nogo-A and NgR in the resulting cerebrocortical microdysgenesis 5–75 days after freezing injury. We observed marked upregulation of Nogo-A and NgR in protein levels. Furthermore, the migration of neural precursor cells (NPCs) derived from the subventricular zone (SVZ) toward the sits of injury was perturbed by treatment of NgR antagonist peptide NEP1-40. In vitro analysis showed that the knockdown of NgR by lentivirus-delivered siRNA promoted in axonal regeneration and SVZ-derived neural stem cell/progenitor cell (SVZ-NPCs) adhesion and migration, findings which were similar to the effects of NEP1-40. Taken together, our results indicate an important role for NgR in regulating the physiological processes of SVZ-NPCs. The observation of upregulated Nogo-A/NgR in lesion sites in the NFL model suggest that the effects of the perturbed Nogo-A are a key feature during the development and/or the progression of cortical malformation. - Highlights: • NFL model is an accurate experimental reproduction of focal microgyria of FCD. • The increase of the Nogo-A Levels occurs in response to freeze-induced focal lesioning. • Nogo-A/NgR may play a critical role for in the pathologic progression of FCD. • Nogo-A is associated with the migration, proliferation and self-renewal of SVZ-NPCs.

  4. Regulated intramembrane proteolysis and degradation of murine epithelial cell adhesion molecule mEpCAM.

    Directory of Open Access Journals (Sweden)

    Matthias Hachmeister

    Full Text Available Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, β-, γ-, and ε-sites to generate soluble ectodomains, soluble Aβ-like-, and intracellular fragments termed mEpEX, mEp-β, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and β-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF are further processed to soluble Aβ-like fragments mEp-β and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.

  5. Neural Cell Adhesion Molecule NrCAM Regulates Semaphorin 3F-Induced Dendritic Spine Remodeling

    Science.gov (United States)

    Demyanenko, Galina P.; Mohan, Vishwa; Zhang, Xuying; Brennaman, Leann H.; Dharbal, Katherine E.S.; Tran, Tracy S.; Manis, Paul B.

    2014-01-01

    Neuron-glial related cell adhesion molecule (NrCAM) is a regulator of axon growth and repellent guidance, and has been implicated in autism spectrum disorders. Here a novel postsynaptic role for NrCAM in Semaphorin3F (Sema3F)-induced dendritic spine remodeling was identified in pyramidal neurons of the primary visual cortex (V1). NrCAM localized to dendritic spines of star pyramidal cells in postnatal V1, where it was coexpressed with Sema3F. NrCAM deletion in mice resulted in elevated spine densities on apical dendrites of star pyramidal cells at both postnatal and adult stages, and electron microscopy revealed increased numbers of asymmetric synapses in layer 4 of V1. Whole-cell recordings in cortical slices from NrCAM-null mice revealed increased frequency of mEPSCs in star pyramidal neurons. Recombinant Sema3F-Fc protein induced spine retraction on apical dendrites of wild-type, but not NrCAM-null cortical neurons in culture, while re-expression of NrCAM rescued the spine retraction response. NrCAM formed a complex in brain with Sema3F receptor subunits Neuropilin-2 (Npn-2) and PlexinA3 (PlexA3) through an Npn-2-binding sequence (TARNER) in the extracellular Ig1 domain. A trans heterozygous genetic interaction test demonstrated that Sema3F and NrCAM pathways interacted in vivo to regulate spine density in star pyramidal neurons. These findings reveal NrCAM as a novel postnatal regulator of dendritic spine density in cortical pyramidal neurons, and an integral component of the Sema3F receptor complex. The results implicate NrCAM as a contributor to excitatory/inhibitory balance in neocortical circuits. PMID:25143608

  6. The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules

    DEFF Research Database (Denmark)

    Halberg, Kenneth Agerlin; Rainey, Stephanie M.; Veland, Iben Rønn

    2016-01-01

    Multicellular organisms rely on cell adhesion molecules to coordinate cell-cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most...... role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border....

  7. Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene.

    Science.gov (United States)

    Prigent-Combaret, C; Brombacher, E; Vidal, O; Ambert, A; Lejeune, P; Landini, P; Dorel, C

    2001-12-01

    The Escherichia coli OmpR/EnvZ two-component regulatory system, which senses environmental osmolarity, also regulates biofilm formation. Up mutations in the ompR gene, such as the ompR234 mutation, stimulate laboratory strains of E. coli to grow as a biofilm community rather than in a planktonic state. In this report, we show that the OmpR234 protein promotes biofilm formation by binding the csgD promoter region and stimulating its transcription. The csgD gene encodes the transcription regulator CsgD, which in turn activates transcription of the csgBA operon encoding curli, extracellular structures involved in bacterial adhesion. Consistent with the role of the ompR gene as part of an osmolarity-sensing regulatory system, we also show that the formation of biofilm by E. coli is inhibited by increasing osmolarity in the growth medium. The ompR234 mutation counteracts adhesion inhibition by high medium osmolarity; we provide evidence that the ompR234 mutation promotes biofilm formation by strongly increasing the initial adhesion of bacteria to an abiotic surface. This increase in initial adhesion is stationary phase dependent, but it is negatively regulated by the stationary-phase-specific sigma factor RpoS. We propose that this negative regulation takes place via rpoS-dependent transcription of the transcription regulator cpxR; cpxR-mediated repression of csgB and csgD promoters is also triggered by osmolarity and by curli overproduction, in a feedback regulation loop.

  8. Deciphering the combinatorial roles of geometric, mechanical, and adhesion cues in regulation of cell spreading.

    Directory of Open Access Journals (Sweden)

    Greg M Harris

    Full Text Available Significant effort has gone towards parsing out the effects of surrounding microenvironment on macroscopic behavior of stem cells. Many of the microenvironmental cues, however, are intertwined, and thus, further studies are warranted to identify the intricate interplay among the conflicting downstream signaling pathways that ultimately guide a cell response. In this contribution, by patterning adhesive PEG (polyethylene glycol hydrogels using Dip Pen Nanolithography (DPN, we demonstrate that substrate elasticity, subcellular elasticity, ligand density, and topography ultimately define mesenchymal stem cells (MSCs spreading and shape. Physical characteristics are parsed individually with 7 kilopascal (kPa hydrogel islands leading to smaller, spindle shaped cells and 105 kPa hydrogel islands leading to larger, polygonal cell shapes. In a parallel effort, a finite element model was constructed to characterize and confirm experimental findings and aid as a predictive tool in modeling cell microenvironments. Signaling pathway inhibition studies suggested that RhoA is a key regulator of cell response to the cooperative effect of the tunable substrate variables. These results are significant for the engineering of cell-extra cellular matrix interfaces and ultimately decoupling matrix bound cues presented to cells in a tissue microenvironment for regenerative medicine.

  9. A role for the retinoblastoma protein as a regulator of mouse osteoblast cell adhesion: implications for osteogenesis and osteosarcoma formation.

    Directory of Open Access Journals (Sweden)

    Bernadette Sosa-García

    Full Text Available The retinoblastoma protein (pRb is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis.

  10. Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin.

    Science.gov (United States)

    Galkina, Svetlana I; Sud'ina, Galina F; Klein, Thomas

    2006-08-01

    Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na(+) and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl(-) efflux through chloride channels and Na(+) influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.

  11. Adhesion GPCRs CD97 and GPR56: From structural regulation to cellular function

    NARCIS (Netherlands)

    Hsiao, C.-C.

    2015-01-01

    Cells correspond with their environment through receptors that translate extracellular signals into intracellular messages. Members of the large superfamily of G protein-coupled receptors (GPCRs) control various physiological functions and have been implicated in numerous diseases. Adhesion GPCRs

  12. JAM-L-mediated leukocyte adhesion to endothelial cells is regulated in cis by alpha4beta1 integrin activation.

    Science.gov (United States)

    Luissint, Anny-Claude; Lutz, Pierre G; Calderwood, David A; Couraud, Pierre-Olivier; Bourdoulous, Sandrine

    2008-12-15

    Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation. However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin activation is required for the dissociation of JAM-L-VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen integrin-dependent adhesion of leukocytes to endothelial cells.

  13. Alpha9beta1 integrin in melanoma cells can signal different adhesion states for migration and anchorage

    DEFF Research Database (Denmark)

    Lydolph, Magnus C; Morgan-Fisher, Marie; Høye, Anette M

    2009-01-01

    Cell surface integrins are the primary receptors for cell migration on extracellular matrix, and exist in several activation states regulated in part by ectodomain conformation. The alpha9 integrin subunit, which pairs only with beta1, has specific roles in the immune system and may regulate cell...... migration. Melanoma cells express abundant alpha9beta1 integrin, and its role in cell migration was assessed. Ligands derived from Tenascin-C and ADAM12 supported alpha9beta1 integrin-mediated cell attachment and GTP-Rac dependent migration, but not focal adhesion formation. Manganese ions induced alpha9......beta1 integrin- and Rho kinase-dependent focal adhesion and stress fibre formation, suggesting that the activation status of alpha9beta1 integrin was altered. The effect of manganese ions in promoting focal adhesion formation was reproduced by beta1 integrin activating antibody. The alpha9beta1...

  14. Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Stopp, Sabine; Bornhäuser, Martin; Ugarte, Fernando; Wobus, Manja; Kuhn, Matthias; Brenner, Sebastian; Thieme, Sebastian

    2013-04-01

    The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.

  15. Cadherin-mediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor Escargot.

    Science.gov (United States)

    Tanaka-Matakatsu, M; Uemura, T; Oda, H; Takeichi, M; Hayashi, S

    1996-12-01

    Coordination of cell motility and adhesion is essential for concerted movement of tissues during animal morphogenesis. The Drosophila tracheal network is formed by branching, migration and fusion of tubular ectodermal epithelia. Tracheal tip cells, located at the end of each branch that is going to fuse, extend filopodia to search for targets and later change their cell shape to a seamless ring to allow passage of lumen. The cell adhesion molecule DE-cadherin accumulates at the site of contact to form a ring that marks the site of lumen entry and is essential for the fusion. DE-cadherin expression in tip cells of a subset of branches is dependent on escargot, a zinc finger gene expressed in all tip cells. Such escargot mutant tip cells failed to adhere to each other and continued to search for alternative targets by extending long filopodia. We present evidence indicating escargot positively regulates transcription of the DE-cadherin gene, shotgun. Overexpression of DE-cadherin rescued the defect in one of the fusion points in escargot mutants, demonstrating an essential role of DE-cadherin in target recognition and identifying escargot as a key regulator of cell adhesion and motility in tracheal morphogenesis.

  16. Mechanisms regulating the degradation of dentin matrices by endogenous dentin proteases and their role in dental adhesion. A review.

    Science.gov (United States)

    Sabatini, Camila; Pashley, David H

    2014-08-01

    This systematic review provides an overview of the different mechanisms proposed to regulate the degradation of dentin matrices by host-derived dentin proteases, particularly as it relates to their role in dental adhesion. Significant developments have taken place over the last few years that have contributed to a better understanding of all the factors affecting the durability of adhesive resin restorations. The complexity of dentin-resin interfaces mandates a thorough understanding of all the mechanical, physical and biochemical aspects that play a role in the formation of hybrid layers. The ionic and hydrophilic nature of current dental adhesives yields permeable, unstable hybrid layers susceptible to water sorption, hydrolytic degradation and resin leaching. The hydrolytic activity of host-derived proteases also contributes to the degradation of the resin-dentin bonds. Preservation of the collagen matrix is critical to the improvement of resin-dentin bond durability. Approaches to regulate collagenolytic activity of dentin proteases have been the subject of extensive research in the last few years. A shift has occurred from the use of proteases inhibitors to the use of collagen cross-linking agents. Data provided by 51 studies published in peer-reviewed journals between January 1999 and December 2013 were compiled in this systematic review. Appraisal of the data provided by the studies included in the present review yielded a summary of the mechanisms which have already proven to be clinically successful and those which need further investigation before new clinical protocols can be adopted.

  17. Smooth muscle hyperplasia due to loss of smooth muscle α-actin is driven by activation of focal adhesion kinase, altered p53 localization and increased levels of platelet-derived growth factor receptor-β.

    Science.gov (United States)

    Papke, Christina L; Cao, Jiumei; Kwartler, Callie S; Villamizar, Carlos; Byanova, Katerina L; Lim, Soon-Mi; Sreenivasappa, Harini; Fischer, Grant; Pham, John; Rees, Meredith; Wang, Miranda; Chaponnier, Christine; Gabbiani, Giulio; Khakoo, Aarif Y; Chandra, Joya; Trache, Andreea; Zimmer, Warren; Milewicz, Dianna M

    2013-08-01

    Mutations in ACTA2, encoding the smooth muscle cell (SMC)-specific isoform of α-actin (α-SMA), cause thoracic aortic aneurysms and dissections and occlusive vascular diseases, including early onset coronary artery disease and stroke. We have shown that occlusive arterial lesions in patients with heterozygous ACTA2 missense mutations show increased numbers of medial or neointimal SMCs. The contribution of SMC hyperplasia to these vascular diseases and the pathways responsible for linking disruption of α-SMA filaments to hyperplasia are unknown. Here, we show that the loss of Acta2 in mice recapitulates the SMC hyperplasia observed in ACTA2 mutant SMCs and determine the cellular pathways responsible for SMC hyperplasia. Acta2(-/-) mice showed increased neointimal formation following vascular injury in vivo, and SMCs explanted from these mice demonstrated increased proliferation and migration. Loss of α-SMA induced hyperplasia through focal adhesion (FA) rearrangement, FA kinase activation, re-localization of p53 from the nucleus to the cytoplasm and increased expression and ligand-independent activation of platelet-derived growth factor receptor beta (Pdgfr-β). Disruption of α-SMA in wild-type SMCs also induced similar cellular changes. Imatinib mesylate inhibited Pdgfr-β activation and Acta2(-/-) SMC proliferation in vitro and neointimal formation with vascular injury in vivo. Loss of α-SMA leads to SMC hyperplasia in vivo and in vitro through a mechanism involving FAK, p53 and Pdgfr-β, supporting the hypothesis that SMC hyperplasia contributes to occlusive lesions in patients with ACTA2 missense mutations.

  18. Inhibition of tumor vasculogenic mimicry and prolongation of host survival in highly aggressive gallbladder cancers by norcantharidin via blocking the ephrin type a receptor 2/focal adhesion kinase/paxillin signaling pathway.

    Directory of Open Access Journals (Sweden)

    Hui Wang

    Full Text Available Vasculogenic mimicry (VM is a newly-defined tumor microcirculation pattern in highly aggressive malignant tumors. We recently reported tumor growth and VM formation of gallbladder cancers through the contribution of the ephrin type a receptor 2 (EphA2/focal adhesion kinase (FAK/Paxillin signaling pathways. In this study, we further investigated the anti-VM activity of norcantharidin (NCTD as a VM inhibitor for gallbladder cancers and the underlying mechanisms. In vivo and in vitro experiments to determine the effects of NCTD on tumor growth, host survival, VM formation of GBC-SD nude mouse xenografts, and vasculogenic-like networks, malignant phenotypes i.e., proliferation, apoptosis, invasion and migration of GBC-SD cells. Expression of VM signaling-related markers EphA2, FAK and Paxillin in vivo and in vitro were examined by immunofluorescence, western blotting and real-time polymerase chain reaction (RT-PCR, respectively. The results showed that after treatment with NCTD, GBC-SD cells were unable to form VM structures when injecting into nude mouse, growth of the xenograft was inhibited and these observations were confirmed by facts that VM formation by three-dimensional (3-D matrix, proliferation, apoptosis, invasion, migration of GBC-SD cells were affected; and survival time of the xenograft mice was prolonged. Furthermore, expression of EphA2, FAK and Paxillin proteins/mRNAs of the xenografts was downregulated. Thus, we concluded that NCTD has potential anti-VM activity against human gallbladder cancers; one of the underlying mechanisms may be via blocking the EphA2/FAK/Paxillin signaling pathway.

  19. 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.

  20. A first-in-Asian phase 1 study to evaluate safety, pharmacokinetics and clinical activity of VS-6063, a focal adhesion kinase (FAK) inhibitor in Japanese patients with advanced solid tumors.

    Science.gov (United States)

    Shimizu, Toshio; Fukuoka, Kazuya; Takeda, Masayuki; Iwasa, Tutomu; Yoshida, Takeshi; Horobin, Joanna; Keegan, Mitchell; Vaickus, Lou; Chavan, Ajit; Padval, Mahesh; Nakagawa, Kazuhiko

    2016-05-01

    VS-6063 (also known as defactinib or PF-04554878) is a second-generation inhibitor of focal adhesion kinase and proline-rich tyrosine kinase-2. This phase 1 study evaluated the safety and tolerability, pharmacokinetics, and clinical activity of VS-6063 in Japanese subjects with advanced solid tumor malignancies in a first-in-Asian study setting. VS-6063 was administered orally twice daily (b.i.d.) in 21-day cycles to cohorts of three subjects each with a standard 3 + 3 dose-escalation design until disease progression or unacceptable toxicity. Blood samples for pharmacokinetics were collected on Day 1 and 15. The assessments were performed using CTCAE v4.0 for adverse events (AEs), and the Response Evaluation Criteria In Solid Tumors, version v1.1 (RECIST v1.1) for tumor response. Nine patients were treated across three dose levels (200-600 mg BID). No dose-limiting toxicities were observed at any dose level. Most frequent treatment-related AEs were Grade 1/2 unconjugated hyperbilirubinemia, fatigue, decreased appetite, and diarrhea. Only one subject in the 200 mg BID cohort experienced reversible and transient Grade 3 unconjugated hyperbilirubinemia. PK analyses confirmed that the exposure at the recommended Phase 2 dose (RP2D) of 400 mg BID was comparable with exposures previously reported in non-Japanese subjects. Durable stable disease of approximately 24 weeks was confirmed in two subjects (malignant mesothelioma and rectal cancer). VS-6063 was well tolerated at all dose levels investigated in this first-in-Asian study. These data support the administration of VS-6063 to Japanese subjects at the RP2D in clinical trials involving solid tumor malignancies.

  1. The Junctional Adhesion Molecule-B regulates JAM-C-dependent melanoma cell metastasis.

    Science.gov (United States)

    Arcangeli, Marie-Laure; Frontera, Vincent; Bardin, Florence; Thomassin, Jeanne; Chetaille, Bruno; Adams, Susanne; Adams, Ralf H; Aurrand-Lions, Michel

    2012-11-16

    Metastasis is a major clinical issue and results in poor prognosis for most cancers. The Junctional Adhesion Molecule-C (JAM-C) expressed by B16 melanoma and endothelial cells has been involved in metastasis of tumor cells through homophilic JAM-C/JAM-C trans-interactions. Here, we show that JAM-B expressed by endothelial cells contributes to murine B16 melanoma cells metastasis through its interaction with JAM-C on tumor cells. We further show that this adhesion molecular pair mediates melanoma cell adhesion to primary Lung Microvascular Endothelial Cells and that it is functional in vivo as demonstrated by the reduced metastasis of B16 cells in Jam-b deficient mice.

  2. Cell adhesion and EGFR activation regulate EphA2 expression in cancer

    DEFF Research Database (Denmark)

    Larsen, Alice Bjerregaard; Stockhausen, Marie-Thérése; Poulsen, Hans Skovgaard

    2010-01-01

    . These effects were however abolished by activation of the EGF-receptor ligand system favoring Ras/MAPK signaling and cell proliferation. Based on our results, we propose a regulatory mechanism where cell adhesion induces EGFR kinase activation and EphA2 expression; and where the effect of ephrinA1 mediated...... largely unknown. Here we show that the expression of EphA2 in in vitro cultured cells, is restricted to cells growing adherently and that adhesion-induced EphA2 expression is dependent upon activation of the epidermal growth factor receptor (EGFR), mitogen activated protein kinase kinase (MEK) and Src...... family kinases (SRC). Moreover, the results show that adhesion-induced EGFR activation and EphA2 expression is affected by interactions with extracellular matrix (ECM) proteins working as integrin ligands. Stimulation with the EphA2 ligand, ephrinA1 inhibited ERK phosphorylation and cancer cell viability...

  3. Caspases and p38 MAPK regulate endothelial cell adhesiveness for mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Irina A Potapova

    Full Text Available Mesenchymal stem cells natively circulating or delivered into the blood stream home to sites of injury. The mechanism of mesenchymal stem cell homing to sites of injury is poorly understood. We have shown that the development of apoptosis in endothelial cells stimulates endothelial cell adhesiveness for mesenchymal stem cells. Adhesion of mesenchymal stem cells to apoptotic endothelial cells depends on the activation of endothelial caspases and p38 MAPK. Activation of p38 MAPK in endothelial cells has a primary effect while the activation of caspases potentiates the mesenchymal stem cell adhesion. Overall, our study of the mesenchymal stem cell interaction with endothelial cells indicates that mesenchymal stem cells recognize and specifically adhere to distressed/apoptotic endothelial cells.

  4. Time-dependent effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in focal cerebral ischemia rats

    Institute of Scientific and Technical Information of China (English)

    Zhuoxin Yang; Lihong Diao; Haibo Yu; Wenshu Luo; Ling Wang; Min Pi; Xiaodan Rao; Junhua Peng

    2008-01-01

    BACKGROUND: The onset of focal cerebral ischemia activates extracellular signal-regulated kinases 1 and 2, regulates cell cycle, promotes cell proliferation and differentiation, and affects the normal stage and function of brain cells.OBJECTIVE: To observe the effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in the lateral cerebral ventricle wall of rats with focal cerebral ischemia. The effects were analyzed at different time points after intervention.DESIGN: Randomized controlled study.SETTING: Department of Anatomy, Sun Yat-Sen University. MATERIALS: A total of 60 healthy adult male Wistar rats weighing (250±10) g were provided by the Experimental Animal Center, Medical College of Sun Yat-Sen University. The animal experiment was conducted with confirmed consent by the local ethics committee. The GB6805-Ⅱ electric acupuncture apparatus was provided by Shanghai Medical Equipment High-techno Company. METHODS: The experiment was performed at the Laboratory of Anatomy, Sun Yat-Sen University, from February to July 2007. All experimental animals were randomly divided into the following groups: normal group (n = 6), sham operation group (n = 18), model group (n = 18), and electroacupuncture group (n = 18). Middle cerebral artery occlusion (MCAO) was performed in the model group and electroacupuncture group. Zea Longa's grading standard was used to assess neurological impairment after reperfusion; animals whose grades were between 1 and 4 were included in this study. The normal control group was not exposed to MCAO. In sham operation animals, the right common carotid artery (CCA) was isolated, and the external carotid artery (ECA) was damaged, but no embolism was induced. The electroacupuncture group was given acupuncture on the second day after surgery. The acupoint locations were chosen according to Experimental Acupuncture (People's Publishing House; 1997; First Edition). The Chengjiang, Qihai, and Guanyuan

  5. Redox regulation of sperm surface thiols modulates adhesion to the fallopian tube epithelium.

    Science.gov (United States)

    Talevi, Riccardo; Zagami, Maria; Castaldo, Marianna; Gualtieri, Roberto

    2007-04-01

    Sperm that adhere to the fallopian tube epithelium are of superior quality and adhesion extends their fertile life. It has been postulated that periovulatory signals, as yet undefined, promote sperm release. In the in vitro studies described here, we examined the effects of several antioxidants, reportedly present within oviductal fluid, on the modulation of sperm-oviduct adhesion in bovine species. Results showed that 1) the cell-permeant thiols (penicillamine, beta mercaptoethanol, cysteine, and dithiotreitol), as well as the nonpermeant thiol, reduced glutathione, cause adhering spermatozoa to release from the epithelium; 2) thiol action is exerted on spermatozoa; and 3) oxidized glutathione, as well as the non-thiol antioxidants (dimethylthiourea, trolox, superoxide dismutase, and catalase) have no effect. Sperm surface sulfhydryls labeled with iodoacetamide fluorescein showed that spermatozoa devoid of sulfhydryls on the head surface adhered to the fallopian epithelium in vitro, whereas thiol-induced release increased the exposure of sulfhydryls on the sperm head surface. Finally, analysis of capacitation status demonstrated that uncapacitated spermatozoa adhered to the oviduct, and that thiol-induced release of spermatozoa was accompanied by capacitation. In conclusion, thiol-reducing agents in the oviductal fluid may modulate the redox status of sperm surface proteins, leading to the release of spermatozoa selected and stored through adhesion to the fallopian tube epithelium in the bovine species.

  6. Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase.

    LENUS (Irish Health Repository)

    McSherry, Elaine A

    2012-02-01

    INTRODUCTION: The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. METHODS: MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and beta1-integrin, we examined activation of the beta1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and beta1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. RESULTS: JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the beta1-integrin substrate fibronectin. This was accompanied by reduced protein expression of beta1-integrin and its binding partners alphaV- and alpha5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and beta1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between

  7. Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase.

    LENUS (Irish Health Repository)

    McSherry, Elaine A

    2011-03-23

    ABSTRACT: INTRODUCTION: The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. METHODS: MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. RESULTS: JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF

  8. Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase

    LENUS (Irish Health Repository)

    McSherry, Elaine A

    2011-03-23

    Abstract Introduction The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. Methods MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. Results JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6

  9. MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development.

    Science.gov (United States)

    Lu, Cecilia S; Zhai, Bo; Mauss, Alex; Landgraf, Matthias; Gygi, Stephen; Van Vactor, David

    2014-09-26

    Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. We find that the localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, we find that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage we find that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins.

  10. Intraosseous transcutaneous amputation prostheses versus dental implants: a comparison between keratinocyte and gingival epithelial cell adhesion in vitro.

    Science.gov (United States)

    Pendegrass, C J; Lancashire, H T; Fontaine, C; Chan, G; Hosseini, P; Blunn, G W

    2015-04-19

    Infection is the primary failure modality for transcutaneous implants because the skin breach provides a route for pathogens to enter the body. Intraosseous transcutaneous amputation prostheses (ITAP) are being developed to overcome this problem by creating a seal at the skin-implant interface. Oral gingival epithelial cell attachment creates an infection-free seal around dental implants. However, this has yet to be achieved consistently outside of the oral environment. Epithelial cells attach to metal substrates by means of hemidesmosomes and focal adhesions. Their density per unit cell is an indicator of attachment strength. We postulate that gingival epithelial cells express more hemidesmosomes and focal adhesions at earlier time points, compared with epidermal keratinocytes, and this increased speed and strength of attachment may be the reason why an infection-free seal is often achieved around dental implants but less frequently around ITAP. The aim of this study was to compare epidermal keratinocyte with oral gingival cell attachment on titanium alloy in vitro, to determine whether these two cell types differ in their speed and strength of attachment. We aimed to test the hypothesis that gingival cells up-regulate focal adhesion and hemidesmosome formation at earlier time points compared with extra-oral keratinocytes. To test this hypothesis we cultured epidermal keratinocytes and oral gingival cells on titanium alloy substrates and assessed cell attachment by focal adhesions and hemidesmosome expression at 4, 24, 48 and 72 hours. Formation and expression of hemidesmosomes temporally lagged behind that of focal adhesions in both cell types. Gingival derived cells up-regulated focal adhesion and hemidesmosome expression at earlier time points compared with epidermal keratinocytes. Hemidesmosome expression in oral gingival cells was 3 times greater compared with epidermal keratinocytes at 4 hours. Our findings indicate that earlier attachment may be key to the

  11. Amygdalin Regulates Apoptosis and Adhesion in Hs578T Triple-Negative Breast Cancer Cells.

    Science.gov (United States)

    Lee, Hye Min; Moon, Aree

    2016-01-01

    Amygdalin, D-mandelonitrile-β-D-glucoside-6-β-glucoside, belongs to aromatic cyanogenic glycoside group derived from rosaceous plant seed. Mounting evidence has supported the anti-cancer effects of amygdalin. However, whether amygdalin indeed acts as an anti-tumor agent against breast cancer cells is not clear. The present study aimed to investigate the effect of amygdalin on the proliferation of human breast cancer cells. Here, we show that amygdalin exerted cytotoxic activities on estrogen receptors (ER)-positive MCF7 cells, and MDA-MB-231 and Hs578T triple-negative breast cancer (TNBC) cells. Amygdalin induced apoptosis of Hs578T TNBC cells. Amygdalin downregulated B-cell lymphoma 2 (Bcl-2), upregulated Bcl-2-associated X protein (Bax), activated of caspase-3 and cleaved poly ADP-ribose polymerase (PARP). Amygdalin activated a pro-apoptotic signaling molecule p38 mitogen-activated protein kinases (p38 MAPK) in Hs578T cells. Treatment of amygdalin significantly inhibited the adhesion of Hs578T cells, in which integrin α5 may be involved. Taken together, this study demonstrates that amygdalin induces apoptosis and inhibits adhesion of breast cancer cells. The results suggest a potential application of amygdalin as a chemopreventive agent to prevent or alleviate progression of breast cancer, especially TNBC.

  12. Chronic Restraint Stress Induces an Isoform-Specific Regulation on the Neural Cell Adhesion Molecule in the Hippocampus

    Science.gov (United States)

    Touyarot, K.; Sandi, C.

    2002-01-01

    Existing evidence indicates that 21-days exposure of rats to restraint stress induces dendritic atrophy in pyramidal cells of the hippocampus. This phenomenon has been related to altered performance in hippocampal-dependent learning tasks. Prior studies have shown that hippocampal expression of cell adhesion molecules is modified by such stress treatment, with the neural cell adhesion molecule (NCAM) decreasing and L1 increasing, their expression, at both the mRNA and protein levels. Given that NCAM comprises several isoforms, we investigated here whether chronic stress might differentially affect the expression of the three major isoforms (NCAM-120, NCAM-140, NCAM-180) in the hippocampus. In addition, as glucocorticoids have been implicated in the deleterious effects induced by chronic stress, we also evaluated plasma corticosterone levels and the hippocampal expression of the corticosteroid mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). The results showed that the protein concentration of the NCAM-140 isoform decreased in the hippoampus of stressed rats. This effect was isoform-specific, because NCAM-120 and NCAM-180 levels were not significantly modified. In addition, whereas basal levels of plasma corticosterone tended to be increased, MR and GR concentrations were not significantly altered. Although possible changes in NCAM-120, NCAM-180 and corticosteroid receptors at earlier time points of the stress period cannot be ignored; this study suggests that a down-regulation of NCAM-140 might be implicated in the structural alterations consistently shown to be induced in the hippocampus by chronic stress exposure. As NCAM-140 is involved in cell-cell adhesion and neurite outgrowth, these findings suggest that this molecule might be one of the molecular mechanisms involved in the complex interactions among neurodegeneration-related events. PMID:12757368

  13. Novel secreted isoform of adhesion molecule ICAM-4: Potential regulator of membrane-associated ICAM-4 interactions

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gloria; Spring, Frances A.; Parons, Stephen F.; Mankelow, Tosti J.; Peters, Luanne L.; Koury, Mark J.; Mohandas, Narla; Anstee, David J.; Chasis, Joel Anne

    2003-02-18

    ICAM-4, a newly characterized adhesion molecule, is expressed early in human erythropoiesis and functions as a ligand for binding a4b1 and aV integrin-expressing cells. Within the bone marrow, erythroblasts surround central macrophages forming erythroblastic islands. Evidence suggests that these islands are highly specialized subcompartments where cell adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. Since erythroblasts express a4b1 and ICAM-4 and macrophages exhibit aV, ICAM-4 is an attractive candidate for mediating cellular interactions within erythroblastic islands. To determine whether ICAM-4 binding properties are conserved across species, we first cloned and sequenced the murine homologue. The translated amino acid sequence showed 68 percent overall identity with human ICAM-4. Using recombinant murine ICAM-4 extracellular domains, we discovered that hematopoietic a4b1-expressing HEL cells and non-hematopoietic aV-expressing FLY cells adhered to mouse ICAM-4. Cell adhesion studies showed that FLY and HEL cells bound to mouse and human proteins with similar avidity. These data strongly suggest conservation of integrin-binding properties across species. Importantly, we characterized a novel second splice cDNA that would be predicted to encode an ICAM-4 isoform, lacking the membrane-spanning domain. Erythroblasts express both isoforms of ICAM-4. COS-7 cells transfected with GFP constructs of prototypic or novel ICAM-4 cDNA showed different cellular localization patterns. Moreover, analysis of tissue culture medium revealed that the novel ICAM-4 cDNA encodes a secreted protein. We postulate that secretion of this newly described isoform, ICAM-4S, may modulate binding of membrane-associated ICAM-4 and could thus play a critical regulatory role in erythroblast molecular attachments.

  14. Engagement of major histocompatibility complex class I and class II molecules up-regulates intercellular adhesion of human B cells via a CD11/CD18-independent mechanism.

    Science.gov (United States)

    Alcover, A; Juillard, V; Acuto, O

    1992-02-01

    We have studied the role of major histocompatibility complex (MHC) molecules in the regulation of intercellular adhesion of human B cells. We found that molecules able to bind to MHC class II molecules, such as monoclonal antibodies or staphylococcal enterotoxins, induced rapid and sustained homotypic adhesion of Epstein-Barr virus (EBV)-transformed B cell lines as well as peripheral blood B lymphocytes. Moreover, anti-MHC class I monoclonal antibodies also stimulated intercellular adherence. Adhesion induced upon MHC engagement was faster and stronger than that triggered by phorbol esters. It needed active metabolism, but divalent cations were not required. Monoclonal antibodies directed against LFA-1 (CD11a/CD18) or its ligand ICAM-1 (CD54) did not inhibit MHC class II-induced homotypic adhesion of various EBV-transformed B cell lines, nor of a variant of the B cell line Raji expressing very low LFA-1 surface levels. Moreover, EBV-transformed B cells from a severe lymphocyte adhesion deficiency patient, lacking surface CD11/CD18, also aggregated in response to anti-MHC class I or class II monoclonal antibodies. Together these data indicate that engagement of MHC molecules may transduce signals to B cells resulting in up-regulation of intercellular adhesion, via an LFA-1-independent mechanism. This may play a role in the stabilization of T cell/antigen-presenting cell conjugates at the moment of antigen recognition.

  15. Ischemic Postconditioning Alleviates Brain Edema After Focal Cerebral Ischemia Reperfusion in Rats Through Down-Regulation of Aquaporin-4.

    Science.gov (United States)

    Han, Dong; Sun, Miao; He, Ping-Ping; Wen, Lu-Lu; Zhang, Hong; Feng, Juan

    2015-07-01

    Cerebral edema is a serious complication associated with cerebral ischemia/reperfusion (I/R). Aquaporin-4 (AQP4) plays a role in generating postischemic edema after reperfusion. Recently, ischemic postconditioning (Postcond) has been shown to produce neuroprotective effects and reduce brain edema in rats after cerebral I/R. It is unclear if ischemic Postcond alleviates brain edema injury through regulation of AQP4. In this study, middle cerebral artery occlusion (MCAO) was induced in rats by filament insertion for 2 h following 24-h reperfusion: ischemic Postcond treatment was performed before reperfusion in the experimental group. We used the wet-dry weight ratio and transmission electron microscopy to evaluate brain edema after 24 h of reperfusion. We used immunohistochemistry and Western blot analyses to evaluate the distribution and expression of AQP4. Ischemic Postcond significantly reduced the water content of the brain tissue and swelling of the astrocytic foot processes. AQP4 expression increased in the I/R and Postcond groups compared to the sham group, but it decreased in the Postcond group compared to the I/R group. The results of our study suggest that ischemic Postcond effectively reduces brain edema after reperfusion by inhibiting AQP4 expression. The data in this study support the use of ischemic Postcond for alleviating brain edema after cerebral I/R.

  16. Virus-activated T cells regulate expression of adhesion molecules on endothelial cells in sites of infection

    DEFF Research Database (Denmark)

    Marker, O; Scheynius, A; Christensen, Jan Pravsgaard

    1995-01-01

    was the inflammatory reaction dependent on the presence of CD8+ cells, but these cells also appeared to be required for maximal upregulation of ICAM-1 and VCAM-1 on the endothelial cells. These results indicate that virus-specific CD8+ T cells are crucially involved in regulating the inflammatory reaction through......To study the role of cell adhesion molecules in the fatal CD8+ T-cell mediated meningitis which is induced by intracerebral infection with lymphocytic choriomeningitis virus, the expression of relevant molecules on inflammatory cells and local endothelium was analyzed immunohistochemically. Most...... inflammatory cells were strongly positive for LFA-1, VLA-4, Pgp-1 and ICAM-1. Expression of ICAM-1 and VCAM-1 was upregulated on the endothelial cells in immunocompetent mice, but not in T-cell deficient nude mice. Analysis of mice deficient in either CD4+ or CD8+ T cells, revealed that not only...

  17. The Drosophila cell adhesion molecule klingon is required for long-term memory formation and is regulated by Notch.

    Science.gov (United States)

    Matsuno, Motomi; Horiuchi, Junjiro; Tully, Tim; Saitoe, Minoru

    2009-01-06

    The ruslan (rus) mutant was previously identified in a behavioral screen for mutants defective in long-lasting memory, which consists of two consolidated memory types, anesthesia-resistant memory, and protein synthesis-dependent long-term memory (LTM). We demonstrate here that rus is a new allele of klingon (klg), which encodes a homophilic cell adhesion molecule. Klg is acutely required for LTM but not anesthesia-resistant memory formation, and Klg expression increases upon LTM induction. LTM formation also requires activity of the Notch cell-surface receptor. Although defects in Notch have been implicated in memory loss because of Alzheimer's disease, downstream signaling linking Notch to memory have not been determined. Strikingly, we found that Notch activity increases upon LTM induction and regulates Klg expression. Furthermore, Notch-induced enhancement of LTM is disrupted by a klg mutation. We propose that Klg is a downstream effector of Notch signaling that links Notch activity to memory.

  18. Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitch-regulated collagen adhesion protein.

    Science.gov (United States)

    Tang, Qing; Yin, Kang; Qian, Hongliang; Zhao, Youwen; Wang, Wen; Chou, Shan-Ho; Fu, Yang; He, Jin

    2016-07-06

    Cyclic di-GMP is a ubiquitous second messenger that regulates diverse cellular processes in bacteria by binding to various protein or riboswitch effectors. In Bacillus thuringiensis BMB171, a c-di-GMP riboswitch termed Bc2 RNA resides in the 5'-untranslated region (5'-UTR) of an mRNA that encodes a collagen adhesion protein (Cap). The expression of cap was strongly repressed in parent strain BMB171 because of the presence of Bc2 RNA but was significantly promoted in the Bc2 RNA markerless deletion mutant. Bc2 RNA acts as a genetic "on" switch, which forms an anti-terminator structure to promote cap read-through transcription upon c-di-GMP binding. As a result, cap transcription was de-repressed under high c-di-GMP levels. Therefore, Bc2 RNA regulates cap expression using a repression/de-repression model. Bc2 RNA-regulated Cap was also found to be tightly associated with motility, aggregation, exopolysaccharide secretion, biofilm formation, and virulence of B. thuringiensis BMB171 against its host insect Helicoverpa armigera.

  19. Ligation of MHC class I and class II molecules can lead to heterologous desensitization of signal transduction pathways that regulate homotypic adhesion in human lymphocytes.

    Science.gov (United States)

    Wagner, N; Engel, P; Vega, M; Tedder, T F

    1994-06-01

    Engagement of lymphocyte MHC class I and class II Ags activates an array of intracellular signal transduction pathways that up-regulates the activity of cell-surface adhesion receptors, resulting in homotypic cell-cell aggregation. In this study, engagement of MHC class I and class II molecules with specific mAbs was shown to also inhibit lymphocyte homotypic adhesion. Two mAbs reactive with class II Ag, homotypic adhesion blocking mAb (HAB)-2, and HAB-3, and one mAb reactive with class I Ag, HAB-4, were generated that inhibited homotypic adhesion of activated lymphocytes and B and T cell lines at concentrations as low as 0.1 microgram/ml. Binding of these mAbs resulted in heterologous desensitization of other surface signal transduction molecules as homotypic adhesion induced through class I, class II, CD19, CD20, CD39, CD40, Leu-13, and PMA was also inhibited. The spontaneous adhesion exhibited by some cell lines was also abrogated by binding of these mAbs. Abs that either induced, blocked, or had no effect on adhesion bound to distinct epitopes on class I, whereas the anti-class II mAbs recognized either distinct or overlapping epitopes. Thus, engagement of distinct epitopes on MHC molecules can result in homologous or heterologous desensitization of cell-surface signaling molecules. The induction or inhibition of homotypic adhesion through class I molecules did not require the presence of the cytoplasmic domain, as deletion of this portion of the class I molecule had no effect. In contrast, the transmembrane region was essential for signal transduction as the mAbs binding to a chimeric molecule in which the transmembrane and cytoplasmic domains of class I were exchanged with those of the HB15 molecule did not induce or inhibit homotypic adhesion. Although this report is the first demonstration that homotypic adhesion can be influenced in a negative manner through MHC molecules, these findings demonstrate a considerable level of cross-talk between MHC molecules

  20. 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.

  1. Annexin II/annexin II receptor axis regulates adhesion, migration, homing, and growth of prostate cancer

    Science.gov (United States)

    Shiozawa, Yusuke; Havens, Aaron M.; Jung, Younghun; Ziegler, Anne M.; Pedersen, Elisabeth A.; Wang, Jingcheng; Wang, Jianhua; Lu, Ganwei; Roodman, G. David; Loberg, Robert D.; Pienta, Kenneth J.; Taichman, Russell S.

    2013-01-01

    One of the most life-threatening complications of prostate cancer is skeletal metastasis. In order to develop treatment for metastasis, it is important to understand its molecular mechanisms. Our work in this field has drawn parallels between hematopoietic stem cell and prostate cancer homing to the marrow. Our recent work demonstrated that annexin II expressed by osteoblasts and endothelial cells plays a critical role in niche selection. In this study, we demonstrate that annexin II and its receptor play a crucial role in establishing metastasis of prostate cancer. Prostate cancer cell lines migrate toward annexin II and the adhesion of prostate cancer to osteoblasts and endothelial cells was inhibited by annexin II. By blocking annexin II or its receptor in animal models, short-term and long-term localization of prostate cancers are limited. Annexin II may also facilitate the growth of prostate cancer in vitro and in vivo by the MAPK pathway. These data strongly suggest annexin II and its receptor axis plays a central role in prostate cancer metastasis, and that prostate cancer utilize the hematopoietic stem cell homing mechanisms to gain access to the niche. PMID:18636554

  2. Prevalence of Adhesion and Regulation of Biofilm-Related Genes in Different Clones of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Salman Sahab Atshan

    2012-01-01

    Full Text Available Clinical information about genotypically different clones of biofilm-producing Staphylococcus aureus is largely unknown. We examined whether different clones of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA differ with respect to staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMMs in biofilm formation. The study used 60 different types of spa and determined the phenotypes, the prevalence of the 13 MSCRAMM, and biofilm genes for each clone. The current investigation was carried out using a modified Congo red agar (MCRA, a microtiter plate assay (MPA, polymerase chain reaction (PCR, and reverse transcriptase polymerase chain reaction (RT-PCR. Clones belonging to the same spa type were found to have similar properties in adheringto thepolystyrene microtiter plate surface. However, their ability to produce slime on MCRA medium was different. PCR experiments showed that 60 clones of MSSA and MRSA were positive for 5 genes (out of 9 MSCRAMM genes. icaADBC genes were found to be present in all the 60 clones tested indicating a high prevalence, and these genes were equally distributed among the clones associated with MSSA and those with MRSA. The prevalence of other MSCRAMM genes among MSSA and MRSA clones was found to be variable. MRSA and MSSA gene expression (MSCRAMM and icaADBC was confirmed by RT-PCR.

  3. Systems analysis of quantitative shRNA-library screens identifies regulators of cell adhesion

    Directory of Open Access Journals (Sweden)

    Huang XiaoDong

    2008-06-01

    Full Text Available Abstract Background High throughput screens with RNA interference technology enable loss-of-function analyses of gene activities in mammalian cells. While the construction of genome-scale shRNA libraries has been successful, results of large-scale screening of those libraries can be difficult to analyze because of the relatively high noise levels and the fact that not all shRNAs in a library are equally effective in silencing gene expression. Results We have screened a library consisting of 43,828 shRNAs directed against 8,500 human genes for functions that are necessary in cell detachment induced by a constitutively activated c-Abl tyrosine kinase. To deal with the issues of noise and uncertainty of knockdown efficiencies, we employed an analytical strategy that combines quantitative data analysis with biological knowledge, i.e. Gene Ontology and pathway information, to increase the power of the RNAi screening technique. Using this strategy we found 16 candidate genes to be involved in Abl-induced disruption of cell adhesion, and verified that the knockdown of IL6ST is associated with enhanced cell attachment. Conclusion Our results suggest that the power of genome-wide quantitative shRNA screens can be significantly increased when analyzed using a systems biology-based approach to identify functional gene networks.

  4. Laminin-511: a multi-functional adhesion protein regulating cell migration, tumor invasion and metastasis.

    Science.gov (United States)

    Pouliot, Normand; Kusuma, Nicole

    2013-01-01

    Laminins are major constituents of basement membranes. At least 16 isoforms have now been described, each with distinct spatio-temporal expression patterns and functions. The laminin-511 heterotrimer (α5β1γ1) is one of the more recent isoforms to be identified and a potent adhesive and pro-migratory substrate for a variety of normal and tumor cell lines in vitro. As our understanding of its precise function in normal tissues and in pathologies is rapidly unraveling, current evidence suggests an important regulatory role in cancer. This review describes published data on laminin-511 expression in several malignancies and experimental evidence from both in vitro and in vivo studies supporting its functional role during tumor progression. A particular emphasis is put on more recent studies from our laboratory and that of others indicating that laminin-511 contributes to tumor dissemination and metastasis in advanced breast carcinomas and other tumor types. Collectively, the experimental evidence suggests that high expression of laminin-511 has prognostic significance and that targeting tumor-laminin-511 interactions may have therapeutic potential in advanced cancer patients.

  5. RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation.

    Science.gov (United States)

    Morales, Mònica; Arenas, Enrique J; Urosevic, Jelena; Guiu, Marc; Fernández, Esther; Planet, Evarist; Fenwick, Robert Bryn; Fernández-Ruiz, Sonia; Salvatella, Xavier; Reverter, David; Carracedo, Arkaitz; Massagué, Joan; Gomis, Roger R

    2014-07-01

    In estrogen receptor-negative breast cancer patients, metastatic relapse usually occurs in the lung and is responsible for the fatal outcome of the disease. Thus, a better understanding of the biology of metastasis is needed. In particular, biomarkers to identify patients that are at risk of lung metastasis could open the avenue for new therapeutic opportunities. Here we characterize the biological activity of RARRES3, a new metastasis suppressor gene whose reduced expression in the primary breast tumors identifies a subgroup of patients more likely to develop lung metastasis. We show that RARRES3 downregulation engages metastasis-initiating capabilities by facilitating adhesion of the tumor cells to the lung parenchyma. In addition, impaired tumor cell differentiation due to the loss of RARRES3 phospholipase A1/A2 activity also contributes to lung metastasis. Our results establish RARRES3 downregulation as a potential biomarker to identify patients at high risk of lung metastasis who might benefit from a differentiation treatment in the adjuvant programme.

  6. Positive and negative regulation by SLP-76/ADAP and Pyk2 of chemokine-stimulated T-lymphocyte adhesion mediated by integrin α4β1

    Science.gov (United States)

    Dios-Esponera, Ana; Isern de Val, Soledad; Sevilla-Movilla, Silvia; García-Verdugo, Rosa; García-Bernal, David; Arellano-Sánchez, Nohemí; Cabañas, Carlos; Teixidó, Joaquin

    2015-01-01

    Stimulation by chemokines of integrin α4β1–dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4β1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4β1 ligands, a transient stimulation in the association of Vav1 with SLP-76, Pyk2, and ADAP was observed. Using T-cells depleted for SLP-76, ADAP, or Pyk2, or expressing Pyk2 kinase–inactive forms, we show that SLP-76 and ADAP stimulate chemokine-activated, α4β1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4β1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4β1-VCAM-1 interaction and cell spreading on VCAM-1 are targets of regulation by these three proteins. GTPase assays, expression of activated or dominant-negative Rac1, or combined ADAP and Pyk2 silencing indicated that Rac1 activation by CXCL12 is a common mediator response in SLP-76–, ADAP-, and Pyk2-regulated cell adhesion involving α4β1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4β1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation. PMID:26202465

  7. Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation

    Science.gov (United States)

    Umar, Sadiq; Hedaya, Omar; Singh, Anil K.; Ahmed, Salahuddin

    2015-01-01

    Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1–5 μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p<0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p<0.05; n=4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p<0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA. PMID:26134265

  8. Neural cell adhesion molecule-180-mediated homophilic binding induces epidermal growth factor receptor (EGFR) down-regulation and uncouples the inhibitory function of EGFR in neurite outgrowth

    DEFF Research Database (Denmark)

    Povlsen, Gro Klitgaard; Berezin, Vladimir; Bock, Elisabeth

    2008-01-01

    The neural cell adhesion molecule (NCAM) plays important roles in neuronal development, regeneration, and synaptic plasticity. NCAM homophilic binding mediates cell adhesion and induces intracellular signals, in which the fibroblast growth factor receptor plays a prominent role. Recent studies...... not require NCAM-mediated fibroblast growth factor receptor activation....... on axon guidance in Drosophila suggest that NCAM also regulates the epidermal growth factor receptor (EGFR) (Molecular and Cellular Neuroscience, 28, 2005, 141). A possible interaction between NCAM and EGFR in mammalian cells has not been investigated. The present study demonstrates for the first time...

  9. Warfarin and coumarin-like Murraya paniculata extract down-regulate EpCAM-mediated cell adhesion: individual components versus mixture for studying botanical metastatic chemopreventives.

    Science.gov (United States)

    Shao, Jingwei; Zhou, Suxia; Jiang, Zhou; Chi, Ting; Ma, Ji; Kuo, Minliang; Lee, Alan Yueh-Luen; Jia, Lee

    2016-08-02

    We recently defined cancer metastatic chemoprevention as utilizing safe and effective molecules to comprehensively prevent the spark of activation-adhesion-extravasation-proliferation metastatic cascade caused by circulating tumor cells (CTCs). The strategy focuses on preventing the most important starting point of the cascade. We identified an extract from a well-known medical plant Murraya paniculata, which inhibited both embryonic implantation to human endometrium as traditionally-used for abortion and CTC adhesion to human endothelium. Here, we separated and characterized five coumarin-containing components (Z1-Z5) from the botanic extract. Flow cytometry revealed that within 1-100 μg/mL, Z3 and Z5 down-regulated EpCAM expression in human colon HCT116, whereas, Z1 and Z2 did oppositely. Warfarin and Z1-Z5 component mixture (CM) also down-regulated EpCAM expression. The down-regulation of EpCAM by Z3, Z5, CM and warfarin was confirmed by western blotting, and caused inhibition on adhesion of cancer cells to human endothelial cells. Rat coagulation study showed that warfarin prolonged prothrombin time, whereas, Z3 did not. The present studies revealed that, for the first time, warfarin and coumarin-like components Z3, Z5 and CM from Murraya paniculata could directly inhibit EpCAM-mediated cell-cell adhesion.

  10. Myocyte enhancer factor 2D regulates ectoderm specification and adhesion properties of animal cap cells in the early Xenopus embryo.

    Science.gov (United States)

    Katz Imberman, Sandra; Kolpakova, Alina; Keren, Aviad; Bengal, Eyal

    2015-08-01

    In Xenopus, animal cap (AC) cells give rise to ectoderm and its derivatives: epidermis and the central nervous system. Ectoderm has long been considered a default pathway of embryonic development, with cells that are not under the influence of vegetal Nodal signaling adopting an ectodermal program of gene expression. In the present study, we describe the involvement of the animally-localized maternal transcription factor myocyte enhancer factor (Mef) 2D in regulating the identity of AC cells. We find that Mef2D is required for the formation of both ectodermal lineages: neural and epidermis. Gain and loss of function experiments indicate that Mef2D regulates early gastrula expression of key ectodermal/epidermal genes in the animal region. Mef2D controls the activity of zygotic bone morphogenetic protein (BMP) signaling known to dictate the epidermal differentiation program. Exogenous expression of Mef2D in vegetal blastomeres was sufficient to induce ectopic expression of ectoderm/epidermal genes in the vegetal half of the embryo, when Nodal signaling was inhibited. Depletion of Mef2D caused a loss of AC cell adhesion that was rescued by the expression of E-cadherin or bone morphogenetic protein 4. In addition, expression of Mef2D in the prospective endoderm caused unusual aggregation of vegetal cells with animal cells in vitro and inappropriate segregation to other germ layers in vivo. Mef2D cooperates with another animally-expressed transcription factor, FoxI1e. Together, they regulate the expression of genes encoding signaling proteins and the transcription factors that control the regional identity of animal cells. Therefore, we describe a new role for the animally-localized Mef2D protein in early ectoderm specification, which is similar to that of the vegetally-localized VegT in endoderm and mesoderm formation. © 2015 FEBS.

  11. Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation.

    Science.gov (United States)

    Umar, Sadiq; Hedaya, Omar; Singh, Anil K; Ahmed, Salahuddin

    2015-09-15

    Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1-5μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (pinhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (pTNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (pTNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA.

  12. The Role of TSC Proteins in Regulating Cell Adhesion and Motility

    Science.gov (United States)

    2006-09-01

    2004). Growth factors, insulin, nutrients , and the cellular energy levels regulate the activity of TSC2 (McManus and Alessi, 2002; Inoki et al., 2003b...growth fac- tors and abundant nutrients , TSC2 activity is suppressed, leading to increasedRheb andmTOR/S6K1 activity. Studies using estab- lished TSC2...Chromosomes Cancer 2003;38:368–375. 45. Jin F, Wienecke R, Xiao G-H, Maize JC Jr, DeClue JE, Yeung RS. Suppression of tumorigenicity by the wild-type

  13. Localized LoxL3-Dependent Fibronectin Oxidation Regulates Myofiber Stretch and Integrin-Mediated Adhesion.

    Science.gov (United States)

    Kraft-Sheleg, Ortal; Zaffryar-Eilot, Shelly; Genin, Olga; Yaseen, Wesal; Soueid-Baumgarten, Sharon; Kessler, Ofra; Smolkin, Tatyana; Akiri, Gal; Neufeld, Gera; Cinnamon, Yuval; Hasson, Peleg

    2016-03-07

    For muscles to function, myofibers have to stretch and anchor at the myotendinous junction (MTJ), a region rich in extracellular matrix (ECM). Integrin signaling is required for MTJ formation, and mutations affecting the cascade lead to muscular dystrophies in mice and humans. Underlying mechanisms for integrin activation at the MTJ and ECM modifications regulating its signaling are unclear. We show that lysyl oxidase-like 3 (LoxL3) is a key regulator of integrin signaling that ensures localized control of the cascade. In LoxL3 mutants, myofibers anchor prematurely or overshoot to adjacent somites, and are loose and lack tension. We find that LoxL3 complexes with and directly oxidizes Fibronectin (FN), an ECM scaffold protein and integrin ligand enriched at the MTJ. We identify a mechanism whereby localized LoxL3 secretion from myofiber termini oxidizes FN, enabling enhanced integrin activation at the tips of myofibers and ensuring correct positioning and anchoring of myofibers along the MTJ. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation

    Energy Technology Data Exchange (ETDEWEB)

    Umar, Sadiq; Hedaya, Omar; Singh, Anil K.; Ahmed, Salahuddin, E-mail: salah.ahmed@wsu.edu

    2015-09-15

    Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1–5 μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p < 0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p < 0.05; n = 4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p < 0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA. - Highlights: • Evolving evidence suggests that ASK1 plays a central role in rheumatic arthritis (RA). • TNF-α activates ASK1, which regulate downstream signaling through JNK/p38 activation in RA-FLS. • ASK1 may be used as a potential therapeutic target in RA. • Thymoquinone was able to selectively inhibit TNF-α-induced phosphorylation of ASK1 in RA-FLS. • Thymoquinone might serve as a potential small

  15. ARF6-regulated endocytosis of growth factor receptors links cadherin-based adhesion to canonical Wnt signaling in epithelia.

    Science.gov (United States)

    Pellon-Cardenas, Oscar; Clancy, James; Uwimpuhwe, Henriette; D'Souza-Schorey, Crislyn

    2013-08-01

    Wnt signaling has an essential role in embryonic development as well as stem/progenitor cell renewal, and its aberrant activation is implicated in many diseases, including several cancers. β-Catenin is a critical component of Wnt-mediated transcriptional activation. Here we show that ARF6 activation during canonical Wnt signaling promotes the intracellular accumulation of β-catenin via a mechanism that involves the endocytosis of growth factor receptors and robust activation of extracellular signal-regulated kinase (ERK). ERK promotes casein kinase 2-mediated phosphorylation of α-catenin, leading to destabilization of the adherens junctions and a subsequent increase in cytoplasmic pools of active β-catenin and E-cadherin. ERK also phosphorylates LRP6 to amplify the Wnt transduction pathway. The aforementioned Wnt-ERK signaling pathway initiates lumen filling of epithelial cysts by promoting cell proliferation in three-dimensional cell cultures. This study elucidates a mechanism responsible for the switch in β-catenin functions in cell adhesion at the adherens junctions and Wnt-induced nuclear signaling.

  16. Breast cancer metastasis suppressor 1 (BRMS1) suppresses attachment and spreading of breast cancer cells on 2D and 3D extracellular matrix components by altering focal adhesion-associated signaling

    Science.gov (United States)

    Metastatic dissemination of cancer cells from primary tumor to secondary sites is a multi-step process that depends heavily on the ability of cancer cells to respond to the microenvironmental cues, such as changes in composition of surrounding extracellular matrix (ECM), by adapting their adhesion a...

  17. Regulation of vascular cell adhesion molecule-1 in dental pulp cells by interleukin-1β: the role of prostanoids.

    Science.gov (United States)

    Chang, Mei-Chi; Lin, Li-Deh; Zwei-Ching Chang, Jenny; Huang, Chiung-Fang; Chuang, Fu-Hsiung; Lee, Jang-Jaer; Jeng, Po-Yuan; Wang, Tong-Mei; Jeng, Jiiang-Huei

    2012-06-01

    Vascular cell adhesion molecule (VCAM-1) plays a critical role in the inflammatory processes by stimulating the recruitment, extravasation, and migration of leukocytes. Its expression and regulation in the dental pulp is not well elucidated. Primary dental pulp cells were exposed to prostaglandin E(2) (PGE(2)), prostaglandin F(2α) (PGF(2α)), or interleukin 1β (IL-1β) with/without aspirin. VCAM-1 messenger RNA expression was analyzed by reverse transcriptase-polymerase chain reaction. Soluble VCAM-1 (sVCAM-1) in the culture medium was determined by enzyme-linked immunosorbent assay, and the number of viable cells was estimated by (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. IL-1β induced VCAM-1 gene expression of pulp cells. IL-1β also stimulated sVCAM-1 production. The IL-1β-induced sVCAM-1 production was not inhibited but rather enhanced by aspirin, a cyclooxygenase (COX) inhibitor. PGE(2) and PGF(2α) decreased the VCAM-1 expression and sVCAM-1 production of pulp cells. U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), a mitogen-activated protein kinase kinase (MEK) inhibitor, attenuated IL-1β-induced sVCAM-1 production. However, no marked cytotoxicity was noted in these experimental conditions as analyzed by MTT assay. IL-1β may be involved in the pulpal inflammatory processes via stimulation of VCAM-1 expression and sVCAM-1 production. This event is not mediated by COX activation and prostanoid production but is associated with MEK signaling. PGE(2) and PGF(2α) may potentially regulate inflammatory processes by the inhibition of VCAM-1. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  18. Association of membrane/lipid rafts with the platelet cytoskeleton and the caveolin PY14: participation in the adhesion process.

    Science.gov (United States)

    Cerecedo, Doris; Martínez-Vieyra, Ivette; Maldonado-García, Deneb; Hernández-González, Enrique; Winder, Steve J

    2015-11-01

    Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and β-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete β-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for β-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics.

  19. A novel role for fibronectin type I domain in the regulation of human hematopoietic cell adhesiveness through binding to follistatin domains of FLRG and follistatin.

    Science.gov (United States)

    Maguer-Satta, Véronique; Forissier, Stéphanie; Bartholin, Laurent; Martel, Sylvie; Jeanpierre, Sandrine; Bachelard, Elodie; Rimokh, Ruth

    2006-02-15

    FLRG and follistatin belong to the family of follistatin proteins involved in the regulation of various biological effects, such as hematopoiesis, mediated by their binding to activin and BMP, both members of the TGFbeta family. To further characterize the function of FLRG, we searched for other possible functional partners using a yeast two-hybrid screen. We identified human fibronectin as a new partner for both FLRG and follistatin. We also demonstrated that their physical interaction is mediated by type I motifs of fibronectin and follistatin domains. We then analyzed the biological consequences of these protein interactions on the regulation of hematopoiesis. For the first time, we associated a biological effect with the regulation of human hematopoietic cell adhesiveness of both the type I motifs of fibronectin and the follistatin domains of FLRG and follistatin. Indeed, we observed a significant and specific dose-dependent increase of cell adhesion to fibronectin in the presence of FLRG or follistatin, using either a human hematopoietic cell line or primary cells. In particular, we observed a significantly increased adhesion of immature hematopoietic precursors (CFC, LTC-IC). Altogether these results highlight a new mechanism by which FLRG and follistatin regulate human hematopoiesis.

  20. The cell adhesion molecules Echinoid and Friend of Echinoid coordinate cell adhesion and cell signaling to regulate the fidelity of ommatidial rotation in the Drosophila eye.

    Science.gov (United States)

    Fetting, Jennifer L; Spencer, Susan A; Wolff, Tanya

    2009-10-01

    Directed cellular movements are a universal feature of morphogenesis in multicellular organisms. Differential adhesion between the stationary and motile cells promotes these cellular movements to effect spatial patterning of cells. A prominent feature of Drosophila eye development is the 90 degrees rotational movement of the multicellular ommatidial precursors within a matrix of stationary cells. We demonstrate that the cell adhesion molecules Echinoid (Ed) and Friend of Echinoid (Fred) act throughout ommatidial rotation to modulate the degree of ommatidial precursor movement. We propose that differential levels of Ed and Fred between stationary and rotating cells at the initiation of rotation create a permissive environment for cell movement, and that uniform levels in these two populations later contribute to stopping the movement. Based on genetic data, we propose that ed and fred impart a second, independent, ;brake-like' contribution to this process via Egfr signaling. Ed and Fred are localized in largely distinct and dynamic patterns throughout rotation. However, ed and fred are required in only a subset of cells - photoreceptors R1, R7 and R6 - for normal rotation, cells that have only recently been linked to a role in planar cell polarity (PCP). This work also provides the first demonstration of a requirement for cone cells in the ommatidial rotation aspect of PCP. ed and fred also genetically interact with the PCP genes, but affect only the degree-of-rotation aspect of the PCP phenotype. Significantly, we demonstrate that at least one PCP protein, Stbm, is required in R7 to control the degree of ommatidial rotation.

  1. Pro-adhesive phenotype of normal endothelial cells responding to metastatic breast cancer cell conditioned medium is linked to NFκB-mediated transcriptomic regulation.

    Science.gov (United States)

    Mejía-Rangel, Janini; Córdova, Emilio; Orozco, Lorena; Ventura-Gallegos, José Luis; Mitre-Aguilar, Irma; Escalona-Guzmán, Alma; Vadillo, Felipe; Vázquez-Prado, José; Gariglio, Patricio; Zentella-Dehesa, Alejandro

    2016-11-01

    Tumor microenvironment is an important promoter of tumorigenesis in all forms of breast cancer and has been associated with the risk of metastasis in the different breast cancer subtypes including the more frequent luminal subtypes that encompass 60% of cancer patients. Adhesive properties of endothelial cells (ECs) are strikingly affected during cancer cell dissemination and are related to functional changes of adhesion receptors. The contribution of tumor secreted factors to tumor‑EC adhesion represents a therapeutic opportunity for breast cancer metastasis. Conditioned medium (CM) of tumor cells can be used as a model to study the role of the secreted molecules to the tumor microenvironment. We explored transcriptomic changes associated to a pro‑adhesive phenotype in primary human umbilical vein endothelial cells (HUVECs) treated with CM of the breast cancer cell line ZR75.30 or with TNF for 3 h. Selected genes were used to validate the microarray through RT‑qPCR. The bioinformatic analysis identified NFκB as the main regulator of the pro-adhesive phenotype and this was confirmed by pharmacological inhibition of NFκB pathway with BAY 11‑7085. The changes induced by ZR75.30‑CM mimic those promoted by TNF and display changes in the expression of genes related to inflammatory response, wound healing, extracellular matrix, cytokines, metabolism and cell communication. Despite the abundance of G‑CSF, IL‑8, IL‑6 and VEGF in the ZR75.30‑CM and the confirmed activation of STAT3 and VEGFR2 pathways, our results suggest dominance of NFκB as a central controller of the transcriptomic response of ECs to breast cancer cells leading to expression of cell adhesion receptors.

  2. [Endothelial cell adhesion molecules].

    Science.gov (United States)

    Ivanov, A N; Norkin, I A; Puchin'ian, D M; Shirokov, V Iu; Zhdanova, O Iu

    2014-01-01

    The review presents current data concerning the functional role of endothelial cell adhesion molecules belonging to different structural families: integrins, selectins, cadherins, and the immunoglobulin super-family. In this manuscript the regulatory mechanisms and factors of adhesion molecules expression and distribution on the surface of endothelial cells are discussed. The data presented reveal the importance of adhesion molecules in the regulation of structural and functional state of endothelial cells in normal conditions and in pathology. Particular attention is paid to the importance of these molecules in the processes of physiological and pathological angiogenesis, regulation of permeability of the endothelial barrier and cell transmigration.

  3. Analysis of TNF-α-induced Leukocyte Adhesion to Vascular Endothelial Cells Regulated by Fluid Shear Stress Using Microfluidic Chip-based Technology

    Institute of Scientific and Technical Information of China (English)

    LI Yuan; YANG De-yu; LIAO Juan; GONG Fang; HE Ping; LIU Bei-zhong

    2015-01-01

    This paper aims to the research of the impact of fluid shear stress on the adhesion between vascular endothelial cells and leukocyte induced by tumor necrosis factor-α(TNF-α) by microfliudic chip technology. Microfluidic chip was fabricated by soft lithograph;Endothelial microfluidic chip was constructed by optimizing types of the extracellular matrix proteins modified in the microchannel and cell incubation time;human umbilical vein endothelial cells EA.Hy926 lined in the microchannel were exposed to fluid shear stress of 1.68 dynes/cm2 and 8.4 dynes/cm2 respectively. Meanwhile, adhesion between EA.Hy926 cells and leukocyte was induced by TNF-αunder a flow condition. EA. Hy926 cell cultured in the static condition was used as control group. The numbers of fluorescently-labeled leukocyte in microchannel were counted to quantize the adhesion level between EA. Hy926 cells and leukocyte; cell immunofluorescence technique was used to detect the intercellular adhesion molecule (ICAM-1) expression. The constructed endothelial microfluidic chip can afford to the fluid shear stress and respond to exogenous stimulus of TNF-α;compared with the adhesion numbers of leukocyte in control group, adhesion between EA. Hy926 cells exposed to low fluid shear stress and leukocyte was reduced under the stimulus of TNF-α at a concentration of 10 ng/ml(P<0.05);leukocyte adhesion with EA. Hy926 cells exposed to high fluid shear stress was reduced significantly than EA. Hy926 cells in control group and EA.1Hy926 cells exposed to low fluid shear stress ( P<0.01); the regulation mechanism of fluid shear stress to the adhesion between EA. Hy926 cells and leukocyte induced by TNF-αwas through the way of ICAM-1. The endothelial microfluidic chip fabricated in this paper could be used to study the functions of endothelial cell in vitro and provide a new technical platform for exploring the pathophysiology of the related cardiovascular system diseases under a flow environment.

  4. The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and filamin A.

    Science.gov (United States)

    Tu, Chia-Ling; Chang, Wenhan; Bikle, Daniel D

    2011-05-01

    Extracellular Ca(2+) (Ca(2+)(o)) functioning through the calcium-sensing receptor (CaR) induces E-cadherin-mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that CaR regulates cell-cell adhesion through Fyn/Src tyrosine kinases. In this study, we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca(2+)(o)-induced association of Fyn with E-cadherin and suppressed the Ca(2+)(o)-induced tyrosine phosphorylation of β-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca(2+)(o)-stimulated expression of terminal differentiation markers. Elevating the Ca(2+)(o) level induced interactions among CaR, Rho A, E-cadherin, and the scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense complementary DNA inhibited Ca(2+)(o)-induced activation of endogenous Rho. Ca(2+)(o) activation of Rho required a direct interaction between CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca(2+)(o)-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca(2+)(o)-induced E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.

  5. Beamlet focal plane diagnostic

    Energy Technology Data Exchange (ETDEWEB)

    Caird, J.A.; Nielsen, N.D.; Patton, H.G.; Seppala, L.G.; Thompson, C.E.; Wegner, P.J.

    1996-12-01

    This paper describes the major optical and mechanical design features of the Beamlet Focal Plane Diagnostic system as well as measurements of the system performance, and typical data obtained to date. We also discuss the NIF requirements on the focal spot that we are interested in measuring, and some of our plans for future work using this system.

  6. Integrin activation and focal complex formation in cardiac hypertrophy

    Science.gov (United States)

    Laser, M.; Willey, C. D.; Jiang, W.; Cooper, G. 4th; Menick, D. R.; Zile, M. R.; Kuppuswamy, D.

    2000-01-01

    Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.

  7. Integrin activation and focal complex formation in cardiac hypertrophy

    Science.gov (United States)

    Laser, M.; Willey, C. D.; Jiang, W.; Cooper, G. 4th; Menick, D. R.; Zile, M. R.; Kuppuswamy, D.

    2000-01-01

    Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.

  8. Met-Independent Hepatocyte Growth Factor-mediated regulation of cell adhesion in human prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Davis Rodney

    2006-07-01

    Full Text Available Abstract Background Prostate cancer cells communicate reciprocally with the stromal cells surrounding them, inside the prostate, and after metastasis, within the bone. Each tissue secretes factors for interpretation by the other. One stromally-derived factor, Hepatocyte Growth Factor (HGF, was found twenty years ago to regulate invasion and growth of carcinoma cells. Working with the LNCaP prostate cancer progression model, we found that these cells could respond to HGF stimulation, even in the absence of Met, the only known HGF receptor. The new HGF binding partner we find on the cell surface may help to clarify conflicts in the past literature about Met expression and HGF response in cancer cells. Methods We searched for Met or any HGF binding partner on the cells of the PC3 and LNCaP prostate cancer cell models, using HGF immobilized on agarose beads. By using mass spectrometry analyses and sequencing we have identified nucleolin protein as a novel HGF binding partner. Antibodies against nucleolin (or HGF were able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. Western blots, RT-PCR, and immunohistochemistry were used to assess nucleolin levels during prostate cancer progression in both LNCaP and PC3 models. Results We have identified HGF as a major signaling component of prostate stromal-conditioned media (SCM and have implicated the protein nucleolin in HGF signal reception by the LNCaP model prostate cancer cells. Antibodies that silence either HGF (in SCM or nucleolin (on the cell surfaces eliminate the adhesion-stimulatory effects of the SCM. Likewise, addition of purified HGF to control media mimics the action of SCM. C4-2, an LNCaP lineage-derived, androgen-independent human prostate cancer cell line, responds to HGF in a concentration-dependent manner by increasing its adhesion and reducing its migration on laminin substratum. These HGF effects are not due to shifts in the expression levels of

  9. Temporal gene expression profile of human precursor B leukemia cells induced by adhesion receptor: identification of pathways regulating B-cell survival.

    Science.gov (United States)

    Astier, Anne Laurence; Xu, Ronghui; Svoboda, Marek; Hinds, Esther; Munoz, Olivier; de Beaumont, Rosalie; Crean, Colin Daniel; Gabig, Theodore; Freedman, Arnold Stephen

    2003-02-01

    The physical interactions between B cells and stromal cells from the lymphoid tissue microenvironment are critical to the survival of normal and malignant B cells. They are principally mediated by integrins expressed on B cells and counterreceptors on stromal cells. Specifically, alpha4beta1 integrin engagement rescues B cells from physiological or drug-induced apoptosis. Therefore, in order to understand the mechanisms by which integrins prevent apoptosis in leukemia B cells, we compared the temporal gene expression profiles induced by beta1-integrin ligation with fibronectin (Fn) or adhesion by poly-L-Lysine in serum-starved precursor B leukemia cells. Among the 38 selected differentially expressed genes, 6 genes involved in adhesion (VAV2, EPB41L1, CORO1A), proliferation (FRAP1, CCT4), and intercellular communication (GJB3) were validated by real-time quantitative polymerase chain reaction (RT-Q-PCR). Gene expression modulation could also be validated at the protein level for 5 other genes. We show that integrin stimulation up-regulated FBI-1 expression but inhibited CD79a, Requiem, c-Fos, and caspase 7 induction when the cells underwent apoptosis. We further demonstrate that Fn stimulation also inhibits caspase 3 activation but increases XIAP and survivin expression. Moreover, integrin stimulation also prevents caspase activation induced by doxorubicin. Therefore, we identified genes modulated by adhesion of human precursor B leukemia cells that regulate proliferation and apoptosis, highlighting new pathways that might provide insights into future therapy aiming at targeting apoptosis of leukemia cells.

  10. Bortezomib interferes with adhesion of B cell precursor acute lymphoblastic leukemia cells through SPARC up-regulation in human bone marrow mesenchymal stromal/stem cells.

    Science.gov (United States)

    Iwasa, Masaki; Miura, Yasuo; Fujishiro, Aya; Fujii, Sumie; Sugino, Noriko; Yoshioka, Satoshi; Yokota, Asumi; Hishita, Terutoshi; Hirai, Hideyo; Andoh, Akira; Ichinohe, Tatsuo; Maekawa, Taira

    2017-05-01

    The poor prognosis of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is attributed to leukemia cells that are protected by the bone marrow (BM) microenvironment. In the present study, we explored the pharmacological targeting of mesenchymal stromal/stem cells in BM (BM-MSCs) to eliminate chemoresistant BCP-ALL cells. Human BCP-ALL cells (NALM-6 cells) that adhered to human BM-MSCs (NALM-6/Ad) were highly resistant to multiple anti-cancer drugs, and exhibited pro-survival characteristics, such as an enhanced Akt/Bcl-2 pathway and increased populations in the G0 and G2/S/M cell cycle stages. Bortezomib, a proteasome inhibitor, interfered with adhesion between BM-MSCs and NALM-6 cells and up-regulated the matricellular protein SPARC (secreted protein acidic and rich in cysteine) in BM-MSCs, thereby reducing the NALM-6/Ad population. Inhibition of SPARC expression in BM-MSCs using a small interfering RNA enhanced adhesion of NALM-6 cells. Conversely, recombinant SPARC protein interfered with adhesion of NALM-6 cells. These results suggest that SPARC disrupts adhesion between BM-MSCs and NALM-6 cells. Co-treatment with bortezomib and doxorubicin prolonged the survival of BCP-ALL xenograft mice, with a significant reduction of leukemia cells in BM. Our findings demonstrate that bortezomib contributes to the elimination of BCP-ALL cells through disruption of their adhesion to BM-MSCs, and offer a novel therapeutic strategy for BCP-ALL through targeting of BM-MSCs.

  11. Interactions between MUC1 and p120 catenin regulate dynamic features of cell adhesion, motility and metastasis

    Science.gov (United States)

    Liu, Xiang; Yi, Chunhui; Wen, Yunfei; Radhakrishnan, Prakash; Tremayne, Jarrod R.; Dao, Thongtan; Johnson, Keith R.; Hollingsworth, Michael A.

    2014-01-01

    The mechanisms by which MUC1 and p120 catenin contribute to progression of cancers from early transformation to metastasis are poorly understood. Here we show that p120 catenin ARM domains 1, 3–5 and 8 mediate interactions between p120 catenin and MUC1, and that these interactions modulate dynamic properties of cell adhesion, motility and metastasis of pancreatic cancer cells. We also show that different isoforms of p120 catenin when co-expressed with MUC1 create cells that exhibit distinct patterns of motility in culture (motility independent of cell adhesion, motility within a monolayer while exchanging contacts with other cells, and unified motility while maintaining static epithelial contacts) and patterns of metastasis. The results provide new insight into the dynamic interplay between cell adhesion and motility and the relationship of these to the metastatic process. PMID:24371222

  12. Mapping cell surface adhesion by rotation tracking and adhesion footprinting

    Science.gov (United States)

    Li, Isaac T. S.; Ha, Taekjip; Chemla, Yann R.

    2017-03-01

    Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell’s surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level.

  13. Bimaxillary Oral Focal Mucinosis.

    Science.gov (United States)

    Yadav, Sunil; Malik, Sunita; Mittal, Hitesh Chander; Singh, Gurdarshan; Kamra, Hemlata

    2016-10-01

    Oral focal mucinosis is considered as oral counterpart of cutaneous focal mucinosis. The preoperative diagnosis of mucinosis is almost impossible because of its rarity and clinical similarity to other lesions of various etiologies. The histological diagnosis of oral mucinosis is important to better understand the etiopathogenesis, treatment modalities, and any recurrence of the lesion besides differentiating from the other soft tissue lesions.The purpose of this paper is to report the first case of bimaxillary involvement with dome-shaped elevated, rounded, asymptomatic, normally colored swelling in left posterior palatal mucosa and left mandibular posterior region in a 25-year old woman who was diagnosed as oral focal mucinosis histopathologically.

  14. Alterations in ovarian cancer cell adhesion drive taxol resistance by increasing microtubule dynamics in a FAK-dependent manner.

    Science.gov (United States)

    McGrail, Daniel J; Khambhati, Niti N; Qi, Mark X; Patel, Krishan S; Ravikumar, Nithin; Brandenburg, Chandler P; Dawson, Michelle R

    2015-04-17

    Chemorefractory ovarian cancer patients show extremely poor prognosis. Microtubule-stabilizing Taxol (paclitaxel) is a first-line treatment against ovarian cancer. Despite the close interplay between microtubules and cell adhesion, it remains unknown if chemoresistance alters the way cells adhere to their extracellular environment, a process critical for cancer metastasis. To investigate this, we isolated Taxol-resistant populations of OVCAR3 and SKOV3 ovarian cancer cell lines. Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics. These changes in microtubule dynamics coincided with faster attachment rates and decreased adhesion strength, which correlated with increased surface β1-integrin expression and decreased focal adhesion formation, respectively. Adhesion strength correlated best with Taxol-sensitivity, and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells. FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics. Taken together, this work demonstrates that Taxol-resistance dramatically alters how ovarian cancer cells adhere to their extracellular environment causing down-stream increases in microtubule dynamics, providing a therapeutic target that may improve prognosis by not only recovering drug sensitivity, but also decreasing metastasis.

  15. Haemodynamic and extracellular matrix cues regulate the mechanical phenotype and stiffness of aortic endothelial cells.

    Science.gov (United States)

    Collins, Caitlin; Osborne, Lukas D; Guilluy, Christophe; Chen, Zhongming; O'Brien, E Tim; Reader, John S; Burridge, Keith; Superfine, Richard; Tzima, Ellie

    2014-06-11

    Endothelial cells (ECs) lining blood vessels express many mechanosensors, including platelet endothelial cell adhesion molecule-1 (PECAM-1), that convert mechanical force into biochemical signals. While it is accepted that mechanical stresses and the mechanical properties of ECs regulate vessel health, the relationship between force and biological response remains elusive. Here we show that ECs integrate mechanical forces and extracellular matrix (ECM) cues to modulate their own mechanical properties. We demonstrate that the ECM influences EC response to tension on PECAM-1. ECs adherent on collagen display divergent stiffening and focal adhesion growth compared with ECs on fibronectin. This is because of protein kinase A (PKA)-dependent serine phosphorylation and inactivation of RhoA. PKA signalling regulates focal adhesion dynamics and EC compliance in response to shear stress in vitro and in vivo. Our study identifies an ECM-specific, mechanosensitive signalling pathway that regulates EC compliance and may serve as an atheroprotective mechanism that maintains blood vessel integrity in vivo.

  16. Phosphoproteome reveals an atlas of protein signaling networks during osteoblast adhesion.

    Science.gov (United States)

    Milani, Renato; Ferreira, Carmen V; Granjeiro, José M; Paredes-Gamero, Edgar J; Silva, Rodrigo A; Justo, Giselle Z; Nader, Helena B; Galembeck, Eduardo; Peppelenbosch, Maikel P; Aoyama, Hiroshi; Zambuzzi, Willian F

    2010-04-01

    Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important regulatory mechanism in eukaryotes. Therefore, the goal of this study is to assess osteoblast adhesion through a molecular prism under a peptide array technology, revealing essential signaling proteins governing adhesion-related events. First, we showed that there are main morphological changes on osteoblast shape during adhesion up to 3 h. Second, besides classical proteins activated upon integrin activation, our results showed a novel network involving signaling proteins such as Rap1A, PKA, PKC, and GSK3beta during osteoblast adhesion on polystyrene. Third, these proteins were grouped in different signaling cascades including focal adhesion establishment, cytoskeleton rearrangement, and cell-cycle arrest. We have thus provided evidence that a global phosphorylation screening is able to yield a systems-oriented look at osteoblast adhesion, providing new insights for understanding of bone formation and improvement of cell-substratum interactions. Altogether, these statements are necessary means for further intervention and development of new approaches for the progress of tissue engineering.

  17. Control of high affinity interactions in the talin C terminus: how talin domains coordinate protein dynamics in cell adhesions.

    Science.gov (United States)

    Himmel, Mirko; Ritter, Anett; Rothemund, Sven; Pauling, Björg V; Rottner, Klemens; Gingras, Alexandre R; Ziegler, Wolfgang H

    2009-05-15

    In cell-extracellular matrix junctions (focal adhesions), the cytoskeletal protein talin is central to the connection of integrins to the actin cytoskeleton. Talin is thought to mediate this connection via its two integrin, (at least) three actin, and several vinculin binding sites. The binding sites are cryptic in the head-to-rod autoinhibited cytoplasmic form of the protein and require (stepwise) conformational activation. This activation process, however, remains poorly understood, and there are contradictory models with respect to the determinants of adhesion site localization. Here, we report turnover rates and protein-protein interactions in a range of talin rod domain constructs varying in helix bundle structure. We conclude that several bundles of the C terminus cooperate to regulate targeting and concomitantly tailor high affinity interactions of the talin rod in cell adhesions. Intrinsic control of ligand binding activities is essential for the coordination of adhesion site function of talin.

  18. Focal neurological deficits

    Science.gov (United States)

    ... Other examples of focal loss of function include: Horner syndrome : small pupil on one side, one-sided ... 403. Read More Alertness - decreased Fine motor control Horner syndrome Hypotonia Movement - uncoordinated Muscle function loss Neurologic ...

  19. Adhesive Categories

    DEFF Research Database (Denmark)

    Lack, Stephen; Sobocinski, Pawel

    2003-01-01

    We introduce adhesive categories, which are categories with structure ensuring that pushouts along monomorphisms are well-behaved. Many types of graphical structures used in computer science are shown to be examples of adhesive categories. Double-pushout graph rewriting generalises well...... to rewriting on arbitrary adhesive categories....

  20. Phosphatidylinositol 4-phosphate in the Golgi apparatus regulates cell-cell adhesion and invasive cell migration in human breast cancer.

    Science.gov (United States)

    Tokuda, Emi; Itoh, Toshiki; Hasegawa, Junya; Ijuin, Takeshi; Takeuchi, Yukiko; Irino, Yasuhiro; Fukumoto, Miki; Takenawa, Tadaomi

    2014-06-01

    Downregulation of cell-cell adhesion and upregulation of cell migration play critical roles in the conversion of benign tumors to aggressive invasive cancers. In this study, we show that changes in cell-cell adhesion and cancer cell migration/invasion capacity depend on the level of phosphatidylinositol 4-phosphate [PI(4)P] in the Golgi apparatus in breast cancer cells. Attenuating SAC1, a PI(4)P phosphatase localized in the Golgi apparatus, resulted in decreased cell-cell adhesion and increased cell migration in weakly invasive cells. In contrast, silencing phosphatidylinositol 4-kinase IIIβ, which generates PI(4)P in the Golgi apparatus, increased cell-cell adhesion and decreased invasion in highly invasive cells. Furthermore, a PI(4)P effector, Golgi phosphoprotein 3, was found to be involved in the generation of these phenotypes in a manner that depends on its PI(4)P-binding ability. Our results provide a new model for breast cancer cell progression in which progression is controlled by PI(4)P levels in the Golgi apparatus.

  1. Ablation of CD11c(hi) dendritic cells exacerbates Japanese encephalitis by regulating blood-brain barrier permeability and altering tight junction/adhesion molecules.

    Science.gov (United States)

    Kim, Jin Hyoung; Hossain, Ferdaus Mohd Altaf; Patil, Ajit Mahadev; Choi, Jin Young; Kim, Seong Bum; Uyangaa, Erdenebelig; Park, Sang-Youel; Lee, John-Hwa; Kim, Bumseok; Kim, Koanhoi; Eo, Seong Kug

    2016-10-01

    Japanese encephalitis (JE), characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV), is becoming a leading cause of viral encephalitis due to rapid changes in climate and demography. The blood-brain barrier (BBB) plays an important role in restricting neuroinvasion of peripheral leukocytes and virus, thereby regulating the progression of viral encephalitis. In this study, we explored the role of CD11c(hi) dendritic cells (DCs) in regulating BBB integrity and JE progression using a conditional depletion model of CD11c(hi) DCs. Transient ablation of CD11c(hi) DCs resulted in markedly increased susceptibility to JE progression along with highly increased neuro-invasion of JEV. In addition, exacerbated JE progression in CD11c(hi) DC-ablated hosts was closely associated with increased expression of proinflammatory cytokines (IFN-β, IL-6, and TNF-α) and CC chemokines (CCL2, CCL3, CXCL2) in the brain. Moreover, our results revealed that the exacerbation of JE progression in CD11c(hi) DC-ablated hosts was correlated with enhanced BBB permeability and reduced expression of tight junction and adhesion molecules (claudin-5, ZO-1, occluding, JAMs). Ultimately, our data conclude that the ablation of CD11c(hi) DCs provided a subsidiary impact on BBB integrity and the expression of tight junction/adhesion molecules, thereby leading to exacerbated JE progression. These findings provide insight into the secondary role of CD11c(hi) DCs in JE progression through regulation of BBB integrity and the expression of tight junction/adhesion molecules.

  2. Role of Adhesion Molecules in Eosinophil Activation: A Comparative Study on the Effect of Adhesion Molecules on Eosinophil Survival

    Directory of Open Access Journals (Sweden)

    Kazutoshi Yamaguchi

    2004-01-01

    Conclusions: The regulation of adhesion molecules, by not only preventing eosinophil adhesion but also eosinophil activation, may be a potential target in the treatment of allergic inflammatory disorders.

  3. Calpain-controlled detachment of major glycoproteins from the cytoskeleton regulates adhesive properties of activated phosphatidylserine-positive platelets.

    Science.gov (United States)

    Artemenko, Elena O; Yakimenko, Alena O; Pichugin, Alexey V; Ataullakhanov, Fazly I; Panteleev, Mikhail A

    2016-02-15

    In resting platelets, adhesive membrane glycoproteins are attached to the cytoskeleton. On strong activation, phosphatidylserine(PS)-positive and -negative platelet subpopulations are formed. Platelet activation is accompanied by cytoskeletal rearrangement, although the glycoprotein attachment status in these two subpopulations is not clear. We developed a new, flow cytometry-based, single-cell approach to investigate attachment of membrane glycoproteins to the cytoskeleton in cell subpopulations. In PS-negative platelets, adhesive glycoproteins integrin αIIbβ3, glycoprotein Ib and, as shown for the first time, P-selectin were associated with the cytoskeleton. In contrast, this attachment was disrupted in PS-positive platelets; it was retained to some extent only in the small convex regions or 'caps'. It correlated with the degradation of talin and filamin observed only in PS-positive platelets. Calpain inhibitors essentially prevented the disruption of membrane glycoprotein attachment in PS-positive platelets, as well as talin and filamin degradation. With the suggestion that detachment of glycoproteins from the cytoskeleton may affect platelet adhesive properties, we investigated the ability of PS-positive platelets to resist shear-induced breakaway from the immobilized fibrinogen. Shear rates of 500/s caused PS-positive platelet breakaway, but their adhesion stability increased more than 10-fold after pretreatment of the platelets with calpain inhibitor. In contrast, the ability of PS-positive platelets to adhere to immobilized von Willebrand's factor at 100/s was low, but this was not affected by the preincubation of platelets with a calpain inhibitor. Our data suggest that calpain-controlled detachment of membrane glycoproteins is a new mechanism that is responsible for the loss of ability of the procoagulant platelets to resist detachment from thrombi by high shear stress.

  4. The Adhesion Molecule KAL-1/anosmin-1 Regulates Neurite Branching through a SAX-7/L1CAM–EGL-15/FGFR Receptor Complex

    Directory of Open Access Journals (Sweden)

    Carlos A. Díaz-Balzac

    2015-06-01

    Full Text Available Neurite branching is essential for correct assembly of neural circuits, yet it remains a poorly understood process. For example, the neural cell adhesion molecule KAL-1/anosmin-1, which is mutated in Kallmann syndrome, regulates neurite branching through mechanisms largely unknown. Here, we show that KAL-1/anosmin-1 mediates neurite branching as an autocrine co-factor with EGL-17/FGF through a receptor complex consisting of the conserved cell adhesion molecule SAX-7/L1CAM and the fibroblast growth factor receptor EGL-15/FGFR. This protein complex, which appears conserved in humans, requires the immunoglobulin (Ig domains of SAX-7/L1CAM and the FN(III domains of KAL-1/anosmin-1 for formation in vitro as well as function in vivo. The kinase domain of the EGL-15/FGFR is required for branching, and genetic evidence suggests that ras-mediated signaling downstream of EGL-15/FGFR is necessary to effect branching. Our studies establish a molecular pathway that regulates neurite branching during development of the nervous system.

  5. Crosstalk between Protease-activated Receptor 1 and Platelet-activating Factor Receptor Regulates Melanoma Cell Adhesion Molecule (MCAM/MUC18) Expression and Melanoma Metastasis*

    Science.gov (United States)

    Melnikova, Vladislava O.; Balasubramanian, Krishnakumar; Villares, Gabriel J.; Dobroff, Andrey S.; Zigler, Maya; Wang, Hua; Petersson, Frederik; Price, Janet E.; Schroit, Alan; Prieto, Victor G.; Hung, Mien-Chie; Bar-Eli, Menashe

    2009-01-01

    The cellular and molecular pathways that regulate platelet activation, blood coagulation, and inflammation are emerging as critical players in cancer progression and metastasis. Here, we demonstrate a novel signaling mechanism whereby protease-activated receptor 1 (PAR1) mediates expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), a critical marker of melanoma metastasis, via activation of platelet-activating factor receptor (PAFR) and cAMP-responsive element-binding protein (CREB). We found that PAR1 silencing with small hairpin RNA inhibits MUC18 expression in metastatic melanoma cells by inhibiting CREB phosphorylation, activity, and binding to the MUC18 promoter. We further demonstrate that the PAF/PAFR pathway mediates MUC18 expression downstream of PAR1. Indeed, PAR1 silencing down-regulates PAFR expression and PAF production, PAFR silencing blocks MUC18 expression, and re-expression of PAFR in PAR1-silenced cells rescues MUC18 expression. We further demonstrate that the PAR1-PAFR-MUC18 pathway mediates melanoma cell adhesion to microvascular endothelial cells, transendothelial migration, and metastatic retention in the lungs. Rescuing PAFR expression in PAR1-silenced cells fully restores metastatic phenotype of melanoma, indicating that PAFR plays critical role in the molecular mechanism of PAR1 action. Our results link the two pro-inflammatory G-protein-coupled receptors, PAR1 and PAFR, with the metastatic dissemination of melanoma and suggest that PAR1, PAFR, and MUC18 are attractive therapeutic targets for preventing melanoma metastasis. PMID:19703903

  6. Transmembrane neural cell-adhesion molecule (NCAM), but not glycosyl-phosphatidylinositol-anchored NCAM, down-regulates secretion of matrix metalloproteinases

    DEFF Research Database (Denmark)

    Edvardsen, K; Chen, W; Rucklidge, G

    1993-01-01

    proteinases, and proteinase inhibitors all participate in the construction, maintenance, and remodeling of extracellular matrix by cells. The neural cell-adhesion molecule (NCAM)-negative rat glioma cell line BT4Cn secretes substantial amounts of metalloproteinases, as compared with its NCAM-positive mother...... cell line BT4C. We have transfected the BT4Cn cell line with cDNAs encoding the human NCAM-B and -C isoforms. We report here that the expression of transmembrane NCAM-B, but not of glycosyl-phosphatidylinositol-linked NCAM-C, induces a down-regulation of 92-kDa gelatinase (matrix metalloproteinase 9......) and interstitial collagenase (matrix metalloproteinase 1), indicating that cellular expression of the recognition molecule NCAM regulates the metabolism of the surrounding matrix....

  7. Kartagener syndrome with focal segmental glomerulosclerosis.

    Science.gov (United States)

    Momeni, Ali; Doroushi, Behzad; Taheri, Nadia

    2013-11-01

    Primary ciliary dyskinesia is characterized by congenital impairment of mucociliary clearance. Kartagener syndrome (KS) is a clinical variant of primary ciliary dyskinesia which is involved in situs inversus associated with chronic respiratory infections. In addition, glomerular disease in KS syndrome is rare and reported cases are limited. We had a 27-year-old female patient with KS who presented with proteinuria, hematuria, normal kidney function, and a family history of systemic lupus erythematosus. Kidney biopsy showed segmental scar with adhesion to Bowman capsule, which was indicative of focal segmental glomerulosclerosis.

  8. Dynamic regulation of a cell adhesion protein complex including CADM1 by combinatorial analysis of FRAP with exponential curve-fitting.

    Science.gov (United States)

    Sakurai-Yageta, Mika; Maruyama, Tomoko; Suzuki, Takashi; Ichikawa, Kazuhisa; Murakami, Yoshinori

    2015-01-01

    Protein components of cell adhesion machinery show continuous renewal even in the static state of epithelial cells and participate in the formation and maintenance of normal epithelial architecture and tumor suppression. CADM1 is a tumor suppressor belonging to the immunoglobulin superfamily of cell adhesion molecule and forms a cell adhesion complex with an actin-binding protein, 4.1B, and a scaffold protein, MPP3, in the cytoplasm. Here, we investigate dynamic regulation of the CADM1-4.1B-MPP3 complex in mature cell adhesion by fluorescence recovery after photobleaching (FRAP) analysis. Traditional FRAP analysis were performed for relatively short period of around 10 min. Here, thanks to recent advances in the sensitive laser detector systems, we examine FRAP of CADM1 complex for longer period of 60 min and analyze the recovery with exponential curve-fitting to distinguish the fractions with different diffusion constants. This approach reveals that the fluorescence recovery of CADM1 is fitted to a single exponential function with a time constant (τ) of approximately 16 min, whereas 4.1B and MPP3 are fitted to a double exponential function with two τs of approximately 40-60 sec and 16 min. The longer τ is similar to that of CADM1, suggesting that 4.1B and MPP3 have two distinct fractions, one forming a complex with CADM1 and the other present as a free pool. Fluorescence loss in photobleaching analysis supports the presence of a free pool of these proteins near the plasma membrane. Furthermore, double exponential fitting makes it possible to estimate the ratio of 4.1B and MPP3 present as a free pool and as a complex with CADM1 as approximately 3:2 and 3:1, respectively. Our analyses reveal a central role of CADM1 in stabilizing the complex with 4.1B and MPP3 and provide insight in the dynamics of adhesion complex formation.

  9. [Focal epithelial hyperplasia].

    Science.gov (United States)

    Vera-Iglesias, E; García-Arpa, M; Sánchez-Caminero, P; Romero-Aguilera, G; Cortina de la Calle, P

    2007-11-01

    Focal epithelial hyperplasia is a rare disease of the oral mucosa caused by the human papilloma virus (HPV). It appears as a benign epithelial growth, usually in the mucosa of the lower lip. It is mainly associated with HPV serotypes 13 and 32 and there is a clear racial predilection for the disease in Native Americans and Eskimos. We describe the case of a 17-year-old girl from Ecuador with multiple papular lesions in both lips that were clinically and histologically consistent with focal epithelial hyperplasia. Analysis by polymerase chain reaction detected HPV serotype 13.

  10. SNAP focal plane

    Energy Technology Data Exchange (ETDEWEB)

    Lampton, Michael L.; Kim, A.; Akerlof, C.W.; Aldering, G.; Amanullah, R.; Astier, P.; Barrelet, E.; Bebek, C.; Bergstrom, L.; Berkovitz, J.; Bernstein, G.; Bester, M.; Bonissent, A.; Bower, C.; Carithers Jr., W.C.; Commins, E.D.; Day, C.; Deustua, S.E.; DiGennaro,R.; Ealet, A.; Ellis, R.S.; Eriksson, M.; Fruchter, A.; Genat, J.-F.; Goldhaber, G.; Goobar, A.; Groom, D.; Harris, S.E.; Harvey, P.R.; Heetderks, H.D.; Holland, S.E.; Huterer, D.; Karcher, A.; Kolbe, W.; Krieger, B.; Lafever, R.; Lamoureux, J.; Levi, M.E.; Levin, D.S.; Linder,E.V.; Loken, S.C.; Malina, R.; Massey, R.; McKay, T.; McKee, S.P.; Miquel, R.; Mortsell, E.; Mostek, N.; Mufson, S.; Musser, J.; Nugent, P.; Oluseyi, H.; Pain, R.; Palaio, N.; Pankow, D.; Perlmutter, S.; Pratt, R.; Prieto, E.; Refregier, A.; Rhodes, J.; Robinson, K.; Roe, N.; Sholl, M.; Schubnell, M.; Smadja, G.; Smoot, G.; Spadafora, A.; Tarle, G.; Tomasch,A.; von der Lippe, H.; Vincent, R.; Walder, J.-P.; Wang, G.

    2002-07-29

    The proposed SuperNova/Acceleration Probe (SNAP) mission will have a two-meter class telescope delivering diffraction-limited images to an instrumented 0.7 square-degree field sensitive in the visible and near-infrared wavelength regime. We describe the requirements for the instrument suite and the evolution of the focal plane design to the present concept in which all the instrumentation--visible and near-infrared imagers, spectrograph, and star guiders--share one common focal plane.

  11. Human TM9SF4 Is a New Gene Down-Regulated by Hypoxia and Involved in Cell Adhesion of Leukemic Cells.

    Directory of Open Access Journals (Sweden)

    Rosa Paolillo

    Full Text Available The transmembrane 9 superfamily protein member 4, TM9SF4, belongs to the TM9SF family of proteins highly conserved through evolution. TM9SF4 homologs, previously identified in many different species, were mainly involved in cellular adhesion, innate immunity and phagocytosis. In human, the function and biological significance of TM9SF4 are currently under investigation. However, TM9SF4 was found overexpressed in human metastatic melanoma and in a small subset of acute myeloid leukemia (AMLs and myelodysplastic syndromes, consistent with an oncogenic function of this gene.In this study, we first analyzed the expression and regulation of TM9SF4 in normal and leukemic cells and identified TM9SF4 as a gene highly expressed in human quiescent CD34+ hematopoietic progenitor cells (HPCs, regulated during monocytic and granulocytic differentiation of HPCs, both lineages giving rise to mature myeloid cells involved in adhesion, phagocytosis and immunity. Then, we found that TM9SF4 is markedly overexpressed in leukemic cells and in AMLs, particularly in M2, M3 and M4 AMLs (i.e., in AMLs characterized by the presence of a more or less differentiated granulocytic progeny, as compared to normal CD34+ HPCs. Proliferation and differentiation of HPCs occurs in hypoxia, a physiological condition in bone marrow, but also a crucial component of cancer microenvironment. Here, we investigated the impact of hypoxia on TM9SF4 expression in leukemic cells and identified TM9SF4 as a direct target of HIF-1α, downregulated in these cells by hypoxia. Then, we found that the hypoxia-mediated downregulation of TM9SF4 expression is associated with a decrease of cell adhesion of leukemic cells to fibronectin, thus demonstrating that human TM9SF4 is a new molecule involved in leukemic cell adhesion.Altogether, our study reports for the first time the expression of TM9SF4 at the level of normal and leukemic hematopoietic cells and its marked expression at the level of AMLs

  12. Adhesion behaviors on superhydrophobic surfaces.

    Science.gov (United States)

    Zhu, Huan; Guo, Zhiguang; Liu, Weimin

    2014-04-18

    The adhesion behaviors of superhydrophobic surfaces have become an emerging topic to researchers in various fields as a vital step in the interactions between materials and organisms/materials. Controlling the chemical compositions and topological structures via various methods or technologies is essential to fabricate and modulate different adhesion properties, such as low-adhesion, high-adhesion and anisotropic adhesion on superhydrophobic surfaces. We summarize the recent developments in both natural superhydrophobic surfaces and artificial superhydrophobic surfaces with various adhesions and also pay attention to superhydrophobic surfaces switching between low- and high-adhesion. The methods to regulate or translate the adhesion of superhydrophobic surfaces can be considered from two perspectives. One is to control the chemical composition and change the surface geometric structure on the surfaces, respectively or simultaneously. The other is to provide external stimulations to induce transitions, which is the most common method for obtaining switchable adhesions. Additionally, adhesion behaviors on solid-solid interfaces, such as the behaviors of cells, bacteria, biomolecules and icing on superhydrophobic surfaces are also noticeable and controversial. This review is aimed at giving a brief and crucial overview of adhesion behaviors on superhydrophobic surfaces.

  13. Control of mesenchymal stem cell phenotype and differentiation depending on cell adhesion mechanism

    Directory of Open Access Journals (Sweden)

    J Kang

    2014-11-01

    Full Text Available Control of cell-matrix adhesion has become an important issue in the regulation of stem cell function. In this study, a maltose-binding protein (MBP-linked basic fibroblast growth factor (FGF2-immobilised polystyrene surface (PS-MBP-FGF2 was applied as an artificial matrix to regulate integrin-mediated signalling. We sought to characterise human mesenchymal-stem cell (hMSC behaviour in response to two different mechanisms of cell adhesion; (i FGF2-heparan sulphate proteoglycan (HSPG-mediated adhesion vs. (ii fibronectin (FN-integrin-mediated adhesion. Heparin inhibited hMSC adhesion to PS-MBP-FGF2 but not to FN-coated surface. The phosphorylation of focal adhesion kinase, cytoskeletal re-organisation, and cell proliferation were restricted in hMSCs adhering to PS-MBP-FGF2 compared to FN-coated surface. Expression of MSC markers, such as CD105, CD90 and CD166, decreased in hMSCs expanded on PS-MBP-FGF2 compared to expression in cells expanded on FN-coated surface. hMSCs that were expanded on FN-coated surface differentiated into osteogenic and adipogenic cells more readily than those that were expanded on PS-MBP-FGF2. Furthermore, we characterised the N-linked glycan structures of hMSCs depending on the cell adhesion mechanism using mass spectrometry (MS-based quantitative techniques. MS analysis revealed that 2,3-sialylated glycans, a potential marker of stem cell function, were more abundant on hMSCs expanded on FN-coated surface than on those expanded on PS-MBP-FGF2. Thus, the differentiation potential of hMSCs is controlled by the type of adhesion substrate that might provide an idea for the design of biomaterials to control stem cell fate. Elucidation of the glycan structure on the cell membrane may help characterise hMSC function.

  14. Control of mesenchymal stem cell phenotype and differentiation depending on cell adhesion mechanism.

    Science.gov (United States)

    Kang, J; Park, H M; Kim, Y W; Kim, Y H; Varghese, S; Seok, H K; Kim, Y G; Kim, S H

    2014-11-25

    Control of cell-matrix adhesion has become an important issue in the regulation of stem cell function. In this study, a maltose-binding protein (MBP)-linked basic fibroblast growth factor (FGF2)-immobilised polystyrene surface (PS-MBP-FGF2) was applied as an artificial matrix to regulate integrin-mediated signalling. We sought to characterise human mesenchymal-stem cell (hMSC) behaviour in response to two different mechanisms of cell adhesion; (i) FGF2-heparan sulphate proteoglycan (HSPG)-mediated adhesion vs. (ii) fibronectin (FN)-integrin-mediated adhesion. Heparin inhibited hMSC adhesion to PS-MBP-FGF2 but not to FN-coated surface. The phosphorylation of focal adhesion kinase, cytoskeletal re-organisation, and cell proliferation were restricted in hMSCs adhering to PS-MBP-FGF2 compared to FN-coated surface. Expression of MSC markers, such as CD105, CD90 and CD166, decreased in hMSCs expanded on PS-MBP-FGF2 compared to expression in cells expanded on FN-coated surface. hMSCs that were expanded on FN-coated surface differentiated into osteogenic and adipogenic cells more readily than those that were expanded on PS-MBP-FGF2. Furthermore, we characterised the N-linked glycan structures of hMSCs depending on the cell adhesion mechanism using mass spectrometry (MS)-based quantitative techniques. MS analysis revealed that 2,3-sialylated glycans, a potential marker of stem cell function, were more abundant on hMSCs expanded on FN-coated surface than on those expanded on PS-MBP-FGF2. Thus, the differentiation potential of hMSCs is controlled by the type of adhesion substrate that might provide an idea for the design of biomaterials to control stem cell fate. Elucidation of the glycan structure on the cell membrane may help characterise hMSC function.

  15. CXCL1 can be regulated by IL-6 and promotes granulocyte adhesion to brain capillaries during bacterial toxin exposure and encephalomyelitis

    Directory of Open Access Journals (Sweden)

    Roy Monica

    2012-01-01

    Full Text Available Abstract Background Granulocytes generally exert protective roles in the central nervous system (CNS, but recent studies suggest that they can be detrimental in experimental autoimmune encephalomyelitis (EAE, the most common model of multiple sclerosis. While the cytokines and adhesion molecules involved in granulocyte adhesion to the brain vasculature have started to be elucidated, the required chemokines remain undetermined. Methods CXCR2 ligand expression was examined in the CNS of mice suffering from EAE or exposed to bacterial toxins by quantitative RT-PCR and in situ hybridization. CXCL1 expression was analyzed in IL-6-treated endothelial cell cultures by quantitative RT-PCR and ELISA. Granulocytes were counted in the brain vasculature after treatment with a neutralizing anti-CXCL1 antibody using stereological techniques. Results CXCL1 was the most highly expressed ligand of the granulocyte receptor CXCR2 in the CNS of mice subjected to EAE or infused with lipopolysaccharide (LPS or pertussis toxin (PTX, the latter being commonly used to induce EAE. IL-6 upregulated CXCL1 expression in brain endothelial cells by acting transcriptionally and mediated the stimulatory effect of PTX on CXCL1 expression. The anti-CXCL1 antibody reduced granulocyte adhesion to brain capillaries in the three conditions under study. Importantly, it attenuated EAE severity when given daily for a week during the effector phase of the disease. Conclusions This study identifies CXCL1 not only as a key regulator of granulocyte recruitment into the CNS, but also as a new potential target for the treatment of neuroinflammatory diseases such as multiple sclerosis.

  16. Neural Cell Adhesion Molecule-Associated Polysialic Acid Regulates Synaptic Plasticity and Learning by Restraining the Signaling through GluN2B-Containing NMDA Receptors

    Science.gov (United States)

    Kochlamazashvili, Gaga; Senkov, Oleg; Grebenyuk, Sergei; Robinson, Catrina; Xiao, Mei-Fang; Stummeyer, Katharina; Gerardy-Schahn, Rita; Engel, Andreas K.; Feig, Larry; Semyanov, Alexey; Suppiramaniam, Vishnu; Schachner, Melitta; Dityatev, Alexander

    2017-01-01

    The neural cell adhesion molecule (NCAM) is the predominant carrier of α2,8 polysialic acid (PSA) in the mammalian brain. Abnormalities in PSA and NCAM expression are associated with schizophrenia in humans and cause deficits in hippocampal synaptic plasticity and contextual fear conditioning in mice. Here, we show that PSA inhibits opening of recombinant NMDA receptors composed of GluN1/2B (NR1/NR2B) or GluN1/2A/2B (NR1/NR2A/NR2B) but not of GluN1/2A (NR1/NR2A) subunits. Deficits in NCAM/PSA increase GluN2B-mediated transmission and Ca2+ transients in the CA1 region of the hippocampus. In line with elevation of GluN2B-mediated transmission, defects in long-term potentiation in the CA1 region and contextual fear memory in NCAM/PSA-deficient mice are abrogated by application of a GluN2B-selective antagonist. Furthermore, treatment with the glutamate scavenger glutamic-pyruvic transaminase, ablation of Ras-GRF1 (a mediator of GluN2B signaling to p38 MAPK), or direct inhibition of hyperactive p38 MAPK can restore impaired synaptic plasticity in brain slices lacking PSA/NCAM. Thus, PSA carried by NCAM regulates plasticity and learning by inhibition of the GluN2B-Ras-GRF1-p38 MAPK signaling pathway. These findings implicate carbohydrates carried by adhesion molecules in modulating NMDA receptor signaling in the brain and demonstrate reversibility of cognitive deficits associated with ablation of a schizophrenia-related adhesion molecule. PMID:20237287

  17. Claudin-5 regulates blood-brain barrier permeability by modifying brain microvascular endothelial cell proliferation, migration, and adhesion to prevent lung cancer metastasis.

    Science.gov (United States)

    Ma, Shun-Chang; Li, Qi; Peng, Jia-Yi; Zhouwen, Jian-Long; Diao, Jin-Fu; Niu, Jian-Xing; Wang, Xi; Guan, Xiu-Dong; Jia, Wang; Jiang, Wen-Guo

    2017-09-29

    To investigate the roles of Claudin-5 (CLDN5) in regulating the permeability of the blood-brain barrier (BBB) during lung cancer brain metastasis. By silencing and overexpressing the CLDN5 gene in human brain vascular endothelial (hCMEC/D3) cells, we demonstrated the attenuation of cell migration ability and CLDN5's significant positive role in cell proliferation in CLDN5-overexpressing hCMEC/D3 cells and observed the opposite result in the CLDN5 knockdown group. The reinforced CLDN5 expression reduced the paracellular permeability of hCMEC/D3 cells and decreased the invasion of lung adenocarcinoma A549 cells. Overall, 1685 genes were found to be differentially expressed between the CLDN5-overexpressing cells and the control cells using the Affymetrix Human Transcriptome Array 2.0 (HTA 2.0), and the function of these genes was determined by Gene Ontology and pathway analyses. The possible biological functions of the 1685 genes include cell proliferation, adhesion molecules, and the Jak-STAT, PI3K-Akt, Wnt, and Notch signaling pathways. The identified sets of mRNAs that were specific to CLDN5-overexpressing hCMEC/D3 cells were verified by a qRT-PCR experiment. CLDN5 regulates the permeability of BBB by regulating the proliferation, migration, and permeability of hCMEC/D3 cells, especially through the cell adhesion molecule signaling pathway, to enhance the function of the tight junctions, which was involved in reducing the formation of lung cancer brain metastasis. © 2017 John Wiley & Sons Ltd.

  18. Adhesive Categories

    DEFF Research Database (Denmark)

    Lack, Stephen; Sobocinski, Pawel

    2004-01-01

    We introduce adhesive categories, which are categories with structure ensuring that pushouts along monomorphisms are well-behaved. Many types of graphical structures used in computer science are shown to be examples of adhesive categories. Double-pushout graph rewriting generalises well to rewrit...

  19. The adaptor protein SAP directly associates with PECAM-1 and regulates PECAM-1-mediated-cell adhesion in T-like cell lines.

    Science.gov (United States)

    Proust, Richard; Crouin, Catherine; Gandji, Leslie Yewakon; Bertoglio, Jacques; Gesbert, Franck

    2014-04-01

    SAP is a small cytosolic adaptor protein expressed in hematopoietic lineages whose main function is to regulate intracellular signaling pathways induced by the triggering of members of the SLAM receptor family. In this paper, we have identified the adhesion molecule PECAM-1 as a new partner for SAP in a conditional yeast two-hybrid screen. PECAM-1 is an immunoglobulin-like molecule expressed by endothelial cells and leukocytes, which possesses both pro- and anti-inflammatory properties. However, little is known about PECAM-1 functions in T cells. We show that SAP directly and specifically interacts with the cytosolic tyrosine 686 of PECAM-1. We generated different T-like cell lines in which SAP or PECAM-1 are expressed or down modulated and we demonstrate that a diminished SAP expression correlates with a diminished PECAM-1-mediated adhesion. Although SAP has mainly been shown to associate with SLAM receptors, we evidence here that SAP is a new actor downstream of PECAM-1. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Alpha-enolase (ENO1 controls alpha v/beta 3 integrin expression and regulates pancreatic cancer adhesion, invasion, and metastasis

    Directory of Open Access Journals (Sweden)

    Moitza Principe

    2017-01-01

    Full Text Available Abstract Background We have previously shown that in pancreatic ductal adenocarcinoma (PDA cells, the glycolytic enzyme alpha-enolase (ENO1 also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability. Methods The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1 PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM. The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model. Results We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN via urokinase plasminogen activator receptor (uPAR. Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell

  1. Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo

    Science.gov (United States)

    Goody, Michelle F.; Kelly, Meghan W.; Lessard, Kevin N.; Khalil, Andre; Henry, Clarissa A.

    2010-01-01

    Cell-matrix adhesion complexes (CMACs) play fundamental roles during morphogenesis. Given the ubiquitous nature of CMACs and their roles in many cellular processes, one question is how specificity of CMAC function is modulated. The clearly defined cell behaviors that generate segmentally reiterated axial skeletal muscle during zebrafish development comprise an ideal system with which to investigate CMAC function during morphogenesis. We found that Nicotinamide riboside kinase 2b (Nrk2b) cell autonomously modulates the molecular composition of CMACs in vivo. Nrk2b is required for normal Laminin polymerization at the myotendinous junction (MTJ). In Nrk2b-deficient embryos, at MTJ loci where Laminin is not properly polymerized, muscle fibers elongate into adjacent myotomes and are abnormally long. In yeast and human cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative salvage pathway. Exogenous NAD+ treatment rescues MTJ development in Nrk2b-deficient embryos, but not in laminin mutant embryos. Both Nrk2b and Laminin are required for localization of Paxillin, but not β-Dystroglycan, to CMACs at the MTJ. Overexpression of Paxillin in Nrk2b-deficient embryos is sufficient to rescue MTJ integrity. Taken together, these data show that Nrk2b plays a specific role in modulating subcellular localization of discrete CMAC components that in turn play roles in musculoskeletal development. Furthermore, these data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development. These results indicate a previously unrecognized complexity to CMAC assembly in vivo and also elucidate a novel role for NAD+ during morphogenesis. PMID:20566368

  2. Focal and generalized alopecia.

    Science.gov (United States)

    O'Dair, H A; Foster, A P

    1995-07-01

    Focal or generalized alopecia is defined as hair loss affecting the ventral, lateral, perineal, and dorsal aspects of the trunk of the cat, usually in a symmetric pattern. This may be attributable to failure of hair coat production, excess loss of hair due to self trauma, or excess shedding of whole hairs. Self trauma is the most common cause of hair loss and is associated particularly with flea allergy dermatitis. Other causes of hair loss are reviewed.

  3. Proximal Focal Femoral Deficiency

    OpenAIRE

    Vishal Kalia, Vibhuti

    2008-01-01

    Proximal focal femoral deficiency (PFFD) is a developmental disorder of the proximal segment of thefemur and of acetabulum resulting in shortening of the affected limb and impairment of the function. It isa spectrum of congenital osseous anomalies characterized by a deficiency in the structure of the proximalfemur. The diagnosis is often made by radiological evaluation which includes identification and descriptionof PFFD and evaluation of associated limb anomalies by plain radiographs. Contra...

  4. Oral focal epithelial hyperplasia.

    Science.gov (United States)

    López-Jornet, Pía; Camacho-Alonso, Fabio; Berdugo, Lucero

    2010-01-01

    Focal epithelial hyperplasia (FEH) is a benign, asymptomatic disease. It appears as papules, principally on the lower lip, although it can also be found on the retro-commissural mucosa and tongue and, less frequently, on the upper lip, gingiva and palate. FEH is caused by human papillomavirus subtype 13 or 32. The condition occurs in many populations and ethnic groups. We present the clinical case of a 31-year-old male with lesions that clinically and histologically corresponded to FEH.

  5. Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

    LENUS (Irish Health Repository)

    Costello, Richard W

    2011-05-01

    The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

  6. Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

    LENUS (Irish Health Repository)

    Costello, Richard W

    2012-02-01

    The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

  7. Focal cortical dysplasia - review.

    Science.gov (United States)

    Kabat, Joanna; Król, Przemysław

    2012-04-01

    Focal cortical dysplasia is a malformation of cortical development, which is the most common cause of medically refractory epilepsy in the pediatric population and the second/third most common etiology of medically intractable seizures in adults.Both genetic and acquired factors are involved in the pathogenesis of cortical dysplasia. Numerous classifications of the complex structural abnormalities of focal cortical dysplasia have been proposed - from Taylor et al. in 1971 to the last modification of Palmini classification made by Blumcke in 2011. In general, three types of cortical dysplasia are recognized.Type I focal cortical dysplasia with mild symptomatic expression and late onset, is more often seen in adults, with changes present in the temporal lobe.Clinical symptoms are more severe in type II of cortical dysplasia usually seen in children. In this type, more extensive changes occur outside the temporal lobe with predilection for the frontal lobes.New type III is one of the above dysplasias with associated another principal lesion as hippocampal sclerosis, tumor, vascular malformation or acquired pathology during early life.Brain MRI imaging shows abnormalities in the majority of type II dysplasias and in only some of type I cortical dysplasias.THE MOST COMMON FINDINGS ON MRI IMAGING INCLUDE: focal cortical thickening or thinning, areas of focal brain atrophy, blurring of the gray-white junction, increased signal on T2- and FLAIR-weighted images in the gray and subcortical white matter often tapering toward the ventricle. On the basis of the MRI findings, it is possible to differentiate between type I and type II cortical dysplasia. A complete resection of the epileptogenic zone is required for seizure-free life. MRI imaging is very helpful to identify those patients who are likely to benefit from surgical treatment in a group of patients with drug-resistant epilepsy.However, in type I cortical dysplasia, MR imaging is often normal, and also in both types

  8. Regulation by gut commensal bacteria of carcinoembryonic antigen-related cell adhesion molecule expression in the intestinal epithelium.

    Science.gov (United States)

    Kitamura, Yasuaki; Murata, Yoji; Park, Jung-Ha; Kotani, Takenori; Imada, Shinya; Saito, Yasuyuki; Okazawa, Hideki; Azuma, Takeshi; Matozaki, Takashi

    2015-07-01

    Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 1 and CEACAM20, immunoglobulin superfamily members, are predominantly expressed in intestinal epithelial cells (IECs) and co-localized at the apical surface of these cells. We here showed that the expression of mouse CEACAM1 and CEACAM20 at both mRNA and protein levels was markedly reduced in IECs of the small intestine by the treatment of mice with antibiotics against Gram-positive bacteria. The expression of both proteins was also decreased in IECs of the small intestine from germ-free mice, compared with that from control specific-pathogen-free mice. Exposure of intestinal organoids to IFN-γ markedly increased the expression of either CEACAM1 or CEACAM20, whereas the exposure to TNF-α increased the expression of the former protein, but not that of the latter. In contrast, the expression of CEACAM20, but not of CEACAM1, in intestinal organoids was markedly increased by exposure to butyrate, a short-chain fatty acid produced by bacterial fermentation in the intestine. Collectively, our results suggest that Gram-positive bacteria promote the mRNA expression of CEACAM1 or CEACAM20 in the small intestine. Inflammatory cytokines or butyrate likely participates in such effects of commensal bacteria.

  9. Regulation of cellular adhesion molecule expression in murine oocytes,peri-implantation and post-implantation embryos

    Institute of Scientific and Technical Information of China (English)

    DAVID; P; LU; LINA; TIAN; CHRIS; O'; NEILL; NICHOLAS; JC; KING

    2002-01-01

    Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, α chain),and CD11a (LFA-1, α chain) on mouse oocytes, and pre- and peri-implantation stage embryos was exam-ined by quantitative indirect immunofluorescence microscopy. ICAM-1 was most strongly expressed at theoocyte stage, gradually declining almost to undetectable levels by the expanded blastocyst stage. NCAM,also expressed maximally on the oocyte, declined to undetectable levels beyond the morula stage. On theother hand, CD44 declined from highest expression at the oocyte stage to show a second maximum at thecompacted 8-cell/morula. This molecule exhibited high expression around contact areas between trophecto-derm and zona pellucida during blastocyst hatching. CD49d was highly expressed in the oocyte, remainedsignificantly expressed throughout and after blastocyst hatching was expressed on the polar trophecto-derm. Like CD44, CD49d declined to undetectable levels at the blastocyst outgrowth stage. Expression ofboth VCAM-1 and CD11a was undetectable throughout. The diametrical temporal expression pattern ofICAM-1 and NCAM compared to CD44 and CD49d suggest that dynamic changes in expression of adhesionmolecules may be important for interaction of the embryo with the maternal cellular environment as wellas for continuing development and survival of the early embryo.

  10. Down-regulation of pigment epithelium-derived factor in uveitic lesion associates with focal vascular endothelial growth factor expression and breakdown of the blood-retinal barrier.

    Science.gov (United States)

    Deeg, Cornelia A; Altmann, Frank; Hauck, Stefanie M; Schoeffmann, Stephanie; Amann, Barbara; Stangassinger, Manfred; Ueffing, Marius

    2007-05-01

    Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Identifying biological markers or pathways associated with this disease may allow the understanding of its pathogenesis at a molecular level. The vitreous is the body fluid closest to the disease-affected tissue and possibly also an effector of pathological processes relevant for ERU. Surgical removal of vitreous leads to cessation of relapses in spontaneous uveitis of both man and horse, therefore vitreous composites are likely to contribute to disease progression. Uveitic vitreous is likely to contain potential biomarkers in relatively undiluted quantities. With the goal to identify these markers, we systematically compared vitreous from healthy and disease-affected eyes by proteomic profiling. Nine differentially expressed proteins were identified, that are functionally related to immune response, inflammation, and maintenance of the blood-retinal barrier. One of these, pigment epithelium-derived factor, a protein involved in maintaining a proper blood-retina barrier as well as protecting from neoangiogenesis was additionally found to be down-regulated within uveitic retinal lesions whereas, conversely, vascular endothelial growth factor was found to be up-regulated at these sites. Together, these changes point to as of yet undiscovered biological pathways involved in the pathogenesis of this autoimmune disease.

  11. Long-term reduction of T-cell intracellular antigens reveals a transcriptome associated with extracellular matrix and cell adhesion components.

    Directory of Open Access Journals (Sweden)

    Mario Núñez

    Full Text Available Knockdown of T-cell intracellular antigens TIA1 and TIAR contributes to a cellular phenotype characterised by uncontrolled proliferation and tumorigenesis. Massive-scale poly(A+ RNA sequencing of TIA1 or TIAR-knocked down HeLa cells reveals transcriptome signatures comprising genes and functional categories potentially able to modulate several aspects of membrane dynamics associated with extracellular matrix and focal/cell adhesion events. The transcriptomic heterogeneity is the result of differentially expressed genes and RNA isoforms generated by alternative splicing and/or promoter usage. These results suggest a role for TIA proteins in the regulation and/or modulation of cellular homeostasis related to focal/cell adhesion, extracellular matrix and membrane and cytoskeleton dynamics.

  12. PED/PEA-15 interacts with the 67 kD laminin receptor and regulates cell adhesion, migration, proliferation and apoptosis

    Science.gov (United States)

    Formisano, Pietro; Ragno, Pia; Pesapane, Ada; Alfano, Daniela; Alberobello, Anna Teresa; Rea, Vincenza Elena Anna; Giusto, Raffaella; Rossi, Francesca W; Beguinot, Francesco; Rossi, Guido; Montuori, Nunzia

    2012-01-01

    Abstract Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kD (PED/PEA-15) is an anti-apoptotic protein whose expression is increased in several human cancers. In addition to apoptosis, PED/PEA-15 is involved in the regulation of other major cellular functions, including cell adhesion, migration, proliferation and glucose metabolism. To further understand the functions of this protein, we performed a yeast two-hybrid screening using PED/PEA-15 as a bait and identified the 67 kD high-affinity laminin receptor (67LR) as an interacting partner. 67 kD laminin receptor is a non-integrin cell-surface receptor for the extracellular matrix (ECM), derived from the dimerization of a 37 kD cytosolic precursor (37LRP). The 67LR is highly expressed in human cancers and widely recognized as a molecular marker of metastatic aggressiveness. The molecular interaction of PED/PEA-15 with 67LR was confirmed by pull-down experiments with recombinant His-tagged 37LRP on lysates of PED/PEA-15 transfected HEK-293 cells. Further, overexpressed or endogenous PED/PEA-15 was co-immunoprecipitated with 67LR in PED/PEA-15-transfected HEK-293 cells and in U-373 glioblastoma cells, respectively. PED/PEA-15 overexpression significantly increased 67LR-mediated HEK-293 cell adhesion and migration to laminin that, in turn, determined PED/PEA-15 phosphorylation both in Ser-104 and Ser-116, thus enabling cell proliferation and resistance to apoptosis. PED/PEA-15 ability to induce cell responses to ECM-derived signals through interaction with 67LR may be of crucial importance for tumour cell survival in a poor microenvironment, thus favouring the metastatic spread and colonization. PMID:21895963

  13. 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.

  14. Sam68 regulates cell proliferation and cell adhesion-mediated drug resistance (CAM-DR) via the AKT pathway in non-Hodgkin's lymphoma.

    Science.gov (United States)

    Wu, Yaxun; Xu, Xiaohong; Miao, Xiaobing; Zhu, Xinghua; Yin, Haibing; He, Yunhua; Li, Chunsun; Liu, Yushan; Chen, Yali; Lu, Xiaoyun; Wang, Yuchan; He, Song

    2015-12-01

    Sam68 (Src-associated in mitosis 68 kDa), a substrate for tyrosine kinase c-Src during mitosis, is up-regulated in a variety of human cancers and acts oncogenically promoting tumour progression. This study has explored biological function and clinical significance of Sam68 in non-Hodgkin's lymphoma (NHL). To examine Sam68 expression in NHL, clinically, eight diffuse large B-cell lymphomas and four reactive lymphoid hyperplasia fresh-frozen tissues were obtained for western blot and quantitative real-time PCR analyses. Using immunohistochemical staining, paraffin wax embedded sections from 164 cases of NHL patients were used to evaluate prognostic value of Sam68. Cell Counting Kit-8 (CCK-8) and soft agar colony assays were conducted to investigate the role of Sam68 in cell viability and cell proliferation respectively. Furthermore, effects of Sam68 on cell adhesion-mediated drug resistance (CAM-DR) was determined by CCK-8 assay and flow cytometric analysis. Expression status of Sam68 inversely correlated with clinical outcomes of patients with NHL, and it was also an independent prognostic factor for the outcomes. In addition, Sam68 was associated with proliferation of NHL cells. Knock-down of its gene inhibited cell proliferation and colony formation by delaying cell cycle progression. Furthermore, OCI-Ly8 and Jeko-1 cells adhering to FN and HS-5 expressed higher Sam68 protein, compared to their suspension counterparts. Sam68 promoted cell adhesion-mediated drug resistance (CAM-DR) via the AKT pathway. Increased Sam68 expression in NHL resulted in poor prognosis, and it promoted CAM-DR in NHL via AKT. © 2015 John Wiley & Sons Ltd.

  15. PED/PEA-15 interacts with the 67 kD laminin receptor and regulates cell adhesion, migration, proliferation and apoptosis.

    Science.gov (United States)

    Formisano, Pietro; Ragno, Pia; Pesapane, Ada; Alfano, Daniela; Alberobello, Anna Teresa; Rea, Vincenza Elena Anna; Giusto, Raffaella; Rossi, Francesca W; Beguinot, Francesco; Rossi, Guido; Montuori, Nunzia

    2012-07-01

    Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kD (PED/PEA-15) is an anti-apoptotic protein whose expression is increased in several human cancers. In addition to apoptosis, PED/PEA-15 is involved in the regulation of other major cellular functions, including cell adhesion, migration, proliferation and glucose metabolism. To further understand the functions of this protein, we performed a yeast two-hybrid screening using PED/PEA-15 as a bait and identified the 67 kD high-affinity laminin receptor (67LR) as an interacting partner. 67 kD laminin receptor is a non-integrin cell-surface receptor for the extracellular matrix (ECM), derived from the dimerization of a 37 kD cytosolic precursor (37LRP). The 67LR is highly expressed in human cancers and widely recognized as a molecular marker of metastatic aggressiveness. The molecular interaction of PED/PEA-15 with 67LR was confirmed by pull-down experiments with recombinant His-tagged 37LRP on lysates of PED/PEA-15 transfected HEK-293 cells. Further, overexpressed or endogenous PED/PEA-15 was co-immunoprecipitated with 67LR in PED/PEA-15-transfected HEK-293 cells and in U-373 glioblastoma cells, respectively. PED/PEA-15 overexpression significantly increased 67LR-mediated HEK-293 cell adhesion and migration to laminin that, in turn, determined PED/PEA-15 phosphorylation both in Ser-104 and Ser-116, thus enabling cell proliferation and resistance to apoptosis. PED/PEA-15 ability to induce cell responses to ECM-derived signals through interaction with 67LR may be of crucial importance for tumour cell survival in a poor microenvironment, thus favouring the metastatic spread and colonization.

  16. Polycystin-1 Induces Cell Migration by Regulating Phosphatidylinositol 3-kinase-dependent Cytoskeletal Rearrangements and GSK3β-dependent Cell–Cell Mechanical Adhesion

    Science.gov (United States)

    Boca, Manila; D'Amato, Lisa; Distefano, Gianfranco; Polishchuk, Roman S.; Germino, Gregory G.

    2007-01-01

    Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1−/− mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and β-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated β-catenin through activation of GSK3β. Expression of a nondegradable form of β-catenin in PC-1 MDCK cells restores strong cell–cell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis. PMID:17671167

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

    Science.gov (United States)

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

    2013-03-01

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

  18. Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5.

    Science.gov (United States)

    Huang, Jiqing; Kast, Juergen

    2015-08-07

    Physiological stimuli, such as thrombin, or pathological stimuli, such as lysophosphatidic acid (LPA), activate platelets circulating in blood. Once activated, platelets bind to monocytes via P-selectin-PSGL-1 interactions but also release the stored contents of their granules. These platelet releasates, in addition to direct platelet binding, activate monocytes and facilitate their recruitment to atherosclerotic sites. Consequently, understanding the changes platelet releasates induce in monocyte membrane proteins is critical. We studied the glyco-proteome changes of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasates. We employed lectin affinity chromatography combined with filter aided sample preparation to achieve high glyco- and membrane protein and protein sequence coverage. Using stable isotope labeling by amino acids in cell culture, we quantified 1715 proteins, including 852 membrane and 500 glycoproteins, identifying the up-regulation of multiple proteins involved in monocyte extracellular matrix binding and transendothelial migration. Flow cytometry indicated expression changes of integrin α5, integrin β1, PECAM-1, and PSGL-1. The observed increase in monocyte adhesion to fibronectin was determined to be mediated by the up-regulation of very late antigen 5 via a P-selectin-PSGL-1 independent mechanism. This novel aspect could be validated on CD14+ human primary monocytes, highlighting the benefits of the improved enrichment method regarding high membrane protein coverage and reliable quantification.

  19. Effect of H2O2,extracellular matrix and out segment of photoreceptor on expression of focal adhesion kinase in RPE cell%氧化衰老、细胞外基质和光感受器外节膜盘对RPE细胞中黏着斑激酶表达的影响

    Institute of Scientific and Technical Information of China (English)

    朱洁; 王雨生; 赵炜; 杨秀梅; 李夏; 姜廷帅

    2011-01-01

    Background The underlying mechanism of choroidal neovascularization(CNV) is multifactorial and complex.Focal adhesion kinase(FAK) plays a crucial role in controlling essential cellular processes and influencing distinct steps of the angiogenic response.But to our knowledge,seldom study on the effect of FAK on CNV formation has been reported previously.Objective In this study,the effect of several CNV risk factors on the expression of FAK in cultured retinal pigment epithelium(RPE) cells was investigated to illuminate effect of FAK on CNV.Methods Human RPE cells were isolated from donor eyes and exposed to H2O2,swallow of outer segment of photoreceptors(POS) and extracellular matrix(ECM) separately with the treating as follows:RPE cells were co-cultured with 10,20,50 and 100μmol/L H2O2 for 20 days;POS(1×106/ml) were co-cultivated with RPE cells for 20 days(setting control group,POS group,hypoxia group with 200μmol/L CoCl2,and POS+hyoxia group);RPE cells were cultured on the plates coated with 100mg/L fibronectin(FN),laminin(LN) or collagen typeⅠfor 30minutes or 1 hour.The expression of FAK and pFAK in RPE cells were examined by Western blot analysis.Results FAK was highly expressed in the 20μmol/L and 50μmol/L H2O2 groups compared with control group(P<0.01);while he expression level of pFAK was reduced after treated with H2O2 in comparison with the control group(P<0.01).After cultured with POS for 20 days,the undigested lysosome could be observed in RPE cells.The expressions of FAK and pFAK in RPE cells were not significantly changed between control group and POS groups(P>0.05),but those in hypoxia group were significantly up-regulated in comparison with control group(P<0.01).Compared with the hypoxia group,the expression amount of pFAK was elevated in POS+hyoxia group(P<0.01).In comparison with control group,the increased pFAK expression was seen in FN,LN and collagen typeⅠtreating for 1-hour groups(P<0.05,P<0.01).Conclusion FAK pathway

  20. EGCG Inhibits Proliferation, Invasiveness and Tumor Growth by Up-Regulation of Adhesion Molecules, Suppression of Gelatinases Activity, and Induction of Apoptosis in Nasopharyngeal Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Chih-Yeu Fang

    2015-01-01

    Full Text Available (−-Epigallocatechin-3-gallate (EGCG, a major green tea polyphenol, has been shown to inhibit the proliferation of a variety of tumor cells. Epidemiological studies have shown that drinking green tea can reduce the incidence of nasopharyngeal carcinoma (NPC, yet the underlying mechanism is not well understood. In this study, the inhibitory effect of EGCG was tested on a set of Epstein Barr virus-negative and -positive NPC cell lines. Treatment with EGCG inhibited the proliferation of NPC cells but did not affect the growth of a non-malignant nasopharyngeal cell line, NP460hTert. Moreover, EGCG treated cells had reduced migration and invasive properties. The expression of the cell adhesion molecules E-cadherin and β-catenin was found to be up-regulated by EGCG treatment, while the down-regulation of matrix metalloproteinases (MMP-2 and MMP-9 were found to be mediated by suppression of extracellular signal-regulated kinase (ERK phosphorylation and AP-1 and Sp1 transactivation. Spheroid formation by NPC cells in suspension was significantly inhibited by EGCG. Oral administration of EGCG was capable of suppressing tumor growth in xenografted mice bearing NPC tumors. Treatment with EGCG was found to elevate the expression of p53 and p21, and eventually led to apoptosis of NPC cells via caspase 3 activation. The nuclear translocation of NF-κB and β-catenin was also suppressed by EGCG treatment. These results indicate that EGCG can inhibit the proliferation and invasiveness, and induce apoptosis, of NPC cells, making it a promising agent for chemoprevention or adjuvant therapy of NPC.

  1. c-Yes regulates cell adhesion at the blood-testis barrier and the apical ectoplasmic specialization in the seminiferous epithelium of rat testes.

    Science.gov (United States)

    Xiao, Xiang; Mruk, Dolores D; Lee, Will M; Cheng, C Yan

    2011-04-01

    During spermatogenesis, extensive junction restructuring takes place at the blood-testis barrier (BTB) and the Sertoli cell-spermatid interface known as the apical ectoplasmic specialization (apical ES, a testis-specific adherens junction) in the seminiferous epithelium. However, the mechanism(s) that regulates these critical events in the testis remains unknown. Based on the current concept in the field, changes in the phosphorylation status of integral membrane proteins at these sites can induce alterations in protein endocytosis and recycling, causing junction restructuring. Herein, c-Yes, a non-receptor protein tyrosine kinase, was found to express abundantly at the BTB and apical ES stage-specifically, coinciding with junction restructuring events at these sites during the seminiferous epithelial cycle of spermatogenesis. c-Yes also structurally associated with adhesion proteins at the BTB (e.g., occludin and N-cadherin) and the apical ES (e.g., β1-integrin, laminins β3 and γ3), possibly to regulate phosphorylation status of proteins at these sites. SU6656, a selective c-Yes inhibitor, was shown to perturb the Sertoli cell tight junction-permeability barrier in vitro, which is mediated by changes in the distribution of occludin and N-cadherin at the cell-cell interface, moving from cell surface to cytosol, thereby destabilizing the tight junction-barrier. However, this disruptive effect of SU6656 on the barrier was blocked by testosterone. Furthermore, c-Yes is crucial to maintain the actin filament network in Sertoli cells since a blockade of c-Yes by SU6656 induced actin filament disorganization. In summary, c-Yes regulates BTB and apical ES integrity by maintaining proper distribution of integral membrane proteins and actin filament organization at these sites.

  2. Systemic focal epileptogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Remler, M.P.; Marcussen, W.H.

    1986-01-01

    Rats that receive radiation to 0.25 cc of one cerebral hemisphere are clinically and electroencephalographically normal until there is a breakdown of the blood-brain barrier (BBB) at 3 to 6 months postradiation. This BBB lesion can be detected by transient focal seizure activity produced by the BBB-excluded systemic convulsant bicuculline methiodide. In two rats the seizure activity induced by this one injection was self-sustaining. In seven of 15 other rats tested, the subsequent administration of repeated 2 mg/kg injections created a chronic focus that continued to spike with great frequency for 3 weeks or more without further administration of any convulsant. In three of eight other rats, implanted minipumps delivering 180 micrograms/h of bicuculline methiodide produced self-sustaining epileptic activity.

  3. Focal femoral condyle resurfacing.

    LENUS (Irish Health Repository)

    Brennan, S A

    2013-03-01

    Focal femoral inlay resurfacing has been developed for the treatment of full-thickness chondral defects of the knee. This technique involves implanting a defect-sized metallic or ceramic cap that is anchored to the subchondral bone through a screw or pin. The use of these experimental caps has been advocated in middle-aged patients who have failed non-operative methods or biological repair techniques and are deemed unsuitable for conventional arthroplasty because of their age. This paper outlines the implant design, surgical technique and biomechanical principles underlying their use. Outcomes following implantation in both animal and human studies are also reviewed. Cite this article: Bone Joint J 2013;95-B:301-4.

  4. Extensive focal epithelial hyperplasia.

    Science.gov (United States)

    Hashemipour, Maryam Alsadat; Shoryabi, Ali; Adhami, Shahrzad; Mehrabizadeh Honarmand, Hoda

    2010-01-01

    Heck's disease or focal epithelial hyperplasia is a benign contagious disease caused by human papillomavirus types 13 or 32. It occurs with low frequency in the Iranian population. This condition is characterized by the occurrence of multiple, small papules or nodules in the oral cavity, especially on the labial and buccal mucosa and tongue. In some populations, up to 39% of children are affected. Conservative surgical excision of lesions may be performed for diagnostic or aesthetic purposes. The risk of recurrence after this therapy is minimal, and there seems to be no malignant transformation potential. In the present work, we presented the clinical case of a 12-year-old Iranian girl with oral lesions that clinically and histologically correspond to Heck's disease.

  5. Proximal Focal Femoral Deficiency

    Directory of Open Access Journals (Sweden)

    Vishal Kalia, Vibhuti

    2008-01-01

    Full Text Available Proximal focal femoral deficiency (PFFD is a developmental disorder of the proximal segment of thefemur and of acetabulum resulting in shortening of the affected limb and impairment of the function. It isa spectrum of congenital osseous anomalies characterized by a deficiency in the structure of the proximalfemur. The diagnosis is often made by radiological evaluation which includes identification and descriptionof PFFD and evaluation of associated limb anomalies by plain radiographs. Contrast arthrography orMagnetic Resonance Imaging is indicated when radiological features are questionable and to disclose thepresence and location of the femoral head and any cartilagenous anlage. The disorder is more commonlyunilateral and is apparent at birth. However, bilateral involvement is rarely seen. Therapy of the disorder isdirected towards satisfactory ambulation and specific treatment depending on the severity of dysplasia.

  6. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    tract to the microbial flocs in waste water treatment facilities. Microbial biofilms may however also cause a wide range of industrial and medical problems, and have been implicated in a wide range of persistent infectious diseases, including implantassociated microbial infections. Bacterial adhesion...... is the first committing step in biofilm formation, and has therefore been intensely scrutinized. Much however, still remains elusive. Bacterial adhesion is a highly complex process, which is influenced by a variety of factors. In this thesis, a range of physico-chemical, molecular and environmental parameters......, which influence the transition from a planktonic lifestyle to a sessile lifestyle, have been studied. Protein conditioning film formation was found to influence bacterial adhesion and subsequent biofilm formation considerable, and an aqueous extract of fish muscle tissue was shown to significantly...

  7. Involvement of rho-gtpases in fibroblast adhesion and fibronectine fibrillogenesis under stretch

    Science.gov (United States)

    Guignandon, A.; Lambert, C.; Rattner, A.; Servotte, S.; Lapiere, C.; Nusgens, B.; Vico, L.

    The Rho family small GTPases play a crucial role in mediating cellular adaptation to mechanical stimulation (MS), and possibly to microgravity (μg), through effects on the cytoskeleton and cell adhesion which is, in turn, mainly regulated by fibronectin fibrillogenesis (FnF). It remains unclear how mechanical stimulation is transduced to the Rho signaling pathways and how it impacts on fibronectin (fbn) fibrillogenesis (FnF). μg (2 days, mission STS-095) led to de-adhesion of fibroblasts and modification of the underlying extracellular matrix. To determine whether GTPases modulated FnF, we generated stable cell lines expressing high level of activated RhoA and Rac1 (QL) as compared to wild type (WI26-WT). After MS application [8% deformation, 1Hz, 15 min., 3 times/day for 1-2 days], we quantified focal adhesion (vinculin, paxillin, FAKY397), f-actin stress fibers (Sf) and FnF with home-developed softwares. We reported that after MS, Sf are more rapidly (30min) formed under the nucleus in Wi26-WT (+100%) and Rac1 (+200%) than in RhoA (+20%). Vinculin & paxillin were only restricted to the cell edge in static conditions and homogeneously distributed after MS in WT and Rac1. The relative area of contacts (vinculin & paxillin) was more dramatically enhanced by MS in Rac1 (+80%) than in WT (+40%) and RhoA (+25%) indicating that new focal contacts are formed under MS and supported the presence of Sf. MS Activation of FAK (FAKY397) was clear in WT and Rac1 and reduced in RhoA. FnF was restricted to cell-cell contacts zone without any change in the relative area of fbn after a 2-days MS. However we found more numerous spots of fbn at the cell center in Rac1 as compared with RhoA & WT suggesting that these fibrillar contacts will grow upon maturation and modulate FnF. The results indicate that MS induces formation of Sf and focal adhesions and enhances FF. RhoA has been shown to induce the formation of Sf and focal adhesions, and Rac1 activation decreases Rho activity in

  8. Integrin-linked kinase regulates cellular mechanics facilitating the motility in 3D extracellular matrices.

    Science.gov (United States)

    Kunschmann, Tom; Puder, Stefanie; Fischer, Tony; Perez, Jeremy; Wilharm, Nils; Mierke, Claudia Tanja

    2017-03-01

    The motility of cells plays an important role for many processes such as wound healing and malignant progression of cancer. The efficiency of cell motility is affected by the microenvironment. The connection between the cell and its microenvironment is facilitated by cell-matrix adhesion receptors and upon their activation focal adhesion proteins such as integrin-linked kinase (ILK) are recruited to sites of focal adhesion formation. In particular, ILK connects cell-matrix receptors to the actomyosin cytoskeleton. However, ILK's role in cell mechanics regulating cellular motility in 3D collagen matrices is still not well understood. We suggest that ILK facilitates 3D motility by regulating cellular mechanical properties such as stiffness and force transmission. Thus, ILK wild-type and knock-out cells are analyzed for their ability to migrate on 2D substrates serving as control and in dense 3D extracellular matrices. Indeed, ILK wild-type cells migrated faster on 2D substrates and migrated more numerous and deeper in 3D matrices. Hence, we analyzed cellular deformability, Young's modulus (stiffness) and adhesion forces. We found that ILK wild-type cells are less deformable (stiffer) and produce higher cell-matrix adhesion forces compared to ILK knock-out cells. Finally, ILK is essential for providing cellular mechanical stiffness regulating 3D motility.

  9. NOV/CCN3 induces adhesion of muscle skeletal cells and cooperates with FGF2 and IGF-1 to promote proliferation and survival.

    Science.gov (United States)

    Lafont, Jerôme; Thibout, Hélène; Dubois, Catherine; Laurent, Maryvonne; Martinerie, Cécile

    2005-01-01

    During mammalian development, expression of the Nephroblastoma overexpressed gene (NOV/CCN3) is tightly regulated in skeletal muscles. Ex vivo, ectopic expression of NOV blocks myogenic differentiation. NOV also supports endothelial cell adhesion and angiogenesis through interactions with integrins. Integrins play fundamental roles during myogenesis. In this study, we show that NOV mediates adhesion and spreading of myoblasts. Myoblasts adhesion to NOV does not require proteoglycans and is dependent on integrin beta1, whereas spreading involves another RGD-sensitive integrin. The C-Terminal part of NOV as well as full-length is able to support adhesion of myoblasts; in addition, both increase focal-adhesion kinase (FAK) phosphorylation. Furthermore, NOV is an adhesive substrate that, combined with FGF2 or IGF-1, promotes cell specific proliferation and survival, respectively, in a better way than fibronectin. Taken together, these results identify NOV as an adhesion substrate for myoblasts which, in concert with growth factors, could play a role in the physiology of muscle cells.

  10. Intercellular Adhesion-Dependent Cell Survival and ROCK-Regulated Actomyosin-Driven Forces Mediate Self-Formation of a Retinal Organoid

    Directory of Open Access Journals (Sweden)

    Albert Lowe

    2016-05-01

    Full Text Available In this study we dissected retinal organoid morphogenesis in human embryonic stem cell (hESC-derived cultures and established a convenient method for isolating large quantities of retinal organoids for modeling human retinal development and disease. Epithelialized cysts were generated via floating culture of clumps of Matrigel/hESCs. Upon spontaneous attachment and spreading of the cysts, patterned retinal monolayers with tight junctions formed. Dispase-mediated detachment of the monolayers and subsequent floating culture led to self-formation of retinal organoids comprising patterned neuroretina, ciliary margin, and retinal pigment epithelium. Intercellular adhesion-dependent cell survival and ROCK-regulated actomyosin-driven forces are required for the self-organization. Our data supports a hypothesis that newly specified neuroretina progenitors form characteristic structures in equilibrium through minimization of cell surface tension. In long-term culture, the retinal organoids autonomously generated stratified retinal tissues, including photoreceptors with ultrastructure of outer segments. Our system requires minimal manual manipulation, has been validated in two lines of human pluripotent stem cells, and provides insight into optic cup invagination in vivo.

  11. The phosphatase PTP-PEST/PTPN12 regulates endothelial cell migration and adhesion, but not permeability, and controls vascular development and embryonic viability.

    Science.gov (United States)

    Souza, Cleiton Martins; Davidson, Dominique; Rhee, Inmoo; Gratton, Jean-Philippe; Davis, Elaine C; Veillette, André

    2012-12-14

    Protein-tyrosine phosphatase (PTP)-PEST (PTPN12) is ubiquitously expressed. It is essential for normal embryonic development and embryonic viability in mice. Herein we addressed the involvement of PTP-PEST in endothelial cell functions using a combination of genetic and biochemical approaches. By generating primary endothelial cells from an inducible PTP-PEST-deficient mouse, we found that PTP-PEST is not needed for endothelial cell differentiation and proliferation or for the control of endothelial cell permeability. Nevertheless, it is required for integrin-mediated adhesion and migration of endothelial cells. PTP-PEST-deficient endothelial cells displayed increased tyrosine phosphorylation of Cas, paxillin, and Pyk2, which were previously also implicated in integrin functions. By eliminating PTP-PEST in endothelial cells in vivo, we obtained evidence that expression of PTP-PEST in endothelial cells is required for normal vascular development and embryonic viability. Therefore, PTP-PEST is a key regulator of integrin-mediated functions in endothelial cells seemingly through its capacity to control Cas, paxillin, and Pyk2. This function explains at least in part the essential role of PTP-PEST in embryonic development and viability.

  12. Molecular clock regulates daily α1-2-fucosylation of the neural cell adhesion molecule (NCAM) within mouse secondary olfactory neurons.

    Science.gov (United States)

    Kondoh, Daisuke; Tateno, Hiroaki; Hirabayashi, Jun; Yasumoto, Yuki; Nakao, Reiko; Oishi, Katsutaka

    2014-12-26

    The circadian clock regulates various behavioral and physiological rhythms in mammals. Circadian changes in olfactory functions such as neuronal firing in the olfactory bulb (OB) and olfactory sensitivity have recently been identified, although the underlying molecular mechanisms remain unknown. We analyzed the temporal profiles of glycan structures in the mouse OB using a high-density microarray that includes 96 lectins, because glycoconjugates play important roles in the nervous system such as neurite outgrowth and synaptogenesis. Sixteen lectin signals significantly fluctuated in the OB, and the intensity of all three that had high affinity for α1-2-fucose (α1-2Fuc) glycan in the microarray was higher during the nighttime. Histochemical analysis revealed that α1-2Fuc glycan is located in a diurnal manner in the lateral olfactory tract that comprises axon bundles of secondary olfactory neurons. The amount of α1-2Fuc glycan associated with the major target glycoprotein neural cell adhesion molecule (NCAM) varied in a diurnal fashion, although the mRNA and protein expression of Ncam1 did not. The mRNA and protein expression of Fut1, a α1-2-specific fucosyltransferase gene, was diurnal in the OB. Daily fluctuation of the α1-2Fuc glycan was obviously damped in homozygous Clock mutant mice with disrupted diurnal Fut1 expression, suggesting that the molecular clock governs rhythmic α1-2-fucosylation in secondary olfactory neurons. These findings suggest the possibility that the molecular clock is involved in the diurnal regulation of olfaction via α1-2-fucosylation in the olfactory system.

  13. Statistical earthquake focal mechanism forecasts

    CERN Document Server

    Kagan, Yan Y

    2013-01-01

    Forecasts of the focal mechanisms of future earthquakes are important for seismic hazard estimates and Coulomb stress and other models of earthquake occurrence. Here we report on a high-resolution global forecast of earthquake rate density as a function of location, magnitude, and focal mechanism. In previous publications we reported forecasts of 0.5 degree spatial resolution, covering the latitude range magnitude, and focal mechanism. In previous publications we reported forecasts of 0.5 degree spatial resolution, covering the latitude range from -75 to +75 degrees, based on the Global Central Moment Tensor earthquake catalog. In the new forecasts we've improved the spatial resolution to 0.1 degree and the latitude range from pole to pole. Our focal mechanism estimates require distance-weighted combinations of observed focal mechanisms within 1000 km of each grid point. Simultaneously we calculate an average rotation angle between the forecasted mechanism and all the surrounding mechanisms, using the method ...

  14. Adhesive plasters

    Science.gov (United States)

    Holcombe, Jr., Cressie E.; Swain, Ronald L.; Banker, John G.; Edwards, Charlene C.

    1978-01-01

    Adhesive plaster compositions are provided by treating particles of Y.sub.2 O.sub.3, Eu.sub.2 O.sub.3, Gd.sub.2 O.sub.3 or Nd.sub.2 O.sub.3 with dilute acid solutions. The resulting compositions have been found to spontaneously harden into rigid reticulated masses resembling plaster of Paris. Upon heating, the hardened material is decomposed into the oxide, yet retains the reticulated rigid structure.

  15. Phosphorylation of actopaxin regulates cell spreading and migration

    Science.gov (United States)

    Clarke, Dominic M.; Brown, Michael C.; LaLonde, David P.; Turner, Christopher E.

    2004-01-01

    Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration. PMID:15353548

  16. SLIT2/ROBO2 signaling pathway inhibits nonmuscle myosin IIA activity and destabilizes kidney podocyte adhesion

    Science.gov (United States)

    Fan, Xueping; Yang, Hongying; Kumar, Sudhir; Tumelty, Kathleen E.; Pisarek-Horowitz, Anna; Sharma, Richa; Chan, Stefanie; Tyminski, Edyta; Shamashkin, Michael; Belghasem, Mostafa; Henderson, Joel M.; Coyle, Anthony J.; Berasi, Stephen P.

    2016-01-01

    The repulsive guidance cue SLIT2 and its receptor ROBO2 are required for kidney development and podocyte foot process structure, but the SLIT2/ROBO2 signaling mechanism regulating podocyte function is not known. Here we report that a potentially novel signaling pathway consisting of SLIT/ROBO Rho GTPase activating protein 1 (SRGAP1) and nonmuscle myosin IIA (NMIIA) regulates podocyte adhesion downstream of ROBO2. We found that the myosin II regulatory light chain (MRLC), a subunit of NMIIA, interacts directly with SRGAP1 and forms a complex with ROBO2/SRGAP1/NMIIA in the presence of SLIT2. Immunostaining demonstrated that SRGAP1 is a podocyte protein and is colocalized with ROBO2 on the basal surface of podocytes. In addition, SLIT2 stimulation inhibits NMIIA activity, decreases focal adhesion formation, and reduces podocyte attachment to collagen. In vivo studies further showed that podocyte-specific knockout of Robo2 protects mice from hypertension-induced podocyte detachment and albuminuria and also partially rescues the podocyte-loss phenotype in Myh9 knockout mice. Thus, we have identified SLIT2/ROBO2/SRGAP1/NMIIA as a potentially novel signaling pathway in kidney podocytes, which may play a role in regulating podocyte adhesion and attachment. Our findings also suggest that SLIT2/ROBO2 signaling might be a therapeutic target for kidney diseases associated with podocyte detachment and loss. PMID:27882344

  17. The independent roles of mechanical, structural and adhesion characteristics of 3D hydrogels on the regulation of cancer invasion and dissemination.

    Science.gov (United States)

    Beck, Jennifer N; Singh, Anirudha; Rothenberg, Ashley R; Elisseeff, Jennifer H; Ewald, Andrew J

    2013-12-01

    Metastasis begins with the escape, or dissemination, of cancer cells from the primary tumor. We recently demonstrated that tumors preferentially disseminate into collagen I and not into basement membrane protein gels (Matrigel). In this study, we used synthetic polymer systems to define material properties that could induce dissemination into Matrigel. We first specifically varied rigidity by varying the crosslinking density of poly(ethylene glycol) (PEG) networks within Matrigel scaffolds. Increased microenvironmental rigidity limited epithelial growth but did not promote dissemination. We next incorporated adhesive signals into the PEG network using peptide-conjugated cyclodextrin (α-CDYRGDS) rings. The α-CDYRGDS rings threaded along the PEG polymers, enabling independent control of matrix mechanics, adhesive peptide composition, and adhesive density. Adhesive PEG networks induced dissemination of normal and malignant mammary epithelial cells at intermediate values of adhesion and rigidity. Our data reveal that microenvironmental signals can induce dissemination of normal and malignant epithelial cells without requiring the fibrillar structure of collagen I or containing collagen I-specific adhesion sequences. Finally, the nanobiomaterials and assays developed in this study are generally useful both in 3D culture of primary mammalian tissues and in the systematic evaluation of the specific role of mechanical and adhesive inputs on 3D tumor growth, invasion, and dissemination. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Actomyosin organisation for adhesion, spreading, growth and movement in chick fibroblasts

    DEFF Research Database (Denmark)

    Couchman, J R; Rees, D A

    1979-01-01

    Examination of the actomyosin structures and their relation to adhesion, movement and growth in the first fibroblasts migrating from chick heart explants shows striking differences with fibroblasts adapted to grow in culture. The latter have focal adhesions which seem to immobilize them for ancho......Examination of the actomyosin structures and their relation to adhesion, movement and growth in the first fibroblasts migrating from chick heart explants shows striking differences with fibroblasts adapted to grow in culture. The latter have focal adhesions which seem to immobilize them...

  19. Cell adhesion to cathodic arc plasma deposited CrAlSiN thin films

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun Kyu, E-mail: skim@ulsan.ac.kr [School of Materials Science and Engineering, University of Ulsan, Ulsan 680-749 (Korea, Republic of); Pham, Vuong-Hung [Department of Materials Science and Engineering, Seoul National University, Seoul 151-744 (Korea, Republic of); Kim, Chong-Hyun [Department of Food Science, Cornell University, Ithaca, NY 14853 (United States)

    2012-07-01

    Osteoblast cell response (cell adhesion, actin cytoskeleton and focal contact adhesion as well as cell proliferation) to CrN, CrAlSiN and Ti thin films was evaluated in vitro. Cell adhesion and actin stress fibers organization depended on the film composition significantly. Immunofluorescent staining of vinculin in osteoblast cells showed good focal contact adhesion on the CrAlSiN and Ti thin films but not on the CrN thin films. Cell proliferation was significantly greater on the CrAlSiN thin films as well as on Ti thin films than on the CrN thin films.

  20. EphrinA/EphA-induced ectodomain shedding of neural cell adhesion molecule regulates growth cone repulsion through ADAM10 metalloprotease.

    Science.gov (United States)

    Brennaman, Leann H; Moss, Marcia L; Maness, Patricia F

    2014-01-01

    EphrinA/EphA-dependent axon repulsion is crucial for synaptic targeting in developing neurons but downstream molecular mechanisms remain obscure. Here, it is shown that ephrinA5/EphA3 triggers proteolysis of the neural cell adhesion molecule (NCAM) by the metalloprotease a disintegrin and metalloprotease (ADAM)10 to promote growth cone collapse in neurons from mouse neocortex. EphrinA5 induced ADAM10 activity to promote ectodomain shedding of polysialic acid-NCAM in cortical neuron cultures, releasing a ~ 250 kDa soluble fragment consisting of most of its extracellular region. NCAM shedding was dependent on ADAM10 and EphA3 kinase activity as shown in HEK293T cells transfected with dominant negative ADAM10 and kinase-inactive EphA3 (K653R) mutants. Purified ADAM10 cleaved NCAM at a sequence within the E-F loop of the second fibronectin type III domain (Leu(671) -Lys(672) /Ser(673) -Leu(674) ) identified by mass spectrometry. Mutations of NCAM within the ADAM10 cleavage sequence prevented EphA3-induced shedding of NCAM in HEK293T cells. EphrinA5-induced growth cone collapse was dependent on ADAM10 activity, was inhibited in cortical cultures from NCAM null mice, and was rescued by WT but not ADAM10 cleavage site mutants of NCAM. Regulated proteolysis of NCAM through the ephrin5/EphA3/ADAM10 mechanism likely impacts synapse development, and may lead to excess NCAM shedding when disrupted, as implicated in neurodevelopmental disorders such as schizophrenia. PSA-NCAM and ephrinA/EphA3 coordinately regulate inhibitory synapse development. Here, we have found that ephrinA5 stimulates EphA3 kinase and ADAM10 activity to promote PSA-NCAM cleavage at a site in its second FNIII repeat, which regulates ephrinA5-induced growth cone collapse in GABAergic and non-GABAergic neurons. These findings identify a new regulatory mechanism which may contribute to inhibitory connectivity.

  1. Vascular endothelial growth factor up-regulates the expression of intracellular adhesion molecule-1 in retinal endothelial cells via reactive oxygen species, but not nitric oxide

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiao-ling; WEN Liang; CHEN Yan-jiong; ZHU Yi

    2009-01-01

    Background The vascular endothelial growth factor (VEGF) is involved in the initiation of retinal vascular leakage and nonperfusion in diabetes. The intracellular adhesion molecule-1 (ICAM-1) is the key mediator of the effect of VEGFs on retinal leukostasis. Although the VEGF is expressed in an early-stage diabetic retina, whether it directly up-regulates ICAM-1 in retinal endothelial cells (ECs) is unknown. In this study, we provided a new mechanism to explain that VEGF does up-regulate the expression of ICAM-1 in retinal ECs.Methods Bovine retinal ECs (BRECs) were isolated and cultured. Immunohistochemical staining was performed to identify BRECs. The cultured cells were divided into corresponding groups. Then, VEGF (100 ng/ml) and other inhibitors were used to treat the cells. Cell lysate and the cultured supernatant were collected, and then, the protein level of ICAM-1 and phosphorylation of the endothelial nitric oxide synthase (eNOS) were detected using Western blotting. Griess reaction was used to detect nitric oxide (NO).Results Western blotting showed that the VEGF up-regulated the expression of ICAM-1 protein and increased phosphorylation of the eNOS in retinal ECs. Neither the block of NO nor protein kinase C (PKC) altered the expression of ICAM-1 or the phosphorylation of eNOS. The result of the Western blotting also showed that inhibition of phosphatidylinositol 3-kinase (PI3K) or reactive oxygen species (ROS) significantly reduced the expression of ICAM-1. Inhibition of PI3K also reduced phosphorylation of eNOS. Griess reaction showed that VEGF significantly increased during NO production. When eNOS was blocked by L-NAME or PI3K was blocked by LY294002, the basal level of NO production and the increment of NO caused by VEGF could be significantly decreased.Conclusion ROS-NO coupling in the retinal endothelium may be a new mechanism that could help to explain why VEGF induces ICAM-1 expression and the resulting leukostasis in diabetic retinopathy.

  2. FOCAL MOTOR SEIZURES WITH TYPICAL AUTOMATISMS (FOCAL AUTOMOTOR SEIZURES

    Directory of Open Access Journals (Sweden)

    M. B. Mironov

    2014-01-01

    Full Text Available The paper deals with the study of a group of patients with focal automotor seizures, by taking into consideration their nosological, anamnestic, clinical, electroencephalographic, and neuroimaging features.

  3. Genetics Home Reference: focal dermal hypoplasia

    Science.gov (United States)

    ... Home Health Conditions focal dermal hypoplasia focal dermal hypoplasia Enable Javascript to view the expand/collapse boxes. ... PDF Open All Close All Description Focal dermal hypoplasia is a genetic disorder that primarily affects the ...

  4. Cell adhesion molecules and sleep.

    Science.gov (United States)

    O'Callaghan, Emma Kate; Ballester Roig, Maria Neus; Mongrain, Valérie

    2017-03-01

    Cell adhesion molecules (CAMs) play essential roles in the central nervous system, where some families are involved in synaptic development and function. These synaptic adhesion molecules (SAMs) are involved in the regulation of synaptic plasticity, and the formation of neuronal networks. Recent findings from studies examining the consequences of sleep loss suggest that these molecules are candidates to act in sleep regulation. This review highlights the experimental data that lead to the identification of SAMs as potential sleep regulators, and discusses results supporting that specific SAMs are involved in different aspects of sleep regulation. Further, some potential mechanisms by which SAMs may act to regulate sleep are outlined, and the proposition that these molecules may serve as molecular machinery in the two sleep regulatory processes, the circadian and homeostatic components, is presented. Together, the data argue that SAMs regulate the neuronal plasticity that underlies sleep and wakefulness. Copyright © 2016 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

  5. Inhibition of Rac and ROCK Signalling Influence Osteoblast Adhesion, Differentiation and Mineralization on Titanium Topographies

    Science.gov (United States)

    Prowse, Paul D. H.; Elliott, Christopher G.; Hutter, Jeff; Hamilton, Douglas W.

    2013-01-01

    Reducing the time required for initial integration of bone-contacting implants with host tissues would be of great clinical significance. Changes in osteoblast adhesion formation and reorganization of the F-actin cytoskeleton in response to altered topography are known to be upstream of osteoblast differentiation, and these processes are regulated by the Rho GTPases. Rac and RhoA (through Rho Kinase (ROCK)). Using pharmacological inhibitors, we tested how inhibition of Rac and ROCK influenced osteoblast adhesion, differentiation and mineralization on PT (Pre-treated) and SLA (sandblasted large grit, acid etched) topographies. Inhibition of ROCK, but not Rac, significantly reduced adhesion number and size on PT, with adhesion size consistent with focal complexes. After 1 day, ROCK, but not Rac inhibition increased osteocalcin mRNA levels on SLA and PT, with levels further increasing at 7 days post seeding. ROCK inhibition also significantly increased bone sialoprotein expression at 7 days, but not BMP-2 levels. Rac inhibition significantly reduced BMP-2 mRNA levels. ROCK inhibition increased nuclear translocation of Runx2 independent of surface roughness. Mineralization of osteoblast cultures was greater on SLA than on PT, but was increased by ROCK inhibition and attenuated by Rac inhibition on both topographies. In conclusion, inhibition of ROCK signalling significantly increases osteoblast differentiation and biomineralization in a topographic dependent manner, and its pharmacological inhibition could represent a new therapeutic to speed bone formation around implanted metals and in regenerative medicine applications. PMID:23505566

  6. Inhibition of Rac and ROCK signalling influence osteoblast adhesion, differentiation and mineralization on titanium topographies.

    Directory of Open Access Journals (Sweden)

    Paul D H Prowse

    Full Text Available Reducing the time required for initial integration of bone-contacting implants with host tissues would be of great clinical significance. Changes in osteoblast adhesion formation and reorganization of the F-actin cytoskeleton in response to altered topography are known to be upstream of osteoblast differentiation, and these processes are regulated by the Rho GTPases. Rac and RhoA (through Rho Kinase (ROCK. Using pharmacological inhibitors, we tested how inhibition of Rac and ROCK influenced osteoblast adhesion, differentiation and mineralization on PT (Pre-treated and SLA (sandblasted large grit, acid etched topographies. Inhibition of ROCK, but not Rac, significantly reduced adhesion number and size on PT, with adhesion size consistent with focal complexes. After 1 day, ROCK, but not Rac inhibition increased osteocalcin mRNA levels on SLA and PT, with levels further increasing at 7 days post seeding. ROCK inhibition also significantly increased bone sialoprotein expression at 7 days, but not BMP-2 levels. Rac inhibition significantly reduced BMP-2 mRNA levels. ROCK inhibition increased nuclear translocation of Runx2 independent of surface roughness. Mineralization of osteoblast cultures was greater on SLA than on PT, but was increased by ROCK inhibition and attenuated by Rac inhibition on both topographies. In conclusion, inhibition of ROCK signalling significantly increases osteoblast differentiation and biomineralization in a topographic dependent manner, and its pharmacological inhibition could represent a new therapeutic to speed bone formation around implanted metals and in regenerative medicine applications.

  7. Modification of Cellular Cholesterol Content Affects Traction Force, Adhesion and Cell Spreading.

    Science.gov (United States)

    Norman, Leann L; Oetama, Ratna J; Dembo, Micah; Byfield, F; Hammer, Daniel A; Levitan, Irena; Aranda-Espinoza, Helim

    2010-06-01

    Cellular cholesterol is a critical component of the plasma membrane, and plays a key role in determining the physical properties of the lipid bilayer, such as elasticity, viscosity, and permeability. Surprisingly, it has been shown that cholesterol depletion increases cell stiffness, not due to plasma membrane stiffening, but rather, due to the interaction between the actin cytoskeleton and the plasma membrane. This indicates that traction stresses of the acto-myosin complex likely increase during cholesterol depletion. Here we use force traction microscopy to quantify the forces individual cells are exerting on the substrate, and total internal reflection fluorescence microscopy as well as interference reflection microscopy to observe cell-substrate adhesion and spreading. We show that single cells depleted of cholesterol produce larger traction forces and have large focal adhesions compared to untreated or cholesterol-enriched cells. Cholesterol depletion also causes a decrease in adhesion area for both single cells and monolayers. Spreading experiments illustrate a decrease in spreading area for cholesterol-depleted cells, and no effect on cholesterol-enriched cells. These results demonstrate that cholesterol plays an important role in controlling and regulating the cell-substrate interactions through the actin-plasma membrane complex, cell-cell adhesion, and spreading.

  8. Variation in one residue associated with the metal ion-dependent adhesion site regulates αIIbβ3 integrin ligand binding affinity.

    Directory of Open Access Journals (Sweden)

    Joel Raborn

    Full Text Available The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala(252 and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala(252 to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion.

  9. Targeting adhesion signaling in KRAS, LKB1 mutant lung adenocarcinoma

    Science.gov (United States)

    Konen, Jessica; Koo, Junghui; Robinson, Brian S.; Wiles, Walter Guy; Huang, Chunzi; Martin, W. David; Behera, Madhusmita; Smith, Geoffrey H.; Hill, Charles E.; Rossi, Michael R.; Sica, Gabriel L.; Rupji, Manali; Chen, Zhengjia; Kowalski, Jeanne; Kasinski, Andrea L.; Ramalingam, Suresh S.; Khuri, Fadlo R.; Marcus, Adam I.

    2017-01-01

    Loss of LKB1 activity is prevalent in KRAS mutant lung adenocarcinoma and promotes aggressive and treatment-resistant tumors. Previous studies have shown that LKB1 is a negative regulator of the focal adhesion kinase (FAK), but in vivo studies testing the efficacy of FAK inhibition in LKB1 mutant cancers are lacking. Here, we took a pharmacologic approach to show that FAK inhibition is an effective early-treatment strategy for this high-risk molecular subtype. We established a lenti-Cre–induced Kras and Lkb1 mutant genetically engineered mouse model (KLLenti) that develops 100% lung adenocarcinoma and showed that high spatiotemporal FAK activation occurs in collective invasive cells that are surrounded by high levels of collagen. Modeling invasion in 3D, loss of Lkb1, but not p53, was sufficient to drive collective invasion and collagen alignment that was highly sensitive to FAK inhibition. Treatment of early, stage-matched KLLenti tumors with FAK inhibitor monotherapy resulted in a striking effect on tumor progression, invasion, and tumor-associated collagen. Chronic treatment extended survival and impeded local lymph node spread. Lastly, we identified focally upregulated FAK and collagen-associated collective invasion in KRAS and LKB1 comutated human lung adenocarcinoma patients. Our results suggest that patients with LKB1 mutant tumors should be stratified for early treatment with FAK inhibitors.

  10. The computed cranial focal point

    NARCIS (Netherlands)

    Jong, G.A. de; Maal, T.J.J.; Delye, H.

    2015-01-01

    INTRODUCTION: Stereophotogrammetry is a radiation-free method for monitoring skull development after craniosynostosis repair. Lack of clear fixed reference points complicate longitudinal comparison of 3D photographs. Therefore we developed the 'computed cranial focal point' (CCFP). METHODS: The CCFP

  11. Adhesion and cohesion.

    Science.gov (United States)

    von Fraunhofer, J Anthony

    2012-01-01

    The phenomena of adhesion and cohesion are reviewed and discussed with particular reference to dentistry. This review considers the forces involved in cohesion and adhesion together with the mechanisms of adhesion and the underlying molecular processes involved in bonding of dissimilar materials. The forces involved in surface tension, surface wetting, chemical adhesion, dispersive adhesion, diffusive adhesion, and mechanical adhesion are reviewed in detail and examples relevant to adhesive dentistry and bonding are given. Substrate surface chemistry and its influence on adhesion, together with the properties of adhesive materials, are evaluated. The underlying mechanisms involved in adhesion failure are covered. The relevance of the adhesion zone and its importance with regard to adhesive dentistry and bonding to enamel and dentin is discussed.

  12. Adhesion and Cohesion

    Directory of Open Access Journals (Sweden)

    J. Anthony von Fraunhofer

    2012-01-01

    Full Text Available The phenomena of adhesion and cohesion are reviewed and discussed with particular reference to dentistry. This review considers the forces involved in cohesion and adhesion together with the mechanisms of adhesion and the underlying molecular processes involved in bonding of dissimilar materials. The forces involved in surface tension, surface wetting, chemical adhesion, dispersive adhesion, diffusive adhesion, and mechanical adhesion are reviewed in detail and examples relevant to adhesive dentistry and bonding are given. Substrate surface chemistry and its influence on adhesion, together with the properties of adhesive materials, are evaluated. The underlying mechanisms involved in adhesion failure are covered. The relevance of the adhesion zone and its importance with regard to adhesive dentistry and bonding to enamel and dentin is discussed.

  13. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    tract to the microbial flocs in waste water treatment facilities. Microbial biofilms may however also cause a wide range of industrial and medical problems, and have been implicated in a wide range of persistent infectious diseases, including implantassociated microbial infections. Bacterial adhesion...... in the formation of highly complex sessile communities, referred to as biofilms. Such microbial communities are often highly dynamic and heterogeneous in nature. Microbial biofilms are of great importance in a wide range of natural processes and industrial settings, from the commensal flora of the gastrointestinal...

  14. Orphan G protein-coupled receptor GPRC5A modulates integrin β1-mediated epithelial cell adhesion.

    Science.gov (United States)

    Bulanova, Daria R; Akimov, Yevhen A; Rokka, Anne; Laajala, Teemu D; Aittokallio, Tero; Kouvonen, Petri; Pellinen, Teijo; Kuznetsov, Sergey G

    2016-10-07

    G-Protein Coupled Receptor (GPCR), Class C, Group 5, Member A (GPRC5A) has been implicated in several malignancies. The underlying mechanisms, however, remain poorly understood. Using a panel of human cell lines, we demonstrate that CRISPR/Cas9-mediated knockout and RNAi-mediated depletion of GPRC5A impairs cell adhesion to integrin substrates: collagens I and IV, fibronectin, as well as to extracellular matrix proteins derived from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (Matrigel). Consistent with the phenotype, knock-out of GPRC5A correlated with a reduced integrin β1 (ITGB1) protein expression, impaired phosphorylation of the focal adhesion kinase (FAK), and lower activity of small GTPases RhoA and Rac1. Furthermore, we provide the first evidence for a direct interaction between GPRC5A and a receptor tyrosine kinase EphA2, an upstream regulator of FAK, although its contribution to the observed adhesion phenotype is unclear. Our findings reveal an unprecedented role for GPRC5A in regulation of the ITGB1-mediated cell adhesion and it's downstream signaling, thus indicating a potential novel role for GPRC5A in human epithelial cancers.

  15. Integrin-linked kinase regulates interphase and mitotic microtubule dynamics.

    Directory of Open Access Journals (Sweden)

    Simin Lim

    Full Text Available Integrin-linked kinase (ILK localizes to both focal adhesions and centrosomes in distinct multiprotein complexes. Its dual function as a kinase and scaffolding protein has been well characterized at focal adhesions, where it regulates integrin-mediated cell adhesion, spreading, migration and signaling. At the centrosomes, ILK regulates mitotic spindle organization and centrosome clustering. Our previous study showed various spindle defects after ILK knockdown or inhibition that suggested alteration in microtubule dynamics. Since ILK expression is frequently elevated in many cancer types, we investigated the effects of ILK overexpression on microtubule dynamics. We show here that overexpressing ILK in HeLa cells was associated with a shorter duration of mitosis and decreased sensitivity to paclitaxel, a chemotherapeutic agent that suppresses microtubule dynamics. Measurement of interphase microtubule dynamics revealed that ILK overexpression favored microtubule depolymerization, suggesting that microtubule destabilization could be the mechanism behind the decreased sensitivity to paclitaxel, which is known to stabilize microtubules. Conversely, the use of a small molecule inhibitor selective against ILK, QLT-0267, resulted in suppressed microtubule dynamics, demonstrating a new mechanism of action for this compound. We further show that treatment of HeLa cells with QLT-0267 resulted in higher inter-centromere tension in aligned chromosomes during mitosis, slower microtubule regrowth after cold depolymerization and the presence of a more stable population of spindle microtubules. These results demonstrate that ILK regulates microtubule dynamics in both interphase and mitotic cells.

  16. Involvement of caveolin-1 in low shear stress-induced breast cancer cell motility and adhesion: Roles of FAK/Src and ROCK/p-MLC pathways.

    Science.gov (United States)

    Xiong, Niya; Li, Shun; Tang, Kai; Bai, Hongxia; Peng, Yueting; Yang, Hong; Wu, Chunhui; Liu, Yiyao

    2017-01-01

    Tumor cells translocating to distant sites are subjected to hemodynamic shear forces during their passage in the blood vessels. Low shear stress (LSS) plays a critical role in the regulation of various aspects of tumor cells functions, including motility and adhesion. Beyond its structural role, caveolin-1 (Cav-1), the important component of caveolae, represents a modulator of several cancer-associated functions as tumor progression and metastasis. However, the role of Cav-1 in regulating tumor cells response to shear stress remains poorly explored. Here, we characterized the role of LSS and Cav-1 in mediating cell motility and adhesion on human breast carcinoma MDA-MB-231 cells. We first showed that LSS exposure promoted cell polarity and focal adhesion (FA) dynamics, thus indicating elevated cell migration. Silencing of Cav-1 leaded to a significantly lower formation of stress fibers. However, LSS exposure was able to rescue it via the alteration of actin-associated proteins expression, including ROCK, p-MLC, cofilin and filamin A. Time-lapse migration assay indicated that Cav-1 expression fostered MDA-MB-231 cells motility and LSS triggered cells to rapidly generate new lamellipodia. Furthermore, Cav-1 and LSS significantly influenced cell adhesion. Taken together, our findings provide insights into mechanisms underlying LSS triggered events mediated by downstream Cav-1, including FAK/Src and ROCK/p-MLC pathways, involved in the reorganization of the cytoskeleton, cell motility, FA dynamics and breast cancer cell adhesion.

  17. Dynamic monitoring of changes in endothelial cell-substrate adhesiveness during leukocyte adhesion by microelectrical impedance assay

    Institute of Scientific and Technical Information of China (English)

    Yakun Ge; Tongle Deng; Xiaoxiang Zheng

    2009-01-01

    Adhesion of leukocytes to endothelial cells in inflammation processes leads to changes of endothelial cell-substrate adhesiveness, and understanding of such changes will provide us with important information of inflammation processes. In this study, we used a non-invasive biosensor system referred to as real-time cell electronic sensor (RT-CES) system to monitor the changes in endothelial cell-substrate adhesiveness induced by human monoblastic cell line U937 cell adhesion in a dynamic and quantitative manner. This assay, which is based on cell-substrate impedance readout, is able to monitor transient changes in cell-substrate adhesiveness as a result of U937 cell adhesion. The U937 cell adhesion to endothelial cells was induced by lipopolysaccharide (LPS) in a dose-dependent manner. Although the number of adherent U937 cells to the endothelial cells was verified by a standard assay, the adhesiveness of endothelial cells after addition of U937 cells was monitored by the RT-CES system. Furthermore, focal adhesion kinase protein decrease and F-actin rearrangement in endothelial cells were observed after addition of U937 cells. Our results indicated that the adhesion of U937 cells to LPS-treated endothelial cells reduced the cell adhesiveness to the substrate, and such reduction might facilitate infiltration of leukocytes.

  18. Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway.

    Science.gov (United States)

    Chakraborty, Sayan; Njah, Kizito; Pobbati, Ajaybabu V; Lim, Ying Bena; Raju, Anandhkumar; Lakshmanan, Manikandan; Tergaonkar, Vinay; Lim, Chwee Teck; Hong, Wanjin

    2017-03-07

    The Hippo pathway effectors YAP and TAZ act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. Here, we uncover a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 signaling and negating the functions of Merlin and LATS1/2. We further show that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. We propose that Agrin acts as a mechanotransduction signal in the ECM.

  19. Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway

    Directory of Open Access Journals (Sweden)

    Sayan Chakraborty

    2017-03-01

    Full Text Available The Hippo pathway effectors YAP and TAZ act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. Here, we uncover a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 signaling and negating the functions of Merlin and LATS1/2. We further show that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. We propose that Agrin acts as a mechanotransduction signal in the ECM.

  20. Reduced Mechanical Stretch Induces Enhanced Endothelin B Receptor-mediated Contractility via Activation of Focal Adhesion Kinase and Extra Cellular-regulated Kinase 1/2 in Cerebral Arteries from Rat

    DEFF Research Database (Denmark)

    Spray, Stine; Rasmussen, Marianne N P; Skovsted, Gry F

    2016-01-01

    Cerebral ischaemia results in enhanced endothelin B (ETB ) receptor-mediated contraction and receptor protein expression in the affected cerebrovascular smooth muscle cells (SMC). Organ culture of cerebral arteries is a method to induce similar alterations in ETB receptor expression. We hypothesize...... expression to SMC expression and 2) an increased calcium sensitivity of the SMCs due to an increased expression of the calcium channel transient receptor potential canonical 1. Collectively, our results present a possible mechanism linking lack of vessel wall stretch/tension to changes in ETB receptor...

  1. Grupo Focal em Pesquisas Sociais

    OpenAIRE

    Maria Lúcia Silva Servo; Pricila Oliveira Araújo

    2012-01-01

    Este artigo tem como objetivo discutir a técnica de grupo focal em pesquisas sociais. Apresenta-se as concepções sobre grupo focal. Traz-se os postulados de Pichon-Rivière sobre grupo operativo, os instrumentos de planificação, os vetores do campo grupal para nortear a dinâmica e a observação do campo grupal, bem como a organização, a operacionalização e a análise dos dados das sessões de gr