Science.gov

Sample records for integrins combinatorial signaling

  1. Integrin endosomal signalling suppresses anoikis

    PubMed Central

    Alanko, Jonna; Mai, Anja; Jacquemet, Guillaume; Schauer, Kristine; Kaukonen, Riina; Saari, Markku; Goud, Bruno; Ivaska, Johanna

    2016-01-01

    Integrin containing focal adhesions (FAs) transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell-ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin-ECM downstream signalling, localises with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 (EEA1) and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage-independence and metastasis. Integrins are heterodimeric cell surface adhesion receptors functioning as integrators of the extra-cellular matrix (ECM) driven cues, the cellular cytoskeleton and the cellular signalling apparatus 1.Upon adhesion, integrins trigger the formation of plasma-membrane proximal large mechanosensing and signal-transmitting protein clusters depicted as “adhesomes” 2, 3. In addition, integrins undergo constant endocytic traffic to facilitate focal adhesion turnover, cell migration, invasion and cytokinesis 4. For other receptor systems it is well established that endocytic membrane traffic regulates bioavailability of cell-surface molecules and therefore the intensity and/or specificity of receptor-initiated signals 5, 6. Although active integrins and their ligands have been detected in endosomes 7–9 and increased integrin recycling to the plasma membrane contributes

  2. Overview of integrin signaling in the immune system.

    PubMed

    Kinashi, Tatsuo

    2012-01-01

    It has been well established that integrins mediate cell-cell and cell-matrix adhesion and play crucial roles in the immune system such as leukocyte-endothelium interactions, immune synapse formation, and effector functions. Since the discovery that integrins undergo dynamic changes of adhesive activities in response to external stimuli, intensive studies have been conducted to elucidate the signaling events that control the activation of integrins (inside-out signaling) and signaling events from the induced integrin-dependent adhesion (outside-in signaling). The molecular characterization of these signaling pathways highlights the importance of integrins as bidirectional signaling receptors. The characteristics of integrin signaling are best exemplified in the immune system. This chapter highlights the recent studies of intracellular signaling pathways that regulate integrins in immunological contexts.

  3. Integrin Signaling in Cancer Cell Survival and Chemoresistance

    PubMed Central

    Aoudjit, Fawzi; Vuori, Kristiina

    2012-01-01

    Resistance to apoptosis and chemotherapy is a hallmark of cancer cells, and it is a critical factor in cancer recurrence and patient relapse. Extracellular matrix (ECM) via its receptors, the integrins, has emerged as a major pathway contributing to cancer cell survival and resistance to chemotherapy. Several studies over the last decade have demonstrated that ECM/integrin signaling provides a survival advantage to various cancer cell types against numerous chemotherapeutic drugs and against antibody therapy. In this paper, we will discuss the major findings on how ECM/integrin signaling protects tumor cells from drug-induced apoptosis. We will also discuss the potential role of ECM in malignant T-cell survival and in cancer stem cell resistance. Understanding how integrins and their signaling partners promote tumor cell survival and chemoresistance will likely lead to the development of new therapeutic strategies and agents for cancer treatment. PMID:22567280

  4. IFATS collection: Combinatorial peptides identify alpha5beta1 integrin as a receptor for the matricellular protein SPARC on adipose stromal cells.

    PubMed

    Nie, Jing; Chang, Benny; Traktuev, Dmitry O; Sun, Jessica; March, Keith; Chan, Lawrence; Sage, E Helene; Pasqualini, Renata; Arap, Wadih; Kolonin, Mikhail G

    2008-10-01

    The biological features of adipose stromal (stem) cells (ASC), which serve as progenitors for differentiated cells of white adipose tissue (WAT), are still largely undefined. In an initiative to identify functional ASC surface receptors, we screened a combinatorial library for peptide ligands binding to patient-derived ASC. We demonstrate that both primary and cultured human and mouse stromal cells express a conserved receptor targeted by peptides found to mimic SPARC, a matricellular protein that is required for normal WAT development. A signaling receptor for SPARC has not as yet been determined. By using the SPARC-mimicking peptides CMLAGWIPC (termed hPep) and CWLGEWLGC (termed mPep), isolated by panning on human and mouse cells, respectively, we identified the alpha5beta1 integrin complex as a candidate receptor for SPARC. On the basis of these results, we evaluated ASC responses to SPARC or SPARC-mimicking peptide exposure. Our results suggest that extracellular SPARC binds to alpha5beta1 integrin at sites of focal adhesions, an interaction disrupting firm attachment of ASC to extracellular matrix. We propose that SPARC-mediated mobilization of ASC through its effect on alpha5beta1 integrin complex provides a functional basis for the regulation of WAT body composition by SPARC. We also show that alpha5beta1 integrin is a potential target for ASC-selective intracellular delivery of bioactive peptides and gene therapy vectors directed by the SPARC-mimicking peptides. Disclosure of potential conflicts of interest is found at the end of this article.

  5. Basic amino-acid side chains regulate transmembrane integrin signalling.

    PubMed

    Kim, Chungho; Schmidt, Thomas; Cho, Eun-Gyung; Ye, Feng; Ulmer, Tobias S; Ginsberg, Mark H

    2011-12-18

    Side chains of Lys/Arg near transmembrane domain (TMD) membrane-water interfaces can 'snorkel', placing their positive charge near negatively charged phospholipid head groups; however, snorkelling's functional effects are obscure. Integrin β TMDs have such conserved basic amino acids. Here we use NMR spectroscopy to show that integrin β(3)(Lys 716) helps determine β(3) TMD topography. The α(ΙΙb)β(3) TMD structure indicates that precise β(3) TMD crossing angles enable the assembly of outer and inner membrane 'clasps' that hold the αβ TMD together to limit transmembrane signalling. Mutation of β(3)(Lys 716) caused dissociation of α(ΙΙb)β(3) TMDs and integrin activation. To confirm that altered topography of β(3)(Lys 716) mutants activated α(ΙΙb)β(3), we used directed evolution of β(3)(K716A) to identify substitutions restoring default state. Introduction of Pro(711) at the midpoint of β(3) TMD (A711P) increased α(ΙΙb)β(3) TMD association and inactivated integrin α(ΙΙb)β(3)(A711P,K716A). β(3)(Pro 711) introduced a TMD kink of 30 ± 1° precisely at the border of the outer and inner membrane clasps, thereby decoupling the tilt between these segments. Thus, widely occurring snorkelling residues in TMDs can help maintain TMD topography and membrane-embedding, thereby regulating transmembrane signalling.

  6. The interaction between uPAR and vitronectin triggers ligand-independent adhesion signalling by integrins

    PubMed Central

    Ferraris, Gian Maria Sarra; Schulte, Carsten; Buttiglione, Valentina; De Lorenzi, Valentina; Piontini, Andrea; Galluzzi, Massimiliano; Podestà, Alessandro; Madsen, Chris D; Sidenius, Nicolai

    2014-01-01

    The urokinase-type plasminogen activator receptor (uPAR) is a non-integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure–function analyses of uPAR, VN and integrins, we document that uPAR-mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin–matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding. Although integrin ligation is dispensable, signalling is crucially dependent upon an active conformation of the integrin and its association with intracellular adaptors such as talin. This non-canonical integrin signalling is not restricted to uPAR as it poses no structural constraints to the receptor mediating cell attachment. In contrast to canonical integrin signalling, where integrins form direct mechanical links between the ECM and the cytoskeleton, the molecular mechanism enabling the crosstalk between non-integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of the molecular clutch. PMID:25168639

  7. Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins

    NASA Technical Reports Server (NTRS)

    Alenghat, Francis J.; Ingber, Donald E.

    2002-01-01

    Mechanical stresses modulate cell function by either activating or tuning signal transduction pathways. Mechanotransduction, the process by which cells convert mechanical stimuli into a chemical response, occurs both in cells specialized for sensing mechanical cues and in parenchymal cells whose primary function is not mechanosensory. However, common among the various responses to mechanical stress is the importance of direct or indirect connections between the internal cytoskeleton, the extracellular matrix (ECM), and traditional signal transducing molecules. In many instances, these elements converge at focal adhesions, sites of structural attachment between the cytoskeleton and ECM that are anchored by cell surface integrin receptors. Alenghat and Ingber discuss the accumulating evidence for the central role of cytoskeleton, ECM, and integrin-anchored focal adhesions in several mechanotransduction pathways.

  8. Hypergravity Stimulates Osteoblast Proliferation Via Matrix-Integrin-Signaling Pathways

    NASA Technical Reports Server (NTRS)

    Vercoutere, W.; Parra, M.; Roden, C.; DaCosta, M.; Wing, A.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

    2003-01-01

    Extensive characterizations of the physiologic consequences of microgravity and gravity indicate that lack of weight-bearing may cause tissue atrophy through cellular and subcellular level mechanisms. We hypothesize that gravity is needed for the efficient transduction of cell growth and survival signals from the extra-cellular matrix (ECM) in mechanosensitive tissues. Recent work from our laboratory and from others shows that an increase of gravity increases bone cell growth and survival. We found that 50-g hypergravity stimulation increased osteoblast proliferation for cells grown on Collagen Type I and Fibronectin, but not on Laminin or uncoated plastic. This may be a tissue-specific response, because 50-g hypergravity stimulation caused no increase in proliferation for primary rat fibroblasts. These results combined with RT-PCR for all possible integrins indicate that beta1 integrin subunit may be involved. The osteoblast proliferation response on Collagen Type I was greater at 25-g than at 10-g or 50-g; 24-h duration of hypergravity was necessary to see an increase in proliferation. Survival was enhanced during hypergravity stimulation by the presence of matrix. Flow cytometry analysis indicated that cell cycle may be altered; BrdU incorporation in proliferating cells showed an increase in the number of actively dividing cells from about 60% at 1-g to over 90% at 25-g. To further investigate the molecular components involved, we applied fluorescence labeling of cytoskeletal and signaling molecules to cells after 2 to 30 minutes of hypergravity stimulation. While structural components did not appear to be altered, phosphorylation increased, indicating that signaling pathways may be activated. These data indicate that gravity mechanostimulation of osteoblast proliferation involves specific matrix-integrin signaling pathways which are sensitive to duration and g-level.

  9. Integrin-mediated adhesion complex: Cooption of signaling systems at the dawn of Metazoa.

    PubMed

    Sebé-Pedrós, Arnau; Ruiz-Trillo, Iñaki

    2010-09-01

    The integrin-mediated adhesion machinery is the primary cell-matrix adhesion mechanism in Metazoa. The integrin adhesion complex, which modulates important aspects of the cell physiology, is composed of integrins (alpha and beta subunits) and several scaffolding and signaling proteins. Integrins appeared to be absent in all non-metazoan eukaryotes so-far analyzed, including fungi, plants and choanoflagellates, the sister-group to Metazoa. Thus, integrins and, therefore, the integrin-mediated adhesion and signaling mechanism was considered a metazoan innovation. Recently, a broad comparative genomic analysis including new genome data from several unicellular organisms closely related to fungi and metazoans shattered previous views. The integrin adhesion and signaling complex is not specific to Metazoa, but rather it is present in apusozoans and holozoan protists. Thus, this important signaling and adhesion system predated the origin of Fungi and Metazoa, and was subsequently lost in fungi and choanoflagellates. This finding suggests that cooption played a more important role in the origin of Metazoa than previously believed. Here, we hypothesize that the integrin adhesome was ancestrally involved in signaling.

  10. β1 integrin-mediated signals are required for platelet granule secretion and hemostasis in mouse.

    PubMed

    Petzold, Tobias; Ruppert, Raphael; Pandey, Dharmendra; Barocke, Verena; Meyer, Hannelore; Lorenz, Michael; Zhang, Lin; Siess, Wolfgang; Massberg, Steffen; Moser, Markus

    2013-10-10

    Integrins are critical for platelet adhesion and aggregation during arterial thrombosis and hemostasis. Although the platelet-specific αIIbβ3 integrin is known to be crucial for these processes, the in vivo role of β1 integrins is a matter of debate. Here we demonstrate that mice expressing reduced levels of β1 integrins or an activation-deficient β1 integrin show strongly reduced platelet adhesion to collagen in vitro and in a carotis ligation model in vivo. Interestingly, hypomorphic mice expressing only 3% of β1 integrins on platelets show normal bleeding times despite reduced platelet adhesion. The residual 3% of β1 integrins are able to trigger intracellular signals driving Rac-1-dependent granule release required for platelet aggregation and hemostasis. Our findings support a model, in which platelet β1 integrins serve as an important signaling receptor rather than an adhesion receptor in vivo and therefore promote β1 integrins as a promising and so far clinically unemployed antithrombotic target.

  11. Beta-1 Integrin Signaling and Function in MLO-Y4 Osteocyte-Like Cells

    NASA Technical Reports Server (NTRS)

    Searby, N. D.; vanderMeulen, M. C. H.; Dovi, J.; Roden, C.; Banerjee, I.; Kim, J.-B.; Damsky, C. D.; Almeida, E. A. C.; Globus, R. K.

    2004-01-01

    In osteocyte-like cells, disruption of beta-1 integrin signaling by the Beta-1 tail construct: 1) Altered cell morphology; 2) Reduced cell motility; 3) Increased proliferation and final cell density; 4) Reduced cell's ability to maintain shape when subjected to uniaxial strain (1%, 30 min). Thus, beta-1 integrin is important in the response of osteocytic cells to mechanical loading.

  12. Mechanical control of cyclic AMP signalling and gene transcription through integrins

    NASA Technical Reports Server (NTRS)

    Meyer, C. J.; Alenghat, F. J.; Rim, P.; Fong, J. H.; Fabry, B.; Ingber, D. E.

    2000-01-01

    This study was carried out to discriminate between two alternative hypotheses as to how cells sense mechanical forces and transduce them into changes in gene transcription. Do cells sense mechanical signals through generalized membrane distortion or through specific transmembrane receptors, such as integrins? Here we show that mechanical stresses applied to the cell surface alter the cyclic AMP signalling cascade and downstream gene transcription by modulating local release of signals generated by activated integrin receptors in a G-protein-dependent manner, whereas distortion of integrins in the absence of receptor occupancy has no effect.

  13. Integrin clustering as a result of local membrane deformations and local signaling feedbacks

    NASA Astrophysics Data System (ADS)

    Felizzi, Federico; Iber, Dagmar

    2014-08-01

    Integrins are essential receptors for the development and functioning of multicellular animals because they mediate cell adhesion and migration, and regulate cell proliferation and apoptosis. Ligand-dependent activation of integrins involves the formation of receptor clusters and this has been accounted both to extracellular forces as mediated by the glycocalyx as well as to intracellular forces mediated by the cytoskeleton. Here we describe a Monte Carlo simulation that considers both the binding processes on the membrane as well as the intracellular signaling processes that stabilize the open integrin conformation. We show that integrin clustering can result both from the effects of integrin avidity, as a result of membrane deformations, as well as from the locally enhanced availability of talins in the open conformation, as a result of local positive feedback signaling via PIPKIγ and PIP2. The model was carefully parameterized based on reported quantitative data and reproduces a wide range of experimental data, including results that previously appeared inconsistent.

  14. Integrin signalling regulates YAP and TAZ to control skin homeostasis

    PubMed Central

    Elbediwy, Ahmed; Vincent-Mistiaen, Zoé I.; Spencer-Dene, Bradley; Stone, Richard K.; Boeing, Stefan; Wculek, Stefanie K.; Cordero, Julia; Tan, Ee H.; Ridgway, Rachel; Brunton, Val G.; Sahai, Erik; Gerhardt, Holger; Behrens, Axel; Malanchi, Ilaria; Sansom, Owen J.; Thompson, Barry J.

    2016-01-01

    ABSTRACT The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib. PMID:26989177

  15. Pairwise agonist scanning predicts cellular signaling responses to combinatorial stimuli.

    PubMed

    Chatterjee, Manash S; Purvis, Jeremy E; Brass, Lawrence F; Diamond, Scott L

    2010-07-01

    Prediction of cellular response to multiple stimuli is central to evaluating patient-specific clinical status and to basic understanding of cell biology. Cross-talk between signaling pathways cannot be predicted by studying them in isolation and the combinatorial complexity of multiple agonists acting together prohibits an exhaustive exploration of the complete experimental space. Here we describe pairwise agonist scanning (PAS), a strategy that trains a neural network model based on measurements of cellular responses to individual and all pairwise combinations of input signals. We apply PAS to predict calcium signaling responses of human platelets in EDTA-treated plasma to six different agonists (ADP, convulxin, U46619, SFLLRN, AYPGKF and PGE(2)) at three concentrations (0.1, 1 and 10 x EC(50)). The model predicted responses to sequentially added agonists, to ternary combinations of agonists and to 45 different combinations of four to six agonists (R = 0.88). Furthermore, we use PAS to distinguish between the phenotypic responses of platelets from ten donors. Training neural networks with pairs of stimuli across the dose-response regime represents an efficient approach for predicting complex signal integration in a patient-specific disease milieu. PMID:20562863

  16. Combinatorial MAPLE gradient thin film assemblies signalling to human osteoblasts.

    PubMed

    Axente, Emanuel; Sima, Felix; Elena Sima, Livia; Erginer, Merve; Eroglu, Mehmet S; Serban, Natalia; Ristoscu, Carmen; Petrescu, Stefana M; Toksoy Oner, Ebru; Mihailescu, Ion N

    2014-09-01

    There is increased interest in smart bioactive materials to control tissue regeneration for the engineering of cell instructive scaffolds. We introduced combinatorial matrix-assisted pulsed laser evaporation (C-MAPLE) as a new method for the fabrication of organic thin films with a compositional gradient. Synchronized C-MAPLE of levan and oxidized levan was employed to assemble a two-compound biopolymer film structure. The gradient of the film composition was validated by fluorescence microscopy. In this study, we investigated the cell response induced by the compositional gradient using imaging of early osteoblast attachment and analysis of signalling phosphoprotein expression. Cells attached along the gradient in direct proportion to oxidized levan concentration. During this process distinct areas of the binary gradient have been shown to modulate the osteoblasts' extracellular signal-regulated kinase signalling with different propensity. The proposed fabrication method results in the preparation of a new bioactive material, which could control the cell signalling response. This approach can be extended to screen new bioactive interfaces for tissue regeneration.

  17. Modeled Microgravity Disrupts Collagen I/Integrin Signaling During Osteoblastic Differentiation of Human Mesenchymal Stem Cells

    NASA Technical Reports Server (NTRS)

    Meyers, Valerie E.; Zayzafoon, Majd; Gonda, Steven R.; Gathings, William E.; McDonald, Jay M.

    2004-01-01

    Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following seven days culture in modeled microgravity. One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of modeled microgravity on integrin expression and function in hMSC. We demonstrate that seven days of culture in modeled microgravity leads to reduced expression of the extracellular matrix protein, type I collagen (Col I). Conversely, modeled microgravity consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin sub-unit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-MAPK pathway is evidenced by a reduction in Ras and ERK activation. Taken together, our findings indicate that modeled microgravity decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.

  18. Feedback Regulation of Cell-Substratum Adhesion by Integrin-Mediated Intracellular Ca2+ Signaling

    NASA Astrophysics Data System (ADS)

    Sjaastad, Michael D.; Angres, Brigitte; Lewis, Richard S.; Nelson, W. James

    1994-08-01

    Integrin binding to extracellular matrix (ECM) regulates cell migration and gene expression in embryogenesis, metastasis, wound healing, and the inflammatory response. In many cases, binding of integrins to ECM triggers intracellular signaling pathways. The regulatory roles of intracellular signaling mechanisms in these events are poorly understood. Using single-cell analysis, we demonstrate that beads coated with peptide containing Arg-Gly-Asp (RGD), an integrin recognition motif found in many ECM proteins, elicit a rapid transient increase in intracellular calcium in Madin-Darby canine kidney (MDCK) epithelial cells. Also, significantly more beads bind to responding cells than to nonresponders. Several independent methods that inhibit RGD-induced Ca2+ signaling decrease both the number of beads bound and the strength of adhesion to an RGD-coated substratum. These results indicate that intracellular Ca2+ signaling participates in a positive feedback loop that enhances integrin-mediated cell adhesion

  19. Integrins as a primary signal transduction molecule regulating monocyte immediate-early gene induction.

    PubMed Central

    Yurochko, A D; Liu, D Y; Eierman, D; Haskill, S

    1992-01-01

    Integrins are cell surface receptors found on monocytes that facilitate adhesion to both cellular and extracellular substrates. These integrins are thought to be involved in the selective gene induction observed after monocyte adhesion to various extracellular matrices. To investigate this hypothesis, we stimulated monocytes with monoclonal antibodies to different integrin receptors to specifically mimic the integrin receptor-ligand interactions. Engagement of the common beta chain of the beta 1 subfamily of integrins resulted in expression of the inflammatory mediator genes, interleukin 1 beta, interleukin 1 receptor antagonist, and monocyte adherence-derived inflammatory gene 6 (MAD-6), whereas engagement of the common beta chain of the beta 2 family did not. Furthermore, to characterize integrin-mediated gene induction, we examined the ability of antibodies to the alpha chain of integrin receptors to regulate gene expression. Engagement of the very late antigen 4 (VLA-4) receptor resulted in induction of all the mediator genes. Receptor crosslinking was required because individual Fab fragments were unable to stimulate gene induction whereas the divalent F(ab')2 fragment and the whole IgG molecule could. Interleukin 1 beta secretion was dependent on the anti-integrin antibody used. Some antibodies required a second signal and, for others, direct engagement was sufficient for protein production. In conclusion, engagement of integrin receptors regulated the production of both inflammatory mediator mRNA and protein. These results suggest that integrin-dependent recognition and adherence may provide the key signals for initiation of the inflammatory response during monocyte diapedesis. Images PMID:1384041

  20. Linear effects models of signaling pathways from combinatorial perturbation data

    PubMed Central

    Szczurek, Ewa; Beerenwinkel, Niko

    2016-01-01

    Motivation: Perturbations constitute the central means to study signaling pathways. Interrupting components of the pathway and analyzing observed effects of those interruptions can give insight into unknown connections within the signaling pathway itself, as well as the link from the pathway to the effects. Different pathway components may have different individual contributions to the measured perturbation effects, such as gene expression changes. Those effects will be observed in combination when the pathway components are perturbed. Extant approaches focus either on the reconstruction of pathway structure or on resolving how the pathway components control the downstream effects. Results: Here, we propose a linear effects model, which can be applied to solve both these problems from combinatorial perturbation data. We use simulated data to demonstrate the accuracy of learning the pathway structure as well as estimation of the individual contributions of pathway components to the perturbation effects. The practical utility of our approach is illustrated by an application to perturbations of the mitogen-activated protein kinase pathway in Saccharomyces cerevisiae. Availability and Implementation: lem is available as a R package at http://www.mimuw.edu.pl/∼szczurek/lem. Contact: szczurek@mimuw.edu.pl; niko.beerenwinkel@bsse.ethz.ch Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307630

  1. A NPxY-independent {beta}5 integrin activation signal regulates phagocytosis of apoptotic cells

    SciTech Connect

    Singh, Sukhwinder; D'mello, Veera; Henegouwen, Paul van Bergen en; Birge, Raymond B.

    2007-12-21

    Integrin receptors are heterodimeric transmembrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the {beta} chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 ({beta}5) integrin cDNA was expressed in {alpha}v positive, {beta}5 and {beta}3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, {beta}5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing {beta}5 mutant (Y750A) abrogated adhesion and {beta}5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of {beta}5 Y750A integrin had a potent gain-of-function effect on apoptotic cell phagocytosis, and further, a {beta}5-Y750A-EGFP fusion integrin readily bound MFG-E8-coated 10 {mu}m diameter microspheres developed as apoptotic cell mimetics. The critical sequences in {beta}5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of {beta}5 integrin is regulated by an unconventional NPxY-talin-independent activation signal and argue for the existence of molecular switches in the {beta}5 cytoplasmic tail for adhesion and phagocytosis.

  2. Integrin activation

    PubMed Central

    Ginsberg, Mark H.

    2014-01-01

    Integrin-mediated cell adhesion is important for development, immune responses, hemostasis and wound healing. Integrins also function as signal transducing receptors that can control intracellular pathways that regulate cell survival, proliferation, and cell fate. Conversely, cells can modulate the affinity of integrins for their ligands a process operationally defined as integrin activation. Analysis of activation of integrins has now provided a detailed molecular understanding of this unique form of “inside-out” signal transduction and revealed new paradigms of how transmembrane domains (TMD) can transmit long range allosteric changes in transmembrane proteins. Here, we will review how talin and mediates integrin activation and how the integrin TMD can transmit these inside out signals. [BMB Reports 2014; 47(12): 655-659] PMID:25388208

  3. Droplet pairing and coalescence control for generation of combinatorial signals

    NASA Astrophysics Data System (ADS)

    Um, Eujin; Rogers, Matthew; Stone, Howard

    2013-03-01

    A co-flowing aqueous phase with an immiscible oil phase in a microchannel generates uniformly spaced, monodisperse droplets, which retain their shape by not touching each other or by being stabilized with surfactants at the oil-water interface. However, droplet coalescence is required in many advanced applications, which can be achieved by a complex channel geometry or size differences in the droplets, and as well as by procedures to reduce the effect of a surfactant. These approaches, again, hinder the stability of droplets further downstream. We designed a microchannel which consistently inserts gas-bubble between droplets so that pairing and coalescence of droplets occurs even in the presence of surfactant, and yet prevents unwanted merging with other droplets. Aqueous droplets placed between the bubbles alter their relative speeds and spacing, and consequently we study the change in the number of droplet pairings in relation to the characteristics of the bubbles and the volume of aqueous droplets. By integrating this approach with droplets of different materials, we can program the output sequence of droplet compositions, and such complex combinatorial signals generated are aimed for concentration gradient generation and dynamic stimulation of biological cells with chemicals.

  4. Enhancing integrin function by VEGF/neuropilin signaling: implications for tumor biology.

    PubMed

    Goel, Hira Lal; Mercurio, Arthur M

    2012-01-01

    This review advances the hypothesis that the ability of integrins to engage their extracellular matrix ligands and signal can be regulated in tumor cells by vascular endothelial growth factor (VEGF), a major angiogenic factor that also has direct effects on the function of tumor cells. More specifically, we will discuss how neuropilins (NRPs), a distinct class of VEGF receptors, enable the function of specific integrins that contribute to tumor initiation and progression.

  5. Role of insulin receptor and insulin signaling on αPS2CβPS integrins' lateral diffusion.

    PubMed

    Mainali, Dipak; Syed, Aleem; Arora, Neha; Smith, Emily A

    2014-12-01

    Integrins are ubiquitous transmembrane receptors with adhesion and signaling properties. The influence of insulin receptor and insulin signaling on αPS2CβPS integrins' lateral diffusion was studied using single particle tracking in S2 cells before and after reducing the insulin receptor expression or insulin stimulation. Insulin signaling was monitored by Western blotting for phospho-Akt expression. The expression of the insulin receptor was reduced using RNA interference (RNAi). After insulin receptor RNAi, four significant changes were measured in integrin diffusion properties: (1) there was a 24% increase in the mobile integrin population, (2) 14% of the increase was represented by integrins with Brownian diffusion, (3) for integrins that reside in confined zones of diffusion, there was a 45% increase in the diameter of the confined zone, and (4) there was a 29% increase in the duration integrins spend in confined zones of diffusion. In contrast to reduced expression of the insulin receptor, which alters integrin diffusion properties, insulin stimulation alone or insulin stimulation under conditions of reduced insulin receptor expression have minimal effects on altering the measured integrin diffusion properties. The differences in integrin diffusion measured after insulin receptor RNAi in the presence or absence of insulin stimulation may be the result of other insulin signaling pathways that are activated at reduced insulin receptor conditions. No change in the average integrin diffusion coefficient was measured for any conditions included in this study.

  6. The involvement of Gab1 and PI 3-kinase in {beta}1 integrin signaling in keratinocytes

    SciTech Connect

    Kuwano, Yoshihiro; Fujimoto, Manabu . E-mail: fujimoto-m@umin.ac.jp; Watanabe, Rei; Ishiura, Nobuko; Nakashima, Hiroko; Komine, Mayumi; Hamazaki, Tatsuo S.; Tamaki, Kunihiko; Okochi, Hitoshi

    2007-09-14

    The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. {beta}1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these {beta}1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of {beta}1 integrins, we established HaCaT cells with high {beta}1integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing {beta}1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high {beta}1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the {beta}1 integrin-mediated regulation of the epidermal stem cell compartment.

  7. Common and Diverging Integrin Signals Downstream of Adhesion and Mechanical Stimuli and Their Interplay with Reactive Oxygen Species

    NASA Astrophysics Data System (ADS)

    Zeller, Kathrin Stephanie; Johansson, Staffan

    The integrin family of adhesion receptors regulates basic functions of cells, and the signals they induce are altered in tumor cells. In this review we discuss how different integrindependent signals are generated during cell adhesion and by physical forces acting on cells. We also describe how reactive oxygen species are integral parts of integrin signaling and highlight a few important questions in the field. Answers to those may improve our understanding of integrins and their role in the development of cancer.

  8. Signals from the surface modulate differentiation of human pluripotent stem cells through glycosaminoglycans and integrins.

    PubMed

    Wrighton, Paul J; Klim, Joseph R; Hernandez, Brandon A; Koonce, Chad H; Kamp, Timothy J; Kiessling, Laura L

    2014-12-23

    The fate decisions of human pluripotent stem (hPS) cells are governed by soluble and insoluble signals from the microenvironment. Many hPS cell differentiation protocols use Matrigel, a complex and undefined substrate that engages multiple adhesion and signaling receptors. Using defined surfaces programmed to engage specific cell-surface ligands (i.e., glycosaminoglycans and integrins), the contribution of specific matrix signals can be dissected. For ectoderm and motor neuron differentiation, peptide-modified surfaces that can engage both glycosaminoglycans and integrins are effective. In contrast, surfaces that interact selectively with glycosaminoglycans are superior to Matrigel in promoting hPS cell differentiation to definitive endoderm and mesoderm. The modular surfaces were used to elucidate the signaling pathways underlying these differences. Matrigel promotes integrin signaling, which in turn inhibits mesendoderm differentiation. The data indicate that integrin-activating surfaces stimulate Akt signaling via integrin-linked kinase (ILK), which is antagonistic to endoderm differentiation. The ability to attribute cellular responses to specific interactions between the cell and the substrate offers new opportunities for revealing and controlling the pathways governing cell fate.

  9. Cross Talk among TGF-β Signaling Pathways, Integrins, and the Extracellular Matrix

    PubMed Central

    Munger, John S.; Sheppard, Dean

    2011-01-01

    The growth factor TGF-β is secreted in a latent complex consisting of three proteins: TGF-β, an inhibitor (latency-associated protein, LAP, which is derived from the TGF-β propeptide) and an ECM-binding protein (one of the latent TGF-β binding proteins, or LTBPs). LTBPs interact with fibrillins and other ECM components and thus function to localize latent TGF-β in the ECM. LAP contains an integrin-binding site (RGD), and several RGD-binding integrins are able to activate latent TGF-β through binding this site. Mutant mice defective in integrin-mediated activators, and humans and mice with fibrillin gene mutations, show the critical role of ECM and integrins in regulating TGF-β signaling. PMID:21900405

  10. Fibronectin-integrin mediated signaling in human cervical cancer cells (SiHa).

    PubMed

    Maity, Gargi; Fahreen, Shabana; Banerji, Aniruddha; Roy Choudhury, Paromita; Sen, Triparna; Dutta, Anindita; Chatterjee, Amitava

    2010-03-01

    Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation, and migration, including tumor development and invasion of tumor cells. Matrix metalloproteinases (MMPs) are a family of metalloproteinases capable of digesting ECM components and are important molecules for cell migration. Binding of ECM to integrins initiates cascades of cell signaling events modulating expression and activity of different MMPs. The aim of this study is to investigate fibronectin-integrin-mediated signaling and modulation of MMPs. Our findings indicated that culture of human cervical cancer cell (SiHa) on fibronectin-coated surface perhaps sends signals via fibronectin-integrin-mediated signaling pathways recruiting focal adhesion kinase (FAK) extracellular signal regulated kinase (ERK), phosphatidyl inositol 3 kinase (PI-3K), integrin-linked kinase (ILK), nuclear factor-kappa B (NF-kappaB), and modulates expression and activation of mainly pro-MMP-9, and moderately pro-MMP-2 in serum-free culture medium.

  11. Neuropilin-2 regulates α6β1 integrin in the formation of focal adhesions and signaling.

    PubMed

    Goel, Hira Lal; Pursell, Bryan; Standley, Clive; Fogarty, Kevin; Mercurio, Arthur M

    2012-01-15

    The neuropilins (NRPs) contribute to the function of cancer cells in their capacity as VEGF receptors. Given that NRP2 is induced in breast cancer and correlates with aggressive disease, we examined the role of NRP2 in regulating the interaction of breast cancer cells with the ECM. Using epithelial cells from breast tumors, we defined NRP2(high) and NRP2(low) populations that differed in integrin expression and adhesion to laminin. Specifically, the NRP2(high) population adhered more avidly to laminin and expressed high levels of the α6β1 integrin than the NRP2(low) population. The NRP2(high) population formed numerous focal adhesions on laminin that were not seen in the NRP2(low) population. These results were substantiated using breast carcinoma cell lines that express NRP2 and α6β1 integrin. Depletion experiments revealed that adhesive strength on laminin but not collagen is dependent on NRP2, and that VEGF is needed for adhesion on laminin. A specific interaction between NRP2 and α6β1 integrin was detected by co-immunoprecipitation. NRP2 is necessary for focal adhesion formation on laminin and for the association of α6β1 integrin with the cytoskeleton. NRP2 also facilitates α6β1-integrin-mediated activation of FAK and Src. Unexpectedly, we discovered that NRP2 is located in focal adhesions on laminin. The mechanism by which NRP2 regulates the interaction of α6β1 integrin with laminin to form focal adhesions involves PKC activation. Together, our data reveal a new VEGF-NRP2 signaling pathway that activates the α6β1 integrin and enables it to form focal adhesions and signal. This pathway is important in the pathogenesis of breast cancer.

  12. Integrin β1 mediates vaccinia virus entry through activation of PI3K/Akt signaling.

    PubMed

    Izmailyan, Roza; Hsao, Jye-Chian; Chung, Che-Sheng; Chen, Chein-Hung; Hsu, Paul Wei-Che; Liao, Chung-Lin; Chang, Wen

    2012-06-01

    Vaccinia virus has a broad range of infectivity in many cell lines and animals. Although it is known that the vaccinia mature virus binds to cell surface glycosaminoglycans and extracellular matrix proteins, whether additional cellular receptors are required for virus entry remains unclear. Our previous studies showed that the vaccinia mature virus enters through lipid rafts, suggesting the involvement of raft-associated cellular proteins. Here we demonstrate that one lipid raft-associated protein, integrin β1, is important for vaccinia mature virus entry into HeLa cells. Vaccinia virus associates with integrin β1 in lipid rafts on the cell surface, and the knockdown of integrin β1 in HeLa cells reduces vaccinia mature virus entry. Additionally, vaccinia mature virus infection is reduced in a mouse cell line, GD25, that is deficient in integrin β1 expression. Vaccinia mature virus infection triggers the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, and the treatment of cells with inhibitors to block P13K activation reduces virus entry in an integrin β1-dependent manner, suggesting that integrin β1-mediates PI3K/Akt activation induced by vaccinia virus and that this signaling pathway is essential for virus endocytosis. The inhibition of integrin β1-mediated cell adhesion results in a reduction of vaccinia virus entry and the disruption of focal adhesion and PI3K/Akt activation. In summary, our results show that the binding of vaccinia mature virus to cells mimics the outside-in activation process of integrin functions to facilitate vaccinia virus entry into HeLa cells.

  13. The Role of Mechanical Force and ROS in Integrin-Dependent Signals

    PubMed Central

    Zeller, Kathrin S.; Riaz, Anjum; Sarve, Hamid; Li, Jia; Tengholm, Anders; Johansson, Staffan

    2013-01-01

    Cells are exposed to several types of integrin stimuli, which generate responses generally referred to as “integrin signals”, but the specific responses to different integrin stimuli are poorly defined. In this study, signals induced by integrin ligation during cell attachment, mechanical force from intracellular contraction, or cell stretching by external force were compared. The elevated phosphorylation levels of several proteins during the early phase of cell attachment and spreading of fibroblast cell lines were not affected by inhibition of ROCK and myosin II activity, i.e. the reactions occurred independently of intracellular contractile force acting on the adhesion sites. The contraction-independent phosphorylation sites included ERK1/2 T202/Y204, AKT S473, p130CAS Y410, and cofilin S3. In contrast to cell attachment, cyclic stretching of the adherent cells induced a robust phosphorylation only of ERK1/2 and the phosphorylation levels of the other investigated proteins were not or only moderately affected by stretching. No major differences between signaling via α5β1 or αvβ3 integrins were detected. The importance of mitochondrial ROS for the integrin-induced signaling pathways was investigated using rotenone, a specific inhibitor of complex I in the respiratory chain. While rotenone only moderately reduced ATP levels and hardly affected the signals induced by cyclic cell stretching, it abolished the activation of AKT and reduced the actin polymerization rate in response to attachment in both cell lines. In contrast, scavenging of extracellular ROS with catalase or the vitamin C analog Asc-2P did not significantly influence the attachment-derived signaling, but caused a selective and pronounced enhancement of ERK1/2 phosphorylation in response to stretching. In conclusion, the results showed that “integrin signals” are composed of separate sets of reactions triggered by different types of integrin stimulation. Mitochondrial ROS and extracellular

  14. A Possible Role for Integrin Signaling in Diffuse Axonal Injury

    PubMed Central

    Kerscher, Lucas; Franck, Christian; Goss, Josue A.; Alford, Patrick W.; Parker, Kevin Kit

    2011-01-01

    Over the past decade, investigators have attempted to establish the pathophysiological mechanisms by which non-penetrating injuries damage the brain. Several studies have implicated either membrane poration or ion channel dysfunction pursuant to neuronal cell death as the primary mechanism of injury. We hypothesized that traumatic stimulation of integrins may be an important etiological contributor to mild Traumatic Brain Injury. In order to study the effects of forces at the cellular level, we utilized two hierarchical, in vitro systems to mimic traumatic injury to rat cortical neurons: a high velocity stretcher and a magnetic tweezer system. In one system, we controlled focal adhesion formation in neurons cultured on a stretchable substrate loaded with an abrupt, one dimensional strain. With the second system, we used magnetic tweezers to directly simulate the abrupt injury forces endured by a focal adhesion on the neurite. Both systems revealed variations in the rate and nature of neuronal injury as a function of focal adhesion density and direct integrin stimulation without membrane poration. Pharmacological inhibition of calpains did not mitigate the injury yet the inhibition of Rho-kinase immediately after injury reduced axonal injury. These data suggest that integrin-mediated activation of Rho may be a contributor to the diffuse axonal injury reported in mild Traumatic Brain Injury. PMID:21799943

  15. Beta 1-integrin-c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes.

    PubMed

    Barrow-McGee, Rachel; Kishi, Naoki; Joffre, Carine; Ménard, Ludovic; Hervieu, Alexia; Bakhouche, Bakhouche A; Noval, Alejandro J; Mai, Anja; Guzmán, Camilo; Robert-Masson, Luisa; Iturrioz, Xavier; Hulit, James; Brennan, Caroline H; Hart, Ian R; Parker, Peter J; Ivaska, Johanna; Kermorgant, Stéphanie

    2016-01-01

    Receptor tyrosine kinases (RTKs) and integrins cooperate to stimulate cell migration and tumour metastasis. Here we report that an integrin influences signalling of an RTK, c-Met, from inside the cell, to promote anchorage-independent cell survival. Thus, c-Met and β1-integrin co-internalize and become progressively recruited on LC3B-positive 'autophagy-related endomembranes' (ARE). In cells growing in suspension, β1-integrin promotes sustained c-Met-dependent ERK1/2 phosphorylation on ARE. This signalling is dependent on ATG5 and Beclin1 but not on ATG13, suggesting ARE belong to a non-canonical autophagy pathway. This β1-integrin-dependent c-Met-sustained signalling on ARE supports anchorage-independent cell survival and growth, tumorigenesis, invasion and lung colonization in vivo. RTK-integrin cooperation has been assumed to occur at the plasma membrane requiring integrin 'inside-out' or 'outside-in' signalling. Our results report a novel mode of integrin-RTK cooperation, which we term 'inside-in signalling'. Targeting integrin signalling in addition to adhesion may have relevance for cancer therapy. PMID:27336951

  16. Mechanical strain promotes osteoblastic differentiation through integrin-β1-mediated β-catenin signaling.

    PubMed

    Yan, Yuxian; Sun, Haoyang; Gong, Yuanwei; Yan, Zhixiong; Zhang, Xizheng; Guo, Yong; Wang, Yang

    2016-08-01

    As integrins are mechanoresponsive, there exists an intimate relationship between integrins and mechanical strain. Integrin-β1 mediates the impact of mechanical strain on bone. Mechanical strain induces bone formation through the activation of β-catenin pathways, which suggests that integrin-β1 mediates β-catenin signaling in osteoblasts in response to mechanical strain. In the present study, we examined the role of integrin-β1 in Wnt/β-catenin signal transduction in mechanically strained osteoblasts. MC3T3-E1 osteoblastic cells were transfected with integrin-β1 small interfering RNA (si-Itgβ1), and exposed to mechanical tensile strain of 2,500 microstrain (µε) using a four-point bending device. The mechanical strain enhanced the mRNA expression of integrin-β1, the protein levels of phosphorylated (p-) glycogen synthase kinase-3β (GSK‑3β) and β-catenin, simultaneously increased the mRNA levels of runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), the protein levels of bone morphogenetic protein (BMP)-2 and -4 and enhanced the alkaline phosphatase (ALP) activity of the ME3T3-E1 cells. The elevations were inhibited by si-Itgβ1. Additionally, the mechanical strain induced the nuclear translocation of β-catenin into the nucleus, which was also inhibited by si-Itgβ1. These findings indicated that mechanical strain promoted osteoblastic differentiation through integrin‑β1‑mediated β-catenin signaling.

  17. Constant Applied Force Stimulates Osteoblast Proliferation Via Matrix-Integrin-Signaling Pathways

    NASA Technical Reports Server (NTRS)

    Vercoutere, W.; Parra, M.; Roden, C.; DaCosta, M.; Wing, A.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E. A. C.

    2003-01-01

    Reduced weight-bearing caused by immobilization, bed-rest or microgravity leads to atrophy in mechanosensitive tissue such as muscle and bone. We hypothesize that bone tissue requires earth s gravity (1-g) for the maintenance of extracellular matrix, integrin, and kinase-mediated cell growth and survival pathways. We investigate the role of matrix-integrin signaling in bone cells using cell culture centrifugation to provide different levels of hypergravity mechanostimulation. The 10-50-g range we use also mimics physiological intermedullary pressure (1.2 - 5 kPa). 24 hours at 50-g increased primary rat osteoblast proliferation on collagen Type I and fibronectin, but not laminin or uncoated plastic. BrdU incorporation in primary osteoblasts over 24 h showed hypergravity increased the number of cells actively synthesizing DNA from about 60% at 1-g to over 90% at 25-g. Primary rat fibroblasts grown at 50-g (24 h) showed no proliferation increase, suggesting this is a tissue-specific phenomenon. These results suggest that the betal and alpha4 integrins may be involved. To further test this, we used osteocytic-like MLO-Y4 cells that showed increased proliferation at 1-g with stable expression of a betal integrin cytoplasmic tail and transmembrane domain construct. At 50-g, MLO-Y4/betal cells showed greater MAPK activation than MLO-Y4 vector controls, suggesting that betal integrin is involved in transducing mitogenic signals in response to hypergravity. Preliminary results also show that interfering with the alpha4 integrin in primary osteoblasts grown on fibronectin blocked the proliferation response. These results indicate that cells from mechanosensitive bone tissue can respond to gravity-generated forces, and this response involves specific matrix and integrin-dependent signaling pathways.

  18. PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

    PubMed Central

    Martin, Katherine; Pritchett, James; Llewellyn, Jessica; Mullan, Aoibheann F.; Athwal, Varinder S.; Dobie, Ross; Harvey, Emma; Zeef, Leo; Farrow, Stuart; Streuli, Charles; Henderson, Neil C.; Friedman, Scott L.; Hanley, Neil A.; Piper Hanley, Karen

    2016-01-01

    Fibrosis due to extracellular matrix (ECM) secretion from myofibroblasts complicates many chronic liver diseases causing scarring and organ failure. Integrin-dependent interaction with scar ECM promotes pro-fibrotic features. However, the pathological intracellular mechanism in liver myofibroblasts is not completely understood, and further insight could enable therapeutic efforts to reverse fibrosis. Here, we show that integrin beta-1, capable of binding integrin alpha-11, regulates the pro-fibrotic phenotype of myofibroblasts. Integrin beta-1 expression is upregulated in pro-fibrotic myofibroblasts in vivo and is required in vitro for production of fibrotic ECM components, myofibroblast proliferation, migration and contraction. Serine/threonine-protein kinase proteins, also known as P21-activated kinase (PAK), and the mechanosensitive factor, Yes-associated protein 1 (YAP-1) are core mediators of pro-fibrotic integrin beta-1 signalling, with YAP-1 capable of perpetuating integrin beta-1 expression. Pharmacological inhibition of either pathway in vivo attenuates liver fibrosis. PAK protein inhibition, in particular, markedly inactivates the pro-fibrotic myofibroblast phenotype, limits scarring from different hepatic insults and represents a new tractable therapeutic target for treating liver fibrosis. PMID:27535340

  19. HLA Class I: an Unexpected Role in Integrin β4 Signaling in Endothelial Cells

    PubMed Central

    Zhang, Xiaohai; Reed, Elaine F.

    2012-01-01

    The production of anti-donor antibodies to HLA class I and class II antigens following transplantation is associated with development of transplant vasculopathy and graft loss. Antibodies against HLA class I (HLA-I) molecules are thought to contribute to transplant vasculopathy by triggering signals that elicit the activation and proliferation of endothelial cells. The proximal molecular events that regulate HLA-I dependent signal transduction are not well understood. We demonstrated a mutual dependency between HLA-I and integrin β4 to stimulate signal transduction and cell proliferation. Similarly, we found that integrin β4-mediated cell migration was dependent upon its interactions with HLA-I molecules. Since integrin β4 has been implicated in angiogenesis and tumor formation, associations between integrin β4 and HLA-I may play an important role in cancer. Further characterization of interactions between HLA-I and integrin β4 may lead to the development of therapeutic strategies for the treatment and prevention of chronic allograft rejection and cancer. PMID:22789625

  20. Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling.

    PubMed

    Zu, Yan; Liang, Xudong; Du, Jing; Zhou, Shuai; Yang, Chun

    2015-11-01

    Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85-89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1β1 up regulated the level of phosphorylated Smad1, 5, 8, which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells.

  1. Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling.

    PubMed

    Zu, Yan; Liang, Xudong; Du, Jing; Zhou, Shuai; Yang, Chun

    2015-11-01

    Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85-89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1β1 up regulated the level of phosphorylated Smad1, 5, 8, which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells. PMID:26475222

  2. α integrin cytoplasmic tails can rescue the loss of Rho-family GTPase signaling in the C. elegans somatic gonad.

    PubMed

    Meighan, Christopher M; Kelly, Victoria E; Krahe, Elena C; Gaeta, Adriel J

    2015-05-01

    Integrin signaling relies on multiple, distinct pathways to impact a diverse set of cell behaviors. The Rho family of GTPases are well-established downstream signaling partners of integrins that regulate cell shape, polarity, and migration. The nematode C. elegans provides a simple in vivo system for studying both integrins and the Rho family. Our previous work showed that the C. elegans α integrin cytoplasmic tails have tissue-specific functions during development. Here, we use chimeric α integrins to show that the cytoplasmic tails can rescue the loss of the Rho family of GTPases in three cell types in the somatic gonad. Knockdown of rho-1 by RNAi causes defects in sheath cell actin organization, ovulation, and vulva morphology. Chimeric α integrin ina-1 with the pat-2 cytoplasmic tail can rescue both actin organization and ovulation after rho-1 RNAi, yet cannot restore vulva morphology. Knockdown of cdc-42 by RNAi causes defects in sheath cell actin organization, ovulation, vulva morphology, and distal tip cell migration. Chimeric α integrin pat-2 with the ina-1 cytoplasmic tail can rescue vulva morphology defects and distal tip cell migration after cdc-42 RNAi, yet cannot restore sheath cell actin organization or ovulation. Disruption of Rac yields the same phenotype in distal tip cells regardless of α integrin cytoplasmic tail composition. Taken together, the cytoplasmic tails of α integrins can bypass signaling from members of the Rho family of GTPases during development. PMID:25576691

  3. A Potential Role for Integrin Signaling in Mechanoelectrical Feedback Progress in Biophysics and Molecular Biology

    PubMed Central

    Dabiri, Borna E.; Lee, Hyungsuk; Parker, Kevin Kit

    2016-01-01

    Certain forms of heart disease involve gross morphological changes to the myocardium that alter its hemodynamic loading conditions. These changes can ultimately lead to the increased deposition of extracellular matrix (ECM) proteins, such as collagen and fibronectin, which together work to pathologically alter the myocardium’s bulk tissue mechanics. In addition to changing the mechanical properties of the heart, this maladaptive remodeling gives rise to changes in myocardium electrical conductivity and synchrony since the tissue’s mechanical properties are intimately tied to its electrical characteristics. This phenomenon, called mechanoelectrical coupling (MEC), can render individuals affected by heart disease arrhythmogenic and susceptible to sudden cardiac death (SCD). The underlying mechanisms of MEC have been attributed to various processes, including the action of stretch activated channels and changes in troponin C-Ca2+ binding affinity. However, changes in the heart post infarction or due to congenital myopathies are also accompanied by shifts in the expression of various molecular components of cardiomyocytes, including the mechanosensitive family of integrin proteins. As transmembrane proteins, integrins mechanically couple the ECM with the intracellular cytoskeleton and have been implicated in mediating ion homeostasis in various cell types, including neurons and smooth muscle. Given evidence of altered integrin expression in the setting of heart disease coupled with the associated increased risk for arrhythmia, we argue in this review that integrin signaling contributes to MEC. In light of the significant mortality associated with arrhythmia and SCD, close examination of all culpable mechanisms, including integrin-mediated MEC, is necessary. PMID:22819851

  4. Claudin-7 indirectly regulates the integrin/FAK signaling pathway in human colon cancer tissue.

    PubMed

    Ding, Lei; Wang, Liyong; Sui, Leiming; Zhao, Huanying; Xu, Xiaoxue; Li, Tengyan; Wang, Xiaonan; Li, Wenjing; Zhou, Ping; Kong, Lu

    2016-08-01

    The claudin family of proteins is integral to the structure and function of tight junctions. The role of claudin-7 (Cldn-7, CLDN7) in regulating the integrin/focal adhesion kinase (FAK)/ERK signaling pathway remains poorly understood. Therefore, we investigated differences in gene expression, primarily focusing on CLDN7 and integrin/FAK/ERK signaling pathway genes, between colon cancer and adjacent normal tissues. Quantitative real-time reverse transcription-PCR and immunohistochemistry were utilized to verify the results of mRNA and protein expression, respectively. In silico analysis was used to predict co-regulation between Cldn-7 and integrin/FAK/ERK signaling pathway components, and the STRING database was used to analyze protein-protein interaction pairs among these proteins. Meta-analysis of expression microarrays in The Cancer Genome Atlas (TCGA) database was used to identify significant correlations between Cldn-7 and components of predicted genes in the integrin/FAK/ERK signaling pathway. Our results showed marked cancer stage-specific decreases in the protein expression of Cldn-7, Gelsolin, MAPK1 and MAPK3 in colon cancer samples, and the observed changes for all proteins except Cldn-7 were in agreement with changes in the corresponding mRNA levels. Cldn-7 might indirectly regulate MAPK3 via KRT8 due to KRT8 co-expression with MAPK3 or CLDN7. Our bioinformatics methods supported the hypothesis that Cldn-7 does not directly regulate any genes in the integrin/FAK/ERK signaling pathway. These factors may participate in a common network that regulates cancer progression in which the MAPK pathway serves as the central node.

  5. α5-Integrin-mediated cellular signaling contributes to the myogenic response of cerebral resistance arteries.

    PubMed

    Colinas, Olaia; Moreno-Domínguez, Alejandro; Zhu, Hai-Lei; Walsh, Emma J; Pérez-García, M Teresa; Walsh, Michael P; Cole, William C

    2015-10-01

    The myogenic response of resistance arterioles and small arteries involving constriction in response to intraluminal pressure elevation and dilation on pressure reduction is fundamental to local blood flow regulation in the microcirculation. Integrins have garnered considerable attention in the context of initiating the myogenic response, but evidence indicative of mechanotransduction by integrin adhesions, for example established changes in tyrosine phosphorylation of key adhesion proteins, has not been obtained to substantiate this interpretation. Here, we evaluated the role of integrin adhesions and associated cellular signaling in the rat cerebral arterial myogenic response using function-blocking antibodies against α5β1-integrins, pharmacological inhibitors of focal adhesion kinase (FAK) and Src family kinase (SFK), an ultra-high-sensitivity western blotting technique, site-specific phosphoprotein antibodies to quantify adhesion and contractile filament protein phosphorylation, and differential centrifugation to determine G-actin levels in rat cerebral arteries at varied intraluminal pressures. Pressure-dependent increases in the levels of phosphorylation of FAK (FAK-Y397, Y576/Y577), SFK (SFK-Y416; Y527 phosphorylation was reduced), vinculin-Y1065, paxillin-Y118 and phosphoinositide-specific phospholipase C-γ1 (PLCγ1)-Y783 were detected. Treatment with α5-integrin function-blocking antibodies, FAK inhibitor FI-14 or SFK inhibitor SU6656 suppressed the changes in adhesion protein phosphorylation, and prevented pressure-dependent phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) at T855 and 20kDa myosin regulatory light chains (LC20) at S19, as well as actin polymerization that are necessary for myogenic constriction. We conclude that mechanotransduction by integrin adhesions and subsequent cellular signaling play a fundamental role in the cerebral arterial myogenic response.

  6. CD99 inhibits CD98-mediated β1 integrin signaling through SHP2-mediated FAK dephosphorylation.

    PubMed

    Lee, Kyoung Jin; Yoo, Yeon Ho; Kim, Min Seo; Yadav, Birendra Kumar; Kim, Yuri; Lim, Dongyoung; Hwangbo, Cheol; Moon, Ki Won; Kim, Daejoong; Jeoung, Dooil; Lee, Hansoo; Lee, Jeong-Hyung; Hahn, Jang-Hee

    2015-08-15

    The human CD99 protein is a 32-kDa type I transmembrane glycoprotein, while CD98 is a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein. It has been previously shown that CD99 and CD98 oppositely regulate β1 integrin signaling, though the mechanisms by which this regulation occurs are not known. Our results revealed that antibody-mediated crosslinking of CD98 induced FAK phosphorylation at Y397 and facilitated the formation of the protein kinase Cα (PKCα)-syntenin-focal adhesion kinase (FAK), focal adhesions (FAs), and IPP-Akt1-syntenin complex, which mediates β1 integrin signaling. In contrast, crosslinking of CD99 disrupted the formation of the PKCα-syntenin-FAK complex as well as FA via FAK dephosphorylation. The CD99-induced dephosphorylation of FAK was apparently mediated by the recruitment of Src homology region 2 domain-containing phosphatase-2 (SHP2) to the plasma membrane and subsequent activation of its phosphatase activity. Further consequences of the activation of SHP2 included the disruption of FAK-talin and talin-β1 integrin interactions and attenuation in the formation of the IPP-Akt1-syntenin complex at the plasma membrane, which resulted in reduced cell-ECM adhesion. This report uncovers the molecular mechanisms underlying the inverse regulation of β1 integrin signaling by CD99 and CD98 and may provide a novel therapeutic approach to treat inflammation and cancer.

  7. Targeting Integrin-Dependent Adhesion and Signaling with 3-Arylquinoline and 3-Aryl-2-Quinolone Derivatives: A new Class of Integrin Antagonists

    PubMed Central

    Fiorucci, Sandrine; Lin, Xiaochen; Sadoul, Karin; Fournet, Guy; Bouvard, Daniel; Vinogradova, Olga; Joseph, Benoît; Block, Marc R.

    2015-01-01

    We previously reported the anti-migratory function of 3-aryl-2-quinolone derivatives, chemically close to flavonoids (Joseph et al., 2002). Herein we show that 3-arylquinoline or 3-aryl-2-quinolone derivatives disrupt cell adhesion in a dose dependent and reversible manner yet antagonized by artificial integrin activation such as manganese. Relying on this anti-adhesive activity, a Structure-Activity Relationship (SAR) study was established on 20 different compounds to throw the bases of future optimization strategies. Active drugs efficiently inhibit platelet spreading, aggregation, and clot retraction, processes that rely on αllbβ3 integrin activation and clustering. In vitro these derivatives interfere with β3 cytoplasmic tail interaction with kindlin-2 in pulldown assays albeit little effect was observed with pure proteins suggesting that the drugs may block an alternative integrin activation process that may not be directly related to kindlin recruitment. Ex vivo, these drugs blunt integrin signaling assayed using focal adhesion kinase auto-phosphorylation as a read-out. Hence, 3-arylquinoline and 3-aryl-2-quinolone series are a novel class of integrin activation and signaling antagonists. PMID:26509443

  8. TM4SF5 suppression disturbs integrin α5-related signalling and muscle development in zebrafish.

    PubMed

    Choi, Yoon-Ju; Kim, Hyun Ho; Kim, Jeong-Gyun; Kim, Hye-Jin; Kang, Minkyung; Lee, Mi-Sook; Ryu, Jihye; Song, Haeng Eun; Nam, Seo Hee; Lee, Doohyung; Kim, Kyu-Won; Lee, Jung Weon

    2014-08-15

    TM4SF5 (transmembrane 4 L six family member 5) is involved in EMT (epithelial-mesenchymal transition) for liver fibrosis and cancer metastasis; however, the function(s) of TM4SF5 during embryogenesis remains unknown. In the present study the effects of TM4SF5 on embryogenesis of zebrafish were investigated. tm4sf5 mRNA was expressed in the posterior somites during somitogenesis and in whole myotome 1 dpf (day post-fertilization). tm4sf5 suppression impaired development of the trunk with aberrant morphology of muscle fibres and altered expression of integrin α5. The arrangement and adhesion of muscle cells were abnormally disorganized in tm4sf5 morphants with reduced muscle fibre masses, where integrin α5-related signalling molecules, including fibronectin, FAK (focal adhesion kinase), vinculin and actin were aberrantly localized, compared with those in control fish. Aberrant muscle developments in tm4sf5 morphants were recovered by additional tm4sf5 or integrin α5 mRNA injection. Such a role for TM4SF5 was observed in the differentiation of C2C12 mouse myoblast cells to multinuclear muscle cells. Taken together, the results show that TM4SF5 controls muscle differentiation via co-operation with integrin α5-related signalling. PMID:24897542

  9. Cordycepin suppresses integrin/FAK signaling and epithelial-mesenchymal transition in hepatocellular carcinoma.

    PubMed

    Yao, Wen-Ling; Ko, Bor-Sheng; Liu, Tzu-An; Liang, Shu-Man; Liu, Chia-Chia; Lu, Yi-Jhu; Tzean, Shean-Shong; Shen, Tang-Long; Liou, Jun-Yang

    2014-01-01

    Cordycepin, also known as 3-deoxyadenosine, is an analogue of adenosine extracted from the traditional Chinese medicine "Dong Chong Xia Cao". Cordycepin is an active small molecular weight compound and is implicated in modulating multiple physiological functions including immune activation, anti-aging and anti-tumor effects. Several studies have indicated that cordycepin suppresses tumor progression. However, the signaling pathways involved in cordycepin regulating cancer cell motility, invasiveness and epithelial-mesenchymal transition (EMT) remain unclear. In this study, we found that cordycepin inhibits hepatocellular carcinoma (HCC) cell proliferation and migration/invasion. Treatment of cordycepin results in the increasing expression of epithelial marker, Ecadherin while no significant effect was found on N-cadherin α-catenin and β-catenin. Furthermore, although the expression of focal adhesion kinase (FAK) was slightly reduced, the level of phosphorylated FAK was significantly reduced by the treatment of cordycepin. In addition, cordycepin significantly suppresses the expression of integrin α3, integrin α6 and integrin β1 which are crucial interacting partners of FAK in regulating the focal adhesion complex. These results suggest cordycepin may contribute to EMT, antimigration/ invasion and growth inhibitory effects of HCC by suppressing E-cadherin and integrin/FAK signaling. Thus, cordycepin is a potential therapeutic or supplementary agent for preventing HCC tumor progression. PMID:23855336

  10. FAK inhibition disrupts a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth

    PubMed Central

    Tancioni, Isabelle; Uryu, Sean; Sulzmaier, Florian J.; Shah, Nina R.; Lawson, Christine; Miller, Nichol L.G.; Jean, Christine; Chen, Xiao Lei; Ward, Kristy K.; Schlaepfer, David D.

    2014-01-01

    Ovarian cancer ascites fluid contains matrix proteins that can impact tumor growth via integrin receptor binding. In human ovarian tumor tissue arrays, we find that activation of the cytoplasmic focal adhesion (FAK) tyrosine kinase parallels increased tumor stage, β5 integrin, and osteopontin (OPN) matrix staining. Elevated OPN, β5 integrin, and FAK mRNA levels are associated with decreased serous ovarian cancer patient survival. FAK remains active within ovarian cancer cells grown as spheroids, and anchorage-independent growth analyses of seven ovarian carcinoma cell lines identified sensitive (HEY, OVCAR8) and resistant (SKOV3-IP, OVCAR10) cells to 0.1 μM FAK inhibitor (VS-4718, formerly PND-1186) treatment. VS-4718 promoted HEY and OVCAR8 G0/G1 cell cycle arrest followed by cell death whereas growth of SKOV3-IP and OVCAR10 cells were resistant to 1.0 μM VS-4718. In HEY cells, genetic or pharmacological FAK inhibition prevented tumor growth in mice with corresponding reductions in β5 integrin and OPN expression. β5 knockdown reduced HEY cell growth in soft agar, tumor growth in mice, and both FAK Y397 phosphorylation and OPN expression in spheroids. FAK inhibitor resistant (SKOV3-IP, OVCAR10) cells exhibited anchorage-independent Akt S473 phosphorylation and expression of membrane-targeted and active Akt in sensitive cells (HEY, OVCAR8) increased growth but did not create a FAK inhibitor resistant phenotype. These results link OPN, β5 integrin, and FAK in promoting ovarian tumor progression.β5 integrin expression may serve as a biomarker for serous ovarian carcinoma cells that possess active FAK signaling. PMID:24899686

  11. Integrin β3 mediates cerebrovascular remodelling through Src/ClC-3 volume-regulated Cl− channel signalling pathway

    PubMed Central

    Zeng, Jia-Wei; Wang, Xiao-Guang; Ma, Ming-Ming; Lv, Xiao-Fei; Liu, Jie; Zhou, Jia-Guo; Guan, Yong-Yuan

    2014-01-01

    BACKGROUND AND PURPOSE Cerebrovascular remodelling is one of the important risk factors of stroke. The underlying mechanisms are unclear. Integrin β3 and volume-regulated ClC-3 Cl− channels have recently been implicated as important contributors to vascular cell proliferation. Therefore, we investigated the role of integrin β3 in cerebrovascular remodelling and related Cl− signalling pathway. EXPERIMENTAL APPROACH Cl− currents were recorded using a patch clamp technique. The expression of integrin β3 in hypertensive animals was examined by Western blot and immunohistochemisty. Immunoprecipitation, cDNA and siRNA transfection were employed to investigate the integrin β3/Src/ClC-3 signalling. KEY RESULTS Integrin β3 expression was up-regulated in stroke-prone spontaneously hypertensive rats, 2-kidney 2-clip hypertensive rats and angiotensin II-infused hypertensive mice. Integrin β3 expression was positively correlated with medial cross-sectional area and ClC-3 expression in the basilar artery of 2-kidney 2-clip hypertensive rats. Knockdown of integrin β3 inhibited the proliferation of rat basilar vascular smooth muscle cells induced by angiotensin II. Co-immunoprecipitation and immunofluorescence experiments revealed a physical interaction between integrin β3, Src and ClC-3 protein. The integrin β3/Src/ClC-3 signalling pathway was shown to be involved in the activation of volume-regulated chloride channels induced by both hypo-osmotic stress and angiotensin II. Tyrosine 284 within a concensus Src phosphorylation site was the key point for ClC-3 channel activation. ClC-3 knockout significantly attenuated angiotensin II-induced cerebrovascular remodelling. CONCLUSIONS AND IMPLICATIONS Integrin β3 mediates cerebrovascular remodelling during hypertension via Src/ClC-3 signalling pathway. PMID:24611720

  12. Ancient origin of the integrin-mediated adhesion and signaling machinery.

    PubMed

    Sebé-Pedrós, Arnau; Roger, Andrew J; Lang, Franz B; King, Nicole; Ruiz-Trillo, Iñaki

    2010-06-01

    The evolution of animals (metazoans) from their unicellular ancestors required the emergence of novel mechanisms for cell adhesion and cell-cell communication. One of the most important cell adhesion mechanisms for metazoan development is integrin-mediated adhesion and signaling. The integrin adhesion complex mediates critical interactions between cells and the extracellular matrix, modulating several aspects of cell physiology. To date this machinery has been considered strictly metazoan specific. Here we report the results of a comparative genomic analysis of the integrin adhesion machinery, using genomic data from several unicellular relatives of Metazoa and Fungi. Unexpectedly, we found that core components of the integrin adhesion complex are encoded in the genome of the apusozoan protist Amastigomonas sp., and therefore their origins predate the divergence of Opisthokonta, the clade that includes metazoans and fungi. Furthermore, our analyses suggest that key components of this apparatus have been lost independently in fungi and choanoflagellates. Our data highlight the fact that many of the key genes that had formerly been cited as crucial for metazoan origins have a much earlier origin. This underscores the importance of gene cooption in the unicellular-to-multicellular transition that led to the emergence of the Metazoa.

  13. Therapeutics targeting CD90-integrin-AMPK-CD133 signal axis in liver cancer

    PubMed Central

    Chen, Wei-Ching; Chang, Yung-Sheng; Hsu, Hui-Ping; Yen, Meng-Chi; Huang, Hau-Lun; Cho, Chien-Yu; Wang, Chih-Yang; Weng, Tzu-Yang; Lai, Po-Ting; Chen, Ching-Shih; Lin, Yih-Jyh; Lai, Ming-Derg

    2015-01-01

    CD90 is used as a marker for cancer stem cell in liver cancer. We aimed to study the mechanism by which CD90 promoted liver cancer progression and identify the new therapeutic targets on CD90 signal pathway. Ectopic expression of CD90 in liver cancer cell lines enhanced anchorage-independent growth and tumor progression. Furthermore, CD90 promoted sphere formation in vitro and upregulated the expression of the cancer stem cell marker CD133. The CD133 expression was higher in CD45-CD90+ cells in liver cancer specimen. The natural carcinogenic molecules TGF-β-1, HGF, and hepatitis B surface antigen increased the expression of CD90 and CD133. Inhibition of CD90 by either shRNA or antibody attenuated the induction of CD133 and anchorage-independent growth. Lentiviral delivery of CD133 shRNA abolished the tumorigenicity induced by CD90. Ectopic expression of CD90 induced mTOR phosphorylation and AMPK dephosphorylation. Mutation of integrin binding-RLD domain in CD90 attenuated the induction of CD133 and anchorage-independent growth. Similar results were observed after silencing β3 integrin. Signaling analyses revealed that AMPK/mTOR and β3 integrin were required for the induction of CD133 and tumor formation by CD90. Importantly, the energy restriction mimetic agent OSU-CG5 reduced the CD90 population in fresh liver tumor sample and repressed the tumor growth. In contrast, sorafenib did not decrease the CD90+ population. In conclusion, the signal axis of CD90-integrin-mTOR/AMPK-CD133 is critical for promoting liver carcinogenesis. Molecules inhibiting the signal axis, including OSU-CG5 and other inhibitors, may serve as potential novel cancer therapeutic targets in liver cancer. PMID:26556861

  14. Therapeutics targeting CD90-integrin-AMPK-CD133 signal axis in liver cancer.

    PubMed

    Chen, Wei-Ching; Chang, Yung-Sheng; Hsu, Hui-Ping; Yen, Meng-Chi; Huang, Hau-Lun; Cho, Chien-Yu; Wang, Chih-Yang; Weng, Tzu-Yang; Lai, Po-Ting; Chen, Ching-Shih; Lin, Yih-Jyh; Lai, Ming-Derg

    2015-12-15

    CD90 is used as a marker for cancer stem cell in liver cancer. We aimed to study the mechanism by which CD90 promoted liver cancer progression and identify the new therapeutic targets on CD90 signal pathway. Ectopic expression of CD90 in liver cancer cell lines enhanced anchorage-independent growth and tumor progression. Furthermore, CD90 promoted sphere formation in vitro and upregulated the expression of the cancer stem cell marker CD133. The CD133 expression was higher in CD45-CD90+ cells in liver cancer specimen. The natural carcinogenic molecules TGF-β-1, HGF, and hepatitis B surface antigen increased the expression of CD90 and CD133. Inhibition of CD90 by either shRNA or antibody attenuated the induction of CD133 and anchorage-independent growth. Lentiviral delivery of CD133 shRNA abolished the tumorigenicity induced by CD90. Ectopic expression of CD90 induced mTOR phosphorylation and AMPK dephosphorylation. Mutation of integrin binding-RLD domain in CD90 attenuated the induction of CD133 and anchorage-independent growth. Similar results were observed after silencing β3 integrin. Signaling analyses revealed that AMPK/mTOR and β3 integrin were required for the induction of CD133 and tumor formation by CD90. Importantly, the energy restriction mimetic agent OSU-CG5 reduced the CD90 population in fresh liver tumor sample and repressed the tumor growth. In contrast, sorafenib did not decrease the CD90+ population. In conclusion, the signal axis of CD90-integrin-mTOR/AMPK-CD133 is critical for promoting liver carcinogenesis. Molecules inhibiting the signal axis, including OSU-CG5 and other inhibitors, may serve as potential novel cancer therapeutic targets in liver cancer.

  15. Dab2IP Regulates Neuronal Positioning, Rap1 Activity and Integrin Signaling in the Developing Cortex.

    PubMed

    Qiao, Shuhong; Homayouni, Ramin

    2015-01-01

    Dab2IP (DOC-2/DAB2 interacting protein) is a GTPase-activating protein which is involved in various aspects of brain development in addition to its roles in tumor formation and apoptosis in other systems. In this study, we carefully examined the expression profile of Dab2IP and investigated its physiological role during brain development using a Dab2IP-knockdown (KD) mouse model created by retroviral insertion of a LacZ-encoding gene-trapping cassette. LacZ staining revealed that Dab2IP is expressed in the ventricular zone as well as the cortical plate and the intermediate zone. Immunohistochemical analysis showed that Dab2IP protein is localized in the leading process and proximal cytoplasmic regions of migrating neurons in the intermediate zone. Bromodeoxyuridine birth dating experiments in combination with immunohistochemical analysis using layer-specific markers showed that Dab2IP is important for proper positioning of a subset of layer II-IV neurons in the developing cortex. Notably, neuronal migration was not completely disrupted in the cerebral cortex of Dab2IP-KD mice and disruption of migration was not strictly layer specific. Previously, we found that Dab2IP regulates multipolar transition in cortical neurons. Others have shown that Rap1 regulates the transition from multipolar to bipolar morphology in migrating postmitotic neurons through N-cadherin signaling and somal translocation in the superficial layer of the cortical plate through integrin signaling. Therefore, we examined whether Rap1 and integrin signaling were affected in Dab2IP-KD brains. We found that Dab2IP-KD resulted in higher levels of activated Rap1 and integrin in the developing cortex. Taken together, our results suggest that Dab2IP plays an important role in the migration and positioning of a subpopulation of later-born (layers II-IV) neurons, likely through the regulation of Rap1 and integrin signaling. PMID:25721469

  16. Dab2IP Regulates Neuronal Positioning, Rap1 Activity and Integrin Signaling in the Developing Cortex.

    PubMed

    Qiao, Shuhong; Homayouni, Ramin

    2015-01-01

    Dab2IP (DOC-2/DAB2 interacting protein) is a GTPase-activating protein which is involved in various aspects of brain development in addition to its roles in tumor formation and apoptosis in other systems. In this study, we carefully examined the expression profile of Dab2IP and investigated its physiological role during brain development using a Dab2IP-knockdown (KD) mouse model created by retroviral insertion of a LacZ-encoding gene-trapping cassette. LacZ staining revealed that Dab2IP is expressed in the ventricular zone as well as the cortical plate and the intermediate zone. Immunohistochemical analysis showed that Dab2IP protein is localized in the leading process and proximal cytoplasmic regions of migrating neurons in the intermediate zone. Bromodeoxyuridine birth dating experiments in combination with immunohistochemical analysis using layer-specific markers showed that Dab2IP is important for proper positioning of a subset of layer II-IV neurons in the developing cortex. Notably, neuronal migration was not completely disrupted in the cerebral cortex of Dab2IP-KD mice and disruption of migration was not strictly layer specific. Previously, we found that Dab2IP regulates multipolar transition in cortical neurons. Others have shown that Rap1 regulates the transition from multipolar to bipolar morphology in migrating postmitotic neurons through N-cadherin signaling and somal translocation in the superficial layer of the cortical plate through integrin signaling. Therefore, we examined whether Rap1 and integrin signaling were affected in Dab2IP-KD brains. We found that Dab2IP-KD resulted in higher levels of activated Rap1 and integrin in the developing cortex. Taken together, our results suggest that Dab2IP plays an important role in the migration and positioning of a subpopulation of later-born (layers II-IV) neurons, likely through the regulation of Rap1 and integrin signaling.

  17. The RGD integrin binding site in human L1-CAM is important for nuclear signaling

    SciTech Connect

    Gast, Daniela; Riedle, Svenja; Kiefel, Helena; Mueerkoester, Susanne Sebens; Schaefer, Heiner; Schaefer, Michael K.E.; Altevogt, Peter

    2008-08-01

    L1 cell adhesion molecule (L1-CAM) is a transmembrane cell adhesion molecule initially defined as a promigratory molecule in the developing nervous system. L1 is also overexpressed in a variety of human carcinomas and is associated with bad prognosis. In carcinoma cell lines L1 augments cell motility and metastasis, tumor growth in nude mice and induces expression of L1-dependent genes. It is not known whether L1-signaling requires ligand binding. The RGD motif in the sixth Ig domain of L1 is a binding site for integrins. In the present study we analyzed the role of RGDs in L1-signaling using site-directed mutagenesis combined with antibody blocking studies. We observed that L1-RGE expressing HEK293 cells showed reduced cell-cell binding, cell motility, invasiveness and tumor growth in NOD/SCID mice. The RGE-mutation impaired L1-dependent gene regulation and antibodies to {alpha}v{beta}5 integrin had similar effects. Mutant L1 was unable to translocate to the nucleus. Our findings highlight the importance of the RGD site in L1 for human tumors and suggest that nuclear signaling of L1 is dependent on integrins.

  18. Maintenance of Stem Cell Niche Integrity by a Novel Activator of Integrin Signaling

    PubMed Central

    Lee, Joo Yeun; Chang, Karen T.

    2016-01-01

    Stem cells depend critically on the surrounding microenvironment, or niche, for their maintenance and self-renewal. While much is known about how the niche regulates stem cell self-renewal and differentiation, mechanisms for how the niche is maintained over time are not well understood. At the apical tip of the Drosophila testes, germline stem cells (GSCs) and somatic stem cells share a common niche formed by hub cells. Here we demonstrate that a novel protein named Shriveled (Shv) is necessary for the maintenance of hub/niche integrity. Depletion of Shv protein results in age-dependent deterioration of the hub structure and loss of GSCs, whereas upregulation of Shv preserves the niche during aging. We find Shv is a secreted protein that modulates DE-cadherin levels through extracellular activation of integrin signaling. Our work identifies Shv as a novel activator of integrin signaling and suggests a new integration model in which crosstalk between integrin and DE-cadherin in niche cells promote their own preservation by maintaining the niche architecture. PMID:27191715

  19. PRG-1 Regulates Synaptic Plasticity via Intracellular PP2A/β1-Integrin Signaling.

    PubMed

    Liu, Xingfeng; Huai, Jisen; Endle, Heiko; Schlüter, Leslie; Fan, Wei; Li, Yunbo; Richers, Sebastian; Yurugi, Hajime; Rajalingam, Krishnaraj; Ji, Haichao; Cheng, Hong; Rister, Benjamin; Horta, Guilherme; Baumgart, Jan; Berger, Hendrik; Laube, Gregor; Schmitt, Ulrich; Schmeisser, Michael J; Boeckers, Tobias M; Tenzer, Stefan; Vlachos, Andreas; Deller, Thomas; Nitsch, Robert; Vogt, Johannes

    2016-08-01

    Alterations in dendritic spine numbers are linked to deficits in learning and memory. While we previously revealed that postsynaptic plasticity-related gene 1 (PRG-1) controls lysophosphatidic acid (LPA) signaling at glutamatergic synapses via presynaptic LPA receptors, we now show that PRG-1 also affects spine density and synaptic plasticity in a cell-autonomous fashion via protein phosphatase 2A (PP2A)/β1-integrin activation. PRG-1 deficiency reduces spine numbers and β1-integrin activation, alters long-term potentiation (LTP), and impairs spatial memory. The intracellular PRG-1 C terminus interacts in an LPA-dependent fashion with PP2A, thus modulating its phosphatase activity at the postsynaptic density. This results in recruitment of adhesome components src, paxillin, and talin to lipid rafts and ultimately in activation of β1-integrins. Consistent with these findings, activation of PP2A with FTY720 rescues defects in spine density and LTP of PRG-1-deficient animals. These results disclose a mechanism by which bioactive lipid signaling via PRG-1 could affect synaptic plasticity and memory formation. PMID:27453502

  20. Lumican induces human corneal epithelial cell migration and integrin expression via ERK 1/2 signaling

    SciTech Connect

    Seomun, Young; Joo, Choun-Ki

    2008-07-18

    Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins {alpha}2 and {beta}1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin {beta}1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.

  1. Combinatorial Fibronectin and Laminin Signaling Promote Highly Efficient Cardiac Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Sa, Silin; Wong, Lian

    2014-01-01

    Abstract Cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs) are a promising and potentially unlimited cell source for myocardial repair and regeneration. Recently, multiple methodologies—primarily based on the optimization of growth factors—have been described for efficient cardiac differentiation of hESCs. However, the role of extracellular matrix (ECM) signaling in CM differentiation has not yet been explored fully. This study examined the role of ECM signaling in the efficient generation of CMs from both H7 and H9 ESCs. The hESCs were differentiated on ECM substrates composed of a range of fibronectin (FN) and laminin (LN) ratios and gelatin and evaluated by the fluorescence activated cell scanning (FACS) analysis on day 14. Of the ECM substrates examined, the 70:30 FN:LN reproducibly generated the greatest numbers of CMs from both hESC lines. Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin. Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway. Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4+ and ITGB5+ cells. PMID:25126479

  2. Nanotopography drives stem cell fate toward osteoblast differentiation through α1β1 integrin signaling pathway.

    PubMed

    Rosa, A L; Kato, R B; Castro Raucci, L M S; Teixeira, L N; de Oliveira, F S; Bellesini, L S; de Oliveira, P T; Hassan, M Q; Beloti, M M

    2014-03-01

    The aim of our study was to investigate the osteoinductive potential of a titanium (Ti) surface with nanotopography, using mesenchymal stem cells (MSCs) and the mechanism involved in this phenomenon. Polished Ti discs were chemically treated with H2 SO4 /H2 O2 to yield nanotopography and rat MSCs were cultured under osteogenic and non-osteogenic conditions on both nanotopography and untreated polished (control) Ti surfaces. The nanotopography increased cell proliferation and alkaline phosphatase (Alp) activity and upregulated the gene expression of key bone markers of cells grown under both osteogenic and non-osteogenic conditions. Additionally, the gene expression of α1 and β1 integrins was higher in cells grown on Ti with nanotopography under non-osteogeneic condition compared with control Ti surface. The higher gene expression of bone markers and Alp activity induced by Ti with nanotopography was reduced by obtustatin, an α1β1 integrin inhibitor. These results indicate that α1β1 integrin signaling pathway determines the osteoinductive effect of nanotopography on MSCs. This finding highlights a novel mechanism involved in nanosurface-mediated MSCs fate and may contribute to the development of new surface modifications aiming to accelerate and/or enhance the process of osseointegration.

  3. Modulation of integrin antagonist signaling by ligand binding of the heparin-binding domain of vitronectin to the alphaVbeta3 integrin.

    PubMed

    Maile, Laura A; Aday, Ariel W; Busby, Walker H; Sanghani, Ravi; Veluvolu, Umadevi; Clemmons, David R

    2008-10-01

    The interaction between the arginine glycine and aspartic acid motif (RGD) of integrin ligands such as vitronectin and the integrin receptor alphaVbeta3 in mediating cell attachment has been well described. Similarly, the ability of disintegrins, small RGD containing peptides, to inhibit cell attachment and other cellular processes has also been studied extensively. Recently, we characterized a second site of interaction between vitronectin and its integrin partner. We determined that amino acids within the heparin-binding domain of vitronectin bind to a cysteine loop (C-loop) region of beta3 and that this interaction is required for the positive effects of alphaVbeta3 ligand occupancy on IGF-I signaling in smooth muscle cells. In this study we examine the signaling events activated following ligand binding of disintegrins to the alphaVbeta3 and the ability of these signals to be regulated by binding of the heparin-binding domain of vitronectin. We demonstrate that disintegrin ligand binding activates a series of events including the sequential activation of the tyrosine kinases c-Src and Syk. This leads to the activation of calpain and the cleavage of the beta3 cytoplasmic tail. Addition of vitronectin or a peptide homologous to the heparin-binding domain inhibited activation of this pathway. Our results suggest that the signaling events that occur following ligand binding to the alphaVbeta3 integrin reflects a balance between the effects mediated through the RGD binding site interaction and the effects mediated by the heparin binding site interaction and that for intact vitronectin the effect of the heparin-binding domain predominates.

  4. Cooperative integrin/ITAM signaling in platelets enhances thrombus formation in vitro and in vivo

    PubMed Central

    Zhi, Huiying; Rauova, Lubica; Hayes, Vincent; Gao, Cunji; Boylan, Brian; Newman, Debra K.; McKenzie, Steven E.; Cooley, Brian C.; Poncz, Mortimer; Newman, Peter J.

    2013-01-01

    The integrin family is composed of a series of 24 αβ heterodimer transmembrane adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. Adaptor molecules bearing immunoreceptor tyrosine-based activation motifs (ITAMs) have recently been shown to cooperate with specific integrins to increase the efficiency of transmitting ligand-binding–induced signals into cells. In human platelets, Fc receptor γ-chain IIa (FcγRIIa) has been identified as an ITAM-bearing transmembrane receptor responsible for mediating “outside-in” signaling through αIIbβ3, the major adhesion receptor on the platelet surface. To explore the importance of FcγRIIa in thrombosis and hemostasis, we subjected FcγRIIa-negative and FcγRIIa-positive murine platelets to a number of well-accepted models of platelet function. Compared with their FcγRIIa-negative counterparts, FcγRIIa-positive platelets exhibited increased tyrosine phosphorylation of Syk and phospholipase Cγ2 and increased spreading upon interaction with immobilized fibrinogen, retracted a fibrin clot faster, and showed markedly enhanced thrombus formation when perfused over a collagen-coated flow chamber under conditions of arterial and venous shear. They also displayed increased thrombus formation and fibrin deposition in in vivo models of vascular injury. Taken together, these data establish FcγRIIa as a physiologically important functional conduit for αIIbβ3-mediated outside-in signaling, and suggest that modulating the activity of this novel integrin/ITAM pair might be effective in controlling thrombosis. PMID:23264598

  5. Ultrasmall integrin-targeted silica nanoparticles modulate signaling events and cellular processes in a concentration-dependent manner.

    PubMed

    Benezra, Miriam; Phillips, Evan; Overholtzer, Michael; Zanzonico, Pat B; Tuominen, Esa; Wiesner, Ulrich; Bradbury, Michelle S

    2015-04-01

    Cellular and molecular-level interactions of nanoparticles with biological systems are a rapidly evolving field requiring an improved understanding of endocytic trafficking as the principal driver and regulator of signaling events and cellular responses. An understanding of these processes is vital to nanomedicine applications. Studies investigating the complex interplay of these processes and their relationship to targeted nanoparticles exploiting endocytic pathways are notably lacking. It is known that integrins traffic through the endosomal pathway and participate in diverse roles controlling signal transduction, cell migration, and proliferation. Here, it is shown that ultrasmall, nontoxic, core-shell silica nanoparticles (C-dots), surface-functionalized with cRGDY peptides, modestly activate integrin-signaling pathways, in turn, promoting the enhancement of cellular functions. First, nanomolar concentrations, two orders of magnitude higher than clinical trial doses, internalize within αvβ3 integrin-expressing melanoma and endothelial cells, predominantly through an integrin receptor-dependent endocytic route. Second, integrin-mediated activation of focal adhesion kinase (FAK) and downstream signaling pathways occurs, in turn, upregulating phosphorylated protein expression levels and promoting concentration-dependent cellular migration and proliferative activity. Inhibiting FAK catalytic activity leads to decreased phosphorylation levels and cellular migration rates. These findings may inform the design of more effectively-targeted nanomedicines and provide insights into endocytic regulation of signal transduction.

  6. Hypergravity Stimulates the Extracellular Matrix/Integrin-Signaling Axis and Proliferation in Primary Osteoblasts

    NASA Technical Reports Server (NTRS)

    Parra, M.; Vercoutere, W.; Roden, C.; Banerjee, I.; Krauser, W.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

    2003-01-01

    We set out to determine the molecular mechanisms involved in the proliferative response of primary rat osteoblasts to mechanical stimulation using cell culture centrifugation as a model for hypergravity. We hypothesized that this proliferative response is mediated by specific integrin/Extracellular Matrix (ECM) interactions. To investigate this question we developed a cell culture centrifuge and an automated system that performs cell fixation during hypergravity loading. We generated expression vectors for various focal adhesion and cytoskeletal proteins fused to GFP or dsRed and visualized these structures in transfected (or infected) osteoblasts. The actin cytoskeleton was also visualized using rhodamine-phalloidin staining and Focal Adhesion Kinase (FAK) levels were assessed biochemically. We observed that a 24 hour exposure to 50-g stimulated proliferation compared to the 1-g control when cells were plated on fibronectin, collagen Type I , and collagen Type IV, but not on uncoated tissue culture plastic surfaces. This proliferative response was greatest for osteoblasts grown on fibronectin (2-fold increase over 1-g control) and collagen Type I (1.4 fold increase over 1-g control), suggesting that specific matrices and integrins are involved in the signaling pathways required for proliferation. Exposing osteoblasts grown on different matrices to 10-g or 25-g showed that effects on proliferation depended on both matrix type and loading level. We found that osteoblasts exposed to a short pulse of hypergravity during adhesion spread further and had more GFP-FAK containing focal adhesions compared to their 1-g controls. While overall levels of FAK did not change, more FAK was in the active (phosphorylated) form under hypergravity than in the 1-g controls. Cytoskeletal F-actin organization into filaments was also more prominent after brief exposures to hypergravity during the first five minutes of adhesion. These results suggest that specific integrins sense

  7. Guidance of Signaling Activations by Cadherins and Integrins in Epithelial Ovarian Cancer Cells

    PubMed Central

    Roggiani, Francesca; Mezzanzanica, Delia; Rea, Katia; Tomassetti, Antonella

    2016-01-01

    Epithelial ovarian cancer (EOC) is the deadliest tumor among gynecological cancer in the industrialized countries. The EOC incidence and mortality have remained unchanged over the last 30 years, despite the progress in diagnosis and treatment. In order to develop novel and more effective therapeutic approaches, the molecular mechanisms involved in EOC progression have been thoroughly investigated in the last few decades. At the late stage, peritoneal metastases originate from the attachment of small clusters of cancer cells that shed from the primary site and carried by the ascites adhere to the abdominal peritoneum or omentum. This behavior suggests that cell–cell or cell–matrix adhesion mechanisms regulate EOC growth and dissemination. Complex downstream signalings, which might be influenced by functional cross-talk between adhesion molecules and co-expressed and activated signaling proteins, can affect the proliferation/survival and the migration/invasion of EOC cells. This review aimed to define the impact of the mechanisms of cell–cell, through cadherins, and cell–extracellular matrix adhesion, through integrins, on the signaling cascades induced by membrane receptors and cytoplasmic proteins known to have a role in the proliferation, migration and invasion of EOC cells. Finally, some novel approaches using peptidomimetic ligands to cadherin and integrins are summarized. PMID:27563880

  8. Ephrin-B1 transduces signals to activate integrin-mediated migration, attachment and angiogenesis.

    PubMed

    Huynh-Do, Uyen; Vindis, Cécile; Liu, Hua; Cerretti, Douglas Pat; McGrew, Jeffrey T; Enriquez, Miriam; Chen, Jin; Daniel, Thomas O

    2002-08-01

    Ephrin-B/EphB family proteins are implicated in bidirectional signaling and were initially defined through the function of their ectodomain sequences in activating EphB receptor tyrosine kinases. Ephrin-B1-3 are transmembrane proteins sharing highly conserved C-terminal cytoplasmic sequences. Here we use a soluble EphB1 ectodomain fusion protein (EphB1/Fc) to demonstrate that ephrin-B1 transduces signals that regulate cell attachment and migration. EphB1/Fc induced endothelial ephrin-B1 tyrosine phosphorylation, migration and integrin-mediated (alpha(v)beta(3) and alpha(5)beta(1)) attachment and promoted neovascularization, in vivo, in a mouse corneal micropocket assay. Activation of ephrin-B1 by EphB1/Fc induced phosphorylation of p46 JNK but not ERK-1/2 or p38 MAPkinases. By contrast, mutant ephrin-B1s bearing either a cytoplasmic deletion (ephrin-B1DeltaCy) or a deletion of four C-terminal amino acids (ephrin-B1DeltaPDZbd) fail to activate p46 JNK. Transient expression of intact ephin-B1 conferred EphB1/Fc migration responses on CHO cells, whereas the ephrin-B1DeltaCy and ephrin-B1DeltaPDZbd mutants were inactive. Thus ephrin-B1 transduces 'outside-in' signals through C-terminal protein interactions that affect integrin-mediated attachment and migration. PMID:12118063

  9. Combinatorial complexity in a transcriptionally centered signaling hub in Arabidopsis.

    PubMed

    Pfeiffer, Anne; Shi, Hui; Tepperman, James M; Zhang, Yu; Quail, Peter H

    2014-11-01

    a framework for probing the mechanisms by which transcription factors with overlapping direct-target genes integrate and selectively transduce signals to their target networks. PMID:25122696

  10. MFG-E8 Activates Proliferation of Vascular Smooth Muscle Cells via Integrin Signaling

    PubMed Central

    Wang, Mingyi; Fu, Zongming; Wu, James; Zhang, Jing; Jiang, Liqun; Khazan, Benjamin; Telljohann, Richard; Zhao, Mingming; Krug, Alexander W.; Pikilidou, Maria; Monticone, Robert E.; Wersto, Robert; Van Eyk, Jennifer; Lakatta, Edward G.

    2012-01-01

    Summary An accumulation of milk fat globule EGF-8 protein (MFG-E8) occurs within the context of arterial wall inflammatory remodeling during aging, hypertension, diabetes mellitus, or atherosclerosis. MFG-E8 induces VSMC invasion, but whether it effects VSMC proliferation, a salient feature of arterial inflammation, is unknown. Here, we show that in the rat arterial wall in vivo, PCNA and Ki67, markers of cell cycle activation, increase with age between 8 and 30-mo. In fresh or early passage VSMC isolated from old aortae, an increase in CDK4 and PCNA, and cell cycle with acceleration of S and G2 phases and reduction of the G1/G0 phase, and an increase in PDGF and its receptors, confer elevated proliferative capacity, compared to young VSMC. Increased co-expression and physical interaction of MFG-E8 and integrin αvβ5 occur with aging in both the rat aortic wall in vivo and in VSMC in vitro. In young VSMC in vitro, MFG-E8 added exogenously, or over-expressed endogenously, triggers phosphorylation of ERK1/2, augmented levels of PCNA and CDK4, increased BrdU incorporation and promotes proliferation, via αvβ5 integrins. MFG-E8 silencing, or its receptor inhibition, or the blockade of ERK1/2 phosphorylation in these cells reduces PCNA and CDK4 levels and decelerates the cell cycle S phase, conferring a reduction in proliferative capacity. Collectively, these results indicate that MFG-E8 in a dose-dependent manner, coordinates the expression of cell cycle molecules and facilitates VSMC proliferation via integrin/ ERK1/2 signaling. Thus, an increase in MFG-E8 signaling is a mechanism of the age-associated increase in aortic VSMC proliferation. PMID:22385834

  11. Neuropilin 1 balances β8 integrin-activated TGFβ signaling to control sprouting angiogenesis in the brain.

    PubMed

    Hirota, Shinya; Clements, Thomas P; Tang, Leung K; Morales, John E; Lee, Hye Shin; Oh, S Paul; Rivera, Gonzalo M; Wagner, Daniel S; McCarty, Joseph H

    2015-12-15

    Angiogenesis in the developing central nervous system (CNS) is regulated by neuroepithelial cells, although the genes and pathways that couple these cells to blood vessels remain largely uncharacterized. Here, we have used biochemical, cell biological and molecular genetic approaches to demonstrate that β8 integrin (Itgb8) and neuropilin 1 (Nrp1) cooperatively promote CNS angiogenesis by mediating adhesion and signaling events between neuroepithelial cells and vascular endothelial cells. β8 integrin in the neuroepithelium promotes the activation of extracellular matrix (ECM)-bound latent transforming growth factor β (TGFβ) ligands and stimulates TGFβ receptor signaling in endothelial cells. Nrp1 in endothelial cells suppresses TGFβ activation and signaling by forming intercellular protein complexes with β8 integrin. Cell type-specific ablation of β8 integrin, Nrp1, or canonical TGFβ receptors results in pathological angiogenesis caused by defective neuroepithelial cell-endothelial cell adhesion and imbalances in canonical TGFβ signaling. Collectively, these data identify a paracrine signaling pathway that links the neuroepithelium to blood vessels and precisely balances TGFβ signaling during cerebral angiogenesis.

  12. Neuropilin 1 balances β8 integrin-activated TGFβ signaling to control sprouting angiogenesis in the brain.

    PubMed

    Hirota, Shinya; Clements, Thomas P; Tang, Leung K; Morales, John E; Lee, Hye Shin; Oh, S Paul; Rivera, Gonzalo M; Wagner, Daniel S; McCarty, Joseph H

    2015-12-15

    Angiogenesis in the developing central nervous system (CNS) is regulated by neuroepithelial cells, although the genes and pathways that couple these cells to blood vessels remain largely uncharacterized. Here, we have used biochemical, cell biological and molecular genetic approaches to demonstrate that β8 integrin (Itgb8) and neuropilin 1 (Nrp1) cooperatively promote CNS angiogenesis by mediating adhesion and signaling events between neuroepithelial cells and vascular endothelial cells. β8 integrin in the neuroepithelium promotes the activation of extracellular matrix (ECM)-bound latent transforming growth factor β (TGFβ) ligands and stimulates TGFβ receptor signaling in endothelial cells. Nrp1 in endothelial cells suppresses TGFβ activation and signaling by forming intercellular protein complexes with β8 integrin. Cell type-specific ablation of β8 integrin, Nrp1, or canonical TGFβ receptors results in pathological angiogenesis caused by defective neuroepithelial cell-endothelial cell adhesion and imbalances in canonical TGFβ signaling. Collectively, these data identify a paracrine signaling pathway that links the neuroepithelium to blood vessels and precisely balances TGFβ signaling during cerebral angiogenesis. PMID:26586223

  13. Neuropilin 1 balances β8 integrin-activated TGFβ signaling to control sprouting angiogenesis in the brain

    PubMed Central

    Hirota, Shinya; Clements, Thomas P.; Tang, Leung K.; Morales, John E.; Lee, Hye Shin; Oh, S. Paul; Rivera, Gonzalo M.; Wagner, Daniel S.; McCarty, Joseph H.

    2015-01-01

    Angiogenesis in the developing central nervous system (CNS) is regulated by neuroepithelial cells, although the genes and pathways that couple these cells to blood vessels remain largely uncharacterized. Here, we have used biochemical, cell biological and molecular genetic approaches to demonstrate that β8 integrin (Itgb8) and neuropilin 1 (Nrp1) cooperatively promote CNS angiogenesis by mediating adhesion and signaling events between neuroepithelial cells and vascular endothelial cells. β8 integrin in the neuroepithelium promotes the activation of extracellular matrix (ECM)-bound latent transforming growth factor β (TGFβ) ligands and stimulates TGFβ receptor signaling in endothelial cells. Nrp1 in endothelial cells suppresses TGFβ activation and signaling by forming intercellular protein complexes with β8 integrin. Cell type-specific ablation of β8 integrin, Nrp1, or canonical TGFβ receptors results in pathological angiogenesis caused by defective neuroepithelial cell-endothelial cell adhesion and imbalances in canonical TGFβ signaling. Collectively, these data identify a paracrine signaling pathway that links the neuroepithelium to blood vessels and precisely balances TGFβ signaling during cerebral angiogenesis. PMID:26586223

  14. Quantitative relationship among integrin-ligand binding, adhesion, and signaling via focal adhesion kinase and extracellular signal-regulated kinase 2.

    PubMed

    Asthagiri, A R; Nelson, C M; Horwitz, A F; Lauffenburger, D A

    1999-09-17

    Because integrin-mediated signals are transferred through a physical architecture and synergistic biochemical network whose properties are not well defined, quantitative relationships between extracellular integrin-ligand binding events and key intracellular responses are poorly understood. We begin to address this by quantifying integrin-mediated FAK and ERK2 responses in CHO cells for varied alpha(5)beta(1) expression level and substratum fibronectin density. Plating cells on fibronectin-coated surfaces initiated a transient, biphasic ERK2 response, the magnitude and kinetics of which depended on integrin-ligand binding properties. Whereas ERK2 activity initially increased with a rate proportional to integrin-ligand bond number for low fibronectin density, the desensitization rate was independent of integrin and fibronectin amount but proportional to the ERK2 activity level with an exponential decay constant of 0.3 (+/- 0.08) min(-1). Unlike the ERK2 activation time course, FAK phosphorylation followed a superficially disparate time course. However, analysis of the early kinetics of the two signals revealed them to be correlated. The initial rates of FAK and ERK2 signal generation exhibited similar dependence on fibronectin surface density, with both rates monotonically increasing with fibronectin amount until saturating at high fibronectin density. Because of this similar initial rate dependence on integrin-ligand bond formation, the disparity in their time courses is attributed to differences in feedback regulation of these signals. Whereas FAK phosphorylation increased to a steady-state level as new integrin-ligand bond formation continued during cell spreading, ERK2 activity was decoupled from the integrin-ligand stimulus and decayed back to a basal level. Accordingly, we propose different functional metrics for representing these two disparate dynamic signals: the steady-state tyrosine phosphorylation level for FAK and the integral of the pulse response for

  15. Trisomy 12 chronic lymphocytic leukemia cells exhibit upregulation of integrin signaling that is modulated by NOTCH1 mutations

    PubMed Central

    Riches, John C.; O’Donovan, Conor J.; Kingdon, Sarah J.; McClanahan, Fabienne; Clear, Andrew J.; Neuberg, Donna S.; Werner, Lillian; Croce, Carlo M.; Ramsay, Alan G.; Rassenti, Laura Z.; Kipps, Thomas J.; Gribben, John G.

    2014-01-01

    The leukocyte adhesion cascade is important in chronic lymphocytic leukemia (CLL), as it controls migration of malignant cells into the pro-survival lymph node microenvironment. Circulating trisomy 12 CLL cells have increased expression of the integrins CD11a and CD49d, as well as CD38, but the tissue expression of these and other molecules, and the functional and clinical sequelae of these changes have not been described. Here, we demonstrate that circulating trisomy 12 CLL cells also have increased expression of the integrins CD11b, CD18, CD29, and ITGB7, and the adhesion molecule CD323. Notably, there was reduced expression of CD11a, CD11b, and CD18 in trisomy 12 cases with NOTCH1 mutations compared with wild type. Trisomy 12 cells also exhibit upregulation of intracellular integrin signaling molecules CALDAG-GEFI, RAP1B, and Ras-related protein ligand, resulting in enhanced very late antigen-4 [VLA-4] directed adhesion and motility. CD38 expression in CLL has prognostic significance, but the increased CD38 expression in trisomy 12 CLL cells must be taken into account in this subgroup, and the threshold of CD38 positivity should be raised to 40% for this marker to retain its prognostic value. In conclusion, trisomy 12 CLL cells exhibit functional upregulation of integrin signaling, with β2-integrin expression being modulated by NOTCH1 mutation status. PMID:24829201

  16. Kindlin-2 directly binds actin and regulates integrin outside-in signaling.

    PubMed

    Bledzka, Kamila; Bialkowska, Katarzyna; Sossey-Alaoui, Khalid; Vaynberg, Julia; Pluskota, Elzbieta; Qin, Jun; Plow, Edward F

    2016-04-11

    Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2(+/-)mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK(47)/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK(47)/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK(47)/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses. PMID:27044892

  17. Combinatorial signals by inflammatory cytokines and chemokines mediate leukocyte interactions with extracellular matrix.

    PubMed

    Vaday, G G; Franitza, S; Schor, H; Hecht, I; Brill, A; Cahalon, L; Hershkoviz, R; Lider, O

    2001-06-01

    On their extravasation from the vascular system into inflamed tissues, leukocytes must maneuver through a complex insoluble network of molecules termed the extracellular matrix (ECM). Leukocytes navigate toward their target sites by adhering to ECM glycoproteins and secreting degradative enzymes, while constantly orienting themselves in response to specific signals in their surroundings. Cytokines and chemokines are key biological mediators that provide such signals for cell navigation. Although the individual effects of various cytokines have been well characterized, it is becoming increasingly evident that the mixture of cytokines encountered in the ECM provides important combinatorial signals that influence cell behavior. Herein, we present an overview of previous and ongoing studies that have examined how leukocytes integrate signals from different combinations of cytokines that they encounter either simultaneously or sequentially within the ECM, to dynamically alter their navigational activities. For example, we describe our findings that tumor necrosis factor (TNF)-alpha acts as an adhesion-strengthening and stop signal for T cells migrating toward stromal cell-derived factor-1alpha, while transforming growth factor-beta down-regulates TNF-alpha-induced matrix metalloproteinase-9 secretion by monocytes. These findings indicate the importance of how one cytokine, such as TNF-alpha, can transmit diverse signals to different subsets of leukocytes, depending on its combination with other cytokines, its concentration, and its time and sequence of exposure. The combinatorial effects of multiple cytokines thus affect leukocytes in a step-by-step manner, whereby cells react to cytokine signals in their immediate vicinity by altering their adhesiveness, directional movement, and remodeling of the ECM. PMID:11404372

  18. Roles of integrin β3 cytoplasmic tail in bidirectional signal transduction in a trans-dominant inhibition model.

    PubMed

    Huang, Jiansong; Zhou, Yulan; Su, Xiaoyu; Lyu, Yuanjing; Tao, Lanlan; Shi, Xiaofeng; Liu, Ping; Long, Zhangbiao; Ruan, Zheng; Xiao, Bing; Xi, Wenda; Zhou, Quansheng; Mao, Jianhua; Xi, Xiaodong

    2016-09-01

    We evaluated the roles of calpain cleavage-related mutations of the integrin β3 cytoplasmic tail in integrin αIIbβ3 bidirectional signaling using a trans-dominant inhibition model. Chimeric Tac-β3 proteins (i.e., Tac-β3, Tac-β3Δ741, Tac-β3Δ747, Tac-β3Δ754, Tac-β3Δ759, and Tac-β3ΔNITY) consisting of the extracellular and transmembrane domains of human IL-2 receptor (Tac) and the human integrin β3 cytoplasmic domain were stably expressed in the 123 CHO cells harboring human glycoprotein Ib-IX and wild-type integrin αIIbβ3. The different cells were assayed for stable adhesion and spreading on immobilized fibrinogen, and for binding soluble fibrinogen representing outside-in and inside-out signaling events, respectively. The chimeric protein Tac-β3 inhibited, and Tac-β3ΔNITY partially attenuated stable adhesion and spreading. Tac-β3, Tac-β3Δ759, Tac-β3ΔNITY, and Tac-β3Δ754, but not Tac-β3Δ747 or Tac-β3Δ741, impaired the soluble fibrinogen binding. Results indicated that the bidirectional signaling was significantly inhibited by Tac-β3 and Tac-β3ΔNITY, albeit to a much lesser extent. Moreover, only inside-out signaling was impaired in the 123/Tac-β3Δ759 and 123/Tac-β3Δ754 cells in contrast to an intact bidirectional signaling in the 123/Tac-β3Δ747 and 123/Tac-β3Δ741 cells. In conclusion, the calpain cleavage of integrin β3 resulted in the regulatory effects on signaling by interrupting its interaction with cytoplasmic proteins rather than altering its conformation, and may thus regulate platelet function.

  19. Mutually Exclusive Roles of SHARPIN in Integrin Inactivation and NF-κB Signaling

    PubMed Central

    De Franceschi, Nicola; Peuhu, Emilia; Parsons, Maddy; Rissanen, Sami; Vattulainen, Ilpo; Salmi, Marko

    2015-01-01

    SHANK-associated RH domain interactor (SHARPIN) inhibits integrins through interaction with the integrin α-subunit. In addition, SHARPIN enhances nuclear factor-kappaB (NF-κB) activity as a component of the linear ubiquitin chain assembly complex (LUBAC). However, it is currently unclear how regulation of these seemingly different roles is coordinated. Here, we show that SHARPIN binds integrin and LUBAC in a mutually exclusive manner. We map the integrin binding site on SHARPIN to the ubiquitin-like (UBL) domain, the same domain implicated in SHARPIN interaction with LUBAC component RNF31 (ring finger protein 31), and identify two SHARPIN residues (V267, L276) required for both integrin and RNF31 regulation. Accordingly, the integrin α-tail is capable of competing with RNF31 for SHARPIN binding in vitro. Importantly, the full SHARPIN RNF31-binding site contains residues (F263A/I272A) that are dispensable for SHARPIN-integrin interaction. Importantly, disrupting SHARPIN interaction with integrin or RNF31 abolishes SHARPIN-mediated regulation of integrin or NF-κB activity, respectively. Altogether these data suggest that the roles of SHARPIN in inhibiting integrin activity and supporting linear ubiquitination are (molecularly) distinct. PMID:26600301

  20. Integrin Targeted Therapeutics

    PubMed Central

    Millard, Melissa; Odde, Srinivas; Neamati, Nouri

    2011-01-01

    Integrins are heterodimeric, transmembrane receptors that function as mechanosensors, adhesion molecules and signal transduction platforms in a multitude of biological processes. As such, integrins are central to the etiology and pathology of many disease states. Therefore, pharmacological inhibition of integrins is of great interest for the treatment and prevention of disease. In the last two decades several integrin-targeted drugs have made their way into clinical use, many others are in clinical trials and still more are showing promise as they advance through preclinical development. Herein, this review examines and evaluates the various drugs and compounds targeting integrins and the disease states in which they are implicated. PMID:21547158

  1. Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor

    PubMed Central

    Tian, Ruijun; Wang, Haopeng; Gish, Gerald D.; Petsalaki, Evangelia; Pasculescu, Adrian; Shi, Yu; Mollenauer, Marianne; Bagshaw, Richard D.; Yosef, Nir; Hunter, Tony; Gingras, Anne-Claude; Weiss, Arthur; Pawson, Tony

    2015-01-01

    Systematic characterization of intercellular signaling approximating the physiological conditions of stimulation that involve direct cell–cell contact is challenging. We describe a proteomic strategy to analyze physiological signaling mediated by the T-cell costimulatory receptor CD28. We identified signaling pathways activated by CD28 during direct cell–cell contact by global analysis of protein phosphorylation. To define immediate CD28 targets, we used phosphorylated forms of the CD28 cytoplasmic region to obtain the CD28 interactome. The interaction profiles of selected CD28-interacting proteins were further characterized in vivo for amplifying the CD28 interactome. The combination of the global phosphorylation and interactome analyses revealed broad regulation of CD28 and its interactome by phosphorylation. Among the cellular phosphoproteins influenced by CD28 signaling, CapZ-interacting protein (CapZIP), a regulator of the actin cytoskeleton, was implicated by functional studies. The combinatorial approach applied herein is widely applicable for characterizing signaling networks associated with membrane receptors with short cytoplasmic tails. PMID:25829543

  2. Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor.

    PubMed

    Tian, Ruijun; Wang, Haopeng; Gish, Gerald D; Petsalaki, Evangelia; Pasculescu, Adrian; Shi, Yu; Mollenauer, Marianne; Bagshaw, Richard D; Yosef, Nir; Hunter, Tony; Gingras, Anne-Claude; Weiss, Arthur; Pawson, Tony

    2015-03-31

    Systematic characterization of intercellular signaling approximating the physiological conditions of stimulation that involve direct cell-cell contact is challenging. We describe a proteomic strategy to analyze physiological signaling mediated by the T-cell costimulatory receptor CD28. We identified signaling pathways activated by CD28 during direct cell-cell contact by global analysis of protein phosphorylation. To define immediate CD28 targets, we used phosphorylated forms of the CD28 cytoplasmic region to obtain the CD28 interactome. The interaction profiles of selected CD28-interacting proteins were further characterized in vivo for amplifying the CD28 interactome. The combination of the global phosphorylation and interactome analyses revealed broad regulation of CD28 and its interactome by phosphorylation. Among the cellular phosphoproteins influenced by CD28 signaling, CapZ-interacting protein (CapZIP), a regulator of the actin cytoskeleton, was implicated by functional studies. The combinatorial approach applied herein is widely applicable for characterizing signaling networks associated with membrane receptors with short cytoplasmic tails. PMID:25829543

  3. Integrins and Integrin-Associated Proteins in the Cardiac Myocyte

    PubMed Central

    Ross, Robert S.

    2014-01-01

    Integrins are heterodimeric, transmembrane receptors that are expressed in all cells, including those in the heart. They participate in multiple critical cellular processes including adhesion, extracellular matrix organization, signaling, survival, and proliferation. Particularly relevant for a contracting muscle cell, integrins are mechanotransducers, translating mechanical to biochemical information. While it is likely that cardiovascular clinicians and scientists have highest recognition of integrins in the cardiovascular system from drugs used to inhibit platelet aggregation, the focus of this article will be on the role of integrins specifically in the cardiac myocyte. Following a general introduction to integrin biology, the manuscript will discuss important work on integrin signaling, mechanotransduction, and lessons learned about integrin function from a range of model organisms. Then we will detail work on integrin-related proteins in the myocyte, how integrins may interact with ion channels and mediate viral uptake into cells, and also play a role in stem cell biology. Finally, we will discuss directions for future study. PMID:24481847

  4. Combinatorial screening of photoelectrocatalytic system with high signal/noise ratio.

    PubMed

    Yuan, Ding; Xiao, Lina; Jia, Jingchun; Zhang, Jie; Han, Lianhuan; Li, Pei; Mao, Bing-Wei; Zhan, Dongping

    2014-12-16

    Solar energy is the most abundant nature resource and plays important roles in the sustainable developments of energy and environment. Scanning photoelectrochemical microscopy provides a high-throughput screening method by introducing the combinatorial technique to prepare the substrate with photoelectrochemical catalyst array. However, the signal/noise (S/N) ratio suffers from the background current of indium-tin oxide or fluorine-doped tin oxide itself, including a transient charge-discharge current of electric double layer and a steady-state photocatalytic current. Here we adopt a facile microfabrication method to isolate the substrate area other than the catalyst array from not only the electrolyte solution but also the light illumination. Consequently, the imaging quality has been promoted dramatically due to suppressed background current. This method provides a high S/N ratio screening method, which will be valuable for the high-throughput optimization of the photoelectrocatalytic system. PMID:25417799

  5. Combinatorial Library of Improved Peptide Aptamers, CLIPs to Inhibit RAGE Signal Transduction in Mammalian Cells

    PubMed Central

    Reverdatto, Sergey; Rai, Vivek; Xue, Jing; Burz, David S.; Schmidt, Ann Marie; Shekhtman, Alexander

    2013-01-01

    Peptide aptamers are small proteins containing a randomized peptide sequence embedded into a stable protein scaffold, such as Thioredoxin. We developed a robust method for building a Combinatorial Library of Improved Peptide aptamers (CLIPs) of high complexity, containing ≥3×1010 independent clones, to be used as a molecular tool in the study of biological pathways. The Thioredoxin scaffold was modified to increase solubility and eliminate aggregation of the peptide aptamers. The CLIPs was used in a yeast two-hybrid screen to identify peptide aptamers that bind to various domains of the Receptor for Advanced Glycation End products (RAGE). NMR spectroscopy was used to identify interaction surfaces between the peptide aptamers and RAGE domains. Cellular functional assays revealed that in addition to directly interfering with known binding sites, peptide aptamer binding distal to ligand sites also inhibits RAGE ligand-induced signal transduction. This finding underscores the potential of using CLIPs to select allosteric inhibitors of biological targets. PMID:23785412

  6. Integrin α6/Akt/Erk signaling is essential for human breast cancer resistance to radiotherapy

    PubMed Central

    Hu, Ting; Zhou, Rui; Zhao, Yanxia; Wu, Gang

    2016-01-01

    Integrin α6 (ITGA6), a transmembrane glycoprotein adhesion receptor protein, is widely upregulated in many types of tumors and promotes migration and invasion in cancer cells. However, the role that the ITGA6-associated signaling network plays in radiosensitivity in breast cancer has not been described. The expression of ITGA6 was examined in human breast cancer and normal breast cell lines using western blot analysis. We also explored the role of ITGA6 in the regulation of radiation sensitivity in breast cancer using the colony formation assays, cell cycle analyses, apoptosis assays and immunofluorescence analyses. The results showed that the protein and mRNA expression levels of ITGA6 was higher in breast cancer cells than in normal cells. ITGA6 protectived responses to radiotherapy in breast cancer cells by altering cell apoptosis, DNA damage repair and cell-cycle regulation. Furthermore, ITGA6 enhanced radiation resistance via PI3K/Akt and MEK/Erk signaling. In addition, overexpressing ITGA6 promoted radiation resistance in cells, and this effect was neutralized by the PI3K inhibitor LY294002 and MEK inhibitor U0126. Taken together, these findings indicate that ITGA6 might be involved in a mechanism that underlies radiation resistance and that ITGA6 could be a potential target for therapies aimed at overcoming radiation resistance in breast cancer. PMID:27624978

  7. Integrin α6/Akt/Erk signaling is essential for human breast cancer resistance to radiotherapy.

    PubMed

    Hu, Ting; Zhou, Rui; Zhao, Yanxia; Wu, Gang

    2016-01-01

    Integrin α6 (ITGA6), a transmembrane glycoprotein adhesion receptor protein, is widely upregulated in many types of tumors and promotes migration and invasion in cancer cells. However, the role that the ITGA6-associated signaling network plays in radiosensitivity in breast cancer has not been described. The expression of ITGA6 was examined in human breast cancer and normal breast cell lines using western blot analysis. We also explored the role of ITGA6 in the regulation of radiation sensitivity in breast cancer using the colony formation assays, cell cycle analyses, apoptosis assays and immunofluorescence analyses. The results showed that the protein and mRNA expression levels of ITGA6 was higher in breast cancer cells than in normal cells. ITGA6 protectived responses to radiotherapy in breast cancer cells by altering cell apoptosis, DNA damage repair and cell-cycle regulation. Furthermore, ITGA6 enhanced radiation resistance via PI3K/Akt and MEK/Erk signaling. In addition, overexpressing ITGA6 promoted radiation resistance in cells, and this effect was neutralized by the PI3K inhibitor LY294002 and MEK inhibitor U0126. Taken together, these findings indicate that ITGA6 might be involved in a mechanism that underlies radiation resistance and that ITGA6 could be a potential target for therapies aimed at overcoming radiation resistance in breast cancer. PMID:27624978

  8. PINCH proteins regulate cardiac contractility by modulating integrin-linked kinase-protein kinase B signaling.

    PubMed

    Meder, Benjamin; Huttner, Inken G; Sedaghat-Hamedani, Farbod; Just, Steffen; Dahme, Tillman; Frese, Karen S; Vogel, Britta; Köhler, Doreen; Kloos, Wanda; Rudloff, Jessica; Marquart, Sabine; Katus, Hugo A; Rottbauer, Wolfgang

    2011-08-01

    Integrin-linked kinase (ILK) is an essential component of the cardiac mechanical stretch sensor and is bound in a protein complex with parvin and PINCH proteins, the so-called ILK-PINCH-parvin (IPP) complex. We have recently shown that inactivation of ILK or β-parvin activity leads to heart failure in zebrafish via reduced protein kinase B (PKB/Akt) activation. Here, we show that PINCH proteins localize at sarcomeric Z disks and costameres in the zebrafish heart and skeletal muscle. To investigate the in vivo role of PINCH proteins for IPP complex stability and PKB signaling within the vertebrate heart, we inactivated PINCH1 and PINCH2 in zebrafish. Inactivation of either PINCH isoform independently leads to instability of ILK, loss of stretch-responsive anf and vegf expression, and progressive heart failure. The predominant cause of heart failure in PINCH morphants seems to be loss of PKB activity, since PKB phosphorylation at serine 473 is significantly reduced in PINCH-deficient hearts and overexpression of constitutively active PKB reconstitutes cardiac function in PINCH morphants. These findings highlight the essential function of PINCH proteins in controlling cardiac contractility by granting IPP/PKB-mediated signaling.

  9. Bi-directional signaling: extracellular matrix and integrin regulation of breast tumor progression.

    PubMed

    Gehler, Scott; Ponik, Suzanne M; Riching, Kristin M; Keely, Patricia J

    2013-01-01

    Cell transformation and tumor progression involve a common set of acquired capabilities, including increased proliferation, failure of cell death, self-sufficiency in growth, angiogenesis, and tumor cell invasion and metastasis. The stromal environment consists of many cell types and various extracellular matrix (ECM) proteins that support normal tissue maintenance and which have been implicated in tumor progression. Both the chemical and mechanical properties of the ECM have been shown to influence normal and malignant cell behavior. For instance, mesenchymal stem cells differentiate into specific lineages that are dependent on matrix stiffness, while tumor cells undergo changes in cell behavior and gene expression in response to matrix stiffness. ECM remodeling is implicated in tumor progression and can result in increased deposition of stromal ECM, enhanced contraction of ECM fibrils, and altered collagen alignment and ECM stiffness. Tumor cells respond to changes in ECM remodeling through altered intracellular signaling and cell cycle control that lead to enhanced proliferation, loss of normal tissue architecture, and local tumor cell migration and invasion. This review focuses on the bi-directional interplay between the mechanical properties of the ECM and integrin-mediated signal transduction events in an effort to elucidate cell behaviors during tumor progression.

  10. The Epithelial αvβ3-Integrin Boosts the MYD88-Dependent TLR2 Signaling in Response to Viral and Bacterial Components

    PubMed Central

    Gianni, Tatiana; Campadelli-Fiume, Gabriella

    2014-01-01

    TLR2 is a cell surface receptor which elicits an immediate response to a wide repertoire of bacteria and viruses. Its response is usually thought to be proinflammatory rather than an antiviral. In monocytic cells TLR2 cooperates with coreceptors, e.g. CD14, CD36 and αMβ2-integrin. In an earlier work we showed that αvβ3-integrin acts in concert with TLR2 to elicit an innate response to HSV, and to lipopolysaccharide. This response is characterized by production of IFN-α and -β, a specific set of cytokines, and NF-κB activation. We investigated the basis of the cooperation between αvβ3-integrin and TLR2. We report that β3-integrin participates by signaling through Y residues located in the C-tail, known to be involved in signaling activity. αvβ3-integrin boosts the MYD88-dependent TLR2 signaling and IRAK4 phosphorylation in 293T and in epithelial, keratinocytic and neuronal cell lines. The replication of ICP0minus HSV is greatly enhanced by DN versions of MYD88, of Akt – a hub of this pathway, or by β3integrin-silencing. αvβ3-integrin enables the recruitment of TLR2, MAL, MYD88 at lipid rafts, the platforms from where the signaling starts. The PAMP of the HSV-induced innate response is the gH/gL virion glycoprotein, which interacts with αvβ3-integrin and TLR2 independently one of the other, and cross-links the two receptors. Given the preferential distribution of αvβ3-integrin to epithelial cells, we propose that αvβ3-integrin serves as coreceptor of TLR2 in these cells. The results open the possibility that TLR2 makes use of coreceptors in a variety of cells to broaden its spectrum of activity and tissue specificity. PMID:25375272

  11. Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands

    PubMed Central

    Moulos, Panagiotis; Samiotaki, Martina; Panayotou, George; Dedos, Skarlatos G.

    2016-01-01

    The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands. PMID:27576083

  12. Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands.

    PubMed

    Moulos, Panagiotis; Samiotaki, Martina; Panayotou, George; Dedos, Skarlatos G

    2016-01-01

    The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands.

  13. Combinatorial signaling codes for the progressive determination of cell fates in the Drosophila embryonic mesoderm.

    PubMed

    Carmena, A; Gisselbrecht, S; Harrison, J; Jiménez, F; Michelson, A M

    1998-12-15

    Mesodermal progenitors arise in the Drosophila embryo from discrete clusters of lethal of scute (l'sc)-expressing cells. Using both genetic loss-of-function and targeted ectopic expression approaches, we demonstrate here that individual progenitors are specified by the sequential deployment of unique combinations of intercellular signals. Initially, the intersection between the Wingless (Wg) and Decapentaplegic (Dpp) expression domains demarcate an ectodermal prepattern that is imprinted on the adjacent mesoderm in the form of a L'sc precluster. All mesodermal cells within this precluster are competent to respond to a subsequent instructive signal mediated by two receptor tyrosine kinases (RTKs), the Drosophila epidermal growth factor receptor (DER) and the Heartless (Htl) fibroblast growth factor receptor. By monitoring the expression of the diphosphorylated form of mitogen-associated protein kinase (MAPK), we found that these RTKs are activated in small clusters of cells within the original competence domain. Each cluster represents an equivalence group because all members initially resemble progenitors in their expression of both L'sc and mesodermal identity genes. Thus, localized RTK activity induces the formation of mesodermal equivalence groups. The RTKs remain active in the single progenitor that emerges from each cluster under the subsequent inhibitory influence of the neurogenic genes. Moreover, DER and Htl are differentially involved in the specification of particular progenitors. We conclude that distinct cellular identity codes are generated by the combinatorial activities of Wg, Dpp, EGF, and FGF signals in the progressive determination of embryonic mesodermal cells.

  14. Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands.

    PubMed

    Moulos, Panagiotis; Samiotaki, Martina; Panayotou, George; Dedos, Skarlatos G

    2016-01-01

    The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands. PMID:27576083

  15. The Ig-ITIM superfamily member PECAM-1 regulates the "outside-in" signaling properties of integrin alpha(IIb)beta3 in platelets.

    PubMed

    Wee, Janet L; Jackson, Denise E

    2005-12-01

    Previous studies have implicated the immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) superfamily member platelet endothelial cell adhesion molecule-1 (PECAM-1) in the regulation of integrin function. While PECAM-1 has been demonstrated to play a role as an inhibitory coreceptor of immunoreceptor tyrosine-based activation motif (ITAM)-associated Fcgamma receptor IIa (FcgammaRIIa) and glycoprotein VI (GPVI)/FcR gamma-chain signaling pathways in platelets, its physiologic role in integrin alpha(IIb)beta3-mediated platelet function is unclear. In this study, we investigate the functional importance of PECAM-1 in murine platelets. Using PECAM-1-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta3 signaling with impaired platelet spreading on fibrinogen, failure to retract fibrin clots in vitro, and reduced tyrosine phosphorylation of focal adhesion kinase p125 (125FAK) following integrin alpha(IIb)beta3-mediated platelet aggregation. This functional integrin alpha(IIb)beta3 defect could not be attributed to altered expression of integrin alpha(IIb)beta3. PECAM-1-/- platelets displayed normal platelet alpha granule secretion, normal platelet aggregation to protease-activated receptor-4 (PAR-4), adenosine diphosphate (ADP), and calcium ionophore, and static platelet adhesion. In addition, PECAM-1-/- platelets displayed normal "inside-out" integrin alpha(IIb)beta3 signaling properties as demonstrated by normal agonist-induced binding of soluble fluoroscein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol (1,4,5)P3 triphosphate (IP3) levels. This study provides direct evidence that PECAM-1 is essential for normal integrin alpha(IIb)beta3-mediated platelet function and that disruption of PECAM-1 induced a moderate "outsidein" integrin alpha(IIb)beta3 signaling defect. PMID:16081692

  16. Integrin-based Therapeutics: Biological Basis, Clinical Use and New Drugs

    PubMed Central

    Ley, Klaus; Rivera-Nieves, Jesus; Sandborn, William J.; Shattil, Sanford

    2016-01-01

    Integrins are activatable adhesion and signaling molecules. Of the 24 known human integrins, three are currently targeted therapeutically by monoclonal antibodies, peptides or small molecules. The platelet αIIbβ3 integrin is targeted by Abciximab, Eptifibatide and Tirofiban, all with indications for preventing thrombotic complications after percutaneous coronary interventions. The lymphocyte α4β1 and α4β7 integrins are targeted by Natalizumab with indications in multiple sclerosis and Crohn’s disease. Although efficacious, use of this antibody is limited by a rare but serious complication, progressive multifocal leukoencephalopathy. Vedolizumab is an antibody to a combinatorial epitope in α4β7 that is approved for use in patients with Crohn’s disease or ulcerative colitis in the United States, Canada and Europe. Progressive multifocal leukoencephalopathy has not been observed in the clinical trials or clinical use of vedolizumab. New antibodies and small molecules targeting β7 integrins (α4β7 and αEβ7) and MAdCAM-1 are in clinical development for treatment of these inflammatory bowel diseases. Overall, integrin-based therapeutics have shown clinically significant benefits in many patients, leading to continued medical interest in the further development of novel integrin inhibitors. Of note, almost all integrin antagonists in use or in late-stage clinical trials target the ligand binding site, or the ligand itself. PMID:26822833

  17. Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

    NASA Technical Reports Server (NTRS)

    Pavalko, F. M.; Chen, N. X.; Turner, C. H.; Burr, D. B.; Atkinson, S.; Hsieh, Y. F.; Qiu, J.; Duncan, R. L.

    1998-01-01

    Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.

  18. Periostin Promotes Atrioventricular Mesenchyme Matrix Invasion and Remodeling Mediated by Integrin Signaling through Rho/PI 3-Kinase

    PubMed Central

    Butcher, Jonathan T.; Norris, Russell A.; Hoffman, Stanley; Mjaatvedt, Corey H.; Markwald, Roger R.

    2007-01-01

    Recent evidence suggests that extracellular matrix components may play a signaling role in embryonic valve development. We have previously identified the spatiotemporal expression patterns of periostin in developing valves, but its function during this process is largely unknown. To evaluate the functional role periostin plays during valvulogenesis, two separate three-dimensional culture assay systems, which model chick atrioventricular cushion development, were employed. These assays demonstrated that cushion mesenchymal cells adhered and spread on purified periostin in a dose responsive manner, similar to collagen I and fibronectin via αvβ3 and β1 integrin pairs. Periostin overexpression resulted in enhanced mesenchyme invasion through 3D collagen gels and increased matrix compaction. This invasion was dependent on αvβ3 more than β1 integrin signaling, and was mediated differentially by Rho kinase and PI 3-kinase. Both matrix invasion and compaction were associated with a colocalization of periostin and β1 integrin expression to migratory cell phenotype in both surface and deep cells. The Rho/PI 3-kinase pathway also differentially mediated matrix compaction. Both Rho and PI 3-kinase were involved in normal cushion mesenchyme matrix compaction, but only PI 3-kinase was required for the enhanced matrix compaction due to periostin. Taken together, these results highlight periostin as a mediator of matrix remodeling by cushion mesenchyme towards a mature valve structure. PMID:17070513

  19. Alteration of Pituitary Tumor Transforming Gene-1 Regulates Trophoblast Invasion via the Integrin/Rho-Family Signaling Pathway

    PubMed Central

    Lim, Seung Mook; Jang, Hee Yeon; Lee, Ji Eun; Shin, Joong Sik; Park, Sun-Hwa; Yoon, Bo Hyun; Kim, Gi Jin

    2016-01-01

    Trophoblast invasion ability is an important factor in early implantation and placental development. Recently, pituitary tumor transforming gene 1 (PTTG1) was shown to be involved in invasion and proliferation of cancer. However, the role of PTTG1 in trophoblast invasion remains unknown. Thus, in this study we analyzed PTTG1 expression in trophoblasts and its effect on trophoblast invasion activity and determined the mechanism through which PTTG1 regulates trophoblast invasion. Trophoblast proliferation and invasion abilities, regardless of PTTG1 expression, were analyzed by quantitative real-time polymerase chain reaction, fluorescence-activated cell sorting analysis, invasion assay, western blot, and zymography after treatment with small interfering RNA against PTTG1 (siPTTG1). Additionally, integrin/Rho-family signaling in trophoblasts by PTTG1 alteration was analyzed. Furthermore, the effect of PTTG1 on trophoblast invasion was evaluated by microRNA (miRNA) mimic and inhibitor treatment. Trophoblast invasion was significantly reduced through decreased matrix metalloproteinase (MMP)-2 and MMP-9 expression when PTTG1 expression was inhibited by siPTTG1 (p < 0.05). Furthermore, knockdown of PTTG1 increased expression of integrin alpha 4 (ITGA4), ITGA5, and integrin beta 1 (ITGB1); otherwise, RhoA expression was significantly decreased (p < 0.05). Treatment of miRNA-186-5p mimic and inhibitor controlled trophoblast invasion ability by altering PTTG1 and MMP expression. PTTG1 can control trophoblast invasion ability via regulation of MMP expression through integrin/Rho-family signaling. In addition, PTTG1 expression and its function were regulated by miRNA-186-5p. These results help in understanding the mechanism through which PTTG1 regulates trophoblast invasion and thereby implantation and placental development. PMID:26900962

  20. Osteoprotegerin (OPG) activates integrin, focal adhesion kinase (FAK), and Akt signaling in ovarian cancer cells to attenuate TRAIL-induced apoptosis

    PubMed Central

    2013-01-01

    Background Resistance to apoptosis is a major problem in ovarian cancer (OC) and correlates with poor prognosis. Osteoprotegerin (OPG) is a soluble secreted factor that acts as a decoy receptor for receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). OPG has been reported to attenuate TRAIL-induced apoptosis in a variety of cancer cells, including OC cells. OPG-mediated protection against TRAIL has been attributed to its decoy receptor function. However, OPG activates integrin/focal adhesion kinase (FAK) signaling in endothelial cells. In OC cells, activation of integrin/FAK signaling inhibits TRAIL-induced apoptosis. Based on these observations, we hypothesized that OPG could attenuate TRAIL-induced apoptosis in OC cells through integrin/FAK signaling. Methods In vitro experiments including immunoblots, colony formation assays, and apoptosis measurements were used to assess the effect of OPG on TRAIL-induced apoptosis. Results Exogenous OPG protected from TRAIL-induced apoptosis in a TRAIL binding-independent manner and OPG protection was αvβ3 and αvβ5 integrin/FAK signaling-dependent. Moreover, OPG-mediated activation of integrin/FAK signaling resulted in the activation of Akt. Inhibition of both integrin/FAK and Akt signaling significantly inhibited OPG-mediated attenuation of TRAIL-induced apoptosis. Although OPG also stimulated ERK1/2 phosphorylation, inhibition of ERK1/2 signaling did not significantly altered OPG protection. Conclusions Our studies provide evidence, for the first time, that OPG can attenuate TRAIL-induced apoptosis in a TRAIL binding-independent manner through the activation of integrin/FAK/Akt signaling in OC cells. PMID:24267510

  1. Roles of lipid rafts in integrin-dependent adhesion and gp130 signalling pathway in mouse embryonic neural precursor cells.

    PubMed

    Yanagisawa, Makoto; Nakamura, Kazuo; Taga, Tetsuya

    2004-09-01

    Neuronal and glial cells organizing the central nervous system are generated from common neural precursor cells present in the neuroepithelium during development. We tried to clarify functions of a cell surface microdomain, lipid raft, in neuroepithelial cells (NECs). NECs are suggested to adhere to fibronectin substratum dependently on integrin molecules. We found that beta1 integrin, a component of fibronectin receptors, was distributed in lipid rafts. Methyl-beta-cyclodextrin (MBCD), an inhibitor of lipid raft formation, inhibited the integrin-fibronectin interaction-dependent adhesion of NECs. However, inhibition of synthesis of glycosphingolipids (GSL), components of lipid rafts, did not affect NEC adhesion. Leukaemia inhibitory factor (LIF), an interleukin 6 type cytokine, induces astrocyte differentiation of NECs via activation of a transcription factor STAT3. We detected gp130, JAK1 and Ras but not STAT3 and ERK2 molecules in lipid rafts of NECs. Disruption of lipid rafts by MBCD inhibited LIF-induced ERK activation but not STAT3 activation. It is thus suggested that LIF-downstream molecules have differential lipid raft-dependency in terms of activation upon LIF-stimulation. In this study, we found functions of lipid rafts in cell adhesion and signal transduction in NECs. This is the first report that characterized functions of lipid rafts in embryonic neural precursor cells.

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

    PubMed

    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.

  3. Reelin promotes the adhesion and drug resistance of multiple myeloma cells via integrin β1 signaling and STAT3

    PubMed Central

    Lv, Meng; Liang, Xiaodong; Dai, Hui; Qin, Xiaodan; Zhang, Yan; Hao, Jie; Sun, Xiuyuan; Yin, Yanhui; Huang, Xiaojun; Zhang, Jun; Lu, Jin; Ge, Qing

    2016-01-01

    Reelin is an extracellular matrix (ECM) protein that is essential for neuron migration and positioning. The expression of reelin in multiple myeloma (MM) cells and its association with cell adhesion and survival were investigated. Overexpression, siRNA knockdown, and the addition of recombinant protein of reelin were used to examine the function of reelin in MM cells. Clinically, high expression of reelin was negatively associated with progression-free survival and overall survival. Functionally, reelin promoted the adhesion of MM cells to fibronectin via activation of α5β1 integrin. The resulting phosphorylation of Focal Adhesion Kinase (FAK) led to the activation of Src/Syk/STAT3 and Akt, crucial signaling molecules involved in enhancing cell adhesion and protecting cells from drug-induced cell apoptosis. These findings indicate reelin's important role in the activation of integrin-β1 and STAT3/Akt pathways in multiple myeloma and highlight the therapeutic potential of targeting reelin/integrin/FAK axis. PMID:26848618

  4. Ganglioside GM2 mediates migration of tumor cells by interacting with integrin and modulating the downstream signaling pathway.

    PubMed

    Kundu, Manjari; Mahata, Barun; Banerjee, Avisek; Chakraborty, Sohini; Debnath, Shibjyoti; Ray, Sougata Sinha; Ghosh, Zhumur; Biswas, Kaushik

    2016-07-01

    The definitive role of ganglioside GM2 in mediating tumor-induced growth and progression is still unknown. Here we report a novel role of ganglioside GM2 in mediating tumor cell migration and uncovered its mechanism. Data shows differential expression levels of GM2-synthase as well as GM2 in different human cancer cells. siRNA mediated knockdown of GM2-synthase in CCF52, A549 and SK-RC-26B cells resulted in significant inhibition of tumor cell migration as well as invasion in vitro without affecting cellular proliferation. Over-expression of GM2-synthase in low-GM2 expressing SK-RC-45 cells resulted in a consequent increase in migration thus confirming the potential role GM2 and its downstream partners play in tumor cell migration and motility. Further, treatment of SK-RC-45 cells with exogenous GM2 resulted in a dramatic increase in migratory and invasive capacity with no change in proliferative capacity, thereby confirming the role of GM2 in tumorigenesis specifically by mediating tumor migration and invasion. Gene expression profiling of GM2-synthase silenced cells revealed altered expression of several genes involved in cell migration primarily those controlling the integrin mediated signaling. GM2-synthase knockdown resulted in decreased phosphorylation of FAK, Src as well as Erk, while over-expression and/or exogenous GM2 treatment caused increased FAK and Erk phosphorylation respectively. Again, GM2 mediated invasion and Erk phosphorylation is blocked in integrin knockdown SK-RC-45 cells, thus confirming that GM2 mediated migration and phosphorylation of Erk is integrin dependent. Finally, confocal microscopy suggested co-localization while co-immunoprecipitation and surface plasmon resonance (SPR) confirmed direct interaction of membrane bound ganglioside, GM2 with the integrin receptor. PMID:27066976

  5. Ganglioside GM2 mediates migration of tumor cells by interacting with integrin and modulating the downstream signaling pathway.

    PubMed

    Kundu, Manjari; Mahata, Barun; Banerjee, Avisek; Chakraborty, Sohini; Debnath, Shibjyoti; Ray, Sougata Sinha; Ghosh, Zhumur; Biswas, Kaushik

    2016-07-01

    The definitive role of ganglioside GM2 in mediating tumor-induced growth and progression is still unknown. Here we report a novel role of ganglioside GM2 in mediating tumor cell migration and uncovered its mechanism. Data shows differential expression levels of GM2-synthase as well as GM2 in different human cancer cells. siRNA mediated knockdown of GM2-synthase in CCF52, A549 and SK-RC-26B cells resulted in significant inhibition of tumor cell migration as well as invasion in vitro without affecting cellular proliferation. Over-expression of GM2-synthase in low-GM2 expressing SK-RC-45 cells resulted in a consequent increase in migration thus confirming the potential role GM2 and its downstream partners play in tumor cell migration and motility. Further, treatment of SK-RC-45 cells with exogenous GM2 resulted in a dramatic increase in migratory and invasive capacity with no change in proliferative capacity, thereby confirming the role of GM2 in tumorigenesis specifically by mediating tumor migration and invasion. Gene expression profiling of GM2-synthase silenced cells revealed altered expression of several genes involved in cell migration primarily those controlling the integrin mediated signaling. GM2-synthase knockdown resulted in decreased phosphorylation of FAK, Src as well as Erk, while over-expression and/or exogenous GM2 treatment caused increased FAK and Erk phosphorylation respectively. Again, GM2 mediated invasion and Erk phosphorylation is blocked in integrin knockdown SK-RC-45 cells, thus confirming that GM2 mediated migration and phosphorylation of Erk is integrin dependent. Finally, confocal microscopy suggested co-localization while co-immunoprecipitation and surface plasmon resonance (SPR) confirmed direct interaction of membrane bound ganglioside, GM2 with the integrin receptor.

  6. Fibrin-mediated integrin signaling plays a critical role in hepatic regeneration after partial hepatectomy in mice

    PubMed Central

    Beier, Juliane I.; Guo, Luping; Joshi-Barve, Swati; Ritzenthaler, Jeffrey D.; Roman, Jesse; Arteel, Gavin E.

    2016-01-01

    Background The regenerative capacity of the liver is critical for proper responses to injury. Fibrin extracellular matrix (ECM) deposition is a common response to insult and contributes to inflammatory liver injury. However, the role of this matrix in hepatic regeneration has not been determined. Objective The purpose of this study was first to determine the role of fibrin ECM in hepatic regeneration followed by the role of the fibrin-binding αvβ3 integrin in mediating this effect. Material and methods C57BI/6J (WT) or PAI-1 knockout (KO) mice underwent 70% partial hepatectomy (PHx); plasma and histologic indices of regeneration were determined, as well as expression of key genes involved in hepatic regeneration. Results PHx promoted transient fibrin deposition by activating coagulation and concomitantly decreasing fibrinolysis. Inhibiting fibrin deposition, either by blocking thrombin (hirudin) in WT mice or by knocking out PAI-1, was associated with a decrease in hepatocyte proliferation after PHx. This strongly suggested a role for fibrin ECM in liver regeneration. To investigate if αvβ3 integrin mediates this action, we tested the effects of the anti-αvβ3 cyclic peptide RGDfV in animals after PHx. As was observed with inhibition of fibrin deposition, competitive inhibition of αvβ3 integrin delayed regeneration after PHx, while not affecting fibrin deposition. These effects of RGDfV correlated with impaired angiogenesis and STAT3 signaling, as well as transient endothelial dysfunction. In conclusion, these data suggest that αvβ3 integrin plays an important role in coordinating hepatocyte division during liver regeneration after PHx via crosstalk with fibrin ECM. PMID:27493116

  7. Epithelial polarity--generating and integrating signals from the ECM with integrins.

    PubMed

    Manninen, Aki

    2015-06-10

    Epithelial cells are important building blocks of most tissues and the corner stone of tissue architectures that allow directional transport of nutrients, ions and waste products in and out of the body. In tissues composed of millions of cells every individual cell needs to make right decisions when to differentiate, migrate, divide or die. Tight control of such fundamental cell-level processes ensures proper tissue morphogenesis, homeostasis and function. Cellular decisions are guided by biochemical and mechanical cues from their immediate microenvironment that consists of the extracellular matrix (ECM), neighboring cells and soluble factors. Generation of two distinct surfaces one facing the outside world (the apical domain) and the other contacting the neighboring cells and basal ECM (basolateral domain) is the most fundamental property of epithelial cells. The cues from the ECM are of particular importance in this process and communication between the cells and the ECM is largely mediated by transmembrane ECM receptors. Integrins constitute the largest family of such receptors binding to the ECM. Integrins have been shown to be essential for the establishment of initial polarity cues that define the position of the basal domain and thereby govern the orientation of the forming apico-basal axis. In this review I will discuss the multifaceted roles of integrins in epithelial cells with a particular focus on recent developments unveiling the specific functions of the different integrin heterodimers in regulating epithelial cell polarization and morphogenesis.

  8. Mutant p53 promotes ovarian cancer cell adhesion to mesothelial cells via integrin β4 and Akt signals

    PubMed Central

    Lee, Jong-Gyu; Ahn, Ji-Hye; Jin Kim, Tae; Ho Lee, Jae; Choi, Jung-Hye

    2015-01-01

    Missense mutations in the TP53 gene resulting in the accumulation of mutant proteins are extremely common in advanced ovarian cancer, which is characterised by peritoneal metastasis. Attachment of cancer cells to the peritoneal mesothelium is regarded as an initial, key step for the metastatic spread of ovarian cancer. In the present study, we investigated the possible role of a p53 mutant in the mesothelial adhesion of ovarian cancer cells. We found that OVCAR-3 cells with the R248 TP53 mutation (p53R248) were more adhesive to mesothelial Met5A cells than were A2780 cells expressing wild-type p53. In addition, ectopic expression of p53R248 in p53-null SKOV-3 cells significantly increased adhesion to Met5A cells. Knockdown of mutant p53 significantly compromised p53R248-induced cell adhesion to Met5A cells. Microarray analysis revealed that several adhesion-related genes, including integrin β4, were markedly up-regulated, and certain signalling pathways, including PI3K/Akt, were activated in p53R248 transfectants of SKOV-3 cells. Inhibition of integrin β4 and Akt signalling using blocking antibody and the inhibitor LY294002, respectively, significantly attenuated p53R248-mediated ovarian cancer-mesothelial adhesion. These data suggest that the p53R248 mutant endows ovarian cancer cells with increased adhesiveness and that integrin β4 and Akt signalling are associated with the mutation-enhanced ovarian cancer-mesothelial cell adhesion. PMID:26223322

  9. Mechanotransduction through Integrins

    NASA Technical Reports Server (NTRS)

    Ingber, Donald

    2004-01-01

    The goal of this project was to characterize the molecular mechanism by which cells recognize and respond to physical forces in their local environment. The project was based on the working hypothesis that cells sense mechanical stresses through cell surface integrin receptors and through their interconnections with the underlying cytoskeleton. Work completed and published in past funding period had provided direct support for this hypothesis. In particular, we demonstrated that application of mechanical stresses to activated integrin receptors (but not inactive integrins or other control transmembrane receptors) resulted in stress-dependent activation of the CAMP signaling pathway leading to gene transcription. We also showed that this form of mechanotransduction requires activation of heterotrimeric G proteins. In this grant, our specific aims included: 1) to characterize the signal processing capabilities of different integrins and other cell surface receptors, 2) to identify heterotrimeric G proteins that mediate CAMP signaling by stresses applied to integrins, 3) to identify molecules that mediate transmembrane mechanochemical coupling between integrins and G proteins, and 4) to use genome-wide gene expression profiling techniques to identify other genes and signaling pathways that are activated by mechanical forces transmitted over specific cell surface receptors. Elucidation of the mechanism by which cells sense mechanical stresses through integrins and translate them into a biochemical response should help us to understand the molecular basis of the cellular response to gravity as well as many other forms of mechanosensation and tissue regulation.

  10. Laminin and integrin expression in the ventral ectodermal ridge of the mouse embryo: implications for regulation of BMP signalling

    PubMed Central

    Lopez-Escobar, Beatriz; de Felipe, Beatriz; Sanchez-Alcazar, Jose Antonio; Sasaki, Takako; Copp, Andrew J.; Ybot-Gonzalez, Patricia

    2013-01-01

    Background The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail-bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear. Results We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co-expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell-matrix interactions regulating BMP signalling at this site of caudal morphogenesis. Conclusions Laminin532 could interact with α6-containing integrin to direct differentiation of the specialised VER cells from surface ectoderm. PMID:22911573

  11. Human liver sinusoidal endothelial cells respond to interaction with Entamoeba histolytica by changes in morphology, integrin signalling and cell death.

    PubMed

    Faust, Daniela M; Marquay Markiewicz, Jacques; Danckaert, Anne; Soubigou, Guillaume; Guillen, Nancy

    2011-07-01

    Invasive infection with Entamoeba histolytica causes intestinal and hepatic amoebiasis. In liver, parasites cross the endothelial barrier before abscess formation in the parenchyma. We focussed on amoebae interactions with human hepatic endothelial cells, the latter potentially playing a dual role in the infection process: as a barrier and as modulators of host defence responses. We characterized early responses of a human liver sinusoidal endothelial cell line to virulent and virulence-attenuated E. histolytica. Within the first minutes human cells start to retract, enter into apoptosis and die. In the presence of virulent amoebae, expression of genes related to cell cycle, cell death and integrin-mediated adhesion signalling was modulated, and actin fibre, focal adhesion kinase and paxillin localizations changed. Effects of inhibitors and amoeba strains not expressing pathogenic factors amoebapore A and cysteine protease A5 indicated that cell death and cytoskeleton disorganization depend upon parasite adhesion and amoebic cysteine proteinase activities. The data establish a relation between cytotoxic effects of E. histolytica and altered human target cell adhesion and suggest that interference with adhesion signalling triggers endothelial cell retraction and death. Understanding the roles of integrin signalling in endothelial cells will provide clues to unravel host-pathogen interactions during amoebic liver infection. PMID:21624031

  12. Collagen regulates transforming growth factor-β receptors of HL-1 cardiomyocytes through activation of stretch and integrin signaling.

    PubMed

    Lu, Yen-Yu; Lin, Yung-Kuo; Kao, Yu-Hsun; Chung, Cheng-Chih; Yeh, Yung-Hsin; Chen, Shih-Ann; Chen, Yi-Jen

    2016-10-01

    The extracellular matrix (ECM) and transforming growth factor-β (TGF)-β are important in cardiac fibrosis, however, the effects of the ECM on TGF‑β signaling remain to be fully elucidated. The aims of the present study were to evaluate the role of collagen in TGF‑β signaling and examine the underlying mechanisms. In the present study, western blot analysis was used to examine TGF‑β signaling in HL‑1 cells treated with and without (control) type I collagen (10 µg/ml), which was co‑administered with either an anti‑β1 integrin antibody (10 µg/ml) or a stretch‑activated channel inhibitor (gadolinium; 50 µM). Cell proliferation and adhesion assays were used to investigate the roles of integrin, mechanical stretch and mitogen‑activated protein kinases (MAPKs) on cell proliferation and adhesion. The type I collagen (10 µg/ml)‑treated HL‑1 cells were incubated with or without anti‑β1 integrin antibody (10 µg/ml), gadolinium (50 µM) or inhibitors of p38 (SB203580; 3 µM), extracellular signal‑regulated kinase (ERK; PD98059; 50 µM) and c‑Jun N‑terminal kinase (JNK; SP600125; 50 µM). Compared with the control cells, the collagen‑treated HL‑1 cells had lower expression levels of type I and type II TGF‑β receptors (TGFβRI and TGFβRII), with an increase in phosphorylated focal adhesion kinase (FAK), p38 and ERK1/2, and a decrease in JNK. Incubation with the anti‑β1 integrin antibody reversed the collagen‑induced downregulation of the expression of TGFβRII and phosphorylated FAK. Gadolinium downregulated the expression levels of TGFβRI and small mothers against decapentaplegic (Smad)2/3, and decreased the levels of phosphorylated p38, ERK1/2 and JNK. In addition, gadolinium reversed the collagen‑induced activation of p38 and ERK1/2. In the presence of gadolinium and anti‑β1 integrin antibody, collagen regulated the expression levels of TGFβRI, TGFβRII and Smad2/3, but did not alter the phosphorylation

  13. Stimulation of Odontogenesis and Angiogenesis via Bioactive Nanocomposite Calcium Phosphate Cements Through Integrin and VEGF Signaling Pathways.

    PubMed

    Lee, Sang-Im; Lee, Eui-Suk; El-Fiqi, Ahmed; Lee, So-Youn; Eun-Cheol Kim; Kim, Hae-Won

    2016-05-01

    Formulating self-setting calcium phosphate cements (CPCs) with secondary phases particularly in the nanoscale order holds great promise to improve biological properties. Here, we focus on the effect that bioactive glass nanoparticles (BGN) incorporated in CPC compositions can have on the proliferation, odontogenic differentiation, and angiogenic stimulation of stem cells derived from human dental pulp (HDPSCs). These odontogenic and angiogenic events are of special importance in the dentin-pulp regeneration processes. In comparison to pure CPCs, nanocomposite cements exhibit a significantly improved proliferation of HDPSCs, and the improvement is more significant as the BGN content increases. The nanocomposite cements substantially enhance the adhesion of cells, and significantly up-regulate odontogenic differentiation, including alkaline phosphatase (ALP) activity and the expressions of odontogenic genes (sialophosphoprotein, dentin matrix protein I, ALP, osteopontin and osteocalcin). Furthermore, the use of nanocomposite cements result in stimulation of angiogenic gene expression (VEGF, FGF-2, VEGFRs, PECAM-1, and VE-cadherin) and protein production (VEGF, VEGFR-1). The angiogenic stimulation by the HDPSCs significantly affects the endothelial cell behaviors, that is, the endothelial cell migration and the tubular network formation are substantially improved when treated with HDPSC-conditioned medium, particularly with the help of nanocomposite cements. The integrin and VEGF signaling pathways are reasoned for the stimulation of the odontogenesis and angiogenesis of cells, where the nanocomposite cements up-regulate the integrin subsets α1, α2, α3, and β1, and activate the integrin downstream signal pathways, such as p-FAK, p-Akt, p-paxillin, JNK, EK, and NF-κB, as well as other nuclear transcriptional factors, including CREB, STAT-3, and ELK-1. The current results indicate that the new formulation of the nanocomposite self-setting cements might provide some

  14. Stimulation of Odontogenesis and Angiogenesis via Bioactive Nanocomposite Calcium Phosphate Cements Through Integrin and VEGF Signaling Pathways.

    PubMed

    Lee, Sang-Im; Lee, Eui-Suk; El-Fiqi, Ahmed; Lee, So-Youn; Eun-Cheol Kim; Kim, Hae-Won

    2016-05-01

    Formulating self-setting calcium phosphate cements (CPCs) with secondary phases particularly in the nanoscale order holds great promise to improve biological properties. Here, we focus on the effect that bioactive glass nanoparticles (BGN) incorporated in CPC compositions can have on the proliferation, odontogenic differentiation, and angiogenic stimulation of stem cells derived from human dental pulp (HDPSCs). These odontogenic and angiogenic events are of special importance in the dentin-pulp regeneration processes. In comparison to pure CPCs, nanocomposite cements exhibit a significantly improved proliferation of HDPSCs, and the improvement is more significant as the BGN content increases. The nanocomposite cements substantially enhance the adhesion of cells, and significantly up-regulate odontogenic differentiation, including alkaline phosphatase (ALP) activity and the expressions of odontogenic genes (sialophosphoprotein, dentin matrix protein I, ALP, osteopontin and osteocalcin). Furthermore, the use of nanocomposite cements result in stimulation of angiogenic gene expression (VEGF, FGF-2, VEGFRs, PECAM-1, and VE-cadherin) and protein production (VEGF, VEGFR-1). The angiogenic stimulation by the HDPSCs significantly affects the endothelial cell behaviors, that is, the endothelial cell migration and the tubular network formation are substantially improved when treated with HDPSC-conditioned medium, particularly with the help of nanocomposite cements. The integrin and VEGF signaling pathways are reasoned for the stimulation of the odontogenesis and angiogenesis of cells, where the nanocomposite cements up-regulate the integrin subsets α1, α2, α3, and β1, and activate the integrin downstream signal pathways, such as p-FAK, p-Akt, p-paxillin, JNK, EK, and NF-κB, as well as other nuclear transcriptional factors, including CREB, STAT-3, and ELK-1. The current results indicate that the new formulation of the nanocomposite self-setting cements might provide some

  15. Essential roles of integrin-mediated signaling for the enhancement of malignant properties of melanomas based on the expression of GD3

    SciTech Connect

    Ohkawa, Yuki; Miyazaki, Sayaka; Miyata, Maiko; Hamamura, Kazunori; Furukawa, Koichi Furukawa, Keiko

    2008-08-15

    We reported that ganglioside GD3 enhances cell proliferation and invasion of melanomas causing stronger tyrosine-phosphorylation of p130Cas and paxillin after stimulation with fetal calf serum. Besides signals via growth factor/receptor, adhesion signals via integrin might be also enhanced by GD3. Here, roles of integrin-mediated signaling in the cell proliferation and invasion, and in the activation of adaptor molecules were examined, showing that integrin was also important for the cell growth and invasion. p130Cas and paxillin underwent stronger tyrosine-phosphorylation in GD3+ cells than in GD3- cells during the adhesion in the absence of serum. On the other hand, no proteins underwent tyrosine phosphorylation in GD3+ and GD3- cells in a suspension state when stimulated with fetal calf serum. These results suggested that integrin-mediated signaling is essential in the effects of GD3 on the malignant properties of melanomas. Co-localization of GD3 and integrin at the focal adhesion supported these results.

  16. Lunasin Sensitivity in Non-Small Cell Lung Cancer Cells Is Linked to Suppression of Integrin Signaling and Changes in Histone Acetylation

    PubMed Central

    Inaba, Junichi; McConnell, Elizabeth J.; Davis, Keith R.

    2014-01-01

    Lunasin is a plant derived bioactive peptide with both cancer chemopreventive and therapeutic activity. We recently showed lunasin inhibits non-small cell lung cancer (NSCLC) cell proliferation in a cell-line-specific manner. We now compared the effects of lunasin treatment of lunasin-sensitive (H661) and lunasin-insensitive (H1299) NSCLC cells with respect to lunasin uptake, histone acetylation and integrin signaling. Both cell lines exhibited changes in histone acetylation, with H661 cells showing a unique increase in H4K16 acetylation. Proximity ligation assays demonstrated lunasin interacted with integrins containing αv, α5, β1 and β3 subunits to a larger extent in the H661 compared to H1299 cells. Moreover, lunasin specifically disrupted the interaction of β1 and β3 subunits with the downstream signaling components phosphorylated Focal Adhesion Kinase (pFAK), Kindlin and Intergrin Linked Kinase in H661 cells. Immunoblot analyses demonstrated lunasin treatment of H661 resulted in reduced levels of pFAK, phosphorylated Akt and phosphorylated ERK1/2 whereas no changes were observed in H1299 cells. Silencing of αv expression in H661 cells confirmed signaling through integrins containing αv is essential for proliferation. Moreover, lunasin was unable to further inhibit proliferation in αv-silenced H661 cells. This indicates antagonism of integrin signaling via αv-containing integrins is an important component of lunasin’s mechanism of action. PMID:25530619

  17. Distinct Integrin-Dependent Signals Define Requirements for Lytic Granule Convergence and Polarization in Natural Killer Cells

    PubMed Central

    Hsu, Hsiang-Ting; Orange, Jordan S.

    2015-01-01

    Lytic granules in natural killer (NK) cells represent a dangerous cargo that is targeted for secretion to destroy diseased cells. The appropriate management of these organelles enables the mounting of a precise and valuable host defense. The process of NK cell adhesion to a target cell through engagement of the integrin LFA-1 (lymphocyte function–associated antigen 1) promotes lytic granule organization through complex cellular mechanics and a signaling pathway characterized by Zhang et al. in this issue of Science Signaling. A limited, partially overlapping set of signaling molecules can be distinguished for their ability to promote the convergence of NK cell lytic granules on the microtubule organizing center and their polarization as they progress en masse toward the interface with a target cell. PMID:25292212

  18. αv Integrins combine with LC3 and atg5 to regulate Toll-like receptor signalling in B cells.

    PubMed

    Acharya, Mridu; Sokolovska, Anna; Tam, Jenny M; Conway, Kara L; Stefani, Caroline; Raso, Fiona; Mukhopadhyay, Subhankar; Feliu, Marianela; Paul, Elahna; Savill, John; Hynes, Richard O; Xavier, Ramnik J; Vyas, Jatin M; Stuart, Lynda M; Lacy-Hulbert, Adam

    2016-01-01

    Integrin signalling triggers cytoskeletal rearrangements, including endocytosis and exocytosis of integrins and other membrane proteins. In addition to recycling integrins, this trafficking can also regulate intracellular signalling pathways. Here we describe a role for αv integrins in regulating Toll-like receptor (TLR) signalling by modulating intracellular trafficking. We show that deletion of αv or β3 causes increased B-cell responses to TLR stimulation in vitro, and αv-conditional knockout mice have elevated antibody responses to TLR-ligand-associated antigens. αv regulates TLR signalling by promoting recruitment of the autophagy component LC3 (microtubule-associated proteins 1 light chain 3) to TLR-containing endosomes, which is essential for progression from NF-κB to IRF signalling, and ultimately for traffic to lysosomes where signalling is terminated. Disruption of LC3 recruitment leads to prolonged NF-κB signalling and increased B-cell proliferation and antibody production. This work identifies a previously unrecognized role for αv and the autophagy components LC3 and atg5 in regulating TLR signalling and B-cell immunity. PMID:26965188

  19. αv Integrins combine with LC3 and atg5 to regulate Toll-like receptor signalling in B cells

    PubMed Central

    Acharya, Mridu; Sokolovska, Anna; Tam, Jenny M.; Conway, Kara L.; Stefani, Caroline; Raso, Fiona; Mukhopadhyay, Subhankar; Feliu, Marianela; Paul, Elahna; Savill, John; Hynes, Richard O.; Xavier, Ramnik J.; Vyas, Jatin M.; Stuart, Lynda M.; Lacy-Hulbert, Adam

    2016-01-01

    Integrin signalling triggers cytoskeletal rearrangements, including endocytosis and exocytosis of integrins and other membrane proteins. In addition to recycling integrins, this trafficking can also regulate intracellular signalling pathways. Here we describe a role for αv integrins in regulating Toll-like receptor (TLR) signalling by modulating intracellular trafficking. We show that deletion of αv or β3 causes increased B-cell responses to TLR stimulation in vitro, and αv-conditional knockout mice have elevated antibody responses to TLR-ligand-associated antigens. αv regulates TLR signalling by promoting recruitment of the autophagy component LC3 (microtubule-associated proteins 1 light chain 3) to TLR-containing endosomes, which is essential for progression from NF-κB to IRF signalling, and ultimately for traffic to lysosomes where signalling is terminated. Disruption of LC3 recruitment leads to prolonged NF-κB signalling and increased B-cell proliferation and antibody production. This work identifies a previously unrecognized role for αv and the autophagy components LC3 and atg5 in regulating TLR signalling and B-cell immunity. PMID:26965188

  20. β3 integrin-mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner.

    PubMed

    Fourel, Laure; Valat, Anne; Faurobert, Eva; Guillot, Raphael; Bourrin-Reynard, Ingrid; Ren, Kefeng; Lafanechère, Laurence; Planus, Emmanuelle; Picart, Catherine; Albiges-Rizo, Corinne

    2016-03-14

    Understanding how cells integrate multiple signaling pathways to achieve specific cell differentiation is a challenging question in cell biology. We have explored the physiological presentation of BMP-2 by using a biomaterial that harbors tunable mechanical properties to promote localized BMP-2 signaling. We show that matrix-bound BMP-2 is sufficient to induce β3 integrin-dependent C2C12 cell spreading by overriding the soft signal of the biomaterial and impacting actin organization and adhesion site dynamics. In turn, αvβ3 integrin is required to mediate BMP-2-induced Smad signaling through a Cdc42-Src-FAK-ILK pathway. β3 integrin regulates a multistep process to control first BMP-2 receptor activity and second the inhibitory role of GSK3 on Smad signaling. Overall, our results show that BMP receptors and β3 integrin work together to control Smad signaling and tensional homeostasis, thereby coupling cell adhesion and fate commitment, two fundamental aspects of developmental biology and regenerative medicine.

  1. Fibronectin-induced proliferation in thyroid cells is mediated by alphavbeta3 integrin through Ras/Raf-1/MEK/ERK and calcium/CaMKII signals.

    PubMed

    Illario, Maddalena; Cavallo, Anna Lina; Monaco, Sara; Di Vito, Ennio; Mueller, Frank; Marzano, Luigi A; Troncone, Giancarlo; Fenzi, Gianfranco; Rossi, Guido; Vitale, Mario

    2005-05-01

    We recently demonstrated in an immortalized thyroid cell line that integrin stimulation by fibronectin (FN) simultaneously activates two signaling pathways: Ras/Raf/MAPK kinase (Mek)/Erk and calcium Ca2+/calcium calmodulin-dependent kinase II (CaMKII). Both signals are necessary to stimulate Erk phosphorylation because CaMKII modulates Ras-induced Raf-1 activity. In this study we present evidence that extends these findings to normal human thyroid cells in primary culture, demonstrating its biological significance in a more physiological cell model. In normal thyroid cells, immobilized FN-induced activation of p21Ras and Erk phosphorylation. This pathway was responsible for FN-induced cell proliferation. Concurrent increase of intracellular Ca2+ concentration and CaMKII activation was observed. Both induction of p21Ras activity and increase of intracellular Ca2+ concentration were mediated by FN binding to alphavbeta3 integrin. Inhibition of the Ca2+/CaMKII signal pathway by calmodulin or CaMKII inhibitors completely abolished the FN-induced Erk phosphorylation. Binding to FN induced Raf-1 and CaMKII to form a protein complex, indicating that intersection between Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways occurred at Raf-1 level. Interruption of the Ca2+/CaMKII signal pathway arrested cell proliferation induced by FN. We also analyzed thyroid tumor cell lines that displayed concomitant aberrant integrin expression and signal transduction. These data confirm that integrin activation by FN in normal thyroid cells generates Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways and that both are necessary to stimulate cell proliferation, whereas in thyroid tumors integrin signaling is altered.

  2. Carbon nanotubes enhance intercalated disc assembly in cardiac myocytes via the β1-integrin-mediated signaling pathway.

    PubMed

    Sun, Hongyu; Lü, Shuanghong; Jiang, Xiao-Xia; Li, Xia; Li, Hong; Lin, Qiuxia; Mou, Yongchao; Zhao, Yuwei; Han, Yao; Zhou, Jin; Wang, Changyong

    2015-07-01

    Carbon nanotubes (CNTs) offer a new paradigm for constructing functional cardiac patches and repairing myocardial infarction (MI). However, little is known about how CNTs enhance the mechanical integrity and electrophysiological function of cardiac myocytes. To address this issue, we investigated the regularity and precise mechanism of the influence of CNTs on the assembly of intercalated disc (IDs). Here, single walled CNTs incorporated into collagen substrates were utilized as growth supports for neonatal cardiomyocytes, which enhanced cardiomyocyte adhesion and maturation. Furthermore, through the use of immunohistochemical staining, western blotting, transmission electron microscopy, and intracellular calcium transient measurement, we discovered that the addition of CNTs remarkably increased ID-related protein expression and enhanced ID assembly and functionality. On that basis, we further explored the underlying mechanism for how CNTs enhanced ID assembly through the use of immunohistochemical staining and western blotting. We found that the β1-integrin-mediated signaling pathway mediated CNT-induced upregulation of electrical and mechanical junction proteins. Notably, CNTs remarkably accelerated gap junction formation via activation of the β1-integrin-mediated FAK/ERK/GATA4 pathway. These findings provide valuable insight into the mechanistic effects that CNTs have on neonatal cardiomyocyte performance and will have a significant impact on the future of nanomedical research.

  3. Reinforcement of integrin-mediated T-Lymphocyte adhesion by TNF-induced Inside-out Signaling

    PubMed Central

    Li, Qian; Huth, Steven; Adam, Dieter; Selhuber-Unkel, Christine

    2016-01-01

    Integrin-mediated leukocyte adhesion to endothelial cells is a crucial step in immunity against pathogens. Whereas the outside-in signaling pathway in response to the pro-inflammatory cytokine tumour necrosis factor (TNF) has already been studied in detail, little knowledge exists about a supposed TNF-mediated inside-out signaling pathway. In contrast to the outside-in signaling pathway, which relies on the TNF-induced upregulation of surface molecules on endothelium, inside-out signaling should also be present in an endothelium-free environment. Using single-cell force spectroscopy, we show here that stimulating Jurkat cells with TNF significantly reinforces their adhesion to fibronectin in a biomimetic in vitro assay for cell-surface contact times of about 1.5 seconds, whereas for larger contact times the effect disappears. Analysis of single-molecule ruptures further demonstrates that TNF strengthens sub-cellular single rupture events at short cell-surface contact times. Hence, our results provide quantitative evidence for the significant impact of TNF-induced inside-out signaling in the T-lymphocyte initial adhesion machinery. PMID:27466027

  4. Reinforcement of integrin-mediated T-Lymphocyte adhesion by TNF-induced Inside-out Signaling.

    PubMed

    Li, Qian; Huth, Steven; Adam, Dieter; Selhuber-Unkel, Christine

    2016-01-01

    Integrin-mediated leukocyte adhesion to endothelial cells is a crucial step in immunity against pathogens. Whereas the outside-in signaling pathway in response to the pro-inflammatory cytokine tumour necrosis factor (TNF) has already been studied in detail, little knowledge exists about a supposed TNF-mediated inside-out signaling pathway. In contrast to the outside-in signaling pathway, which relies on the TNF-induced upregulation of surface molecules on endothelium, inside-out signaling should also be present in an endothelium-free environment. Using single-cell force spectroscopy, we show here that stimulating Jurkat cells with TNF significantly reinforces their adhesion to fibronectin in a biomimetic in vitro assay for cell-surface contact times of about 1.5 seconds, whereas for larger contact times the effect disappears. Analysis of single-molecule ruptures further demonstrates that TNF strengthens sub-cellular single rupture events at short cell-surface contact times. Hence, our results provide quantitative evidence for the significant impact of TNF-induced inside-out signaling in the T-lymphocyte initial adhesion machinery. PMID:27466027

  5. Reinforcement of integrin-mediated T-Lymphocyte adhesion by TNF-induced Inside-out Signaling

    NASA Astrophysics Data System (ADS)

    Li, Qian; Huth, Steven; Adam, Dieter; Selhuber-Unkel, Christine

    2016-07-01

    Integrin-mediated leukocyte adhesion to endothelial cells is a crucial step in immunity against pathogens. Whereas the outside-in signaling pathway in response to the pro-inflammatory cytokine tumour necrosis factor (TNF) has already been studied in detail, little knowledge exists about a supposed TNF-mediated inside-out signaling pathway. In contrast to the outside-in signaling pathway, which relies on the TNF-induced upregulation of surface molecules on endothelium, inside-out signaling should also be present in an endothelium-free environment. Using single-cell force spectroscopy, we show here that stimulating Jurkat cells with TNF significantly reinforces their adhesion to fibronectin in a biomimetic in vitro assay for cell-surface contact times of about 1.5 seconds, whereas for larger contact times the effect disappears. Analysis of single-molecule ruptures further demonstrates that TNF strengthens sub-cellular single rupture events at short cell-surface contact times. Hence, our results provide quantitative evidence for the significant impact of TNF-induced inside-out signaling in the T-lymphocyte initial adhesion machinery.

  6. DNA-mediated gold nanoparticle signal transducers for combinatorial logic operations and heavy metal ions sensing.

    PubMed

    Zhang, Yuhuan; Liu, Wei; Zhang, Wentao; Yu, Shaoxuan; Yue, Xiaoyue; Zhu, Wenxin; Zhang, Daohong; Wang, Yanru; Wang, Jianlong

    2015-10-15

    Herein, the structure of two DNA strands which are complementary except fourteen T-T and C-C mismatches was programmed for the design of the combinatorial logic operation by utilizing the different protective capacities of single chain DNA, part-hybridized DNA and completed-hybridized DNA on unmodified gold nanoparticles. In the presence of either Hg(2+) or Ag(+), the T-Hg(2+)-T or C-Ag(+)-C coordination chemistry could lead to the formation of part-hybridized DNA which keeps gold nanoparticles from clumping after the addition of 40 μL 0.2M NaClO4 solution, but the protection would be screened by 120 μL 0.2M NaClO4 solution. While the coexistence of Hg(2+), Ag(+) caused the formation of completed-hybridized DNA and the protection for gold nanoparticles lost in either 40 μL or 120 μL NaClO4 solutions. Benefiting from sharing of the same inputs of Hg(2+) and Ag(+), OR and AND logic gates were easily integrated into a simple colorimetric combinatorial logic operation in one system, which make it possible to execute logic gates in parallel to mimic arithmetic calculations on a binary digit. Furthermore, two other logic gates including INHIBIT1 and INHIBIT2 were realized to integrated with OR logic gate both for simultaneous qualitative discrimination and quantitative determination of Hg(2+) and Ag(+). Results indicate that the developed logic system based on the different protective capacities of DNA structure on gold nanoparticles provides a new pathway for the design of the combinatorial logic operation in one system and presents a useful strategy for development of advanced sensors, which may have potential applications in multiplex chemical analysis and molecular-scale computer design. PMID:25985196

  7. DNA-mediated gold nanoparticle signal transducers for combinatorial logic operations and heavy metal ions sensing.

    PubMed

    Zhang, Yuhuan; Liu, Wei; Zhang, Wentao; Yu, Shaoxuan; Yue, Xiaoyue; Zhu, Wenxin; Zhang, Daohong; Wang, Yanru; Wang, Jianlong

    2015-10-15

    Herein, the structure of two DNA strands which are complementary except fourteen T-T and C-C mismatches was programmed for the design of the combinatorial logic operation by utilizing the different protective capacities of single chain DNA, part-hybridized DNA and completed-hybridized DNA on unmodified gold nanoparticles. In the presence of either Hg(2+) or Ag(+), the T-Hg(2+)-T or C-Ag(+)-C coordination chemistry could lead to the formation of part-hybridized DNA which keeps gold nanoparticles from clumping after the addition of 40 μL 0.2M NaClO4 solution, but the protection would be screened by 120 μL 0.2M NaClO4 solution. While the coexistence of Hg(2+), Ag(+) caused the formation of completed-hybridized DNA and the protection for gold nanoparticles lost in either 40 μL or 120 μL NaClO4 solutions. Benefiting from sharing of the same inputs of Hg(2+) and Ag(+), OR and AND logic gates were easily integrated into a simple colorimetric combinatorial logic operation in one system, which make it possible to execute logic gates in parallel to mimic arithmetic calculations on a binary digit. Furthermore, two other logic gates including INHIBIT1 and INHIBIT2 were realized to integrated with OR logic gate both for simultaneous qualitative discrimination and quantitative determination of Hg(2+) and Ag(+). Results indicate that the developed logic system based on the different protective capacities of DNA structure on gold nanoparticles provides a new pathway for the design of the combinatorial logic operation in one system and presents a useful strategy for development of advanced sensors, which may have potential applications in multiplex chemical analysis and molecular-scale computer design.

  8. Integrating with integrins

    NASA Technical Reports Server (NTRS)

    Schwartz, M. A.; Ingber, D. E.

    1994-01-01

    Our central claim is that signaling by integrins provides a mechanism by which signals generated in response to adhesion, soluble hormones, and mechanical forces can interact. Such interactions permit cells to integrate these different classes of external stimuli and hence to orchestrate an efficient response. This integrating function of integrins is likely to be essential for much of development and physiology, as well as complex pathologies such as cancer. Understanding in detail how these signals are transduced and processed is likely to be an important area of research in the near future.

  9. Integrin CD11b positively regulates TLR4-induced signalling pathways in dendritic cells but not in macrophages.

    PubMed

    Ling, Guang Sheng; Bennett, Jason; Woollard, Kevin J; Szajna, Marta; Fossati-Jimack, Liliane; Taylor, Philip R; Scott, Diane; Franzoso, Guido; Cook, H Terence; Botto, Marina

    2014-01-01

    Tuned and distinct responses of macrophages and dendritic cells to Toll-like receptor 4 (TLR4) activation induced by lipopolysaccharide (LPS) underpin the balance between innate and adaptive immunity. However, the molecule(s) that confer these cell-type-specific LPS-induced effects remain poorly understood. Here we report that the integrin α(M) (CD11b) positively regulates LPS-induced signalling pathways selectively in myeloid dendritic cells but not in macrophages. In dendritic cells, which express lower levels of CD14 and TLR4 than macrophages, CD11b promotes MyD88-dependent and MyD88-independent signalling pathways. In particular, in dendritic cells CD11b facilitates LPS-induced TLR4 endocytosis and is required for the subsequent signalling in the endosomes. Consistent with this, CD11b deficiency dampens dendritic cell-mediated TLR4-triggered responses in vivo leading to impaired T-cell activation. Thus, by modulating the trafficking and signalling functions of TLR4 in a cell-type-specific manner CD11b fine tunes the balance between adaptive and innate immune responses initiated by LPS. PMID:24423728

  10. Integrin CD11b positively regulates TLR4-induced signalling pathways in dendritic cells but not in macrophages

    NASA Astrophysics Data System (ADS)

    Ling, Guang Sheng; Bennett, Jason; Woollard, Kevin J.; Szajna, Marta; Fossati-Jimack, Liliane; Taylor, Philip R.; Scott, Diane; Franzoso, Guido; Cook, H. Terence; Botto, Marina

    2014-01-01

    Tuned and distinct responses of macrophages and dendritic cells to Toll-like receptor 4 (TLR4) activation induced by lipopolysaccharide (LPS) underpin the balance between innate and adaptive immunity. However, the molecule(s) that confer these cell-type-specific LPS-induced effects remain poorly understood. Here we report that the integrin αM (CD11b) positively regulates LPS-induced signalling pathways selectively in myeloid dendritic cells but not in macrophages. In dendritic cells, which express lower levels of CD14 and TLR4 than macrophages, CD11b promotes MyD88-dependent and MyD88-independent signalling pathways. In particular, in dendritic cells CD11b facilitates LPS-induced TLR4 endocytosis and is required for the subsequent signalling in the endosomes. Consistent with this, CD11b deficiency dampens dendritic cell-mediated TLR4-triggered responses in vivo leading to impaired T-cell activation. Thus, by modulating the trafficking and signalling functions of TLR4 in a cell-type-specific manner CD11b fine tunes the balance between adaptive and innate immune responses initiated by LPS.

  11. WISP1-αvβ3 integrin signaling positively regulates TLR-triggered inflammation response in sepsis induced lung injury

    PubMed Central

    Chen, Zhixia; Ding, Xibing; Jin, Shuqing; Pitt, Bruce; Zhang, Liming; Billiar, Timothy; Li, Quan

    2016-01-01

    We recently noted that the matricellular protein WISP1 contributes to sepsis induced acute lung injury (ALI) via integrin β6. In the current study, we pursued further aspects of WISP1 modulation of TLR signaling in lungs of mice after sepsis and TLR4 mediated release of TNF-α in macrophages. After confirming that TLR4 and CD14 are critical in transducing sepsis mediated ALI, we now demonstrate that intrapulmonary αvβ3 is increased by polymicrobrial sepsis in a TLR4, CD14 dependent fashion. Comparison of cultured macrophages revealed that WISP1 increased release of TNF-α from RAW264.7 cells with baseline expression of αvβ3, but primary cultures of peritoneal macrophages (PMø) required activation of TLR4 to induce de novo synthesis of αvβ3 enabling WISP1 to stimulate release of TNF-α. The specific requirement for β3 integrin was apparent when the effect of WISP1 was lost in PMø isolated from β3−/− mice. WISP1 enhanced TLR4 mediated ERK signaling and U0126 (an ERK inhibitor) blocked LPS induced β3 integrin expression and WISP1 enhanced TNF-α release. Collectively these data suggest that WISP1-αvβ3 integrin signaling is involved in TLR4 pathways in macrophages and may be an important contributor to TLR4/CD14 mediated inflammation in sepsis induced lung injury. PMID:27349568

  12. A novel role for integrin-linked kinase in periodic mechanical stress-mediated ERK1/2 mitogenic signaling in rat chondrocytes.

    PubMed

    Song, Huanghe; Liang, Wenwei; Xu, Shun; Li, Zeng; Chen, Zhefeng; Cui, Weiding; Zhou, Jinchun; Wang, Qing; Liu, Feng; Fan, Weimin

    2016-07-01

    In recent years, a variety of studies have been performed to investigate the cellular responses of periodic mechanical stress on chondrocytes. Integrin β1-mediated ERK1/2 activation was proven to be indispensable in periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. However, other signal proteins responsible for the mitogenesis of chondrocytes under periodic mechanical stress remain incompletely understood. In the current investigation, we probed the roles of integrin-linked kinase (ILK) signaling in periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. We found that upon periodic mechanical stress induction, ILK activity increased significantly. Depletion of ILK with targeted shRNA strongly inhibited periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. In addition, pretreatment with a blocking antibody against integrin β1 resulted in a remarkable decrease in ILK activity in cells exposed to periodic mechanical stress. Furthermore, inhibition of ILK with its target shRNA significantly suppressed ERK1/2 activation in relation to periodic mechanical stress. Based on the above results, we identified ILK as a crucial regulator involved in the integrin β1-ERK1/2 signal cascade responsible for periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis.

  13. Silibinin inhibits fibronectin induced motility, invasiveness and survival in human prostate carcinoma PC3 cells via targeting integrin signaling.

    PubMed

    Deep, Gagan; Kumar, Rahul; Jain, Anil K; Agarwal, Chapla; Agarwal, Rajesh

    2014-10-01

    Prostate cancer (PCA) is the 2nd leading cause of cancer-related deaths among men in the United States. Preventing or inhibiting metastasis-related events through non-toxic agents could be a useful approach for lowering high mortality among PCA patients. We have earlier reported that natural flavonoid silibinin possesses strong anti-metastatic efficacy against PCA however, mechanism/s of its action still remains largely unknown. One of the major events during metastasis is the replacement of cell-cell interaction with integrins-based cell-matrix interaction that controls motility, invasiveness and survival of cancer cells. Accordingly, here we examined silibinin effect on advanced human PCA PC3 cells' interaction with extracellular matrix component fibronectin. Silibinin (50-200 μM) treatment significantly decreased the fibronectin (5 μg/ml)-induced motile morphology via targeting actin cytoskeleton organization in PC3 cells. Silibinin also decreased the fibronectin-induced cell proliferation and motility but significantly increased cell death in PC3 cells. Silibinin also inhibited the PC3 cells invasiveness in Transwell invasion assays with fibronectin or cancer associated fibroblasts (CAFs) serving as chemoattractant. Importantly, PC3-luc cells cultured on fibronectin showed rapid dissemination and localized in lungs following tail vein injection in athymic male nude mice; however, in silibinin-treated PC3-luc cells, dissemination and lung localization was largely compromised. Molecular analyses revealed that silibinin treatment modulated the fibronectin-induced expression of integrins (α5, αV, β1 and β3), actin-remodeling (FAK, Src, GTPases, ARP2 and cortactin), apoptosis (cPARP and cleaved caspase 3), EMT (E-cadherin and β-catenin), and cell survival (survivin and Akt) related signaling molecules in PC3 cells. Furthermore, PC3-xenograft tissue analyses confirmed the inhibitory effect of silibinin on fibronectin and integrins expression. Together, these

  14. CD151-α3β1 integrin complexes suppress ovarian tumor growth by repressing slug-mediated EMT and canonical Wnt signaling.

    PubMed

    Baldwin, Lauren A; Hoff, John T; Lefringhouse, Jason; Zhang, Michael; Jia, Changhe; Liu, Zeyi; Erfani, Sonia; Jin, Hongyan; Xu, Mei; She, Qing-Bai; van Nagell, John R; Wang, Chi; Chen, Li; Plattner, Rina; Kaetzel, David M; Luo, Jia; Lu, Michael; West, Dava; Liu, Chunming; Ueland, Fred R; Drapkin, Ronny; Zhou, Binhua P; Yang, Xiuwei H

    2014-12-15

    Human ovarian cancer is diagnosed in the late, metastatic stages but the underlying mechanisms remain poorly understood. We report a surprising functional link between CD151-α3β1 integrin complexes and the malignancy of serous-type ovarian cancer. Analyses of clinical specimens indicate that CD151 expression is significantly reduced or diminished in 90% of metastatic lesions, while it remains detectable in 58% of primary tumors. These observations suggest a putative tumor-suppressing role of CD151 in ovarian cancer. Indeed, our analyses show that knocking down CD151 or α3 integrin enhances tumor cell proliferation, growth and ascites production in nude mice. These changes are accompanied by impaired cell-cell contacts and aberrant expression of E-cadherin, Mucin 5AC and fibronectin, largely reminiscent of an epithelial to mesenchymal transition (EMT)-like change. Importantly, Slug, a master regulator of EMT, is markedly elevated. Knocking down Slug partially restores CD151-α3β1 integrin complex-dependent suppression of cell proliferation. Moreover, disruption of these adhesion protein complexes is accompanied by a concomitant activation of canonical Wnt signaling, including elevated levels of β-catenin and Axin-2 as well as resistance to the inhibition in β-catenin-dependent transcriptional complexes. Together, our study demonstrates that CD151-α3β1 integrin complexes regulate ovarian tumor growth by repressing Slug-mediated EMT and Wnt signaling. PMID:25356755

  15. CD151-α3β1 integrin complexes suppress ovarian tumor growth by repressing slug-mediated EMT and canonical Wnt signaling

    PubMed Central

    Zhang, Michael; Jia, Changhe; Liu, Zeyi; Erfani, Sonia; Jin, Hongyan; Xu, Mei; She, Qing-Bai; van Nagell, John R.; Wang, Chi; Chen, Li; Plattner, Rina; Kaetzel, David M.; Luo, Jia; Lu, Michael; West, Dava; Liu, Chunming; Ueland, Fred R.; Drapkin, Ronny; Zhou, Binhua P.; Yang, Xiuwei H.

    2014-01-01

    Human ovarian cancer is diagnosed in the late, metastatic stages but the underlying mechanisms remain poorly understood. We report a surprising functional link between CD151-α3β1 integrin complexes and the malignancy of serous-type ovarian cancer. Analyses of clinical specimens indicate that CD151 expression is significantly reduced or diminished in 90% of metastatic lesions, while it remains detectable in 58% of primary tumors. These observations suggest a putative tumor-suppressing role of CD151 in ovarian cancer. Indeed, our analyses show that knocking down CD151 or α3 integrin enhances tumor cell proliferation, growth and ascites production in nude mice. These changes are accompanied by impaired cell-cell contacts and aberrant expression of E-cadherin, Mucin 5AC and fibronectin, largely reminiscent of an epithelial to mesenchymal transition (EMT)-like change. Importantly, Slug, a master regulator of EMT, is markedly elevated. Knocking down Slug partially restores CD151-α3β1 integrin complex-dependent suppression of cell proliferation. Moreover, disruption of these adhesion protein complexes is accompanied by a concomitant activation of canonical Wnt signaling, including elevated levels of β-catenin and Axin-2 as well as resistance to the inhibition in β-catenin-dependent transcriptional complexes. Together, our study demonstrates that CD151-α3β1 integrin complexes regulate ovarian tumor growth by repressing Slug-mediated EMT and Wnt signaling. PMID:25356755

  16. Urinary-Type Plasminogen Activator Receptor (uPA/R)/α3β1 Integrin Signaling, Altered Gene Expression, and Oral Tumor Progression

    PubMed Central

    Ghosh, Supurna; Koblinski, Jennifer; Johnson, Jeffrey; Liu, Yueying; Ericsson, Aaron; Davis, J. Wade; Shi, Zonggao; Ravosa, Matthew J.; Crawford, Susan; Frazier, Shellaine; Stack, M. Sharon

    2009-01-01

    Oral squamous cell carcinoma (OSCC) has 50% 5-year survival rate, highlighting our limited understanding of the molecular events that contribute to disease progression. Microarray analyses of primary oral tumors have identified urinary type plasminogen activator (uPA) and its receptor (uPAR) as key genes associated with human OSCC progression. The uPAR functions both as a proteinase receptor and an integrin ligand, modifying proteolysis, migration, integrin signaling and cellular transcription. In the current study, uPAR expression levels were modified in OSCC cells, followed by analysis of tumor growth in an in vivo orthotopic xenograft model and by transcriptional profiling. Overexpression of uPAR resulted in more infiltrative and less differentiated tumors, with ill-defined borders, cytologic atypia, and enhanced vascularity. Analysis of serial sections of both murine experimental tumors and microarrayed human OSCC demonstrated a statistically significant association between uPAR and α3 integrin co-localization in areas exhibiting ERK phosphorylation, suggesting that uPAR/α3 integrin interaction potentiates ERK signaling in vivo. This is supported by cDNA microarray analysis which showed differential expression of 148 genes (113 up, 35 down). Validation of gene expression changes in human OSCC using immunohistochemistry and quantitative real-time PCR showed increased growth factors, proteinases/inhibitor and matrix components in uPAR-overexpressing tumors. Together these results support a model wherein increased uPAR expression promotes α3β1 integrin association, resulting in increased MAPK signaling and transcriptional activation, leading to the formation of more aggressive tongue tumors. This combined approach has efficacy to identify additional biomarkers and/or prognostic indicators associated with aggressive human OSCC. PMID:20145038

  17. Integrin traffic – the update

    PubMed Central

    De Franceschi, Nicola; Hamidi, Hellyeh; Alanko, Jonna; Sahgal, Pranshu; Ivaska, Johanna

    2015-01-01

    ABSTRACT Integrins are a family of transmembrane cell surface molecules that constitute the principal adhesion receptors for the extracellular matrix (ECM) and are indispensable for the existence of multicellular organisms. In vertebrates, 24 different integrin heterodimers exist with differing substrate specificity and tissue expression. Integrin–extracellular-ligand interaction provides a physical anchor for the cell and triggers a vast array of intracellular signalling events that determine cell fate. Dynamic remodelling of adhesions, through rapid endocytic and exocytic trafficking of integrin receptors, is an important mechanism employed by cells to regulate integrin–ECM interactions, and thus cellular signalling, during processes such as cell migration, invasion and cytokinesis. The initial concept of integrin traffic as a means to translocate adhesion receptors within the cell has now been expanded with the growing appreciation that traffic is intimately linked to the cell signalling apparatus. Furthermore, endosomal pathways are emerging as crucial regulators of integrin stability and expression in cells. Thus, integrin traffic is relevant in a number of pathological conditions, especially in cancer. Nearly a decade ago we wrote a Commentary in Journal of Cell Science entitled ‘Integrin traffic’. With the advances in the field, we felt it would be appropriate to provide the growing number of researchers interested in integrin traffic with an update. PMID:25663697

  18. Odontogenic Ameloblast-associated Protein (ODAM) Mediates Junctional Epithelium Attachment to Teeth via Integrin-ODAM-Rho Guanine Nucleotide Exchange Factor 5 (ARHGEF5)-RhoA Signaling.

    PubMed

    Lee, Hye-Kyung; Ji, Suk; Park, Su-Jin; Choung, Han-Wool; Choi, Youngnim; Lee, Hyo-Jung; Park, Shin-Young; Park, Joo-Cheol

    2015-06-01

    Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown. We investigated ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontitis and peri-implantitis. ODAM was expressed in the normal JE of healthy teeth but absent in the pathologic pocket epithelium of diseased periodontium. In periodontitis and peri-implantitis, ODAM was extruded from the JE following onset with JE attachment loss and detected in gingival crevicular fluid. ODAM induced RhoA activity and the expression of downstream factors, including ROCK (Rho-associated kinase), by interacting with Rho guanine nucleotide exchange factor 5 (ARHGEF5). ODAM-mediated RhoA signaling resulted in actin filament rearrangement. Reduced ODAM and RhoA expression in integrin β3- and β6-knockout mice revealed that cytoskeleton reorganization in the JE occurred via integrin-ODAM-ARHGEF5-RhoA signaling. Fibronectin and laminin activated RhoA signaling via the integrin-ODAM pathway. Finally, ODAM was re-expressed with RhoA in regenerating JE after gingivectomy in vivo. These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. We also propose that ODAM could be used as a biomarker of periodontitis and peri-implantitis.

  19. Biosynthesis of B2-integrin, intracellular calcium signalling and functional responses of normal and CD18-deficient bovine neutrophils.

    PubMed

    Nagahata, H; Higuchi, H; Nochi, H; Tamoto, K; Araiso, T; Noda, H; Kociba, G J

    1996-09-01

    1Biosynthesis of CD11/CD18 in bovine leucocytes, intracellular Ca2+ ([Ca2+]i) signalling, chemiluminescent responses and membrane fluidity of neutrophils and the effects of D-mannose on neutrophils from control heifers and a heifer with bovine leucocyte adhesion deficiency (BLAD) were measured. The synthesis of CD11/CD18 complex was clearly detected in leucocytes from a normal heifer, but not in a BLAD-affected heifer. The transient phase of increased [Ca2+]i was clearly detected in neutrophils from a heifer with BLAD stimulated with opsonised zymosan, aggregated bovine immunoglobulin G or concanavalin A, whereas the sustained phase was deficient or significantly decreased compared with control heifers. [Ca2+]i signalling of neutrophils from control heifers and a heifer with BLAD stimulated with phorbol myristate acetate via an 11b/CD18-independent pathway showed no transient phase, and the subsequent increase in [Ca2+]i was almost identical in neutrophils from affected and control heifers. [Ca2+]i concentration and chemiluminescent responses of neutrophils from a control heifer were clearly decreased by treatment with anti-CD18 and anti-IgG antibodies. No differences in membrane fluidity were detected between neutrophils derived from control and CD18-deficient cattle. D-mannose binds mainly to Fc rather than CD18 receptors, and decreased Agg-IgG induced [Ca2+]i and the chemiluminescent response of neutrophils. The [Ca2+]i responses and Agg-IgG induced chemiluminescent responses of neutrophils from control heifers and a BLAD-affected heifer were inhibited by D-mannose. The characteristic changes of [Ca2+]i signalling and functional responses of B2-integrin-deficient neutrophils were demonstrated. PMID:8880976

  20. Chronic TGFβ stimulation promotes the metastatic potential of lung cancer cells by Snail protein stabilization through integrin β3-Akt-GSK3β signaling

    PubMed Central

    Park, Jeong-Rak; Kwon, Ok-Seon; Kim, Keun-Tae; Koo, JaeHyung; Oh, Ensel; Cha, Hyuk-Jin

    2016-01-01

    Chronic exposure to TGFβ, a frequent occurrence for tumor cells in the tumor microenvironment, confers more aggressive phenotypes on cancer cells by promoting their invasion and migration while at the same time increasing their resistance to the growth-inhibitory effect of TGFβ. In this study, a transdifferentiated (TD) A549 cell model, established by chronically exposing A549 cells to TGFβ, showed highly invasive phenotypes in conjunction with attenuation of Smad-dependent signaling. We show that Snail protein, the mRNA expression of which strongly correlates with a poor prognosis in lung cancer patients, was highly stable in TD cells after TGFβ stimulation. The increased protein stability of Snail in TD cells correlated with elevated inhibitory phosphorylation of GSK3β, resulting from the high Akt activity. Notably, integrin β3, whose expression was markedly increased upon sustained exposure to TGFβ, was responsible for the high Akt activity as well as the increased Snail protein stability in TD cells. Consistently, clinical database analysis on lung cancer patients revealed a negative correlation between overall survival and integrin β3 mRNA levels. Therefore, we suggest that the integrin β3-Akt-GSK3β signaling axis plays an important role in non-canonical TGFβ signaling, determining the invasive properties of tumor cells chronically exposed to TGFβ. PMID:27015122

  1. Fgfr1 regulates development through the combinatorial use of signaling proteins

    PubMed Central

    Brewer, J. Richard; Molotkov, Andrei; Mazot, Pierre; Hoch, Renée V.; Soriano, Philippe

    2015-01-01

    Fibroblast growth factor (Fgf) signaling governs multiple processes important in development and disease. Many lines of evidence have implicated Erk1/2 signaling induced through Frs2 as the predominant effector pathway downstream from Fgf receptors (Fgfrs), but these receptors can also signal through other mechanisms. To explore the functional significance of the full range of signaling downstream from Fgfrs in mice, we engineered an allelic series of knock-in point mutations designed to disrupt Fgfr1 signaling functions individually and in combination. Analysis of each mutant indicates that Frs2 binding to Fgfr1 has the most pleiotropic functions in development but also that the receptor uses multiple proteins additively in vivo. In addition to Frs2, Crk proteins and Plcγ also contribute to Erk1/2 activation, affecting axis elongation and craniofacial and limb development and providing a biochemical mechanism for additive signaling requirements. Disruption of all known signaling functions diminished Erk1/2 and Plcγ activation but did not recapitulate the peri-implantation Fgfr1-null phenotype. This suggests that Erk1/2-independent signaling pathways are functionally important for Fgf signaling in vivo. PMID:26341559

  2. Modified Panax ginseng Extract Inhibits uPAR-Mediated α[Formula: see text]β1-Integrin Signaling by Modulating Caveolin-1 to Induce Early Apoptosis in Lung Cancer Cells.

    PubMed

    Hwang, In-Hu; Kwon, Yong-Kyun; Cho, Chong-Kwan; Lee, Yeon-Weol; Sung, Jung-Suk; Joo, Jong-Cheon; Lee, Kyung-Bok; Yoo, Hwa-Seung; Jang, Ik-Soon

    2016-01-01

    Urokinase receptor (uPAR) is enhanced in many human cancer cells and is frequently an indicator of poor prognosis. Activation of [Formula: see text]1-integrin requires caveolin-1 and is regulated by uPAR. However, the underlying molecular mechanism responsible for the interaction between uPAR and [Formula: see text]1-integrin remains obscure. We found that modified regular Panax ginseng extract (MRGX) had a negative modulating effect on the uPAR/[Formula: see text]1-integrin interaction, disrupted the uPAR/integrin interaction by modulating caveoline-1, and caused early apoptosis in cancer cells. Additionally, we found that siRNA-mediated caveoline-1 downregulation inhibited uPAR-mediated [Formula: see text]1-integrin signaling, whereas caveoline-1 up-regulation stimulated the signaling, which suppressed p53 expression, thereby indicating negative crosstalk exists between the integrin [Formula: see text]1 and the p53 pathways. Thus, these findings identify a novel mechanism whereby the inhibition of [Formula: see text]1 integrin and the activation of p53 modulate the expression of the anti-apoptotic proteins that are crucially involved in inducing apoptosis in A549 lung cancer cells. Furthermore, MRGX causes changes in the expressions of members of the Bcl-2 family (Bax and Bcl-2) in a pro-apoptotic manner. In addition, MGRX-mediated inhibition of [Formula: see text]1 integrin attenuates ERK phosphorylation (p-ERK), which up-regulates caspase-8 and Bax. Therefore, ERK may affect mitochondria through a negative regulation of caspase-8 and Bax. Taken together, these findings reveal that MRGX is involved in uPAR-[Formula: see text]1-integrin signaling by modulating caveolin-1 signaling to induce early apoptosis in A549 lung-cancer cells and strongly indicate that MRGX might be useful as a herbal medicine and may lead to the development of new herbal medicine that would suppress the growth of lung-cancer cells.

  3. Magnetic Nanocomposite Scaffold-Induced Stimulation of Migration and Odontogenesis of Human Dental Pulp Cells through Integrin Signaling Pathways.

    PubMed

    Yun, Hyung-Mun; Lee, Eui-Suk; Kim, Mi-joo; Kim, Jung-Ju; Lee, Jung-Hwan; Lee, Hae-Hyoung; Park, Kyung-Ran; Yi, Jin-Kyu; Kim, Hae-Won; Kim, Eun-cheol

    2015-01-01

    Magnetism is an intriguing physical cue that can alter the behaviors of a broad range of cells. Nanocomposite scaffolds that exhibit magnetic properties are thus considered useful 3D matrix for culture of cells and their fate control in repair and regeneration processes. Here we produced magnetic nanocomposite scaffolds made of magnetite nanoparticles (MNPs) and polycaprolactone (PCL), and the effects of the scaffolds on the adhesion, growth, migration and odontogenic differentiation of human dental pulp cells (HDPCs) were investigated. Furthermore, the associated signaling pathways were examined in order to elucidate the molecular mechanisms in the cellular events. The magnetic scaffolds incorporated with MNPs at varying concentrations (up to 10%wt) supported cellular adhesion and multiplication over 2 weeks, showing good viability. The cellular constructs in the nanocomposite scaffolds played significant roles in the stimulation of adhesion, migration and odontogenesis of HDPCs. Cells were shown to adhere to substantially higher number when affected by the magnetic scaffolds. Cell migration tested by in vitro wound closure model was significantly enhanced by the magnetic scaffolds. Furthermore, odontogenic differentiation of HDPCs, as assessed by the alkaline phosphatase activity, mRNA expressions of odontogenic markers (DMP-1, DSPP,osteocalcin, and ostepontin), and alizarin red staining, was significantly stimulated by the magnetic scaffolds. Signal transduction was analyzed by RT-PCR, Western blotting, and confocal microscopy. The magnetic scaffolds upregulated the integrin subunits (α1, α2, β1 and β3) and activated downstream pathways, such as FAK, paxillin, p38, ERK MAPK, and NF-κB. The current study reports for the first time the significant impact of magnetic scaffolds in stimulating HDPC behaviors, including cell migration and odontogenesis, implying the potential usefulness of the magnetic scaffolds for dentin-pulp tissue engineering.

  4. Binding of Alphaherpesvirus Glycoprotein H to Surface α4β1-Integrins Activates Calcium-Signaling Pathways and Induces Phosphatidylserine Exposure on the Plasma Membrane

    PubMed Central

    Gramatica, Andrea; Herrmann, Andreas; Osterrieder, Nikolaus

    2015-01-01

    ABSTRACT Intracellular signaling connected to integrin activation is known to induce cytoplasmic Ca2+ release, which in turn mediates a number of downstream signals. The cellular entry pathways of two closely related alphaherpesviruses, equine herpesviruses 1 and 4 (EHV-1 and EHV-4), are differentially regulated with respect to the requirement of interaction of glycoprotein H (gH) with α4β1-integrins. We show here that binding of EHV-1, but not EHV-4, to target cells resulted in a rapid and significant increase in cytosolic Ca2+ levels. EHV-1 expressing EHV-4 gH (gH4) in lieu of authentic gH1 failed to induce Ca2+ release, while EHV-4 with gH1 triggered significant Ca2+ release. Blocking the interaction between gH1 and α4β1-integrins, inhibiting phospholipase C (PLC) activation, or blocking binding of inositol 1,4,5-triphosphate (IP3) to its receptor on the endoplasmic reticulum (ER) abrogated Ca2+ release. Interestingly, phosphatidylserine (PS) was exposed on the plasma membrane in response to cytosolic calcium increase after EHV-1 binding through a scramblase-dependent mechanism. Inhibition of both Ca2+ release from the ER and scramblase activation blocked PS scrambling and redirected virus entry to the endocytic pathway, indicating that PS may play a role in facilitating virus entry directly at the plasma membrane. PMID:26489864

  5. Extracellular signal-regulated kinase and c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts.

    PubMed Central

    MacKenna, D A; Dolfi, F; Vuori, K; Ruoslahti, E

    1998-01-01

    Integrins, which connect the cytoskeleton to the extracellular matrix and mediate a variety of signaling cascades, may transduce mechanical stimuli into biochemical signals. We studied integrin- and matrix-dependent activation of extracellular signal-regulated kinase (ERK2), c-Jun NH2-terminal kinase (JNK1), and p38 in response to 4% static biaxial stretch in rat cardiac fibroblasts. ERK2 and JNK1, but not p38, were rapidly activated by stretch when the fibroblasts were allowed to synthesize their own matrices. When the cells were limited to specific matrix substrates, ERK2 and JNK1 were differentially activated: ERK2 was only activated when the cells were plated on fibronectin, while JNK1 was activated when the cells were plated on fibronectin, vitronectin, or laminin. Plating cells on collagen before stretching did not activate either kinase. Adhesion to all matrices was integrin-dependent because it could be blocked by inhibitors of specific integrins. ERK2 activation could be blocked with a combination of anti-alpha4 and -alpha5 antibodies and an arginine-glycine-aspartic acid (RGD) peptide, while the antibodies or peptide used separately failed to block ERK2 activation. This result suggests that at least two integrins, alpha4beta1 and an RGD-directed, non-alpha5beta1 integrin, activate ERK2 in response to mechanical stimulation. Activation of JNK1 could not be blocked with the inhibitors, suggesting that an RGD-independent integrin or integrins other than alpha4beta1 can activate JNK1 in cells adherent to fibronectin. This study demonstrates that integrins act as mechanotransducers, providing insight into potential mechanisms for in vivo responses to mechanical stimuli. PMID:9435301

  6. Phosphorylation and glycosylation interplay: protein modifications at hydroxy amino acids and prediction of signaling functions of the human beta3 integrin family.

    PubMed

    Ahmad, Ishtiaq; Hoessli, Daniel C; Walker-Nasir, Evelyne; Choudhary, M Iqbal; Rafik, Saleem M; Shakoori, Abdul Rauf

    2006-10-15

    Protein functions are determined by their three-dimensional structures and the folded 3-D structure is in turn governed by the primary structure and post-translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post-translational modifications, like phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer-assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi-directional (outside-in and inside-out) signaling events. The beta3 integrin family, including, alpha(IIb)beta3 and alpha(v)beta3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the beta3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside-out and outside-in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin-associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay.

  7. Cyclooxygenase-2 induced β1-integrin expression in NSCLC and promoted cell invasion via the EP1/MAPK/E2F-1/FoxC2 signal pathway

    PubMed Central

    Pan, Jinshun; Yang, Qinyi; Shao, Jiaofang; Zhang, Li; Ma, Juan; Wang, Yipin; Jiang, Bing-Hua; Leng, Jing; Bai, Xiaoming

    2016-01-01

    Cyclooxygenase-2 (COX-2) has been implicated in cell invasion in non-small-cell lung cancer (NSCLC). However, the mechanism is unclear. The present study investigated the effect of COX-2 on β1-integrin expression and cell invasion in NSCLC. COX-2 and β1-integrin were co-expressed in NSCLC tissues. COX-2 overexpression or Prostaglandin E2 (PGE2) treatment increased β1-integrin expression in NSCLC cell lines. β1-integrin silencing suppressed COX-2-mediated tumour growth and cancer cell invasion in vivo and in vitro. Prostaglandin E Receptor EP1 transfection or treatment with EP1 agonist mimicked the effect of PGE2 treatment. EP1 siRNA blocked PGE2-mediated β1-integrin expression. EP1 agonist treatment promoted Erk1/2, p38 phosphorylation and E2F-1 expression. MEK1/2 and p38 inhibitors suppressed EP1-mediated β1-integrin expression. E2F-1 silencing suppressed EP1-mediated FoxC2 and β1-integrin upregulation. ChIP and Luciferase Reporter assays identified that EP1 agonist treatment induced E2F-1 binding to FoxC2 promotor directly and improved FoxC2 transcription. FoxC2 siRNA suppressed β1-integrin expression and EP1-mediated cell invasion. Immunohistochemistry showed E2F-1, FoxC2, and EP1R were all highly expressed in the NSCLC cases. This study suggested that COX-2 upregulates β1-integrin expression and cell invasion in NSCLC by activating the MAPK/E2F-1 signalling pathway. Targeting the COX-2/EP1/PKC/MAPK/E2F-1/FoxC2/β1-integrin pathway might represent a new therapeutic strategy for the prevention and treatment of this cancer. PMID:27654511

  8. Cell cycle arrest or survival signaling through αv integrins, activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids.

    PubMed

    Carduner, Ludovic; Picot, Cédric R; Leroy-Dudal, Johanne; Blay, Lyvia; Kellouche, Sabrina; Carreiras, Franck

    2014-01-15

    Ovarian cancer is the most lethal gynecologic cancer mainly due to spheroids organization of cancer cells that disseminate within the peritoneal cavity. We have investigated the molecular mechanisms by which ovarian cancer spheroids resist anoikis, choosing as models the 2 well-characterized human ovarian cancer cell lines IGROV1 and SKOV3. These cell lines have the propensity to float as clusters, and were isolated from tumor tissue and ascites, respectively. To form spheroids, IGROV1 and SKOV3 ovarian adenocarcinoma cells were maintained under anchorage-independent culture conditions, in which both lines survive at least a week. A short apoptotic period prior to a survival signaling commitment was observed for IGROV1 cells whereas SKOV3 cells entered G0/G1 phase of the cell cycle. This difference in behavior was due to different signals. With regard to SKOV3 cells, activation of p38 and an increase in p130/Rb occurred once anchorage-independent culture was established. Analyses of the survival signaling pathway switched on by IGROV1 cells showed that activation of ERK1/2 was required to evade apoptosis, an effect partly dependent on PKC activation and αv integrins. αv-integrin expression is essential for survival through activation of ERK1/2 phosphorylation. The above data indicate that ovarian cancer cells can resist anoikis in the spheroid state by arrest in the cell cycle or through activation of αv-integrin-ERK-mediated survival signals. Such signaling might result in the selection of resistant cells within disseminating spheroids, favoring further relapse in ovarian cancers. PMID:24291221

  9. Signal-dependent Slow Leukocyte Rolling Does Not Require Cytoskeletal Anchorage of P-selectin Glycoprotein Ligand-1 (PSGL-1) or Integrin αLβ2*

    PubMed Central

    Shao, Bojing; Yago, Tadayuki; Coghill, Phillip A.; Klopocki, Arkadiusz G.; Mehta-D'souza, Padmaja; Schmidtke, David W.; Rodgers, William; McEver, Rodger P.

    2012-01-01

    In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin αLβ2 in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate αLβ2, leading to arrest on ICAM-1. Cytoskeletal anchorage of αLβ2 has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether αLβ2 must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal β2 integrin-dependent slow rolling on P-selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the β2-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, αLβ2-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, αLβ2-mediated arrest. PMID:22511754

  10. Combinatorial Fgf and Bmp signalling patterns the gastrula ectoderm into prospective neural and epidermal domains

    PubMed Central

    Kudoh, Tetsuhiro; Concha, Miguel L.; Houart, Corinne; Dawid, Igor B.; Wilson, Stephen W.

    2009-01-01

    Summary Studies in fish and amphibia have shown that graded Bmp signalling activity regulates dorsal-to-ventral (DV) patterning of the gastrula embryo. In the ectoderm, it is thought that high levels of Bmp activity promote epidermal development ventrally, whereas secreted Bmp antagonists emanating from the organiser induce neural tissue dorsally. However, in zebrafish embryos, the domain of cells destined to contribute to the spinal cord extends all the way to the ventral side of the gastrula, a long way from the organiser. We show that in vegetal (trunk and tail) regions of the zebrafish gastrula, neural specification is initiated at all DV positions of the ectoderm in a manner that is unaffected by levels of Bmp activity and independent of organiser-derived signals. Instead, we find that Fgf activity is required to induce vegetal prospective neural markers and can do so without suppressing Bmp activity. We further show that Bmp signalling does occur within the vegetal prospective neural domain and that Bmp activity promotes the adoption of caudal fate by this tissue. PMID:15262889

  11. Liquid-diet with alcohol alters maternal, fetal and placental weights and the expression of molecules involved in integrin signaling in the fetal cerebral cortex.

    PubMed

    Rout, Ujjwal K; Dhossche, Julie M

    2010-11-01

    Maternal alcohol consumption during pregnancy causes wide range of behavioral and structural deficits in children, commonly known as Fetal Alcohol Syndrome (FAS). Children with FAS may suffer behavioral deficits in the absence of obvious malformations. In rodents, the exposure to alcohol during gestation changes brain structures and weights of offspring. The mechanism of FAS is not completely understood. In the present study, an established rat (Long-Evans) model of FAS was used. The litter size and the weights of mothers, fetuses and placentas were examined on gestation days 18 or 20. On gestation day 18, the effects of chronic alcohol on the expression levels of integrin receptor subunits, phospholipase-Cγ and N-cadherin were examined in the fetal cerebral cortices. Presence of alcohol in the liquid-diet reduced the consumption and decreased weights of mothers and fetuses but increased the placental weights. Expression levels of β(1) and α(3) integrin subunits and phospholipase-Cγ(2) were significantly altered in the fetal cerebral cortices of mothers on alcohol containing diet. Results show that alcohol consumption during pregnancy even with protein, mineral and vitamin enriched diet may affect maternal and fetal health, and alter integrin receptor signaling pathways in the fetal cerebral cortex disturbing the development of fetal brains.

  12. Inhibition of the integrin signal constitutes a mouse iPS cell niche.

    PubMed

    Higuchi, Sayaka; Yoshina, Sawako; Mitani, Shohei

    2016-09-01

    Stem cells are regulated by their surrounding microenvironments, called niche, such as cell-cell interaction and extracellular matrix. Classically, feeder cells as a niche have been used in the culture of iPS cells from both the mouse and the human. However, the regulation mechanism of stem cells by feeder cells as a niche still have been partially unclear. In this study, we used three murine iPS cell lines, iPS-MEF-Ng-20D-17, iPS-MEF-Ng-178B-5 and iPS-MEF-Fb/Ng-440A-3, which were generated by different reprogramming methods. In general, these cell lines commonly need the feeder cells as a niche to culture. Recently, the effect of substrate stiffness is known in stem cell study. First, we focused on the mechanical properties of feeder cells, and then we speculated that feeder-less culture might be made possible by using molecules in place of the mechanical properties of the niche. Finally, we found that the combination of disintegrin (echistatin) and 2i (GSK3 inhibitor and MEK inhibitor) is a sufficient condition for three murine iPS culture. This novel method of mimicking the murine iPS cell niche may be useful to understand signaling pathways to maintain the pluripotency of stem cells. PMID:27633818

  13. Integrins as therapeutic targets: lessons and opportunities.

    PubMed

    Cox, Dermot; Brennan, Marian; Moran, Niamh

    2010-10-01

    The integrins are a large family of cell adhesion molecules that are essential for the regulation of cell growth and function. The identification of key roles for integrins in a diverse range of diseases, including cancer, infection, thrombosis and autoimmune disorders, has revealed their substantial potential as therapeutic targets. However, so far, pharmacological inhibitors for only three integrins have received marketing approval. This article discusses the structure and function of integrins, their roles in disease and the chequered history of the approved integrin antagonists. Recent advances in the understanding of integrin function, ligand interaction and signalling pathways suggest novel strategies for inhibiting integrin function that could help harness their full potential as therapeutic targets. PMID:20885411

  14. Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway.

    PubMed

    You, Yang; Zheng, Qiongdan; Dong, Yinying; Wang, Yaohui; Zhang, Lan; Xue, Tongchun; Xie, Xiaoying; Hu, Chao; Wang, Zhiming; Chen, Rongxin; Wang, Yanhong; Cui, Jiefeng; Ren, Zhenggang

    2015-01-01

    Increased stromal stiffness is associated with hepatocellular carcinoma (HCC) development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN) expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells.

  15. Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway

    PubMed Central

    You, Yang; Zheng, Qiongdan; Dong, Yinying; Wang, Yaohui; Zhang, Lan; Xue, Tongchun; Xie, Xiaoying; Hu, Chao; Wang, Zhiming; Chen, Rongxin; Wang, Yanhong; Cui, Jiefeng; Ren, Zhenggang

    2015-01-01

    Increased stromal stiffness is associated with hepatocellular carcinoma (HCC) development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN) expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells. PMID:26280346

  16. The Role of Integrins in the Trabecular Meshwork

    PubMed Central

    Gagen, Debjani; Faralli, Jennifer A.; Filla, Mark S.

    2014-01-01

    Abstract Integrins are a family of heterodimeric transmembrane receptors that mediate adhesion to the extracellular matrix (ECM). However, integrins are not just adhesion receptors. They can act as “bidirectional signal transducers” that coordinate a large number of cellular activities in response to the extracellular environment and intracellular signaling events. Among the activities regulated by integrins are cell adhesion, assembly of the ECM, growth factor signaling, apoptosis, organization of the cytoskeleton, and cytoskeleton-mediated processes such as contraction, endocytosis, and phagocytosis. Integrins regulate these activities through a complex network of intracellular signaling kinases and adaptor proteins that associate with the transmembrane and cytoplasmic domains of the integrin subunits. In this review, we will discuss how some of the known integrin-mediated activities can control the function of the trabecular meshwork. We will also discuss how integrin activity is a tightly regulated process that involves conformation changes within the heterodimer which are mediated by specific integrin-binding proteins. PMID:24266581

  17. Dysregulation of Semaphorin7A/β1-integrin signaling leads to defective GnRH-1 cell migration, abnormal gonadal development and altered fertility.

    PubMed

    Messina, Andrea; Ferraris, Nicoletta; Wray, Susan; Cagnoni, Gabriella; Donohue, Duncan E; Casoni, Filippo; Kramer, Phillip R; Derijck, Alwin A; Adolfs, Youri; Fasolo, Aldo; Pasterkamp, Ronald J; Giacobini, Paolo

    2011-12-15

    Reproduction in mammals is dependent on the function of specific neurons that secrete gonadotropin-releasing hormone-1 (GnRH-1). These neurons originate prenatally in the nasal placode and migrate into the forebrain along the olfactory-vomeronasal nerves. Alterations in this migratory process lead to defective GnRH-1 secretion, resulting in heterogeneous genetic disorders such as idiopathic hypogonadotropic hypogonadism (IHH), and other reproductive diseases characterized by the reduction or failure of sexual competence. Combining mouse genetics with in vitro models, we demonstrate that Semaphorin 7A (Sema7A) is essential for the development of the GnRH-1 neuronal system. Loss of Sema7A signaling alters the migration of GnRH-1 neurons, resulting in significantly reduced numbers of these neurons in the adult brain as well as in reduced gonadal size and subfertility. We also show that GnRH-1 cells differentially express the Sema7 receptors β1-integrin and Plexin C1 as a function of their migratory stage, whereas the ligand is robustly expressed along developing olfactory/vomeronasal fibers. Disruption of Sema7A function in vitro inhibits β1-integrin-mediated migration. Analysis of Plexin C1(-/-) mice did not reveal any difference in the migratory process of GnRH-1 neurons, indicating that Sema7A mainly signals through β1-integrin to regulate GnRH-1 cell motility. In conclusion, we have identified Sema7A as a gene implicated in the normal development of the GnRH-1 system in mice and as a genetic marker for the elucidation of some forms of GnRH-1 deficiency in humans. PMID:21903667

  18. Delivery of dexamethasone from bioactive nanofiber matrices stimulates odontogenesis of human dental pulp cells through integrin/BMP/mTOR signaling pathways.

    PubMed

    Lim, Hyun-Chang; Nam, Ok Hyung; Kim, Mi-Joo; El-Fiqi, Ahmed; Yun, Hyung-Mun; Lee, Yoo-Mi; Jin, Guang-Zhen; Lee, Hae-Hyoung; Kim, Hae-Won; Kim, Eun-Cheol

    2016-01-01

    Therapeutically relevant design of scaffolds is of special importance in the repair and regeneration of tissues including dentin and pulp. Here we exploit nanofiber matrices that incorporate bioactive glass nanoparticles (BGNs) and deliver the odontogenic drug dexamethasone (DEX) to stimulate the odontogenic differentiation of human dental pulp cells (HDPCs). DEX molecules were first loaded onto the BGN, and then the DEX-BGN complex was incorporated within the biopolymer nanofiber matrix through electrospinning. The release of DEX continued over a month, showing a slow releasing profile. HDPCs cultured on the DEX-releasing BGN matrices were viable, proliferating well up to 14 days. The odontogenic differentiation, as assessed by alkaline phosphatase activity, mRNA expression of genes, and mineralization, was significantly stimulated on the matrices incorporating BGN and further on those releasing DEX. The DEX-releasing BGN matrices highly upregulated the expression of the integrin subsets α1, α5, and β3 as well as integrin downstream signaling molecules, including focal adhesion kinase (FAK), Paxillin, and RhoA, and activated bone morphogenetic protein mRNA and phosphorylation of Smad1/5/8. Furthermore, the DEX-releasing BGN-matrices stimulated Akt and mammalian target of rapamycin (mTOR), which was proven by the inhibition study. Collectively, the designed therapeutic nanofiber matrices that incorporate BGN and deliver DEX were demonstrated to promote odontogenesis of HDPCs, and the integrins, bone morphogenetic protein, and mTOR signaling pathways are proposed to be the possible molecular mechanisms. While further in vivo studies are still needed, the DEX-releasing bioactive scaffolds are considered as a potential therapeutic nanomatrix for regenerative endodontics and tissue engineering. PMID:27354790

  19. Essential function for PDLIM2 in cell polarization in three-dimensional cultures by feedback regulation of the β1-integrin-RhoA signaling axis.

    PubMed

    Deevi, Ravi Kiran; Cox, Orla T; O'Connor, Rosemary

    2014-05-01

    PDLIM2 is a cytoskeletal and nuclear PDZ-LIM domain protein that regulates the stability of Nuclear Factor kappa-B (NFκB) and other transcription factors, and is required for polarized cell migration. PDLIM2 expression is suppressed by methylation in different cancers, but is strongly expressed in invasive breast cancer cells that have undergone an Epithelial Mesenchymal Transition (EMT). PDLIM2 is also expressed in non-transformed breast myoepithelial MCF10A cells and here we asked whether it is important for maintaining the polarized, epithelial phenotype of these cells. Suppression of PDLIM2 in MCF10A cells was sufficient to disrupt cell polarization and acini formation with increased proliferation and reduced apoptosis in the luminal space compared to control acini with hollow lumina. Spheroids with suppressed PDLIM2 exhibited increased expression of cell-cell and cell-matrix adhesion proteins including beta 1 (β1) integrin. Interestingly, levels of the Insulin-like growth factor 1 receptor (IGF-1 R) and Receptor of activated protein kinase C 1 (RACK1), which scaffolds IGF-1R to β1 integrin, were also increased, indicating a transformed phenotype. Focal Adhesion Kinase (FAK) and cofilin phosphorylation, and RhoA Guanosine Triphosphatase (GTPase) activity were all enhanced in these spheroids compared to control acini. Importantly, inhibition of either FAK or Rho Kinase (ROCK) was sufficient to rescue the polarity defect. We conclude that PDLIM2 expression is essential for feedback regulation of the β1-integrin-RhoA signalling axis and integration of cellular microenvironment signals with gene expression to control the polarity of breast epithelial acini structures. This is a mechanism by which PDLIM2 could mediate tumour suppression in breast epithelium. PMID:24863845

  20. Delivery of dexamethasone from bioactive nanofiber matrices stimulates odontogenesis of human dental pulp cells through integrin/BMP/mTOR signaling pathways.

    PubMed

    Lim, Hyun-Chang; Nam, Ok Hyung; Kim, Mi-Joo; El-Fiqi, Ahmed; Yun, Hyung-Mun; Lee, Yoo-Mi; Jin, Guang-Zhen; Lee, Hae-Hyoung; Kim, Hae-Won; Kim, Eun-Cheol

    2016-01-01

    Therapeutically relevant design of scaffolds is of special importance in the repair and regeneration of tissues including dentin and pulp. Here we exploit nanofiber matrices that incorporate bioactive glass nanoparticles (BGNs) and deliver the odontogenic drug dexamethasone (DEX) to stimulate the odontogenic differentiation of human dental pulp cells (HDPCs). DEX molecules were first loaded onto the BGN, and then the DEX-BGN complex was incorporated within the biopolymer nanofiber matrix through electrospinning. The release of DEX continued over a month, showing a slow releasing profile. HDPCs cultured on the DEX-releasing BGN matrices were viable, proliferating well up to 14 days. The odontogenic differentiation, as assessed by alkaline phosphatase activity, mRNA expression of genes, and mineralization, was significantly stimulated on the matrices incorporating BGN and further on those releasing DEX. The DEX-releasing BGN matrices highly upregulated the expression of the integrin subsets α1, α5, and β3 as well as integrin downstream signaling molecules, including focal adhesion kinase (FAK), Paxillin, and RhoA, and activated bone morphogenetic protein mRNA and phosphorylation of Smad1/5/8. Furthermore, the DEX-releasing BGN-matrices stimulated Akt and mammalian target of rapamycin (mTOR), which was proven by the inhibition study. Collectively, the designed therapeutic nanofiber matrices that incorporate BGN and deliver DEX were demonstrated to promote odontogenesis of HDPCs, and the integrins, bone morphogenetic protein, and mTOR signaling pathways are proposed to be the possible molecular mechanisms. While further in vivo studies are still needed, the DEX-releasing bioactive scaffolds are considered as a potential therapeutic nanomatrix for regenerative endodontics and tissue engineering.

  1. Delivery of dexamethasone from bioactive nanofiber matrices stimulates odontogenesis of human dental pulp cells through integrin/BMP/mTOR signaling pathways

    PubMed Central

    Lim, Hyun-Chang; Nam, Ok Hyung; Kim, Mi-joo; El-Fiqi, Ahmed; Yun, Hyung-Mun; Lee, Yoo-Mi; Jin, Guang-Zhen; Lee, Hae-Hyoung; Kim, Hae-Won; Kim, Eun-Cheol

    2016-01-01

    Therapeutically relevant design of scaffolds is of special importance in the repair and regeneration of tissues including dentin and pulp. Here we exploit nanofiber matrices that incorporate bioactive glass nanoparticles (BGNs) and deliver the odontogenic drug dexamethasone (DEX) to stimulate the odontogenic differentiation of human dental pulp cells (HDPCs). DEX molecules were first loaded onto the BGN, and then the DEX-BGN complex was incorporated within the biopolymer nanofiber matrix through electrospinning. The release of DEX continued over a month, showing a slow releasing profile. HDPCs cultured on the DEX-releasing BGN matrices were viable, proliferating well up to 14 days. The odontogenic differentiation, as assessed by alkaline phosphatase activity, mRNA expression of genes, and mineralization, was significantly stimulated on the matrices incorporating BGN and further on those releasing DEX. The DEX-releasing BGN matrices highly upregulated the expression of the integrin subsets α1, α5, and β3 as well as integrin downstream signaling molecules, including focal adhesion kinase (FAK), Paxillin, and RhoA, and activated bone morphogenetic protein mRNA and phosphorylation of Smad1/5/8. Furthermore, the DEX-releasing BGN-matrices stimulated Akt and mammalian target of rapamycin (mTOR), which was proven by the inhibition study. Collectively, the designed therapeutic nanofiber matrices that incorporate BGN and deliver DEX were demonstrated to promote odontogenesis of HDPCs, and the integrins, bone morphogenetic protein, and mTOR signaling pathways are proposed to be the possible molecular mechanisms. While further in vivo studies are still needed, the DEX-releasing bioactive scaffolds are considered as a potential therapeutic nanomatrix for regenerative endodontics and tissue engineering. PMID:27354790

  2. Integrin-linked kinase as a novel molecular switch of the IL-6-NF-κB signaling loop in breast cancer.

    PubMed

    Hsu, En-Chi; Kulp, Samuel K; Huang, Han-Li; Tu, Huang-Ju; Chao, Min-Wu; Tseng, Yu-Chou; Yang, Ming-Chen; Salunke, Santosh B; Sullivan, Nicholas J; Chen, Wen-Chung; Zhang, Jianying; Teng, Che-Ming; Fu, Wen-Mei; Sun, Duxin; Wicha, Max S; Shapiro, Charles L; Chen, Ching-Shih

    2016-04-01

    Substantial evidence has clearly demonstrated the role of the IL-6-NF-κB signaling loop in promoting aggressive phenotypes in breast cancer. However, the exact mechanism by which this inflammatory loop is regulated remains to be defined. Here, we report that integrin-linked kinase (ILK) acts as a molecular switch for this feedback loop. Specifically, we show that IL-6 induces ILK expression via E2F1 upregulation, which, in turn, activates NF-κB signaling to facilitate IL-6 production. shRNA-mediated knockdown or pharmacological inhibition of ILK disrupted this IL-6-NF-κB signaling loop, and blocked IL-6-induced cancer stem cells in vitro and estrogen-independent tumor growth in vivo Together, these findings establish ILK as an intermediary effector of the IL-6-NF-κB feedback loop and a promising therapeutic target for breast cancer.

  3. Small GTPase Rho signaling is involved in {beta}1 integrin-mediated up-regulation of intercellular adhesion molecule 1 and receptor activator of nuclear factor {kappa}B ligand on osteoblasts and osteoclast maturation

    SciTech Connect

    Hirai, Fumihiko; Nakayamada, Shingo; Okada, Yosuke; Saito, Kazuyoshi; Kurose, Hitoshi; Mogami, Akira; Tanaka, Yoshiya . E-mail: tanaka@med.uoeh-u.ac.jp

    2007-04-27

    We assessed the characteristics of human osteoblasts, focusing on small GTPase Rho signaling. {beta}1 Integrin were highly expressed on osteoblasts. Engagement of {beta}1 integrins by type I collagen augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor {kappa}B ligand (RANKL) on osteoblasts. Rho was activated by {beta}1 stimulation in osteoblasts. {beta}1 Integrin-induced up-regulation of ICAM-1 and RANKL was inhibited by transfection with adenoviruses encoding C3 transferase or pretreated with Y-27632, specific Rho and Rho-kinase inhibitors. Engagement of {beta}1 integrin on osteoblasts induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNC) in a coculture system of osteoblasts and peripheral monocytes, but this action was completely abrogated by transfection of C3 transferase. Our results indicate the direct involvement of Rho-mediated signaling in {beta}1 integrin-induced up-regulation of ICAM-1 and RANKL and RANKL-dependent osteoclast maturation. Thus, Rho-mediated signaling in osteoblasts seems to introduce major biases to bone resorption.

  4. Mediation of the migration of endothelial cells and fibroblasts on polyurethane nanocomposites by the activation of integrin-focal adhesion kinase signaling.

    PubMed

    Hung, Huey-Shan; Chu, Mei-Yun; Lin, Chien-Hsun; Wu, Chia-Ching; Hsu, Shan-hui

    2012-01-01

    Model surfaces of polyurethane-gold nanocomposites (PU-Au) were used to examine cell behavior on nanophase-segregated materials. Previously we showed that endothelial cell (EC) migration on these materials was modulated by the PI3K/Akt/eNOS pathway. The present study, investigated the expressions of alpha5/beta3 (α5β3) integrin, focal adhesion kinase (FAK), and other downstream signal molecules such as the Rho family and matrix metalloproteinases 2 (MMP-2) induced by the materials in two different cells, that is bovine arterial endothelial cells (BAEC) and human skin fibroblasts (HSF). Both cells proliferated better on the more phase-separated PU-Au 43.5 ppm than on the less phase-separated controls (PU and PU-Au 174 ppm). On PU-Au 43.5 ppm, BAEC compared to HSF had denser actin fibers and were more extended. BAEC became rounded with Y-27632 treatment and shrunk with LY294002 treatment. Treatment by inhibitors only caused slight changes in HSF. The migration distance of BAEC on PU-Au 43.5 ppm was greater than that of HSF, and was significantly reduced by LY294002 or Y-27632 but not SU-1498. The expressions of p-FAK, p-RhoA, p-Rac/Cdc42, MMP2, and α5β3 integrin induced by PU-Au 43.5 ppm were more pronounced in BAEC versus HSF. Further enhancement in MMP2 and α5β3 integrin expressions by FAK-GFP transfection was more remarkable for cells on PU-Au 43.5 ppm. Our findings suggested that the integrin α5β3/FAK pathway may be induced by nanophase-separated materials in both ECs and fibroblasts to promote their proliferation/migration, while the crosstalk between the PI3K/Akt/eNOS pathway and FAK/Rho-GTPase activation may account for the greater effect in ECs than in fibroblasts.

  5. The final steps of integrin activation: the end game

    PubMed Central

    Shattil, Sanford J.; Kim, Chungho; Ginsberg, Mark H.

    2014-01-01

    Cell-directed changes in the ligand-binding affinity (‘activation’) of integrins regulate cell adhesion and migration, extracellular matrix assembly and mechanotransduction, thereby contributing to embryonic development and diseases such as atherothrombosis and cancer. Integrin activation comprises triggering events, intermediate signalling events and, finally, the interaction of integrins with cytoplasmic regulators, which changes an integrin’s affinity for its ligands. The first two events involve diverse interacting signalling pathways, whereas the final steps are immediately proximal to integrins, thus enabling integrin-focused therapeutic strategies. Recent progress provides insight into the structure of integrin transmembrane domains, and reveals how the final steps of integrin activation are mediated by integrin-binding proteins such as talins and kindlins. PMID:20308986

  6. DNA methylation-mediated silencing of matricellular protein dermatopontin promotes hepatocellular carcinoma metastasis by α3β1 integrin-Rho GTPase signaling.

    PubMed

    Fu, Ying; Feng, Ming-Xuan; Yu, Jian; Ma, Ming-Ze; Liu, Xiao-Jin; Li, Jun; Yang, Xiao-Mei; Wang, Ya-Hui; Zhang, Yan-Li; Ao, Jun-Ping; Xue, Feng; Qin, Wenxin; Gu, Jianren; Xia, Qiang; Zhang, Zhi-Gang

    2014-08-30

    Dermatopontin (DPT), a tyrosine-rich, acidic matricellular protein, has been implicated in several human cancers. However, its biological functions and molecular mechanisms in cancer progression, particular hepatocellular carcinoma (HCC), remain unknown. We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis. The overexpression of DPT dramatically suppressed HCC cell migration in vitro and intrahepatic metastasis in vivo. We further revealed that the down-regulation of DPT in HCC was due to epigenetic silencing by promoter DNA methylation. And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling. Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3β1 integrin. Our study provides new evidence for epigenetic control of tumor microenvironment, and suggests matricellular protein DPT may serve as a novel prognostic marker and act as a HCC metastasis suppressor. PMID:25149533

  7. Tie2 Signaling Enhances Mast Cell Progenitor Adhesion to Vascular Cell Adhesion Molecule-1 (VCAM-1) through α4β1 Integrin

    PubMed Central

    Kanemaru, Kazumasa; Noguchi, Emiko; Tokunaga, Takahiro; Nagai, Kei; Hiroyama, Takashi; Nakamura, Yukio; Tahara-Hanaoka, Satoko; Shibuya, Akira

    2015-01-01

    Mast cell (MC) activation contributes considerably to immune responses, such as host protection and allergy. Cell surface immunoreceptors expressed on MCs play an important role in MC activation. Although various immunoreceptors on MCs have been identified, the regulatory mechanism of MC activation is not fully understood. To understand the regulatory mechanisms of MC activation, we used gene expression analyses of human and mouse MCs to identify a novel immunoreceptor expressed on MCs. We found that Tek, which encodes Tie2, was preferentially expressed in the MCs of both humans and mice. However, Tie2 was not detected on the cell surface of the mouse MCs of the peritoneal cavity, ear skin, or colon lamina propria. In contrast, it was expressed on mouse bone marrow–derived MCs and bone marrow MC progenitors (BM-MCps). Stimulation of Tie2 by its ligand angiopoietin-1 induced tyrosine phosphorylation of Tie2 in MEDMC-BRC6, a mouse embryonic stem cell-derived mast cell line, and enhanced MEDMC-BRC6 and mouse BM-MCp adhesion to vascular cell adhesion molecule-1 (VCAM-1) through α4β1 integrin. These results suggest that Tie2 signaling induces α4β1 integrin activation on BM-MCps for adhesion to VCAM-1. PMID:26659448

  8. DNA methylation-mediated silencing of matricellular protein dermatopontin promotes hepatocellular carcinoma metastasis by α3β1 integrin-Rho GTPase signaling.

    PubMed

    Fu, Ying; Feng, Ming-Xuan; Yu, Jian; Ma, Ming-Ze; Liu, Xiao-Jin; Li, Jun; Yang, Xiao-Mei; Wang, Ya-Hui; Zhang, Yan-Li; Ao, Jun-Ping; Xue, Feng; Qin, Wenxin; Gu, Jianren; Xia, Qiang; Zhang, Zhi-Gang

    2014-08-30

    Dermatopontin (DPT), a tyrosine-rich, acidic matricellular protein, has been implicated in several human cancers. However, its biological functions and molecular mechanisms in cancer progression, particular hepatocellular carcinoma (HCC), remain unknown. We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis. The overexpression of DPT dramatically suppressed HCC cell migration in vitro and intrahepatic metastasis in vivo. We further revealed that the down-regulation of DPT in HCC was due to epigenetic silencing by promoter DNA methylation. And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling. Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3β1 integrin. Our study provides new evidence for epigenetic control of tumor microenvironment, and suggests matricellular protein DPT may serve as a novel prognostic marker and act as a HCC metastasis suppressor.

  9. Combinatorial Origami

    NASA Astrophysics Data System (ADS)

    Dieleman, Peter; Waitukaitis, Scott; van Hecke, Martin

    To design rigidly foldable quadrilateral meshes one generally needs to solve a complicated set of constraints. Here we present a systematic, combinatorial approach to create rigidly foldable quadrilateral meshes with a limited number of different vertices. The number of discrete, 1 degree-of-freedom folding branches for some of these meshes scales exponentially with the number of vertices on the edge, whilst other meshes generated this way only have two discrete folding branches, regardless of mesh size. We show how these two different behaviours both emerge from the two folding branches present in a single generic 4-vertex. Furthermore, we model generic 4-vertices as a spherical linkage and exploit a previously overlooked symmetry to create non-developable origami patterns using the same combinatorial framework.

  10. Research advances on structure and biological functions of integrins.

    PubMed

    Pan, Li; Zhao, Yuan; Yuan, Zhijie; Qin, Guixin

    2016-01-01

    Integrins are an important family of adhesion molecules that were first discovered two decades ago. Integrins are transmembrane heterodimeric glycoprotein receptors consisting of α and β subunits, and are comprised of an extracellular domain, a transmembrane domain, and a cytoplasmic tail. Therein, integrin cytoplasmic domains may associate directly with numerous cytoskeletal proteins and intracellular signaling molecules, which are crucial for modulating fundamental cell processes and functions including cell adhesion, proliferation, migration, and survival. The purpose of this review is to describe the unique structure of each integrin subunit, primary cytoplasmic association proteins, and transduction signaling pathway of integrins, with an emphasis on their biological functions. PMID:27468395

  11. Furanodiene presents synergistic anti-proliferative activity with paclitaxel via altering cell cycle and integrin signaling in 95-D lung cancer cells.

    PubMed

    Xu, Wen-Shan; Dang, Yuan-Ye; Chen, Xiu-Ping; Lu, Jin-Jian; Wang, Yi-Tao

    2014-02-01

    Furanodiene (FUR) is a natural terpenoid isolated from Rhizoma Curcumae, a well-known Chinese medicinal herb that presents anti-proliferative activities in several cancer cell lines. Recently, we found that the combined treatment of FUR with paclitaxel (TAX) showed synergetic anti-proliferative activities in 95-D lung cancer cells. Herein, we showed that FUR reduced the cell numbers distributed in mitosis phase induced by TAX while increased those in G1 phase. The protein levels of cyclin D1, cyclin B1, CDK6 and c-Myc were all down-regulated in the group of combined treatment. The dramatically down-regulated expression of integrin β4, focal adhesion kinase and paxillin might partially contribute to the synergic effect. Though FUR alone obviously induced endoplasmic reticulum stress, this signaling pathway may not contribute to the synergetic anti-proliferative effect as the protein expression of CHOP and BIP was similar in FUR alone and combined treatment group.

  12. The microRNA-200/Zeb1 axis regulates ECM-dependent β1-integrin/FAK signaling, cancer cell invasion and metastasis through CRKL

    PubMed Central

    Ungewiss, Christin; Rizvi, Zain H.; Roybal, Jonathon D.; Peng, David H.; Gold, Kathryn A.; Shin, Dong-Hoon; Creighton, Chad J.; Gibbons, Don L.

    2016-01-01

    Tumor cell metastasis is a complex process that has been mechanistically linked to the epithelial-mesenchymal transition (EMT). The double-negative feedback loop between the microRNA-200 family and the Zeb1 transcriptional repressor is a master EMT regulator, but there is incomplete understanding of how miR-200 suppresses invasion. Our recent efforts have focused on the tumor cell-matrix interactions essential to tumor cell activation. Herein we utilized both our Kras/p53 mutant mouse model and human lung cancer cell lines to demonstrate that upon miR-200 loss integrin β1-collagen I interactions drive 3D in vitro migration/invasion and in vivo metastases. Zeb1-dependent EMT enhances tumor cell responsiveness to the ECM composition and activates FAK/Src pathway signaling by de-repression of the direct miR-200 target, CRKL. We demonstrate that CRKL serves as an adaptor molecule to facilitate focal adhesion formation, mediates outside-in signaling through Itgβ1 to drive cell invasion, and inside-out signaling that maintains tumor cell-matrix contacts required for cell invasion. Importantly, CRKL levels in pan-cancer TCGA analyses were predictive of survival and CRKL knockdown suppressed experimental metastases in vivo without affecting primary tumor growth. Our findings highlight the critical ECM-tumor cell interactions regulated by miR-200/Zeb1-dependent EMT that activate intracellular signaling pathways responsible for tumor cell invasion and metastasis. PMID:26728244

  13. The microRNA-200/Zeb1 axis regulates ECM-dependent β1-integrin/FAK signaling, cancer cell invasion and metastasis through CRKL.

    PubMed

    Ungewiss, Christin; Rizvi, Zain H; Roybal, Jonathon D; Peng, David H; Gold, Kathryn A; Shin, Dong-Hoon; Creighton, Chad J; Gibbons, Don L

    2016-01-01

    Tumor cell metastasis is a complex process that has been mechanistically linked to the epithelial-mesenchymal transition (EMT). The double-negative feedback loop between the microRNA-200 family and the Zeb1 transcriptional repressor is a master EMT regulator, but there is incomplete understanding of how miR-200 suppresses invasion. Our recent efforts have focused on the tumor cell-matrix interactions essential to tumor cell activation. Herein we utilized both our Kras/p53 mutant mouse model and human lung cancer cell lines to demonstrate that upon miR-200 loss integrin β1-collagen I interactions drive 3D in vitro migration/invasion and in vivo metastases. Zeb1-dependent EMT enhances tumor cell responsiveness to the ECM composition and activates FAK/Src pathway signaling by de-repression of the direct miR-200 target, CRKL. We demonstrate that CRKL serves as an adaptor molecule to facilitate focal adhesion formation, mediates outside-in signaling through Itgβ1 to drive cell invasion, and inside-out signaling that maintains tumor cell-matrix contacts required for cell invasion. Importantly, CRKL levels in pan-cancer TCGA analyses were predictive of survival and CRKL knockdown suppressed experimental metastases in vivo without affecting primary tumor growth. Our findings highlight the critical ECM-tumor cell interactions regulated by miR-200/Zeb1-dependent EMT that activate intracellular signaling pathways responsible for tumor cell invasion and metastasis. PMID:26728244

  14. Combinatorial therapy with tamoxifen and trifluoperazine effectively inhibits malignant peripheral nerve sheath tumor growth by targeting complementary signaling cascades.

    PubMed

    Brosius, Stephanie N; Turk, Amy N; Byer, Stephanie J; Longo, Jody Fromm; Kappes, John C; Roth, Kevin A; Carroll, Steven L

    2014-11-01

    Chemotherapeutic agents effective against malignant peripheral nerve sheath tumors (MPNSTs) are urgently needed. We recently found that tamoxifen potently impedes xenograft growth. In vitro, tamoxifen inhibits MPNST proliferation and survival in an estrogen receptor-independent manner; these effects are phenocopied by the calmodulin inhibitor trifluoperazine. The present study was performed to establish the mechanism of action of tamoxifen in vivo and optimize its therapeutic effectiveness. To determine if tamoxifen has estrogen receptor-dependent effects in vivo, we grafted MPNST cells in castrated and ovariectomized mice; xenograft growth was unaffected by reductions in sex hormones. To establish whether tamoxifen and trifluoperazine additively or synergistically impede MPNST growth, mice xenografted with neurofibromatosis type 1-associated or sporadic MPNST cells were treated with tamoxifen, trifluoperazine, or both drugs for 30 days. Both monotherapies inhibited graft growth by 50%, whereas combinatorial treatment maximally reduced graft mass by 90% and enhanced decreases in proliferation and survival. Kinomic analyses showed that tamoxifen and trifluoperazine have both shared and distinct targets in MPNSTs. In addition, trifluoperazine prevented tamoxifen-induced increases in serum/glucocorticoid regulated kinase 1, a protein linked to tamoxifen resistance. These findings suggest that combinatorial therapy with tamoxifen and trifluoperazine is effective against MPNSTs because these agents target complementary pathways that are essential for MPNST pathogenesis.

  15. Lunasin potentiates the effect of oxaliplatin preventing outgrowth of colon cancer metastasis, binds to α5β1 integrin and suppresses FAK/ERK/NF-κB signaling.

    PubMed

    Dia, Vermont P; Gonzalez de Mejia, Elvira

    2011-12-27

    The effect of lunasin on colon cancer metastasis was studied using three human colon cancer cell lines in vitro and a liver metastasis model of colon cancer in vivo. Lunasin bound with α5β1 integrin and internalized into the nucleus of KM12L4 human colon cancer cells. Lunasin (10 μM) inhibited the activation of focal adhesion kinase (FAK) by 28%, 39% and 60% in RKO, HCT-116 and KM12L4 human colon cancer cells, respectively. Lunasin caused an increase in the expression of the inhibitor of kappa B alpha (IκB-α), a decrease in nuclear p50 NF-κB and a reduction in the migration of cancer cells. Lunasin (4 mg/kg bw) inhibited metastasis and potentiated the effect of oxaliplatin by reducing the expression of proliferating cell nuclear antigen. Liver metastatic nodules were reduced from 28 (PBS) to 14 (lunasin, P = 0.047) while combination of lunasin and oxaliplatin to 5 (P = 0.004). The tumor burden was reduced from 0.13 (PBS) to 0.10 (lunasin, P = 0.039) to 0.04 (lunasin + oxaliplatin, P < 0.0001). Moreover, lunasin potentiated the effect of oxaliplatin in modifying expression of proteins involved in apoptosis and metastasis including Bax, Bcl-2, IKK-α and p-p65. Lunasin inhibited metastasis of human colon cancer cells by direct binding with α5β1 integrin suppressing FAK/ERK/NF-κB signaling, and potentiated the effect of oxaliplatin in preventing the outgrowth of metastasis.

  16. Control of Integrin αIIbβ3 Outside-In Signaling and Platelet Adhesion by Sensing the Physical Properties of Fibrin(ogen) Substrates†

    PubMed Central

    Podolnikova, Nataly P.; Yermolenko, Ivan S.; Fuhrmann, Alexander; Lishko, Valeryi K.; Magonov, Sergei; Bowen, Benjamin; Enderlein, Joerg; Podolnikov, Andriy V.; Ros, Robert; Ugarova, Tatiana P.

    2015-01-01

    The physical properties of substrates are known to control cell adhesion via integrin-mediated signaling. Fibrin and fibrinogen, the principal components of hemostatic and pathological thrombi, may represent biologically relevant substrates whose variable physical properties control adhesion of leukocytes and platelets. In our previous work, we have shown that binding of fibrinogen to the surface of fibrin clot prevents cell adhesion by creating an antiadhesive fibrinogen layer. Furthermore, fibrinogen immobilized on various surfaces at high density supports weak cell adhesion whereas at low density it is highly adhesive. To explore the mechanism underlying differential cell adhesion, we examined the structural and physical properties of surfaces prepared by deposition of various concentrations of fibrinogen using atomic force microscopy and force spectroscopy. Fibrinogen deposition at high density resulted in an aggregated multilayered material characterized by low adhesion forces. In contrast, immobilization of fibrinogen at low density produced a single layer in which molecules were directly attached to the solid surface, resulting in higher adhesion forces. Consistent with their distinct physical properties, low- but not high-density fibrinogen induced strong αIIbβ3-mediated outside-in signaling in platelets, resulting in their spreading. Moreover, while intact fibrin gels induced strong signaling in platelets, deposition of fibrinogen on the surface of fibrin resulted in diminished cell signaling. The data suggest that deposition of a multilayered fibrinogen matrix prevents stable cell adhesion by modifying the physical properties of surfaces, which results in reduced force generation and insufficient signaling. The mechanism whereby circulating fibrinogen alters adhesive properties of fibrin clots may have important implications for control of thrombus formation and thrombogenicity of biomaterials. PMID:19929007

  17. Transmembrane/cytoplasmic, rather than catalytic, domains of Mmp14 signal to MAPK activation and mammary branching morphogenesis via binding to integrin β1

    PubMed Central

    Mori, Hidetoshi; Lo, Alvin T.; Inman, Jamie L.; Alcaraz, Jordi; Ghajar, Cyrus M.; Mott, Joni D.; Nelson, Celeste M.; Chen, Connie S.; Zhang, Hui; Bascom, Jamie L.; Seiki, Motoharu; Bissell, Mina J.

    2013-01-01

    Epithelial cell invasion through the extracellular matrix (ECM) is a crucial step in branching morphogenesis. The mechanisms by which the mammary epithelium integrates cues from the ECM with intracellular signaling in order to coordinate invasion through the stroma to make the mammary tree are poorly understood. Because the cell membrane-bound matrix metalloproteinase Mmp14 is known to play a key role in cancer cell invasion, we hypothesized that it could also be centrally involved in integrating signals for mammary epithelial cells (MECs) to navigate the collagen 1 (CL-1)-rich stroma of the mammary gland. Expression studies in nulliparous mice that carry a NLS-lacZ transgene downstream of the Mmp14 promoter revealed that Mmp14 is expressed in MECs at the tips of the branches. Using both mammary organoids and 3D organotypic cultures, we show that MMP activity is necessary for invasion through dense CL-1 (3 mg/ml) gels, but dispensable for MEC branching in sparse CL-1 (1 mg/ml) gels. Surprisingly, however, Mmp14 without its catalytic activity was still necessary for branching. Silencing Mmp14 prevented cell invasion through CL-1 and disrupted branching altogether; it also reduced integrin β1 (Itgb1) levels and attenuated MAPK signaling, disrupting Itgb1-dependent invasion/branching within CL-1 gels. FRET imaging revealed that Mmp14 associates directly with Itgb1. We identified a domain of Mmp14 that is required for modulating the levels of Itgb1, MEC signaling and the rate of invasion within CL-1. These results shed light on hitherto undescribed non-proteolytic activities of Mmp14 that are necessary for the Itgb1-dependent biochemical and mechanical signals that regulate branching in the mammary epithelium. PMID:23250208

  18. Bioinert Anodic Alumina Nanotubes for Targeting of Endoplasmic Reticulum Stress and Autophagic Signaling: A Combinatorial Nanotube-Based Drug Delivery System for Enhancing Cancer Therapy.

    PubMed

    Wang, Ye; Kaur, Gagandeep; Chen, Yuting; Santos, Abel; Losic, Dusan; Evdokiou, Andreas

    2015-12-16

    Although nanoparticle-based targeted delivery systems have gained promising achievements for cancer therapy, the development of sophisticated strategies with effective combinatorial therapies remains an enduring challenge. Herein, we report the fabrication of a novel nanomaterial, so-called anodic alumina nanotubes (AANTs) for proof-of-concept cancer therapy by targeting cell signaling networks. This strategy is to target autophagic and endoplasmic reticulum (ER) stress signaling by using thapsigargin (TG)-loaded AANTs cotreated with an autophagy inhibitor 3-methyladenine (3-MA). We first show that AANTs are nontoxic and can activate autophagy in different cell types including human fibroblast cells (HFF), human monocyte cells (THP-1), and human breast cancer cells (MDA-MB 231-TXSA). Treatment with 3-MA at a nontoxic dose reduced the level of autophagy induced by AANTs, and consequently sensitized breast cancer cells to AANTs-induced cellular stresses. To target autophagic and ER stress signaling networking, breast cancer cells were treated with 3-MA together with AANTs loaded with the prototype ER stress inducer TG. We demonstrated that 3-MA enhanced the cancer cell killing effect of AANTs loaded with TG. This effect was associated with enhanced ER stress signaling due to the combination effect of TG and 3-MA. These findings not only demonstrate the excellent biocompatibility of AANTs as novel biomaterials but also provide new opportunities for developing ER- and autophagy-targeted delivery systems for future clinical cancer therapy.

  19. Basement membrane protein ladinin-1 and the MIF-CD44-β1 integrin signaling axis are implicated in laryngeal cancer metastasis.

    PubMed

    Klobučar, Marko; Sedić, Mirela; Gehrig, Peter; Grossmann, Jonas; Bilić, Mario; Kovač-Bilić, Lana; Pavelić, Krešimir; Kraljević Pavelić, Sandra

    2016-10-01

    Laryngeal squamous cell carcinoma (LSCC) is the most common form of malignant disease in the head and neck region characterized by frequent occurrence of metastases in the neck lymph nodes early in the disease onset. In the presented study, we performed quantitative proteomic profiling of patient-matched primary tumor and adjacent non-tumorous tissues derived from metastatic LSCC as to identify new protein candidates with potential diagnostic and therapeutic significance. Obtained results revealed for the first time involvement of the basement membrane protein ladinin-1 in laryngeal cancer metastases. Alterations in the cellular microenvironment that propel metastatic events in laryngeal cancer include activation of MIF-CD44-β1 integrin signal transduction pathway and induction of downstream signaling mediated by NF-κB and Src tyrosine kinase, which ultimately impinge on cytoskeletal dynamics and architecture resulting in increased cellular motility and invasiveness. In this context, particularly interesting finding is upregulation of several actin-binding proteins novel to laryngeal cancer pathogenesis including coronin-1C and plastin-2, whose functional significance in laryngeal carcinogenesis has yet to be established. We also detected for the first time a complete loss of afamin in metastatic laryngeal cancer tissues, which warrants further studies into its use as a possible marker for monitoring disease progression and/or treatment outcome. PMID:27460703

  20. Carbon nanotube-based substrates promote cardiogenesis in brown adipose-derived stem cells via β1-integrin-dependent TGF-β1 signaling pathway

    PubMed Central

    Sun, Hongyu; Mou, Yongchao; Li, Yi; Li, Xia; Chen, Zi; Duval, Kayla; Huang, Zhu; Dai, Ruiwu; Tang, Lijun; Tian, Fuzhou

    2016-01-01

    Stem cell-based therapy remains one of the promising approaches for cardiac repair and regeneration. However, its applications are restricted by the limited efficacy of cardiac differentiation. To address this issue, we examined whether carbon nanotubes (CNTs) would provide an instructive extracellular microenvironment to facilitate cardiogenesis in brown adipose-derived stem cells (BASCs) and to elucidate the underlying signaling pathways. In this study, we systematically investigated a series of cellular responses of BASCs due to the incorporation of CNTs into collagen (CNT-Col) substrates that promoted cell adhesion, spreading, and growth. Moreover, we found that CNT-Col substrates remarkably improved the efficiency of BASCs cardiogenesis by using fluorescence staining and quantitative real-time reverse transcription-polymerase chain reaction. Critically, CNTs in the substrates accelerated the maturation of BASCs-derived cardiomyocytes. Furthermore, the underlying mechanism for promotion of BASCs cardiac differentiation by CNTs was determined by immunostaining, quantitative real-time reverse transcription-polymerase chain reaction, and Western blotting assay. It is notable that β1-integrin-dependent TGF-β1 signaling pathway modulates the facilitative effect of CNTs in cardiac differentiation of BASCs. Therefore, it is an efficient approach to regulate cardiac differentiation of BASCs by the incorporation of CNTs into the native matrix. Importantly, our findings can not only facilitate the mechanistic understanding of molecular events initiating cardiac differentiation in stem cells, but also offer a potentially safer source for cardiac regenerative medicine.

  1. Carbon nanotube-based substrates promote cardiogenesis in brown adipose-derived stem cells via β1-integrin-dependent TGF-β1 signaling pathway

    PubMed Central

    Sun, Hongyu; Mou, Yongchao; Li, Yi; Li, Xia; Chen, Zi; Duval, Kayla; Huang, Zhu; Dai, Ruiwu; Tang, Lijun; Tian, Fuzhou

    2016-01-01

    Stem cell-based therapy remains one of the promising approaches for cardiac repair and regeneration. However, its applications are restricted by the limited efficacy of cardiac differentiation. To address this issue, we examined whether carbon nanotubes (CNTs) would provide an instructive extracellular microenvironment to facilitate cardiogenesis in brown adipose-derived stem cells (BASCs) and to elucidate the underlying signaling pathways. In this study, we systematically investigated a series of cellular responses of BASCs due to the incorporation of CNTs into collagen (CNT-Col) substrates that promoted cell adhesion, spreading, and growth. Moreover, we found that CNT-Col substrates remarkably improved the efficiency of BASCs cardiogenesis by using fluorescence staining and quantitative real-time reverse transcription-polymerase chain reaction. Critically, CNTs in the substrates accelerated the maturation of BASCs-derived cardiomyocytes. Furthermore, the underlying mechanism for promotion of BASCs cardiac differentiation by CNTs was determined by immunostaining, quantitative real-time reverse transcription-polymerase chain reaction, and Western blotting assay. It is notable that β1-integrin-dependent TGF-β1 signaling pathway modulates the facilitative effect of CNTs in cardiac differentiation of BASCs. Therefore, it is an efficient approach to regulate cardiac differentiation of BASCs by the incorporation of CNTs into the native matrix. Importantly, our findings can not only facilitate the mechanistic understanding of molecular events initiating cardiac differentiation in stem cells, but also offer a potentially safer source for cardiac regenerative medicine. PMID:27660434

  2. Agglucetin, a tetrameric C-type lectin-like venom protein, regulates endothelial cell survival and promotes angiogenesis by activating integrin {alpha}v{beta}3 signaling

    SciTech Connect

    Wang, W.-J.

    2008-05-02

    Agglucetin, a platelet glycoprotein (GP)Ib binding protein from Formosan Agkistrodon acutus (A. acutus) venom, could sustain human umbilical vein endothelial cell (HUVEC) proliferation and HUVEC adhering to immobilized agglucetin showed extensive spreading, which was strongly abrogated by integrin antagonists 7E3 and triflavin. Flow cytometric analyses confirmed the expression of GPIb complex on HUVEC is absent and fluorescein isothiocyanate (FITC)-agglucetin binds to HUVEC in a dose-dependent and saturable manner. Furthermore, native agglucetin specifically and dose-dependently inhibited the binding of FITC-23C6, an anti-{alpha}v{beta}3 monoclonal antibody (mAb), but not antibodies against {alpha}2 and {alpha}5, toward HUVEC and purified {alpha}v{beta}3 also bound to immobilized agglucetin-{beta} in a dose-dependent manner. Moreover, agglucetin exhibited a pro-angiogenic effect in vitro, as well as the focal adhesion kinase (FAK)-associated signaling molecules responsible for HUVEC activation were initiated by agglucetin. In conclusion, agglucetin, acting as a survival factor, promotes endothelial adhesion and angiogenesis by triggering {alpha}v{beta}3 signaling through FAK/phosphatidylinositol 3-kinase (PI3K)/Akt pathway.

  3. Why Integrin as a Primary Target for Imaging and Therapy

    PubMed Central

    Niu, Gang; Chen, Xiaoyuan

    2011-01-01

    Integrin-mediated cell adhesion is involved in many essential normal cellular and pathological functions including cell survival, growth, differentiation, migration, inflammatory responses, platelet aggregation, tissue repair and tumor invasion. 24 different heterodimerized transmembrane integrin receptors are combined from 18 different α and 8 different β subunits. Each integrin subunit contains a large extracellular domain, a single transmembrane domain and a usually short cytoplasmic domain. Integrins bind extracellular matrix (ECM) proteins through their large extracellular domain, and engage the cytoskeleton via their short cytoplasmic tails. These integrin-mediated linkages on either side of the plasma membrane are dynamically linked. Thus, integrins communicate over the plasma membrane in both directions, i.e., outside-in and inside-out signaling. In outside-in signaling through integrins, conformational changes of integrin induced by ligand binding on the extracellular domain altered the cytoplasmic domain structures to elicit various intracellular signaling pathways. Inside-out signaling originates from non-integrin cell surface receptors or cytoplasmic molecules and it activates signaling pathways inside the cells, ultimately resulting in the activation/deactivation of integrins. Integrins are one of key family proteins for cell adhesion regulation through binding to a large number of ECM molecules and cell membrane proteins. Lack of expression of integrins may result in a wide variety of effects ranging from blockage in pre-implantation to embryonic or perinatal lethality and developmental defects. Based on both the key role they played in angiogenesis, leukocytes function and tumor development and easy accessibility as cell surface receptors interacting with extracellular ligands, the integrin superfamily represents the best opportunity of targeting both antibodies and small-molecule antagonists for both therapeutic and diagnostic utility in various

  4. Integrin-linked kinase affects signaling pathways and migration in thyroid cancer cells and is a potential therapeutic target

    PubMed Central

    Shirley, Lawrence A.; McCarty, Samantha; Yang, Ming-Chen; Saji, Motoyasu; Zhang, Xiaoli; Phay, John; Ringel, Matthew D.; Chen, Ching-Shih

    2016-01-01

    Background Integrin-linked kinase (ILK) is a serine-threonine kinase that regulates interactions between the cell and the extracellular matrix. In many cancers, overexpression of ILK leads to increased cell proliferation, motility, and invasion. We hypothesized that ILK functions as a regulator of viability and migration in thyroid cancer cells. Methods Eleven human thyroid cancer cell lines were screened for ILK protein expression. The cell lines with the greatest expression were treated with either ILK small interfering RNA (siRNA) or a novel ILK inhibitor, T315, and the effects were evaluated via Western blot and migration assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assays were performed to assess cell viability. Results siRNA against ILK decreased phosphorylation of downstream effectors Akt and MLC, as well as decreased migration. Treatment with T315 showed a dose-related decrease in both Akt and MLC phosphorylation, as well as decreased migration. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assays showed T315 to have an half maximal inhibitory concentration of less than 1 µM in cell lines with high ILK expression. Conclusion ILK is expressed differentially in thyroid cancer cell lines. Both ILK siRNA and T315 inhibit motility of thyroid cancer cell lines, and T315 is shown to be cytotoxic at low concentrations. Altogether, our study suggests that ILK may represent an important kinase in aggressive thyroid cancers. PMID:26549818

  5. Integrin αvβ3 mediates the synergetic regulation of core-binding factor α1 transcriptional activity by gravity and insulin-like growth factor-1 through phosphoinositide 3-kinase signaling.

    PubMed

    Dai, Zhongquan; Guo, Feima; Wu, Feng; Xu, Hongjie; Yang, Chao; Li, Jinqiao; Liang, Peilong; Zhang, Hongyu; Qu, Lina; Tan, Yingjun; Wan, Yumin; Li, Yinghui

    2014-12-01

    Mechanical stimulation and biological factors coordinately regulate bone development and regeneration; however, the underlying mechanisms are poorly understood. Microgravity induces bone loss, which may be partly related to the development of resistance to local cytokines, including insulin-like growth factor 1 (IGF-1). Here, we report the involvement of integrin αvβ3 in microgravity-associated bone loss. An established OSE-3T3 cell model was stably transfected with a 6OSE2 (Osteoblast-Specific Element 2)-luciferase reporter and cultured under simulated microgravity (SMG) and hypergravity (HG) conditions in the presence or absence of IGF-1, the disintegrin echistatin, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, or combinations of these agents. Activity of core-binding factor α1 (Cbfa1), an essential transcription factor for osteoblastic differentiation and osteogenesis, was reflected by luciferase activity. Different gravity conditions affected the induction of IGF-1 and subsequent effects on Cbfa1 transcription activity. SMG and HG influenced the expression and activity of integrin αvβ3 and phosphorylation level of p85. LY294002 inhibited the effects of HG or IGF-1 on Cbfa1 activity, indicating that HG and IGF-1 could increase Cbfa1 activity via PI3K signaling. Inhibition of integrin αvβ3 by echistatin attenuated the induction of IGF-1 and thus its effect on Cbfa1 activity under normal and HG conditions. Co-immunoprecipitation demonstrated that integrin β3 interacted with insulin receptor substrate 1, and that this interaction was decreased under SMG and increased under HG conditions. These results suggest that integrin αvβ3 mediates the synergetic regulation of Cbfa1 transcription activity by gravity and IGF-1 via PI3K signaling.

  6. β1-Integrin and integrin linked kinase regulate astrocytic differentiation of neural stem cells.

    PubMed

    Pan, Liuliu; North, Hilary A; Sahni, Vibhu; Jeong, Su Ji; Mcguire, Tammy L; Berns, Eric J; Stupp, Samuel I; Kessler, John A

    2014-01-01

    Astrogliosis with glial scar formation after damage to the nervous system is a major impediment to axonal regeneration and functional recovery. The present study examined the role of β1-integrin signaling in regulating astrocytic differentiation of neural stem cells. In the adult spinal cord β1-integrin is expressed predominantly in the ependymal region where ependymal stem cells (ESCs) reside. β1-integrin signaling suppressed astrocytic differentiation of both cultured ESCs and subventricular zone (SVZ) progenitor cells. Conditional knockout of β1-integrin enhanced astrogliogenesis both by cultured ESCs and by SVZ progenitor cells. Previous studies have shown that injection into the injured spinal cord of a self-assembling peptide amphiphile that displays an IKVAV epitope (IKVAV-PA) limits glial scar formation and enhances functional recovery. Here we find that injection of IKVAV-PA induced high levels of β1-integrin in ESCs in vivo, and that conditional knockout of β1-integrin abolished the astroglial suppressive effects of IKVAV-PA in vitro. Injection into an injured spinal cord of PAs expressing two other epitopes known to interact with β1-integrin, a Tenascin C epitope and the fibronectin epitope RGD, improved functional recovery comparable to the effects of IKVAV-PA. Finally we found that the effects of β1-integrin signaling on astrogliosis are mediated by integrin linked kinase (ILK). These observations demonstrate an important role for β1-integrin/ILK signaling in regulating astrogliosis from ESCs and suggest ILK as a potential target for limiting glial scar formation after nervous system injury.

  7. Regulation of oncogenic KRAS signaling via a novel KRAS-integrin-linked kinase-hnRNPA1 regulatory loop in human pancreatic cancer cells.

    PubMed

    Chu, P-C; Yang, M-C; Kulp, S K; Salunke, S B; Himmel, L E; Fang, C-S; Jadhav, A M; Shan, Y-S; Lee, C-T; Lai, M-D; Shirley, L A; Bekaii-Saab, T; Chen, C-S

    2016-07-28

    Integrin-linked kinase (ILK) is a mediator of aggressive phenotype in pancreatic cancer. On the basis of our finding that knockdown of either KRAS or ILK has a reciprocal effect on the other's expression, we hypothesized the presence of an ILK-KRAS regulatory loop that enables pancreatic cancer cells to regulate KRAS expression. This study aimed to elucidate the mechanism by which this regulatory circuitry is regulated and to investigate the translational potential of targeting ILK to suppress oncogenic KRAS signaling in pancreatic cancer. Interplay between KRAS and ILK and the roles of E2F1, c-Myc and heterogeneous nuclear ribonucleoprotein as intermediary effectors in this feedback loop was interrogated by genetic manipulations through small interfering RNA/short hairpin RNA knockdown and ectopic expression, western blotting, PCR, promoter-luciferase reporter assays, chromatin immunoprecipitation and pull-down analyses. In vivo efficacy of ILK inhibition was evaluated in two murine xenograft models. Our data show that KRAS regulated the expression of ILK through E2F1-mediated transcriptional activation, which, in turn, controlled KRAS gene expression via hnRNPA1-mediated destabilization of the G-quadruplex on the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven epithelial-mesenchymal transition and growth factor-stimulated KRAS expression. The knockdown or pharmacological inhibition of ILK suppressed pancreatic tumor growth, in part, by suppressing KRAS signaling. These studies suggest that this KRAS-E2F1-ILK-hnRNPA1 regulatory loop enables pancreatic cancer cells to promote oncogenic KRAS signaling and to interact with the tumor microenvironment to promote aggressive phenotypes. This regulatory loop provides a mechanistic rationale for targeting ILK to suppress oncogenic KRAS signaling, which might foster new therapeutic strategies for pancreatic cancer.

  8. Combinatorial signal integration by APETALA2/Ethylene Response Factor (ERF)-transcription factors and the involvement of AP2-2 in starvation response.

    PubMed

    Vogel, Marc Oliver; Gomez-Perez, Deborah; Probst, Nina; Dietz, Karl-Josef

    2012-01-01

    Transcription factors of the APETALA 2/Ethylene Response Factor (AP2/ERF)- family have been implicated in diverse processes during development, stress acclimation and retrograde signaling. Fifty-three leaf-expressed AP2/ERFs were screened for their transcriptional response to abscisic acid (ABA), 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), methylviologen (MV), sucrose and high or low light, respectively, and revealed high reactivity to these effectors. Six of them (AP2-2, ARF14, CEJ1, ERF8, ERF11, RAP2.5) were selected for combinatorial response analysis to ABA, DCMU and high light. Additive, synergistic and antagonistic effects demonstrated that these transcription factors are components of multiple signaling pathways. AP2-2 (At1g79700) was subjected to an in depth study. AP2-2 transcripts were high under conditions linked to limited carbohydrate availability and stress and down-regulated in extended light phase, high light or in the presence of sugar. ap2-2 knock out plants had unchanged metabolite profiles and transcript levels of co-expressed genes in extended darkness. However, ap2-2 revealed more efficient germination and faster early growth under high sugar, osmotic or salinity stress, but the difference was abolished in the absence of sugar or during subsequent growth. It is suggested that AP2-2 is involved in mediating starvation-related and hormonal signals.

  9. Pairwise agonist scanning-flow cytometry (PAS-FC) measures inside-out signaling and patient-specific response to combinatorial platelet agonists.

    PubMed

    Jaeger, Daniel T L; Diamond, Scott L

    2013-05-01

    Understanding the response of cells to multiple stimuli is vital for predicting donor specific responses and better understanding the signaling pathways involved. This is of particular importance in platelets because exposure of phosphatidylserine (PS) occurs upon costimulation but not with a single agonist. Here, we describe a multiplexed pairwise agonist scanning-flow cytometry (PAS-FC) method of measuring platelet inside-out responses to all pairs of six platelet agonists (convulxin, SFLLRN, AYPGKF, ADP, U46619, and PGE(2)) used at their EC(50) concentrations. These agonists allowed exploration of platelet signaling downstream of GPVI, PAR-1, PAR-4, P2Y(1), P2Y(12), TP, and IP receptors. The three-color flow cytometry method simultaneously measured integrin α(IIb)β(3) activation with PAC-1 antibody, P-selectin exposure (via α granule release) with anti-P-selectin, and PS exposure with annexin V. These responses were consistent across a healthy male donor pool. In duplicate measurements with each donor, 4 of the 10 donors had a sufficiently unique 45-parameter (15 pairs × 3 colors) phenotype to self-cluster (P < 0.001). This method has the potential for efficiently scanning for patient specific responses across a broad agonist-receptor space.

  10. Pairwise agonist scanning-flow cytometry (PAS-FC) measures inside-out signaling and patient-specific response to combinatorial platelet agonists.

    PubMed

    Jaeger, Daniel T L; Diamond, Scott L

    2013-05-01

    Understanding the response of cells to multiple stimuli is vital for predicting donor specific responses and better understanding the signaling pathways involved. This is of particular importance in platelets because exposure of phosphatidylserine (PS) occurs upon costimulation but not with a single agonist. Here, we describe a multiplexed pairwise agonist scanning-flow cytometry (PAS-FC) method of measuring platelet inside-out responses to all pairs of six platelet agonists (convulxin, SFLLRN, AYPGKF, ADP, U46619, and PGE(2)) used at their EC(50) concentrations. These agonists allowed exploration of platelet signaling downstream of GPVI, PAR-1, PAR-4, P2Y(1), P2Y(12), TP, and IP receptors. The three-color flow cytometry method simultaneously measured integrin α(IIb)β(3) activation with PAC-1 antibody, P-selectin exposure (via α granule release) with anti-P-selectin, and PS exposure with annexin V. These responses were consistent across a healthy male donor pool. In duplicate measurements with each donor, 4 of the 10 donors had a sufficiently unique 45-parameter (15 pairs × 3 colors) phenotype to self-cluster (P < 0.001). This method has the potential for efficiently scanning for patient specific responses across a broad agonist-receptor space. PMID:23662898

  11. The Biomechanical Integrin

    PubMed Central

    Baker, Erin L.; Zaman, Muhammad H.

    2009-01-01

    The integrin lies at the center of our efforts to understand mechanotransduction in the human body. Over the past two decades, a wealth of information has yielded important insights into integrin structure and functioning in biochemical pathways; however, relatively little emphasis has been placed on mechanics. In this article, we review the current knowledge base of integrin mechanobiology by examining the role of integrins in stabilizing tissue structure, the mechanisms of integrin force transfer, the process of cell migration, and the pathology of cancer. In order to successfully address the gaps in cancer and other disease research going forward, future efforts of integrin mechanobiology must focus on examining cells in 3D environments and integrating our current understanding into computational models that predict the behavior of integrins in non-equilibrium interactions. PMID:19811786

  12. Integrins, tensegrity, and mechanotransduction

    NASA Technical Reports Server (NTRS)

    Ingber, D. E.

    1997-01-01

    Physical forces, such as those due to gravity, play an important role in tissue development and remodeling. Yet, little is known about how individual cells sense mechanical signals or how they transduce them into a chemical response. Rather than listing the numerous signal pathways that have been found to be sensitive to mechanical stimulation, we need to place potential molecular signaling mechanisms within the context of the entire cell. The model presented is based on the concept that cells use tensegrity architecture to organize their cytoskeleton and stabilize their form. Studies with stick and string tensegrity cell models predict that living cells are hard-wired to respond immediately to external mechanical stresses. This hard-wiring exists in the form of discrete cytoskeletal filament networks that mechanically couple specific cell surface receptors, such as integrins, to nuclear matrix scaffolds and to potential transducing molecules that physically associate with the cytoskeleton. If these signaling molecules do function in a "solid-state", then mechanical stresses may be transduced into biochemical responses through force-dependent changes in cytoskeletal geometry or through local alterations in thermodynamic or kinetic parameters. Changes in cytoskeletal tension (prestress) also may play a role in signal amplification and adaptation. Recent experimental results are described which provide direct support for the tensegrity theory.

  13. Laminin-6 assembles into multimolecular fibrillar complexes with perlecan and participates in mechanical-signal transduction via a dystroglycan-dependent, integrin-independent mechanism

    PubMed Central

    Jones, Jonathan C. R.; Lane, Kimberly; Hopkinson, Susan B.; Lecuona, Emilia; Geiger, Robert C.; Dean, David A.; Correa-Meyer, Eduardo; Gonzales, Meredith; Campbell, Kevin; Sznajder, Jacob I.; Budinger, Scott

    2010-01-01

    Summary Mechanical ventilation is a valuable treatment regimen for respiratory failure. However, mechanical ventilation (especially with high tidal volumes) is implicated in the initiation and/or exacerbation of lung injury. Hence, it is important to understand how the cells that line the inner surface of the lung [alveolar epithelial cells (AECs)] sense cyclic stretching. Here, we tested the hypothesis that matrix molecules, via their interaction with surface receptors, transduce mechanical signals in AECs. We first determined that rat AECs secrete an extracellular matrix (ECM) rich in anastamosing fibers composed of the α3 laminin subunit, complexed with β1 and γ1 laminin subunits (i.e. laminin-6), and perlecan by a combination of immunofluorescence microscopy and immunoblotting analyses. The fibrous network exhibits isotropic expansion when exposed to cyclic stretching (30 cycles per minute, 10% strain). Moreover, this same stretching regimen activates mitogen-activated-protein kinase (MAPK) in AECs. Stretch-induced MAPK activation is not inhibited in AECs treated with antagonists to α3 or β1 integrin. However, MAPK activation is significantly reduced in cells treated with function-inhibiting antibodies against the α3 laminin subunit and dystroglycan, and when dystroglycan is knocked down in AECs using short hairpin RNA. In summary, our results support a novel mechanism by which laminin-6, via interaction with dystroglycan, transduces a mechanical signal initiated by stretching that subsequently activates the MAPK pathway in rat AECs. These results are the first to indicate a function for laminin-6. They also provide novel insight into the role of the pericellular environment in dictating the response of epithelial cells to mechanical stimulation and have broad implications for the pathophysiology of lung injury. PMID:15928048

  14. Perlecan Domain V Induces VEGf Secretion in Brain Endothelial Cells through Integrin α5β1 and ERK-Dependent Signaling Pathways

    PubMed Central

    Clarke, Douglas N.; Al Ahmad, Abraham; Lee, Boyeon; Parham, Christi; Auckland, Lisa; Fertala, Andrezj; Kahle, Michael; Shaw, Courtney S.; Roberts, Jill; Bix, Gregory J.

    2012-01-01

    Perlecan Domain V (DV) promotes brain angiogenesis by inducing VEGF release from brain endothelial cells (BECs) following stroke. In this study, we define the specific mechanism of DV interaction with the α5β1 integrin, identify the downstream signal transduction pathway, and further investigate the functional significance of resultant VEGF release. Interestingly, we found that the LG3 portion of DV, which has been suggested to possess most of DV’s angio-modulatory activity outside of the brain, binds poorly to α5β1 and induces less BEC proliferation compared to full length DV. Additionally, we implicate DV’s DGR sequence as an important element for the interaction of DV with α5β1. Furthermore, we investigated the importance of AKT and ERK signaling in DV-induced VEGF expression and secretion. We show that DV increases the phosphorylation of ERK, which leads to subsequent activation and stabilization of eIF4E and HIF-1α. Inhibition of ERK activity by U0126 suppressed DV-induced expression and secretion of VEGR in BECs. While DV was capable of phosphorylating AKT we show that AKT phosphorylation does not play a role in DV’s induction of VEGF expression or secretion using two separate inhibitors, LY294002 and Akt IV. Lastly, we demonstrate that VEGF activity is critical for DV increases in BEC proliferation, as well as angiogenesis in a BEC-neuronal co-culture system. Collectively, our findings expand our understanding of DV’s mechanism of action on BECs, and further support its potential as a novel stroke therapy. PMID:23028886

  15. Fc receptor-mediated phagocytosis, superoxide production and calcium signaling of beta 2 integrin-deficient bovine neutrophils.

    PubMed

    Nagahata, H; Sawada, C; Higuchi, H; Teraoka, H; Yamaguchi, M

    1997-01-01

    Fc receptor for immunoglobulin G-mediated phagocytosis, superoxide production and intracellular calcium ([Ca2+]i) signaling of complement receptor type 3 (CR3)-deficient neutrophils from a heifer with leukocyte adhesion deficiency (BLAD) were compared to those of control heifers. The mean phagocytic activity of IgG-coated yeasts and aggregated bovine IgG (Agg-IgG)-induced superoxide production of CR3-deficient neutrophils were 10% and 77.9%, respectively, of those of control neutrophils. The [Ca2+]i signals in CR3-deficient neutrophils stimulated with Agg-IgG or concanavalin A were different with mean peak [Ca2+]i concentrations of 78% and 41.9%, respectively, of those of control neutrophils. These findings suggest that Fc receptor-mediated neutrophil functions are closely dependent on the presence of CR3 (CD11b/CD18) on the neutrophil cell surfaces. PMID:9343828

  16. Antagonizing Integrin β3 Increases Immunosuppression in Cancer.

    PubMed

    Su, Xinming; Esser, Alison K; Amend, Sarah R; Xiang, Jingyu; Xu, Yalin; Ross, Michael H; Fox, Gregory C; Kobayashi, Takayuki; Steri, Veronica; Roomp, Kirsten; Fontana, Francesca; Hurchla, Michelle A; Knolhoff, Brett L; Meyer, Melissa A; Morgan, Elizabeth A; Tomasson, Julia C; Novack, Joshua S; Zou, Wei; Faccio, Roberta; Novack, Deborah V; Robinson, Stephen D; Teitelbaum, Steven L; DeNardo, David G; Schneider, Jochen G; Weilbaecher, Katherine N

    2016-06-15

    Integrin β3 is critical for tumor invasion, neoangiogenesis, and inflammation, making it a promising cancer target. However, preclinical and clinical data of integrin β3 antagonists have demonstrated no benefit or worse outcomes. We hypothesized that integrin β3 could affect tumor immunity and evaluated tumors in mice with deletion of integrin β3 in macrophage lineage cells (β3KOM). β3KOM mice had increased melanoma and breast cancer growth with increased tumor-promoting M2 macrophages and decreased CD8(+) T cells. Integrin β3 antagonist, cilengitide, also enhanced tumor growth and increased M2 function. We uncovered a negative feedback loop in M2 myeloid cells, wherein integrin β3 signaling favored STAT1 activation, an M1-polarizing signal, and suppressed M2-polarizing STAT6 activation. Finally, disruption of CD8(+) T cells, macrophages, or macrophage integrin β3 signaling blocked the tumor-promoting effects of integrin β3 antagonism. These results suggest that effects of integrin β3 therapies on immune cells should be considered to improve outcomes. Cancer Res; 76(12); 3484-95. ©2016 AACR.

  17. Alpha8 Integrin (Itga8) Signalling Attenuates Chronic Renal Interstitial Fibrosis by Reducing Fibroblast Activation, Not by Interfering with Regulation of Cell Turnover

    PubMed Central

    Marek, Ines; Lichtneger, Till; Cordasic, Nada; Hilgers, Karl F.; Volkert, Gudrun; Fahlbusch, Fabian; Rascher, Wolfgang; Hartner, Andrea; Menendez-Castro, Carlos

    2016-01-01

    The α8 integrin (Itga8) chain contributes to the regulation of cell proliferation and apoptosis in renal glomerular cells. In unilateral ureteral obstruction Itga8 is de novo expressed in the tubulointerstitium and a deficiency of Itga8 results in more severe renal fibrosis after unilateral ureteral obstruction. We hypothesized that the increased tubulointerstitial damage after unilateral ureteral obstruction observed in mice deficient for Itga8 is associated with altered tubulointerstitial cell turnover and apoptotic mechanisms resulting from the lack of Itga8 in cells of the tubulointerstitium. Induction of unilateral ureteral obstruction was achieved by ligation of the right ureter in mice lacking Itga8. Unilateral ureteral obstruction increased proliferation and apoptosis rates of tubuloepithelial and interstitial cells, however, no differences were observed in the tubulointerstitium of mice lacking Itga8 and wild type controls regarding fibroblast or proliferating cell numbers as well as markers of endoplasmic reticulum stress and apoptosis after unilateral ureteral obstruction. In contrast, unilateral ureteral obstruction in mice lacking Itga8 led to more pronounced tubulointerstitial cell activation i.e. to the appearance of more phospho-SMAD2/3-positive cells and more α-smooth muscle actin-positive cells in the tubulointerstitium. Furthermore, a more severe macrophage and T-cell infiltration was observed in these animals compared to controls. Thus, Itga8 seems to attenuate tubulointerstitial fibrosis in unilateral ureteral obstruction not via regulation of cell turnover, but via regulation of TGF-β signalling, fibroblast activation and/or immune cell infiltration. PMID:26938996

  18. A Novel Interaction of the Catalytic Subunit of Protein Phosphatase 2A with the Adaptor Protein CIN85 Suppresses Phosphatase Activity and Facilitates Platelet Outside-in αIIbβ3 Integrin Signaling.

    PubMed

    Khatlani, Tanvir; Pradhan, Subhashree; Da, Qi; Shaw, Tanner; Buchman, Vladimir L; Cruz, Miguel A; Vijayan, K Vinod

    2016-08-12

    The transduction of signals generated by protein kinases and phosphatases are critical for the ability of integrin αIIbβ3 to support stable platelet adhesion and thrombus formation. Unlike kinases, it remains unclear how serine/threonine phosphatases engage the signaling networks that are initiated following integrin ligation. Because protein-protein interactions form the backbone of signal transduction, we searched for proteins that interact with the catalytic subunit of protein phosphatase 2A (PP2Ac). In a yeast two-hybrid study, we identified a novel interaction between PP2Ac and an adaptor protein CIN85 (Cbl-interacting protein of 85 kDa). Truncation and alanine mutagenesis studies revealed that PP2Ac binds to the P3 block ((396)PAIPPKKPRP(405)) of the proline-rich region in CIN85. The interaction of purified PP2Ac with CIN85 suppressed phosphatase activity. Human embryonal kidney 293 αIIbβ3 cells overexpressing a CIN85 P3 mutant, which cannot support PP2Ac binding, displayed decreased adhesion to immobilized fibrinogen. Platelets contain the ∼85 kDa CIN85 protein along with the PP2Ac-CIN85 complex. A myristylated cell-permeable peptide derived from residues 395-407 of CIN85 protein (P3 peptide) disrupted the platelet PP2Ac-CIN85 complex and decreased αIIbβ3 signaling dependent functions such as platelet spreading on fibrinogen and thrombin-mediated fibrin clot retraction. In a phospho-profiling study P3 peptide treated platelets also displayed decreased phosphorylation of several signaling proteins including Src and GSK3β. Taken together, these data support a role for the novel PP2Ac-CIN85 complex in supporting integrin-dependent platelet function by dampening the phosphatase activity. PMID:27334924

  19. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

    ABSTRACT Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell–matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical–basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: ‘ERM proteins at a glance’ by Andrea McClatchey (J. Cell Sci. 127, 3199–3204). ‘Establishment of epithelial polarity – GEF who's minding the GAP?’ by Siu Ngok et al. (J. Cell Sci. 127, 3205–3215). PMID:24994933

  20. Integrin-linked kinase overexpression promotes epithelial-mesenchymal transition via nuclear factor-κB signaling in colorectal cancer cells

    PubMed Central

    Shen, Hong; Ma, Jun-Li; Zhang, Yan; Deng, Gan-Lu; Qu, Yan-Ling; Wu, Xiao-Ling; He, Jing-Xuan; Zhang, Sai; Zeng, Shan

    2016-01-01

    AIM: To investigate the effect of integrin-linked kinase (ILK) on proliferation, metastasis, and invasion of the colorectal cancer cell line SW480. METHODS: In this study, the colorectal cancer cell line SW480 was stably transfected with ILK plasmids, and small interfering RNA (siRNA) was used to knockdown expression of nuclear factor (NF)-κB/p65. Methylthiazole tetrazolium (MTT) assay was performed to measure proliferation, and the wound healing migration assay and matrigel invasion assay were used to test the metastasis and invasion ability of SW480 cells. To explore the epithelial-mesenchymal transition (EMT) process, embryonic development, and the invasion and metastasis of tumors, the protein level of E-cadherin, vimentin, snail, and slug was detected by western blot. Immunofluorescence was also used to detect E-cadherin expression. Western blot was used to determine the level of phosphorylated-inhibitor of kappa B (IκB)a, inhibitor of gamma B (IγB)a, and nuclear factor kappa B (NF-κB) expressions and to explore the ILK signaling pathway. RESULTS: Western blot results revealed that ILK expression significantly increased when ILK was overexpressed in SW480 cells (P < 0.05). Proliferation, metastasis, and invasion ability were improved in the vector-ILK group compared to the vector group (P < 0.05). Immunofluorescence results revealed that E-cadherin fluorescence intensity decreased after ILK was overexpressed (P < 0.05). Western blot results revealed that the protein expression of E-cadherin was reduced, while vimentin, snail, and slug were upregulated when ILK was overexpressed in SW480 cells (P < 0.05). In order to determine the role of the NF-κB signaling pathway in ILK overexpression promoted EMT occurrence, we overexpressed ILK in SW480 cells and found that levels of NF-κB/p65 and cytoplasmic phosphorylated-IκBa were increased and that cytoplasmic IкBa levels were decreased compared to the control group (P < 0.05). Furthermore, NF-κB/p65 knockout

  1. Integrin receptors and ligand-gated channels.

    PubMed

    Morini, Raffaella; Becchetti, Andrea

    2010-01-01

    Plastic expression of different integrin subunits controls the different stages of neural development, whereas in the adult integrins regulate synaptic stability. Evidence of integrin-channel crosstalk exists for ionotropic glutamate receptors. As is often the case in other tissues, integrin engagement regulates channel activity through complex signaling pathways that often include tyrosine phosphorylation cascades. The specific pathways recruited by integrin activation depend on cerebral region and cell type. In turn, ion channels control integrin expression onto the plasma membrane and their ligand binding affinity. The most extensive studies concern the hippocampus and suggest implications for neuronal circuit plasticity. The physiological relevance of these findings depends on whether adhesion molecules, aside from determining tissue stability, contribute to synaptogenesis and the responsiveness of mature synapses, thus contributing to long-term circuit consolidation. Little evidence is available for other ligand-gated channels, with the exception of nicotinic receptors. These exert a variety of functions in neurons and non neural tissue, both in development and in the adult, by regulating cell cycle, synaptogenesis and synaptic circuit refinement. Detailed studies in epidermal keratinocytes have shed some light on the possible mechanisms through which ACh can regulate cell motility, which may be of general relevance for morphogenetic processes. As to the control of mature synapses, most results concern the integrinic control of nicotinic receptors in the neuromuscular junction. Following this lead, a few studies have addressed similar topics in adult cerebral synapses. However, pursuing and interpreting these results in the brain is especially difficult because of the complexity of the nicotinic roles and the widespread contribution of nonsynaptic, paracrine transmission. From a pathological point of view, considering the well-known contribution of both

  2. Method and apparatus for combinatorial logic signal processor in a digitally based high speed x-ray spectrometer

    DOEpatents

    Warburton, W.K.

    1999-02-16

    A high speed, digitally based, signal processing system is disclosed which accepts a digitized input signal and detects the presence of step-like pulses in the this data stream, extracts filtered estimates of their amplitudes, inspects for pulse pileup, and records input pulse rates and system lifetime. The system has two parallel processing channels: a slow channel, which filters the data stream with a long time constant trapezoidal filter for good energy resolution; and a fast channel which filters the data stream with a short time constant trapezoidal filter, detects pulses, inspects for pileups, and captures peak values from the slow channel for good events. The presence of a simple digital interface allows the system to be easily integrated with a digital processor to produce accurate spectra at high count rates and allow all spectrometer functions to be fully automated. Because the method is digitally based, it allows pulses to be binned based on time related values, as well as on their amplitudes, if desired. 31 figs.

  3. Method and apparatus for combinatorial logic signal processor in a digitally based high speed x-ray spectrometer

    DOEpatents

    Warburton, William K.; Zhou, Zhiquing

    1999-01-01

    A high speed, digitally based, signal processing system which accepts a digitized input signal and detects the presence of step-like pulses in the this data stream, extracts filtered estimates of their amplitudes, inspects for pulse pileup, and records input pulse rates and system livetime. The system has two parallel processing channels: a slow channel, which filters the data stream with a long time constant trapezoidal filter for good energy resolution; and a fast channel which filters the data stream with a short time constant trapezoidal filter, detects pulses, inspects for pileups, and captures peak values from the slow channel for good events. The presence of a simple digital interface allows the system to be easily integrated with a digital processor to produce accurate spectra at high count rates and allow all spectrometer functions to be fully automated. Because the method is digitally based, it allows pulses to be binned based on time related values, as well as on their amplitudes, if desired.

  4. Integrin Targeted MR Imaging.

    PubMed

    Tan, Mingqian; Lu, Zheng-Rong

    2011-01-19

    Magnetic resonance imaging (MRI) is a powerful medical diagnostic imaging modality for integrin targeted imaging, which uses the magnetic resonance of tissue water protons to display tissue anatomic structures with high spatial resolution. Contrast agents are often used in MRI to highlight specific regions of the body and make them easier to visualize. There are four main classes of MRI contrast agents based on their different contrast mechanisms, including T(1), T(2), chemical exchange saturation transfer (CEST) agents, and heteronuclear contrast agents. Integrins are an important family of heterodimeric transmembrane glycoproteins that function as mediators of cell-cell and cell-extracellular matrix interactions. The overexpressed integrins can be used as the molecular targets for designing suitable integrin targeted contrast agents for MR molecular imaging. Integrin targeted contrast agent includes a targeting agent specific to a target integrin, a paramagnetic agent and a linker connecting the targeting agent with the paramagnetic agent. Proper selection of targeting agents is critical for targeted MRI contrast agents to effectively bind to integrins for in vivo imaging. An ideal integrin targeted MR contrast agent should be non-toxic, provide strong contrast enhancement at the target sites and can be completely excreted from the body after MR imaging. An overview of integrin targeted MR contrast agents based on small molecular and macromolecular Gd(III) complexes, lipid nanoparticles and superparamagnetic nanoparticles is provided for MR molecular imaging. By using proper delivery systems for loading sufficient Gd(III) chelates or superparamagnetic nanoparticles, effective molecular imaging of integrins with MRI has been demonstrated in animal models.

  5. Integrin β1 regulates leiomyoma cytoskeletal integrity and growth

    PubMed Central

    Malik, Minnie; Segars, James; Catherino, William H.

    2014-01-01

    Uterine leiomyomas are characterized by an excessive extracellular matrix, increased mechanical stress, and increased active RhoA. Previously, we observed that mechanical signaling was attenuated in leiomyoma, but the mechanisms responsible remain unclear. Integrins, especially integrin β1, are transmembrane adhesion receptors that couple extracellular matrix stresses to the intracellular cytoskeleton to influence cell proliferation and differentiation. Here we characterized integrin and laminin to signaling in leiomyoma cells. We observed a 2.25 ± 0.32 fold increased expression of integrin β1 in leiomyoma cells, compared to myometrial cells. Antibody-mediated inhibition of integrin β1 led to significant growth inhibition in leiomyoma cells and a loss of cytoskeletal integrity. Specifically, polymerization of actin filaments and formation of focal adhesions were reduced by inhibition of integrin p1. Inhibition of integrin β1 in leiomyoma cells led to 0.81 ± 0.02 fold decrease in active RhoA, and resembled levels found in serum-starved cells. Likewise, inhibition of integrin β1 was accompanied by a decrease in phospho-ERK. Compared to myometrial cells, leiomyoma cells demonstrated increased expression of integrin α6 subunit to laminin receptor (1.91 ± 0.11 fold), and increased expression of laminin 5α (1.52±0.02), laminin 5β (3.06±0.92), and laminin 5γ (1.66 ± 0.06). Of note, leiomyoma cells grown on laminin matrix appear to realign themselves. Taken together, the findings reveal that the attenuated mechanical signaling in leiomyoma cells is accompanied by an increased expression and a dependence on integrin β1 signaling in leiomyoma cells, compared to myometrial cells. PMID:23023061

  6. Stem cells and combinatorial science.

    PubMed

    Fang, Yue Qin; Wong, Wan Qing; Yap, Yan Wen; Orner, Brendan P

    2007-09-01

    Stem cell-based technologies have the potential to help cure a number of cell degenerative diseases. Combinatorial and high throughput screening techniques could provide tools to control and manipulate the self-renewal and differentiation of stem cells. This review chronicles historic and recent progress in the stem cell field involving both pluripotent and multipotent cells, and it highlights relevant cellular signal transduction pathways. This review further describes screens using libraries of soluble, small-molecule ligands, and arrays of molecules immobilized onto surfaces while proposing future trends in similar studies. It is hoped that by reviewing both the stem cell and the relevant high throughput screening literature, this paper can act as a resource to the combinatorial science community.

  7. [Implication of integrin alpha5beta1 signal pathways in proliferation and apoptosis of MCF-7/Dox human breast carcinoma cells].

    PubMed

    Kozlova, N I; Morozevich, G E; Ushakova, N A; Gevorkian, N M; Berman, A E

    2016-03-01

    In MCF-7/Dox human breast carcinoma cells, down-regulation of integrin alpha5beta1 and inhibition of epidermal growth factor receptor (EGFR) markedly reduced rates of cell proliferation. Mitotic cycle analysis showed that alpha5beta1 down-regulation resulted in cell cycle arrest at the S phase, followed by a significant increase in the population of apoptotic cells (subG1 population). Inhibition of EGFR activity also caused cell cycle arrest at the S-phase but without any increase in the subG1 population. Down-regulation of alpha5beta1 and EGFR inhibition resulted in a significant decrease of cell content of the active (phosphorylated) forms of FAK and Erk protein kinases. The data obtained suggest that alpha5beta1 integrin is implicated in cell growth control via inhibition of apoptotic cell death and through EGFR activation. PMID:27420618

  8. Leader cells regulate collective cell migration via Rac activation in the downstream signaling of integrin β1 and PI3K.

    PubMed

    Yamaguchi, Naoya; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

    2015-01-07

    Collective cell migration plays a crucial role in several biological processes, such as embryonic development, wound healing, and cancer metastasis. Here, we focused on collectively migrating Madin-Darby Canine Kidney (MDCK) epithelial cells that follow a leader cell on a collagen gel to clarify the mechanism of collective cell migration. First, we removed a leader cell from the migrating collective with a micromanipulator. This then caused disruption of the cohesive migration of cells that followed in movement, called "follower" cells, which showed the importance of leader cells. Next, we observed localization of active Rac, integrin β1, and PI3K. These molecules were clearly localized in the leading edge of leader cells, but not in follower cells. Live cell imaging using active Rac and active PI3K indicators was performed to elucidate the relationship between Rac, integrin β1, and PI3K. Finally, we demonstrated that the inhibition of these molecules resulted in the disruption of collective migration. Our findings not only demonstrated the significance of a leader cell in collective cell migration, but also showed that Rac, integrin β1, and PI3K are upregulated in leader cells and drive collective cell migration.

  9. Pinocembrin suppresses TGF-β1-induced epithelial-mesenchymal transition and metastasis of human Y-79 retinoblastoma cells through inactivating αvβ3 integrin/FAK/p38α signaling pathway

    PubMed Central

    2014-01-01

    Background Pinocembrin is the most abundant flavonoid in propolis. In this study, we investigated the antimetastatic effect of pinocembrin on TGF-β1-induced epithelial-mesenchymal transition (EMT) and metastasis of human Y-79 retinoblastoma cells. Results Firstly, the results showed that pinocembrin significantly suppresses the TGF-β1-induced abilities of the invasion and migration of Y-79 cells under non-cytotoxic concentration. Pinocembrin decreased TGF-β1-induced expression of vimentin, N-cadherin, αv and β3 integrin in Y-79 cells. Molecular data also showed pinocembrin inhibits the activation of focal adhesion kinase (FAK) and p38α signal involved in the downregulation of enzyme activities, protein and messenger RNA levels of matrix metalloproteinase-2/9 (MMP-2/-9) induced by TGF-β1. Next, pinocembrin also strongly inhibited the degradation of inhibitor of kappaBα (IκBα) and the nuclear levels of nuclear factor kappa B (NF-κB). Also, a dose-dependent inhibition on the binding ability of NF-κB was further observed under pinocembrin treatment. Conclusions Presented results indicated that pinocembrin inhibits TGF-β1-induced epithelial-mesenchymal transition (EMT) and metastasis of Y-79 cells by inactivating the αvβ3 integrin/FAK/p38α signaling pathway. Thus, our findings point to the anticancer potential of pinocembrin against retinoblastoma cells. PMID:25949790

  10. Mulberry water extracts inhibit atherosclerosis through suppression of the integrin-β₃/focal adhesion kinase complex and downregulation of nuclear factor κB signaling in vivo and in vitro.

    PubMed

    Chan, Kuei-Chuan; Ho, Hsieh-Hsun; Lin, Ming-Cheng; Yen, Chi-Hua; Huang, Chien-Ning; Huang, Hui-Pei; Wang, Chau-Jong

    2014-10-01

    Previous studies have shown that mulberry water extracts (MWEs), which contain polyphenolic compounds, have an antiatherosclerotic effect in vivo and in vitro through stimulating apoptosis of vascular smooth muscle cells (VSMCs). Histological analysis was performed on atherosclerotic lesions from high-cholesterol diet (HCD)-fed rabbits after treatment with 0.5-1% MWEs for 10 weeks. Immunohistochemistry showed that the expressions of SMA, Ras, and matrix metalloproteinase-2 in the VSMCs were dose-dependently inhibited after MWE treatment. The antimigratory effects of MWEs on A7r5 VSMCs were assessed by western blot analysis of migration-related proteins, visualization of F-actin cytoskeleton, and reverse transcription polymerase chain reaction. The results showed that MWEs inhibited VSMC migration through reducing interactions of the integrin-β3/focal adhesion kinase complex, alterations of the cytoskeleton, and downregulation of glycogen synthase kinase 3β/nuclear factor κB signaling. Taken together, MWEs inhibited HCD-induced rabbit atherogenesis through blocking VSMC migration via reducing interactions of integrin-β3 and focal adhesion kinase and downregulating migration-related proteins.

  11. Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

    PubMed

    Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

    2014-04-01

    Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking.

  12. Combinatorial Geometry Printer Plotting.

    1987-01-05

    Picture generates plots of two-dimensional slices through the three-dimensional geometry described by the combinatorial geometry (CG) package used in such codes as MORSE and QAD-CG. These plots are printed on a standard line printer.

  13. Nonparametric Combinatorial Sequence Models

    NASA Astrophysics Data System (ADS)

    Wauthier, Fabian L.; Jordan, Michael I.; Jojic, Nebojsa

    This work considers biological sequences that exhibit combinatorial structures in their composition: groups of positions of the aligned sequences are "linked" and covary as one unit across sequences. If multiple such groups exist, complex interactions can emerge between them. Sequences of this kind arise frequently in biology but methodologies for analyzing them are still being developed. This paper presents a nonparametric prior on sequences which allows combinatorial structures to emerge and which induces a posterior distribution over factorized sequence representations. We carry out experiments on three sequence datasets which indicate that combinatorial structures are indeed present and that combinatorial sequence models can more succinctly describe them than simpler mixture models. We conclude with an application to MHC binding prediction which highlights the utility of the posterior distribution induced by the prior. By integrating out the posterior our method compares favorably to leading binding predictors.

  14. ISOLATION OF INTEGRIN-BASED ADHESION COMPLEXES

    PubMed Central

    Jones, Matthew C.; Humphries, Jonathan D.; Byron, Adam; Millon-Frémillon, Angelique; Robertson, Joseph; Paul, Nikki R.; Ng, Daniel H. J.; Askari, Janet A.; Humphries, Martin J.

    2015-01-01

    The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signalling across the plasma membrane and as such help to coordinate and / or modulate the multitude of physical or chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes together with their local plasma membrane / cytosolic environments from cells in culture. In the first protocol integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components by subsequent downstream analysis by Western blotting or mass spectrometry. PMID:25727331

  15. Immunolocalization of integrin-like proteins in Arabidopsis and Chara

    NASA Technical Reports Server (NTRS)

    Katembe, W. J.; Swatzell, L. J.; Makaroff, C. A.; Kiss, J. Z.

    1997-01-01

    Integrins are a large family of integral plasma membrane proteins that link the extracellular matrix to the cytoskeleton in animal cells. As a first step in determining if integrin-like proteins are involved in gravitropic signal transduction pathways, we have used a polyclonal antibody against the chicken beta1 integrin subunit in western blot analyses and immunofluorescence microscopy to gain information on the size and location of these proteins in plants. Several different polypeptides are recognized by the anti-integrin antibody in roots and shoots of Arabidopsis and in the internodal cells and rhizoids of Chara. These cross-reactive polypeptides are associated with cellular membranes, a feature which is consistent with the known location of integrins in animal systems. In immunofluorescence studies of Arabidopsis roots, a strong signal was obtained from labeling integrin-like proteins in root cap cells, and there was little or no immunolabel in other regions of the root tip. While the antibody stained throughout Chara rhizoids, the highest density of immunolabel was at the tip. Thus, in both Arabidopsis roots and Chara rhizoids, the sites of gravity perception/transduction appear to be enriched in integrin-like molecules.

  16. Integrin adhesions suppress syncytium formation in the Drosophila larval epidermis

    PubMed Central

    Wang, Yan; Antunes, Marco; Anderson, Aimee E.; Kadrmas, Julie L.; Jacinto, Antonio; Galko, Michael J.

    2015-01-01

    Summary Integrins are critical for barrier epithelial architecture. Integrin loss in vertebrate skin leads to blistering and wound healing defects. However, how Integrins and associated proteins maintain the regular morphology of epithelia is not well understood. We found that targeted knockdown of the integrin focal adhesion (FA) complex components βIntegrin, PINCH, and Integrin-linked kinase (ILK), caused formation of multinucleate epidermal cells within the Drosophila larval epidermis. This phenotype was specific to the Integrin FA complex and not due to secondary effects on polarity or junctional structures. The multinucleate cells resembled the syncytia caused by physical wounding. Live imaging of wound-induced syncytium formation in the pupal epidermis suggested direct membrane breakdown leading to cell-cell fusion and consequent mixing of cytoplasmic contents. Activation of Jun N-terminal kinase (JNK) signaling, which occurs upon wounding, also correlated with syncytium formation induced by PINCH knockdown. Further, ectopic JNK activation directly caused epidermal syncytium formation. No mode of syncytium formation including that induced by wounding, genetic loss-of FA-proteins, or local JNK hyperactivation, involved misregulation of mitosis or apoptosis. Finally, the mechanism of epidermal syncytium formation following JNK hyperactivation and wounding appeared to be direct disassembly of FA complexes. In conclusion, the loss of function phenotype of Integrin FA components in the larval epidermis resembles a wound. Integrin FA loss in mouse and human skin also causes a wound-like appearance. Our results reveal a novel and unexpected role for proper Integrin-based adhesion in suppressing larval epidermal cell-cell fusion– a role that may be conserved in other epithelia. PMID:26255846

  17. Integrin Adhesions Suppress Syncytium Formation in the Drosophila Larval Epidermis.

    PubMed

    Wang, Yan; Antunes, Marco; Anderson, Aimee E; Kadrmas, Julie L; Jacinto, Antonio; Galko, Michael J

    2015-08-31

    Integrins are critical for barrier epithelial architecture. Integrin loss in vertebrate skin leads to blistering and wound healing defects. However, how integrins and associated proteins maintain the regular morphology of epithelia is not well understood. We found that targeted knockdown of the integrin focal adhesion (FA) complex components β-integrin, PINCH, and integrin-linked kinase (ILK) caused formation of multinucleate epidermal cells within the Drosophila larval epidermis. This phenotype was specific to the integrin FA complex and not due to secondary effects on polarity or junctional structures. The multinucleate cells resembled the syncytia caused by physical wounding. Live imaging of wound-induced syncytium formation in the pupal epidermis suggested direct membrane breakdown leading to cell-cell fusion and consequent mixing of cytoplasmic contents. Activation of Jun N-terminal kinase (JNK) signaling, which occurs upon wounding, also correlated with syncytium formation induced by PINCH knockdown. Further, ectopic JNK activation directly caused epidermal syncytium formation. No mode of syncytium formation, including that induced by wounding, genetic loss of FA proteins, or local JNK hyperactivation, involved misregulation of mitosis or apoptosis. Finally, the mechanism of epidermal syncytium formation following JNK hyperactivation and wounding appeared to be direct disassembly of FA complexes. In conclusion, the loss-of-function phenotype of integrin FA components in the larval epidermis resembles a wound. Integrin FA loss in mouse and human skin also causes a wound-like appearance. Our results reveal a novel and unexpected role for proper integrin-based adhesion in suppressing larval epidermal cell-cell fusion--a role that may be conserved in other epithelia.

  18. Integrin α3 Mutations with Kidney, Lung, and Skin Disease

    PubMed Central

    Has, Cristina; Spartà, Giuseppina; Kiritsi, Dimitra; Weibel, Lisa; Moeller, Alexander; Vega-Warner, Virginia; Waters, Aoife; He, Yinghong; Anikster, Yair; Esser, Philipp; Straub, Beate K.; Hausser, Ingrid; Bockenhauer, Detlef; Dekel, Benjamin; Hildebrandt, Friedhelm; Bruckner-Tuderman, Leena; Laube, Guido F.

    2012-01-01

    SUMMARY Integrin α3 is a transmembrane integrin receptor subunit that mediates signals between the cells and their microenvironment. We identified three patients with homozygous mutations in the integrin α3 gene that were associated with disrupted basement-membrane structures and compromised barrier functions in kidney, lung, and skin. The patients had a multiorgan disorder that included congenital nephrotic syndrome, interstitial lung disease, and epidermolysis bullosa. The renal and respiratory features predominated, and the lung involvement accounted for the lethal course of the disease. Although skin fragility was mild, it provided clues to the diagnosis. PMID:22512483

  19. Full length talin stimulates integrin activation and axon regeneration

    PubMed Central

    Tan, Chin Lik; Kwok, Jessica C.F.; Heller, Janosch P.D.; Zhao, Rongrong; Eva, Richard; Fawcett, James W.

    2015-01-01

    Integrin function is regulated by activation involving conformational changes that modulate ligand-binding affinity and downstream signaling. Activation is regulated through inside-out signaling which is controlled by many signaling pathways via a final common pathway through kindlin and talin, which bind to the intracellular tail of beta integrins. Previous studies have shown that the axon growth inhibitory molecules NogoA and chondroitin sulfate proteoglycans (CSPGs) inactivate integrins. Overexpressing kindlin-1 in dorsal root ganglion (DRG) neurons activates integrins, enabling their axons to overcome inhibitory molecules in the environment, and promoting regeneration in vivo following dorsal root crush. Other studies have indicated that expression of the talin head alone or with kindlin can enhance integrin activation. Here, using adult rat DRG neurons, we investigate the effects of overexpressing various forms of talin on axon growth and integrin signaling. We found that overexpression of the talin head activated axonal integrins but inhibited downstream signaling via FAK, and did not promote axon growth. Similarly, co-expression of the talin head and kindlin-1 prevented the growth-promoting effect of kindlin-1, suggesting that the talin head acts as a form of dominant negative for integrin function. Using full-length talin constructs in PC12 cells we observed that neurite growth was enhanced by the expression of wild-type talin and more so by two ‘activated’ forms of talin produced by point mutation (on laminin and aggrecan–laminin substrates). Nevertheless, co-expression of full-length talin with kindlin did not promote neurite growth more than either molecule alone. In vivo, we find that talin is present in PNS axons (sciatic nerve), and also in CNS axons of the corticospinal tract. PMID:25771432

  20. Integrin α5β1, the Fibronectin Receptor, as a Pertinent Therapeutic Target in Solid Tumors

    PubMed Central

    Schaffner, Florence; Ray, Anne Marie; Dontenwill, Monique

    2013-01-01

    Integrins are transmembrane heterodimeric proteins sensing the cell microenvironment and modulating numerous signalling pathways. Changes in integrin expression between normal and tumoral cells support involvement of specific integrins in tumor progression and aggressiveness. This review highlights the current knowledge about α5β1 integrin, also called the fibronectin receptor, in solid tumors. We summarize data showing that α5β1 integrin is a pertinent therapeutic target expressed by tumoral neovessels and tumoral cells. Although mainly evaluated in preclinical models, α5β1 integrin merits interest in particular in colon, breast, ovarian, lung and brain tumors where its overexpression is associated with a poor prognosis for patients. Specific α5β1 integrin antagonists will be listed that may represent new potential therapeutic agents to fight defined subpopulations of particularly aggressive tumors. PMID:24216697

  1. Integrins regulate apical constriction via microtubule stabilization in the Drosophila eye disc epithelium.

    PubMed

    Fernandes, Vilaiwan M; McCormack, Kasandra; Lewellyn, Lindsay; Verheyen, Esther M

    2014-12-24

    During morphogenesis, extracellular signals trigger actomyosin contractility in subpopulations of cells to coordinate changes in cell shape. To illuminate the link between signaling-mediated tissue patterning and cytoskeletal remodeling, we study the progression of the morphogenetic furrow (MF), the wave of apical constriction that traverses the Drosophila eye imaginal disc preceding photoreceptor neurogenesis. Apical constriction depends on actomyosin contractility downstream of the Hedgehog (Hh) and bone morphogenetic protein (BMP) pathways. We identify a role for integrin adhesion receptors in MF progression. We show that Hh and BMP regulate integrin expression, the loss of which disrupts apical constriction and slows furrow progression; conversely, elevated integrins accelerate furrow progression. We present evidence that integrins regulate MF progression by promoting microtubule stabilization, since reducing microtubule stability rescues integrin-mediated furrow acceleration. Thus, integrins act as a genetic link between tissue-level signaling events and morphological change at the cellular level, leading to morphogenesis and neurogenesis in the eye.

  2. Integrin adhesion in regulation of lacrimal gland acinar cell secretion.

    PubMed

    Andersson, Sofia V; Hamm-Alvarez, Sarah F; Gierow, J Peter

    2006-09-01

    The extracellular microenvironment regulates lacrimal gland acinar cell secretion. Culturing isolated rabbit lacrimal gland acinar cells on different extracellular matrix proteins revealed that laminin enhances carbachol-stimulated secretion to a greater extent than other extracellular matrix proteins investigated. Furthermore, immunofluorescence indicated that integrin subunits, potentially functioning as laminin receptors are present in acinar cells. Among these, the integrin alpha6 and beta1 subunit mRNA expression was also confirmed by RT-PCR and sequence analysis. Secretion assays, which measured beta-hexosaminidase activity released in the culture media, demonstrated that function-blocking integrin alpha6 and beta1 monoclonal antibodies (mAbs) induce a rapid, transient and dose-dependent secretory response in cultured cells. To determine the intracellular pathways by which integrin alpha6 and beta1 mAbs could induce secretion, selected second messenger molecules were inhibited. Although inhibitors of protein kinase C and IP(3)-induced Ca(2+) mobilization attenuated carbachol-stimulated secretion, no effect on integrin mAb-induced release was observed. In addition, protein tyrosine kinases do not appear to have a role in transducing signals arising from mAb interactions. Our data clearly demonstrate, though, that cell adhesion through integrins regulates secretion from lacrimal gland acinar cells. The fact that the integrin mAbs affect the cholinergic response differently and that the integrin beta1 mAb secretion, but not the alpha6, was attenuated by the phosphatase inhibitor, sodium orthovanadate, suggests that each subunit utilizes separate intracellular signaling pathways to induce exocytosis. The results also indicate that the secretory response triggered by the beta1 integrin mAb is generated through dephosphorylation events.

  3. Integrin dynamics produce a delayed stage of long-term potentiation and memory consolidation.

    PubMed

    Babayan, Alex H; Kramár, Enikö A; Barrett, Ruth M; Jafari, Matiar; Häettig, Jakob; Chen, Lulu Y; Rex, Christopher S; Lauterborn, Julie C; Wood, Marcelo A; Gall, Christine M; Lynch, Gary

    2012-09-12

    Memory consolidation theory posits that newly acquired information passes through a series of stabilization steps before being firmly encoded. We report here that in rat and mouse, hippocampus cell adhesion receptors belonging to the β1-integrin family exhibit dynamic properties in adult synapses and that these contribute importantly to a previously unidentified stage of consolidation. Quantitative dual immunofluorescence microscopy showed that induction of long-term potentiation (LTP) by theta burst stimulation (TBS) activates β1 integrins, and integrin-signaling kinases, at spine synapses in adult hippocampal slices. Neutralizing antisera selective for β1 integrins blocked these effects. TBS-induced integrin activation was brief (<7 min) and followed by an ∼45 min period during which the adhesion receptors did not respond to a second application of TBS. Brefeldin A, which blocks integrin trafficking to the plasma membrane, prevented the delayed recovery of integrin responses to TBS. β1 integrin-neutralizing antisera erased LTP when applied during, but not after, the return of integrin responsivity. Similarly, infusions of anti-β1 into rostral mouse hippocampus blocked formation of long-term, object location memory when started 20 min after learning but not 40 min later. The finding that β1 integrin neutralization was effective in the same time window for slice and behavioral experiments strongly suggests that integrin recovery triggers a temporally discrete, previously undetected second stage of consolidation for both LTP and memory. PMID:22973009

  4. Canine malignant melanoma alpha-3 integrin binding peptides

    PubMed Central

    Aina, Olulanu H.; Maeda, Yoshiko; Harrison, Matthew; Zwingenberger, Allison L.; Walker, Naomi J.; Lam, Kit S.; Kent, Michael S.

    2014-01-01

    There is a need to develop novel targeted imaging and therapeutic agents that can aid in early diagnosis, detection of metastasis and treatment of melanoma. Alpha-3 integrin is overexpressed in 82% of metastatic melanomas in humans and may be a potential target for peptide ligands carrying therapeutic agents. Five melanoma cell lines were generated from canine primary oral and metastatic canine tumors, grown in mice, and validated with melanoma markers Melan A, S-100, Micropthalmia transcription factor (MITF), Tyrosinase, and MART-1. The melanoma cell lines were tested for binding affinity to previously published alpha-3 integrin-binding peptides containing the cdGXGXXc motif. Fluorescent conjugates of the alpha-3 integrin binding OA02 peptide were used to quantify receptor affinity in the cell lines, a specimen of canine primary oral melanoma, and melanoma xenografts. Alpha-3 integrin was expressed by all 5 canine melanoma cell lines. Four of the 5 lines as well as the primary canine tumor showed affinity to alpha-3 integrin binding peptides with the cdGXGXXc motif. Optical imaging of canine melanoma xenografts in nude mice indicates rapid, strong uptake of the optical tracer in the tumor with an average persistence of approximately 48 hours. Ex vivo images showed high tumor-to-background ratio, with tumor signals more than twice that of the kidney and other vital organs. We propose that integrin alpha-3 integrin binding ligands could potentially become useful probes for imaging and delivery of cytotoxic agents for the treatment of melanoma. PMID:21722969

  5. Tenascin-C and integrins in cancer

    PubMed Central

    Yoshida, Toshimichi; Akatsuka, Tatsuya; Imanaka-Yoshida, Kyoko

    2015-01-01

    Tenascin-C (TNC) is highly expressed in cancer tissues. Its cellular sources are cancer and stromal cells, including fibroblasts/myofibroblasts, and also vascular cells. TNC expressed in cancer tissues dominantly contains large splice variants. Deposition of the stroma promotes the epithelial-mesenchymal transition, proliferation, and migration of cancer cells. It also facilitates the formation of cancer stroma including desmoplasia and angiogenesis. Integrin receptors that mediate the signals of TNC have also been discussed. PMID:25793576

  6. Tenascin-C and integrins in cancer.

    PubMed

    Yoshida, Toshimichi; Akatsuka, Tatsuya; Imanaka-Yoshida, Kyoko

    2015-01-01

    Tenascin-C (TNC) is highly expressed in cancer tissues. Its cellular sources are cancer and stromal cells, including fibroblasts/myofibroblasts, and also vascular cells. TNC expressed in cancer tissues dominantly contains large splice variants. Deposition of the stroma promotes the epithelial-mesenchymal transition, proliferation, and migration of cancer cells. It also facilitates the formation of cancer stroma including desmoplasia and angiogenesis. Integrin receptors that mediate the signals of TNC have also been discussed. PMID:25793576

  7. GLUT10 deficiency leads to oxidative stress and non-canonical αvβ3 integrin-mediated TGFβ signalling associated with extracellular matrix disarray in arterial tortuosity syndrome skin fibroblasts.

    PubMed

    Zoppi, Nicoletta; Chiarelli, Nicola; Cinquina, Valeria; Ritelli, Marco; Colombi, Marina

    2015-12-01

    Arterial tortuosity syndrome (ATS) is an autosomal recessive connective tissue disorder caused by loss-of-function mutations in SLC2A10, which encodes facilitative glucose transporter 10 (GLUT10). The role of GLUT10 in ATS pathogenesis remains an enigma, and the transported metabolite(s), i.e. glucose and/or dehydroascorbic acid, have not been clearly elucidated. To discern the molecular mechanisms underlying the ATS aetiology, we performed gene expression profiling and biochemical studies on skin fibroblasts. Transcriptome analyses revealed the dysregulation of several genes involved in TGFβ signalling and extracellular matrix (ECM) homeostasis as well as the perturbation of specific pathways that control both the cell energy balance and the oxidative stress response. Biochemical and functional studies showed a marked increase in ROS-induced lipid peroxidation sustained by altered PPARγ function, which contributes to the redox imbalance and the compensatory antioxidant activity of ALDH1A1. ATS fibroblasts also showed activation of a non-canonical TGFβ signalling due to TGFBRI disorganization, the upregulation of TGFBRII and connective tissue growth factor, and the activation of the αvβ3 integrin transduction pathway, which involves p125FAK, p60Src and p38 MAPK. Stable GLUT10 expression in patients' fibroblasts normalized redox homeostasis and PPARγ activity, rescued canonical TGFβ signalling and induced partial ECM re-organization. These data add new insights into the ATS dysregulated biological pathways and definition of the pathomechanisms involved in this disorder. PMID:26376865

  8. GLUT10 deficiency leads to oxidative stress and non-canonical αvβ3 integrin-mediated TGFβ signalling associated with extracellular matrix disarray in arterial tortuosity syndrome skin fibroblasts

    PubMed Central

    Zoppi, Nicoletta; Chiarelli, Nicola; Cinquina, Valeria; Ritelli, Marco; Colombi, Marina

    2015-01-01

    Arterial tortuosity syndrome (ATS) is an autosomal recessive connective tissue disorder caused by loss-of-function mutations in SLC2A10, which encodes facilitative glucose transporter 10 (GLUT10). The role of GLUT10 in ATS pathogenesis remains an enigma, and the transported metabolite(s), i.e. glucose and/or dehydroascorbic acid, have not been clearly elucidated. To discern the molecular mechanisms underlying the ATS aetiology, we performed gene expression profiling and biochemical studies on skin fibroblasts. Transcriptome analyses revealed the dysregulation of several genes involved in TGFβ signalling and extracellular matrix (ECM) homeostasis as well as the perturbation of specific pathways that control both the cell energy balance and the oxidative stress response. Biochemical and functional studies showed a marked increase in ROS-induced lipid peroxidation sustained by altered PPARγ function, which contributes to the redox imbalance and the compensatory antioxidant activity of ALDH1A1. ATS fibroblasts also showed activation of a non-canonical TGFβ signalling due to TGFBRI disorganization, the upregulation of TGFBRII and connective tissue growth factor, and the activation of the αvβ3 integrin transduction pathway, which involves p125FAK, p60Src and p38 MAPK. Stable GLUT10 expression in patients' fibroblasts normalized redox homeostasis and PPARγ activity, rescued canonical TGFβ signalling and induced partial ECM re-organization. These data add new insights into the ATS dysregulated biological pathways and definition of the pathomechanisms involved in this disorder. PMID:26376865

  9. The role of integrins in primary and secondary brain tumors.

    PubMed

    Schittenhelm, Jens; Tabatabai, Ghazaleh; Sipos, Bence

    2016-10-01

    The tumor environment plays an integral part in the biology of cancer, participating in tumor initiation, progression, and response to therapy. Integrins, a family of cell surface receptors, bridge the extracellular matrix to the intracellular cytoskeleton. Since their first characterization 25 years ago, a vast amount of work has been performed to understand the essential role of integrins in cell development, tissue organization, tumor growth, vessel development and their signaling mechanisms. Their potential as therapeutic targets in various types of cancer is intensively studied. In this review, we discuss the expression patterns and functional role of integrin in primary brain tumors and brain metastases, provide an overview of clinical data on integrin inhibition and their potential application in imaging and therapy of these tumors. PMID:27097828

  10. The β-glucan receptor Dectin-1 activates the integrin Mac-1 in neutrophils via Vav protein signaling to promote Candida albicans clearance.

    PubMed

    Li, Xun; Utomo, Ahmad; Cullere, Xavier; Choi, Myunghwan Mark; Milner, Danny A; Venkatesh, Deepak; Yun, Seok-Hyun; Mayadas, Tanya N

    2011-12-15

    Resistance to fungal infections is attributed to engagement of host pattern-recognition receptors, notably the β-glucan receptor Dectin-1 and the integrin Mac-1, which induce phagocytosis and antifungal immunity. However, the mechanisms by which these receptors coordinate fungal clearance are unknown. We show that upon ligand binding, Dectin-1 activates Mac-1 to also recognize fungal components, and this stepwise process is critical for neutrophil cytotoxic responses. Both Mac-1 activation and Dectin-1- and Mac-1-induced neutrophil effector functions require Vav1 and Vav3, exchange factors for RhoGTPases. Mac-1- or Vav1,3-deficient mice have increased susceptibility to systemic candidiasis that is not due to impaired neutrophil recruitment but defective intracellular killing of C. albicans yeast forms, and Mac-1 or Vav1,3 reconstitution in hematopoietic cells restores resistance. Our results demonstrate that antifungal immunity depends on Dectin-1-induced activation of Mac-1 functions that is coordinated by Vav proteins, a pathway that may localize cytotoxic responses of circulating neutrophils to infected tissues.

  11. Integrin Dynamics and Matrix Assembly

    PubMed Central

    Pankov, Roumen; Cukierman, Edna; Katz, Ben-Zion; Matsumoto, Kazue; Lin, Diane C.; Lin, Shin; Hahn, Cornelia; Yamada, Kenneth M.

    2000-01-01

    Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, we developed an antibody-chasing technique for simultaneous tracking of two integrin populations by different antibodies. We established that whereas the vitronectin receptor αvβ3 remains within focal contacts, the fibronectin receptor α5β1 translocates from focal contacts into and along extracellular matrix (ECM) contacts. This escalator-like translocation occurs relative to the focal contacts at 6.5 ± 0.7 μm/h and is independent of cell migration. It is induced by ligation of α5β1 integrins and depends on interactions with a functional actin cytoskeleton and vitronectin receptor ligation. During cell spreading, translocation of ligand-occupied α5β1 integrins away from focal contacts and along bundles of actin filaments generates ECM contacts. Tensin is a primary cytoskeletal component of these ECM contacts, and a novel dominant-negative inhibitor of tensin blocked ECM contact formation, integrin translocation, and fibronectin fibrillogenesis without affecting focal contacts. We propose that translocating α5β1 integrins induce initial fibronectin fibrillogenesis by transmitting cytoskeleton-generated tension to extracellular fibronectin molecules. Blocking this integrin translocation by a variety of treatments prevents the formation of ECM contacts and fibronectin fibrillogenesis. These studies identify a localized, directional, integrin translocation mechanism for matrix assembly. PMID:10704455

  12. Cooperativity between Integrin Activation and Mechanical Stress Leads to Integrin Clustering

    PubMed Central

    Ali, O.; Guillou, H.; Destaing, O.; Albigès-Rizo, C.; Block, M.R.; Fourcade, B.

    2011-01-01

    Integrins are transmembrane receptors involved in crucial cellular biological functions such as migration, adhesion, and spreading. Upon the modulation of integrin affinity toward their extracellular ligands by cytoplasmic proteins (inside-out signaling) these receptors bind to their ligands and cluster into nascent adhesions. This clustering results in the increase in the mechanical linkage among the cell and substratum, cytoskeleton rearrangements, and further outside-in signaling. Based on experimental observations of the distribution of focal adhesions in cells attached to micropatterned surfaces, we introduce a physical model relying on experimental numerical constants determined in the literature. In this model, allosteric integrin activation works in synergy with the stress build by adhesion and the membrane rigidity to allow the clustering to nascent adhesions independently of actin but dependent on the integrin diffusion onto adhesive surfaces. The initial clustering could provide a template to the mature adhesive structures. Predictions of our model for the organization of focal adhesions are discussed in comparison with experiments using adhesive protein microarrays. PMID:21641304

  13. A dual role for integrin-linked kinase and β1-integrin in modulating cardiac aging

    PubMed Central

    Nishimura, Mayuko; Kumsta, Caroline; Kaushik, Gaurav; Diop, Soda B; Ding, Yun; Bisharat-Kernizan, Jumana; Catan, Hannah; Cammarato, Anthony; Ross, Robert S; Engler, Adam J; Bodmer, Rolf; Hansen, Malene; Ocorr, Karen

    2014-01-01

    Cardiac performance decreases with age, which is a major risk factor for cardiovascular disease and mortality in the aging human population, but the molecular mechanisms underlying cardiac aging are still poorly understood. Investigating the role of integrin-linked kinase (ilk) and β1-integrin (myospheroid, mys) in Drosophila, which colocalize near cardiomyocyte contacts and Z-bands, we find that reduced ilk or mys function prevents the typical changes of cardiac aging seen in wildtype, such as arrhythmias. In particular, the characteristic increase in cardiac arrhythmias with age is prevented in ilk and mys heterozygous flies with nearly identical genetic background, and they live longer, in line with previous findings in Caenorhabditis elegans for ilk and in Drosophila for mys. Consistent with these findings, we observed elevated β1-integrin protein levels in old compared with young wild-type flies, and cardiac-specific overexpression of mys in young flies causes aging-like heart dysfunction. Moreover, moderate cardiac-specific knockdown of integrin-linked kinase (ILK)/integrin pathway-associated genes also prevented the decline in cardiac performance with age. In contrast, strong cardiac knockdown of ilk or ILK-associated genes can severely compromise cardiac integrity, including cardiomyocyte adhesion and overall heart function. These data suggest that ilk/mys function is necessary for establishing and maintaining normal heart structure and function, and appropriate fine-tuning of this pathway can retard the age-dependent decline in cardiac performance and extend lifespan. Thus, ILK/integrin-associated signaling emerges as an important and conserved genetic mechanism in longevity, and as a new means to improve age-dependent cardiac performance, in addition to its vital role in maintaining cardiac integrity. PMID:24400780

  14. Manipulating Combinatorial Structures.

    ERIC Educational Resources Information Center

    Labelle, Gilbert

    This set of transparencies shows how the manipulation of combinatorial structures in the context of modern combinatorics can easily lead to interesting teaching and learning activities at every level of education from elementary school to university. The transparencies describe: (1) the importance and relations of combinatorics to science and…

  15. Research on universal combinatorial coding.

    PubMed

    Lu, Jun; Zhang, Zhuo; Mo, Juan

    2014-01-01

    The conception of universal combinatorial coding is proposed. Relations exist more or less in many coding methods. It means that a kind of universal coding method is objectively existent. It can be a bridge connecting many coding methods. Universal combinatorial coding is lossless and it is based on the combinatorics theory. The combinational and exhaustive property make it closely related with the existing code methods. Universal combinatorial coding does not depend on the probability statistic characteristic of information source, and it has the characteristics across three coding branches. It has analyzed the relationship between the universal combinatorial coding and the variety of coding method and has researched many applications technologies of this coding method. In addition, the efficiency of universal combinatorial coding is analyzed theoretically. The multicharacteristic and multiapplication of universal combinatorial coding are unique in the existing coding methods. Universal combinatorial coding has theoretical research and practical application value.

  16. Research on universal combinatorial coding.

    PubMed

    Lu, Jun; Zhang, Zhuo; Mo, Juan

    2014-01-01

    The conception of universal combinatorial coding is proposed. Relations exist more or less in many coding methods. It means that a kind of universal coding method is objectively existent. It can be a bridge connecting many coding methods. Universal combinatorial coding is lossless and it is based on the combinatorics theory. The combinational and exhaustive property make it closely related with the existing code methods. Universal combinatorial coding does not depend on the probability statistic characteristic of information source, and it has the characteristics across three coding branches. It has analyzed the relationship between the universal combinatorial coding and the variety of coding method and has researched many applications technologies of this coding method. In addition, the efficiency of universal combinatorial coding is analyzed theoretically. The multicharacteristic and multiapplication of universal combinatorial coding are unique in the existing coding methods. Universal combinatorial coding has theoretical research and practical application value. PMID:24772019

  17. Integrins and bone metastasis: integrating tumor cell and stromal cell interactions.

    PubMed

    Schneider, Jochen G; Amend, Sarah R; Weilbaecher, Katherine N

    2011-01-01

    Integrins on both tumor cells and the supporting host stromal cells in bone (osteoclasts, new blood vessels, inflammatory cells, platelets and bone marrow stromal cells) play key roles in enhancing bone metastasis. Tumor cells localize to specific tissues through integrin-mediated contacts with extracellular matrix and stromal cells. Integrin expression and signaling are perturbed in cancer cells, allowing them to "escape" from cell-cell and cell-matrix tethers, invade, migrate and colonize within new tissues and matrices. Integrin signaling through αvβ3 and VLA-4 on tumor cells can promote tumor metastasis to and proliferation in the bone microenvironment. Osteoclast (OC) mediated bone resorption is a critical component of bone metastasis and can promote tumor growth in bone and αvβ3 integrins are critical to OC function and development. Tumors in the bone microenvironment can recruit new blood vessel formation, platelets, pro-tumor immune cells and bone marrow stromal cells that promote tumor growth and invasion in bone. Integrins and their ligands play critical roles in platelet aggregation (αvβ3 and αIIbβ3), hematopoietic cell mobilization (VLA-4 and osteopontin), neoangiogenesis (αvβ3, αvβ5, α6β4, and β1 integrin) and stromal function (osteopontin and VLA-4). Integrins are involved in the pathogenesis of bone metastasis at many levels and further study to define integrin dysregulation by cancer will yield new therapeutic targets for the prevention and treatment of bone metastasis.

  18. Human macrophage differentiation involves an interaction between integrins and fibronectin

    SciTech Connect

    Laouar, A.; Chubb, C.B.H.; Collart, F.; Huberman, E.

    1997-03-14

    The authors have examined the role of integrins and extracellular matrix (ECM) proteins in macrophage differentiation of (1) human HL-60 myeloid leukemia cells induced by phorbol 12-myristate 13-acetate (PMA) and (2) human peripheral blood monocytes induced by either PMA or macrophage-colony stimulating factor (M-CSF). Increased {beta}{sub 1} integrin and fibronectin (FN) gene expression was observed in PMA-treated HL-60 cells and PMA- or M-CSF-treated monocytes, even at a time preceding the manifestation of macrophage markers. Treated HL-60 cells and monocytes also released and deposited FN on the culture dishes. An HL-60 cell variant, HL-525, which is deficient in protein kinase C {beta} (PKC{beta}) and resistant to PMA-induced differentiation, failed to express FN after PMA treatment. Restoration of PKC{beta} resulted in PMA-induced FN gene expression and macrophage differentiation. The macrophage phenotype induced in HL-60 cells or monocytes was attenuated by anti-{beta}{sub 1} integrin or anti-FN MAbs. The authors suggest that macrophage differentiation involves activation of PKC and expression of specific integrins and ECM proteins. The stimulated cells, through their integrins, attach and spread on these substrates by binding to the deposited ECM proteins. This attachment and spreading in turn, through integrin signaling, leads to the macrophage phenotype.

  19. Novel monoclonal antibody against beta 1 integrin enhances cisplatin efficacy in human lung adenocarcinoma cells

    PubMed Central

    Kim, Min-Young; Cho, Woon-Dong; Hong, Kwon Pyo; Choi, Da Bin; Hong, Jeong won; Kim, Soseul; Moon, Yoo Ri; Son, Seung-Myoung; Lee, Ok-Jun; Lee, Ho-Chang; Song, Hyung Geun

    2016-01-01

    Abstract The use of anti-beta 1 integrin monoclonal antibody in lung cancer treatment has proven beneficial. Here, we developed a novel monoclonal antibody (mAb), called P5, by immunizing mice with human peripheral blood mononuclear cells (PBMC). Its anti-tumor effect is now being tested, in a clinical phase III trial, in combinatorial treatments with various chemical drugs. To confirm that P5 indeed binds to beta 1 integrin, cell lysates were immunoprecipitated with commercial anti-beta 1 integrin mAb (TS2/16) and immunoblotted against P5 to reveal a 140 kDa molecular weight band, as expected. Immunoprecipitation with P5 followed by LC/MS protein sequence analysis further verified P5 antigen to be beta 1 integrin. Cisplatin treatment upregulated cell surface expression of beta 1 integrin in A549 cells, while causing inhibition of cell growth. When cells were co-treated with different concentrations of P5 mAb, the cisplatin-mediated inhibitory effect was enhanced in a dose-dependent manner. Our findings show that a combinatorial treatment of P5 mAb and cisplatin in A549 cells resulted in a 30% increase in apoptosis, compared to baseline, and significantly more when compared to either the cisplatin or P5 alone group. The entire peptide sequences in CDR from variable region of Ig heavy and light chain gene for P5 mAb are also disclosed. Together, these results provide evidence of the beneficial effect of P5 mAb in combinatorial treatment of human lung adenocarcinoma. PMID:27533932

  20. Combinatorial Effects of Curcumin with an Anti-Neoplastic Agent on Head and Neck Squamous Cell Carcinoma Through the Regulation of EGFR-ERK1/2 and Apoptotic Signaling Pathways.

    PubMed

    Sivanantham, Banudevi; Sethuraman, Swaminathan; Krishnan, Uma Maheswari

    2016-01-11

    Globally, head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer and represents approximately 6% of all diagnosed cancers. The use of anti-cancer drugs, such as docetaxel, doxorubicin (DOX), 5-fluorouracil (5-FU), and cisplatin (diammine dichloroplatinum(II), CDDP), is limited due to their non-specificity, drug resistance, and toxicity. A combinatorial approach may improve the efficacy of these chemotherapeutic drugs and reduce their non-specific toxicities. In the present study, curcumin, an anti-cancer phytochemical, was used in combination with 5-FU, doxorubicin, and cisplatin and their combinatorial effect on the HNSCC cell line NT8e was investigated. Our results showed that the combination of 5-FU or DOX with curcumin exhibited significant growth inhibition and enhanced apoptosis in NT8e cancer cells. Treatment with 5-FU or DOX in combination with curcumin induced apoptosis by inhibiting Bcl-2 and increasing Bax, caspase-3, and poly-ADP ribose polymerase (PARP) in NT8e cells. This was further confirmed through apoptotic characteristic features in cells, such as membrane blebbing, nuclear condensation, and cell shrinkage, as observed by DAPI staining and through decreased red/green fluorescence by JC-1. These two combinations also exhibited cell cycle growth arrest at the G1/S phase, which was confirmed by downregulation of cyclins (D1, E2, B1, and A2), CDK2, and increased p21 levels. In addition, curcumin exposure along with 5-FU or DOX inhibited cell proliferation through the downregulation of EGFR-ERK1/2 signaling molecules. Overall, our data demonstrates the promising therapeutic potential and underlying mechanisms of curcumin with 5-FU/DOX combinations as a new treatment modality for head and neck cancer management.

  1. Optimization of Combinatorial Mutagenesis

    NASA Astrophysics Data System (ADS)

    Parker, Andrew S.; Griswold, Karl E.; Bailey-Kellogg, Chris

    Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (stability, activity, immunogenicity, etc.). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries - a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 107 variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and

  2. Bifurcation-based approach reveals synergism and optimal combinatorial perturbation.

    PubMed

    Liu, Yanwei; Li, Shanshan; Liu, Zengrong; Wang, Ruiqi

    2016-06-01

    Cells accomplish the process of fate decisions and form terminal lineages through a series of binary choices in which cells switch stable states from one branch to another as the interacting strengths of regulatory factors continuously vary. Various combinatorial effects may occur because almost all regulatory processes are managed in a combinatorial fashion. Combinatorial regulation is crucial for cell fate decisions because it may effectively integrate many different signaling pathways to meet the higher regulation demand during cell development. However, whether the contribution of combinatorial regulation to the state transition is better than that of a single one and if so, what the optimal combination strategy is, seem to be significant issue from the point of view of both biology and mathematics. Using the approaches of combinatorial perturbations and bifurcation analysis, we provide a general framework for the quantitative analysis of synergism in molecular networks. Different from the known methods, the bifurcation-based approach depends only on stable state responses to stimuli because the state transition induced by combinatorial perturbations occurs between stable states. More importantly, an optimal combinatorial perturbation strategy can be determined by investigating the relationship between the bifurcation curve of a synergistic perturbation pair and the level set of a specific objective function. The approach is applied to two models, i.e., a theoretical multistable decision model and a biologically realistic CREB model, to show its validity, although the approach holds for a general class of biological systems.

  3. The anti-tumor NC1 domain of collagen XIX inhibits the FAK/ PI3K/Akt/mTOR signaling pathway through αvβ3 integrin interaction.

    PubMed

    Oudart, Jean-Baptiste; Doué, Manon; Vautrin, Alexia; Brassart, Bertrand; Sellier, Christèle; Dupont-Deshorgue, Aurelie; Monboisse, Jean-Claude; Maquart, François-Xavier; Brassart-Pasco, Sylvie; Ramont, Laurent

    2016-01-12

    Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvβ3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3β activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs. PMID:26621838

  4. The anti-tumor NC1 domain of collagen XIX inhibits the FAK/ PI3K/Akt/mTOR signaling pathway through αvβ3 integrin interaction

    PubMed Central

    Oudart, Jean-Baptiste; Doué, Manon; Vautrin, Alexia; Brassart, Bertrand; Sellier, Christèle; Dupont-Deshorgue, Aurelie; Monboisse, Jean-Claude; Maquart, François-Xavier; Brassart-Pasco, Sylvie; Ramont, Laurent

    2016-01-01

    Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvβ3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3β activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs. PMID:26621838

  5. CCN1 acutely increases nitric oxide production via integrin αvβ3-Akt-S6K-phosphorylation of endothelial nitric oxide synthase at the serine 1177 signaling axis.

    PubMed

    Hwang, Soojin; Lee, Hyeon-Ju; Kim, Gyungah; Won, Kyung-Jong; Park, Yoon Shin; Jo, Inho

    2015-12-01

    Although CCN1 (also known as cysteine-rich, angiogenic inducer 61, CYR61) has been reported to promote angiogenesis and neovascularization in endothelial cells (ECs), its effects on endothelial nitric oxide (NO) production have never been studied. Using human umbilical vein ECs, we investigated whether and how CCN1 regulates NO production. CCN1 acutely increased NO production in a time- and dose-dependent manner, which was accompanied by increased phosphorylation of endothelial NO synthase (eNOS) at serine 1177 (eNOS-Ser(1177)), but not that of eNOS-Thr(495) or eNOS-Ser(114). The level of total eNOS expression was unaltered. Treatment with either LY294002, a selective inhibitor of phosphoinositide 3-kinase known as an upstream kinase of Akt, or H-89, an inhibitor of protein kinase A, mitogen- and stress-activated protein kinase 1, Rho-associated protein kinase 2, and ribosomal protein S6 kinase (S6K), inhibited CCN1-stimulated eNOS-Ser(1177) phosphorylation and subsequent NO production. Ectopic expression of small interfering RNA against Akt and S6K significantly inhibited the effects of CCN1. Consistently, CCN1 increased the phosphorylation of Akt-Ser(473) and S6K-Thr(389). However, CCN1 did not alter the expression or secretion of VEGF, a known downstream factor of CCN1 and a potential upstream factor of Akt-mediated eNOS-Ser(1177) phosphorylation. Furthermore, neutralization of integrin αvβ3 with corresponding antibody completely reversed all of the observed effects of CCN1. Moreover, CCN1 increased acetylcholine-induced relaxation in the rat aortas. Finally, we also found that CCN1-stimulated eNOS-Ser(1177) phosphorylation and NO production are true for other types of EC tested. In conclusion, CCN1 acutely increases NO production via activation of a signaling axis in integrin αvβ3-Akt-S6K-eNOS-Ser(1177) phosphorylation, suggesting an important role for CCN1 in vasodilation.

  6. Leukocyte integrins: role in leukocyte recruitment and as therapeutic targets in inflammatory disease.

    PubMed

    Mitroulis, Ioannis; Alexaki, Vasileia I; Kourtzelis, Ioannis; Ziogas, Athanassios; Hajishengallis, George; Chavakis, Triantafyllos

    2015-03-01

    Infection or sterile inflammation triggers site-specific attraction of leukocytes. Leukocyte recruitment is a process comprising several steps orchestrated by adhesion molecules, chemokines, cytokines and endogenous regulatory molecules. Distinct adhesive interactions between endothelial cells and leukocytes and signaling mechanisms contribute to the temporal and spatial fine-tuning of the leukocyte adhesion cascade. Central players in the leukocyte adhesion cascade include the leukocyte adhesion receptors of the β2-integrin family, such as the αLβ2 and αMβ2 integrins, or of the β1-integrin family, such as the α4β1-integrin. Given the central involvement of leukocyte recruitment in different inflammatory and autoimmune diseases, the leukocyte adhesion cascade in general, and leukocyte integrins in particular, represent key therapeutic targets. In this context, the present review focuses on the role of leukocyte integrins in the leukocyte adhesion cascade. Experimental evidence that has implicated leukocyte integrins as targets in animal models of inflammatory disorders, such as experimental autoimmune encephalomyelitis, psoriasis, inflammatory bone loss and inflammatory bowel disease as well as preclinical and clinical therapeutic applications of antibodies that target leukocyte integrins in various inflammatory disorders are presented. Finally, we review recent findings on endogenous inhibitors that modify leukocyte integrin function, which could emerge as promising therapeutic targets.

  7. Regulation of Cell Migration and β1 Integrin Trafficking by the Endosomal Adaptor GGA3.

    PubMed

    Ratcliffe, Colin D H; Sahgal, Pranshu; Parachoniak, Christine A; Ivaska, Johanna; Park, Morag

    2016-06-01

    The integrin family of cell adhesion receptors link extracellular matrices to intracellular signaling pathways and the actin cytoskeleton; and regulate cell migration, proliferation and survival in normal and diseased tissues. The subcellular location of integrin receptors is critical for their function and deregulated trafficking is implicated in various human diseases. Here we identify a role for Golgi-localized gamma-ear containing Arf-binding protein 3 (GGA3), in regulating trafficking of β1 integrin. GGA3 knockdown reduces cell surface and total levels of α2, α5 and β1 integrin subunits, inhibits cell spreading, reduces focal adhesion number, as well as cell migration. In the absence of GGA3, integrins are increasingly retained inside the cell, traffic toward the perinuclear lysosomal compartment and their degradation is enhanced. Integrin traffic and maintenance of integrin levels are dependent on the integrity of the Arf binding site of GGA3. Furthermore, sorting nexin 17 (SNX17), a critical regulator of integrin recycling, becomes mislocalized to enlarged late endosomes upon GGA3 depletion. These data support a model whereby GGA3, through its ability to regulate SNX17 endosomal localization and through interaction with Arf6 diverts integrins from the degradative pathway supporting cell migration. PMID:26935970

  8. Integrin α5/β1 Mediates Fibronectin-dependent Epithelial Cell Proliferation through Epidermal Growth Factor Receptor Activation

    PubMed Central

    Kuwada, Scott K.; Li, Xiufen

    2000-01-01

    Human integrin α5 was transfected into the integrin α5/β1–negative intestinal epithelial cell line Caco-2 to study EGF receptor (EGFR) and integrin α5/β1 signaling interactions involved in epithelial cell proliferation. On uncoated or fibronectin-coated plastic, the integrin α5 and control (vector only) transfectants grew at similar rates. In the presence of the EGFR antagonistic mAb 225, the integrin α5 transfectants and controls were significantly growth inhibited on plastic. However, when cultured on fibronectin, the integrin α5 transfectants were not growth inhibited by mAb 225. The reversal of mAb 225–mediated growth inhibition on fibronectin for the integrin α5 transfectants correlated with activation of the EGFR, activation of MAPK, and expression of proliferating cell nuclear antigen. EGFR kinase activity was necessary for both MAPK activation and integrin α5/β1–mediated cell proliferation. Although EGFR activation occurred when either the integrin α5–transfected or control cells were cultured on fibronectin, coprecipitation of the EGFR with SHC could be demonstrated only in the integrin α5–transfected cells. These results suggest that integrin α5/β1 mediates fibronectin-induced epithelial cell proliferation through activation of the EGFR. PMID:10888683

  9. Targeting ILK and {beta}4 integrin abrogates the invasive potential of ovarian cancer

    SciTech Connect

    Choi, Yoon Pyo; Kim, Baek Gil; Gao, Ming-Qing; Kang, Suki; Cho, Nam Hoon

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer The potential of targeting ILK and integrins for highly aggressive ovarian cancer. Black-Right-Pointing-Pointer Unanticipated synergistic effect for the combination of ILK/{beta}4 integrin. Black-Right-Pointing-Pointer Combination of ILK/{beta}4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade. Black-Right-Pointing-Pointer Targeting of {beta}4 integrin/ILK had potent inhibitory effects in ovarian cancer. -- Abstract: Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of {beta}1 and {beta}4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expression of {beta}1 and {beta}4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of {beta}4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of {beta}4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting {beta}4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.

  10. Optimization of combinatorial mutagenesis.

    PubMed

    Parker, Andrew S; Griswold, Karl E; Bailey-Kellogg, Chris

    2011-11-01

    Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (e.g., stability, activity, immunogenicity). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries--a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 10⁷ variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and

  11. DNA-Encoded Dynamic Combinatorial Chemical Libraries.

    PubMed

    Reddavide, Francesco V; Lin, Weilin; Lehnert, Sarah; Zhang, Yixin

    2015-06-26

    Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA-encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal-to-noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA-encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high-affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA-templated chemical reactions.

  12. Beta2-integrins contribute to skeletal muscle hypertrophy in mice.

    PubMed

    Marino, Joseph S; Tausch, Brian J; Dearth, Christopher L; Manacci, Marc V; McLoughlin, Thomas J; Rakyta, Samuel J; Linsenmayer, Matthew P; Pizza, Francis X

    2008-10-01

    We tested the contribution of beta(2)-integrins, which are important for normal function of neutrophils and macrophages, to skeletal muscle hypertrophy after mechanical loading. Using the synergist ablation model of hypertrophy and mice deficient in the common beta-subunit of beta(2)-integrins (CD18(-/-)), we found that overloaded muscles of wild-type mice had greater myofiber size, dry muscle mass, and total protein content compared with CD18(-/-) mice. The hypertrophy in wild-type mice was preceded by elevations in neutrophils, macrophages, satellite cell/myoblast proliferation (5'-bromo-2'-deoxyuridine- and desmin-positive cells), markers of muscle differentiation (MyoD1 and myogenin gene expression and formation and size of regenerating myofibers), signaling for protein synthesis [phosphorylation of Akt and 70-kDa ribosomal protein S6 kinase (p70S6k)], and reduced signaling for protein degradation (decreased gene expression of muscle atrophy F box/atrogin-1). The deficiency in beta(2)-integrins, however, altered the accumulation profile of neutrophils and macrophages, disrupted the temporal profile of satellite cell/myoblast proliferation, reduced the markers of muscle differentiation, and impaired the p70S6k signaling, all of which could serve as mechanisms for the impaired hypertrophy in overloaded CD18(-/-) mice. In conclusion, our findings indicate that beta(2)-integrins contribute to the hypertrophic response to muscle overload by temporally regulating satellite cells/myoblast proliferation and by enhancing muscle differentiation and p70S6k signaling.

  13. Cryptographic Combinatorial Securities Exchanges

    NASA Astrophysics Data System (ADS)

    Thorpe, Christopher; Parkes, David C.

    We present a useful new mechanism that facilitates the atomic exchange of many large baskets of securities in a combinatorial exchange. Cryptography prevents information about the securities in the baskets from being exploited, enhancing trust. Our exchange offers institutions who wish to trade large positions a new alternative to existing methods of block trading: they can reduce transaction costs by taking advantage of other institutions’ available liquidity, while third party liquidity providers guarantee execution—preserving their desired portfolio composition at all times. In our exchange, institutions submit encrypted orders which are crossed, leaving a “remainder”. The exchange proves facts about the portfolio risk of this remainder to third party liquidity providers without revealing the securities in the remainder, the knowledge of which could also be exploited. The third parties learn either (depending on the setting) the portfolio risk parameters of the remainder itself, or how their own portfolio risk would change if they were to incorporate the remainder into a portfolio they submit. In one setting, these third parties submit bids on the commission, and the winner supplies necessary liquidity for the entire exchange to clear. This guaranteed clearing, coupled with external price discovery from the primary markets for the securities, sidesteps difficult combinatorial optimization problems. This latter method of proving how taking on the remainder would change risk parameters of one’s own portfolio, without revealing the remainder’s contents or its own risk parameters, is a useful protocol of independent interest.

  14. Osteopontin involvement in integrin-mediated cell signaling and regulation of expression of alkaline phosphatase during early differentiation of UMR cells.

    PubMed

    Liu, Y K; Uemura, T; Nemoto, A; Yabe, T; Fujii, N; Ushida, T; Tateishi, T

    1997-12-22

    To clarify the function of osteopontin in osteoblast differentiation, we have examined the signal transduction pathway in an osteoblastic cell line (UMR106-6) bound to osteopontin, fibronectin, vitronectin and collagen type I surfaces. This was done by investigating the production and autophosphorylation of focal adhesion kinase (FAK) and the expression of alkaline phosphatase (ALP) at the transcription level. Results suggest that osteopontin was not only responsible for the autophosphorylation of FAK but regulated the expression of ALP, which was strongly correlated with FAK activity. These results suggest that osteopontin might act as a trigger in the early differentiation of osteoblasts.

  15. The modulation of MiR-155 and MiR-23a manipulates Klebsiella pneumoniae Adhesion on Human pulmonary Epithelial cells via Integrin α5β1 Signaling

    PubMed Central

    Teng, Yan; Miao, Junming; Shen, Xiaofei; Yang, Xiaolong; Wang, Xinyuan; Ren, Laibin; Wang, Xiaoying; Chen, Junli; Li, Jingyu; Chen, Shanze; Wang, Yi; Huang, Ning

    2016-01-01

    Micro-RNAs (miRNAs) critically regulate several host defense mechanisms, but their roles in the bacteria-epithelium interplay remain unclear. Our results displayed that the expression of miR-155 and miR-23a were down-regulated in K. pneumoniae-infected pulmonary epithelial cells. The elevated bacterial adhesion on A549 cells followed the enhancement of the cellular levels of these two miRNAs. Meanwhile, a mechanistic study demonstrated that miR-155 promoted integrin α5β1 function and resulted in the increased actin polymerization. Moreover, a non-histone nuclear protein, high mobility group nucleosomal-binding domain 2 (HMGN2) served as the potential target of miR-155 and miR-23a to regulate the integrin α5β1 expression and K. pneumoniae adhesion. Furthermore, the expression of a known integrin transcription suppressor-Nuclear Factor-I (NFI) was also repressed by miR-155, which paralleled with its chromatin location in the promoter regions of integrin α5 and β1. These results uncover novel links between miRNAs and integrin function to regulate bacterial adhesion, indicating a potential mechanism of host cell autonomous immune response to K. pneumoniae infection. PMID:27534887

  16. Integrin α3β1 regulates kidney collecting duct development via TRAF6-dependent K63-linked polyubiquitination of Akt

    PubMed Central

    Yazlovitskaya, Eugenia M.; Tseng, Hui-Yuan; Viquez, Olga; Tu, Tianxiang; Mernaugh, Glenda; McKee, Karen K.; Riggins, Karen; Quaranta, Vito; Pathak, Amrita; Carter, Bruce D.; Yurchenco, Peter; Sonnenberg, Arnoud; Böttcher, Ralph T.; Pozzi, Ambra; Zent, Roy

    2015-01-01

    The collecting system of the kidney develops from the ureteric bud (UB), which undergoes branching morphogenesis, a process regulated by multiple factors, including integrin–extracellular matrix interactions. The laminin (LM)-binding integrin α3β1 is crucial for this developmental program; however, the LM types and LM/integrin α3β1–dependent signaling pathways are poorly defined. We show that α3 chain–containing LMs promote normal UB branching morphogenesis and that LM-332 is a better substrate than LM-511 for stimulating integrin α3β1–dependent collecting duct cell functions. We demonstrate that integrin α3β1–mediated cell adhesion to LM-332 modulates Akt activation in the developing collecting system and that Akt activation is PI3K independent but requires decreased PTEN activity and K63-linked polyubiquitination. We identified the ubiquitin-modifying enzyme TRAF6 as an interactor with the integrin β1 subunit and regulator of integrin α3β1–dependent Akt activation. Finally, we established that the developmental defects of TRAF6- and integrin α3–null mouse kidneys are similar. Thus K63-linked polyubiquitination plays a previously unrecognized role in integrin α3β1–dependent cell signaling required for UB development and may represent a novel mechanism whereby integrins regulate signaling pathways. PMID:25808491

  17. Integrin-associated protein: a 50-kD plasma membrane antigen physically and functionally associated with integrins

    PubMed Central

    1990-01-01

    Phagocytosis by monocytes or neutrophils can be enhanced by interaction with several proteins or synthetic peptides containing the Arg-Gly-Asp sequence. Recently we showed that an mAb, B6H12, specifically inhibited this enhancement of neutrophil phagocytosis by inhibiting Arg-Gly-Asp binding to the leukocyte response integrin (Gresham, H. D., J. L. Goodwin, P. M. Allen, D. C. Anderson, and E. J. Brown. 1989. J. Cell Biol. 108:1935-1943). Now, we have purified the antigen recognized by B6H12 to homogeneity. Surprisingly, it is a 50-kD molecule that is expressed on the plasma membranes of all hematopoietic cells, including erythrocytes, which express no known integrins. On platelets and placenta, but not on erythrocytes, this protein is associated with an integrin that can be recognized by an anti-beta 3 antibody. In addition, both the anti-beta 3 and several mAbs recognizing the 50-kD protein inhibit Arg-Gly-Asp stimulation of phagocytosis. These data demonstrate an association between integrins and the 50-kD protein on several cell types. For this reason, we call it Integrin-associated Protein (IAP). We hypothesize that IAP may play a role in signal transduction for enhanced phagocytosis by Arg-Gly-Asp ligands. PMID:2277087

  18. αVβ3 Integrin Regulation of Respiratory Burst in Fibrinogen Adherent Human Neutrophils

    PubMed Central

    Kim, Hye-Yeong; Skokos, Eleni A.; Myer, Deborah J.; Agaba, Perez; Gonzalez, Anjelica L.

    2015-01-01

    In response to inflammatory stimuli, microvascular endothelial cells become activated, initiating the capture and exit of neutrophils from the blood vessel and into the extravascular extracellular matrix (ECM). In the extravascular space, neutrophils bind to ECM proteins, regulating cellular functions via signaling through adhesion molecules known as integrins. The αVβ3 integrin is an important mediator of neutrophil adhesion to ECM proteins containing the Arg-Gly-Asp (RGD) peptide sequence, including fibrinogen and fibronectin. Despite the abundance of RGD sequence in the ECM, adhesion molecule-mediated neutrophil activity has been focused on the β2 (Mac-1, CD11b/CD18) and β1 integrin response to matrix proteins. Here we investigated αVβ3 integrin-mediated reactive oxidant suppression as a consequence of human neutrophil adhesion to RGD containing proteins. Using integrin ligand-modified (poly)ethylene glycol hydrogels and reactive oxygen species (ROS) sensitive fluorescent probes (dihydrotetramethylrhosamine, H2TMRos), we evaluated integrin–peptide interactions that effectively regulate ROS generation. This study demonstrates that neutrophil adhesion suppresses ROS production in an αVβ3-dependent manner. Additionally, we determine that p38 mitogen-activated protein kinase in the respiratory burst signaling pathway is interrupted by integrin-mediated adhesion. These data indicate that ECM/integrin interactions can induce αVβ3-mediated adhesion dependent downstream signaling of ROS regulation via a Mac-1 independent mechanism. PMID:25632307

  19. Combinatorial optimization games

    SciTech Connect

    Deng, X.; Ibaraki, Toshihide; Nagamochi, Hiroshi

    1997-06-01

    We introduce a general integer programming formulation for a class of combinatorial optimization games, which immediately allows us to improve the algorithmic result for finding amputations in the core (an important solution concept in cooperative game theory) of the network flow game on simple networks by Kalai and Zemel. An interesting result is a general theorem that the core for this class of games is nonempty if and only if a related linear program has an integer optimal solution. We study the properties for this mathematical condition to hold for several interesting problems, and apply them to resolve algorithmic and complexity issues for their cores along the line as put forward in: decide whether the core is empty; if the core is empty, find an imputation in the core; given an imputation x, test whether x is in the core. We also explore the properties of totally balanced games in this succinct formulation of cooperative games.

  20. Integrins activate trimeric G proteins via the nonreceptor protein GIV/Girdin

    PubMed Central

    Leyme, Anthony; Marivin, Arthur; Perez-Gutierrez, Lorena; Nguyen, Lien T.

    2015-01-01

    Signal transduction via integrins and G protein–coupled receptors is critical to control cell behavior. These two receptor classes have been traditionally believed to trigger distinct and independent signaling cascades in response to extracellular cues. Here, we report a novel mechanism of integrin signaling that requires activation of the trimeric G protein Gαi by the nonreceptor guanine nucleotide exchange factor (GEF) GIV (also known as Girdin), a metastasis-associated protein. We demonstrate that GIV enhances integrin-dependent cell responses upon extracellular matrix stimulation and makes tumor cells more invasive. These responses include remodeling of the actin cytoskeleton and PI3K-dependent signaling, resulting in enhanced haptotaxis and invasion. We show that both GIV and its substrate Gαi3 are recruited to active integrin complexes and that tumor cells engineered to express GEF-deficient GIV fail to transduce integrin signals into proinvasive responses via a Gβγ-PI3K axis. Our discoveries delineate a novel mechanism by which integrin signaling is rewired during metastasis to result in increased tumor invasiveness. PMID:26391662

  1. Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes

    NASA Technical Reports Server (NTRS)

    Miyamoto, Yuko J.; Mitchell, Jason S.; McIntyre, Bradley W.

    2003-01-01

    CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

  2. Structural insights into the extra cellular segment of integrinβ5 and molecular interaction studies.

    PubMed

    Setti, Aravind; Sankati, Harsha Sagar; Devi, T A Phazna; Sekhar, A Chandra; Rao, J Venkateshwar; Pawar, Smita C

    2013-10-01

    Primary tumor cells often spread to other organs by metastasis. Despite of it, primary tumor cells break their surrounding extra cellular matrix (ECM) proteins and reach the destination organ by the process of intravasation and extravasation. Metastasized tumor cells induce the process of angiogenesis, this highly regulated process involves several ECM proteins. However, integrins are primarily involved in the blood vessel growth and repair. Therefore, integrins are promising angiogenesis targets. Integrins are receptors on cell surface, involved in signal transduction and attachments in extra cellular matrix (ECM). IntegrinαVβ3 and αVβ5 are implicated in tumor angiogenesis, metastasis, inflammation and bone resorption. The crystal structure of integrinαvβ5 is not available in protein structural databases, therefore; molecular model of integrinβ5 structure was prepared and stereo chemical model quality was checked. Integrin β5 active sites were identified based on insilico analysis tools. Further, molecular level interactions between integrinβ5 and ECM proteins were predicted. In the present study ECM proteins such as focal adhesion kinase 1 (FAK1), annexin A5 and P21 activated kinase 4 (PAK4) were considered for protein-protein docking, to understand inter molecular interactions. The predicted model is conceived to be stereo chemically good and can be used for molecular interaction studies of angiogenic inhibitors. PMID:23962022

  3. Integrin β3 Is Required in Infection and Proliferation of Classical Swine Fever Virus

    PubMed Central

    Li, Weiwei; Wang, Gang; Liang, Wulong; Kang, Kai; Guo, Kangkang; Zhang, Yanming

    2014-01-01

    Classical Swine Fever (CSF) is a highly infectious fatal pig disease, resulting in huge economic loss to the swine industry. Integrins are membrane-bound signal mediators, expressed on a variety of cell surfaces and are known as receptors or co-receptors for many viruses. However, the role of integrin β3 in CSFV infection is unknown. Here, through quantitive PCR, immunofluorescence (IFC) and immunocytohistochemistry (ICC), we revealed that ST (swine testicles epithelial) cells have a prominent advantage in CSFV proliferation as compared to EC (swine umbilical vein endothelial cell), IEC (swine intestinal epithelial cell) and PK (porcine kidney epithelial) cells. Meanwhile, ST cells had remarkably more integrin β3 expression as compared to EC, IEC and PK cells, which was positively correlated with CSFV infection and proliferation. Integrin β3 was up-regulated post CSFV infection in all the four cell lines, while the CSFV proliferation rate was decreased in integrin β3 function-blocked cells. ShRNA1755 dramatically decreased integrin β3, with a deficiency of 96% at the mRNA level and 80% at the protein level. CSFV proliferation was dramatically reduced in integrin β3 constantly-defected cells (ICDC), with the deficiencies of 92.6%, 99% and 81.7% at 24 h, 48 h and 72 h post CSFV infection, respectively. These results demonstrate that integrin β3 is required in CSFV infection and proliferation, which provide a new insight into the mechanism of CSFV infection. PMID:25340775

  4. Integrin αv in the mechanical response of osteoblast lineage cells

    SciTech Connect

    Kaneko, Keiko; Ito, Masako; Naoe, Yoshinori; Lacy-Hulbert, Adam; Ikeda, Kyoji

    2014-05-02

    Highlights: • Deletion of integrin αv in osteoblast lineage results in an impaired SOST response to loading in vivo. • c-Src–p130Cas–JNK–YAP/TAZ is activated via integrin αv on osteoblasts in response to FSS. • Deletion of integrin αv in osteoblasts results in impaired responses to mechanical stimulation. • Integrin αv is a key component of the mechanosensing machinery in bone. - Abstract: Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation with fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src–JNK–YAP/TAZ pathway in response to mechanical stimulation.

  5. Why is combinatorial communication rare in the natural world, and why is language an exception to this trend?

    PubMed Central

    Scott-Phillips, Thomas C.; Blythe, Richard A.

    2013-01-01

    In a combinatorial communication system, some signals consist of the combinations of other signals. Such systems are more efficient than equivalent, non-combinatorial systems, yet despite this they are rare in nature. Why? Previous explanations have focused on the adaptive limits of combinatorial communication, or on its purported cognitive difficulties, but neither of these explains the full distribution of combinatorial communication in the natural world. Here, we present a nonlinear dynamical model of the emergence of combinatorial communication that, unlike previous models, considers how initially non-communicative behaviour evolves to take on a communicative function. We derive three basic principles about the emergence of combinatorial communication. We hence show that the interdependence of signals and responses places significant constraints on the historical pathways by which combinatorial signals might emerge, to the extent that anything other than the most simple form of combinatorial communication is extremely unlikely. We also argue that these constraints can be bypassed if individuals have the socio-cognitive capacity to engage in ostensive communication. Humans, but probably no other species, have this ability. This may explain why language, which is massively combinatorial, is such an extreme exception to nature's general trend for non-combinatorial communication. PMID:24047871

  6. Why is combinatorial communication rare in the natural world, and why is language an exception to this trend?

    PubMed

    Scott-Phillips, Thomas C; Blythe, Richard A

    2013-11-01

    In a combinatorial communication system, some signals consist of the combinations of other signals. Such systems are more efficient than equivalent, non-combinatorial systems, yet despite this they are rare in nature. Why? Previous explanations have focused on the adaptive limits of combinatorial communication, or on its purported cognitive difficulties, but neither of these explains the full distribution of combinatorial communication in the natural world. Here, we present a nonlinear dynamical model of the emergence of combinatorial communication that, unlike previous models, considers how initially non-communicative behaviour evolves to take on a communicative function. We derive three basic principles about the emergence of combinatorial communication. We hence show that the interdependence of signals and responses places significant constraints on the historical pathways by which combinatorial signals might emerge, to the extent that anything other than the most simple form of combinatorial communication is extremely unlikely. We also argue that these constraints can be bypassed if individuals have the socio-cognitive capacity to engage in ostensive communication. Humans, but probably no other species, have this ability. This may explain why language, which is massively combinatorial, is such an extreme exception to nature's general trend for non-combinatorial communication.

  7. Cells on the run: shear-regulated integrin activation in leukocyte rolling and arrest on endothelial cells.

    PubMed

    Alon, Ronen; Ley, Klaus

    2008-10-01

    The arrest of rolling leukocytes on various target vascular beds is mediated by specialized leukocyte integrins and their endothelial immunoglobulin superfamily (IgSF) ligands. These integrins are kept in largely inactive states and undergo in situ activation upon leukocyte-endothelial contact by both biochemical and mechanical signals from flow-derived shear forces. In vivo and in vitro studies suggest that leukocyte integrin activation involves conformational alterations through inside-out signaling followed by ligand-induced rearrangements accelerated by external forces. This activation process takes place within fractions of seconds by in situ signals transduced to the rolling leukocyte as it encounters specialized endothelial-displayed chemoattractants, collectively termed arrest chemokines. In neutrophils, selectin rolling engagements trigger intermediate affinity integrins to support reversible adhesions before chemokine-triggered arrest. Different leukocyte subsets appear to use different modalities of integrin activation during rolling and arrest at distinct endothelial sites.

  8. Integrin β4 regulates SPARC protein to promote invasion.

    PubMed

    Gerson, Kristin D; Shearstone, Jeffrey R; Maddula, V S R Krishna; Seligmann, Bruce E; Mercurio, Arthur M

    2012-03-23

    The α6β4 integrin (referred to as "β4" integrin) is a receptor for laminins that promotes carcinoma invasion through its ability to regulate key signaling pathways and cytoskeletal dynamics. An analysis of published Affymetrix GeneChip data to detect downstream effectors involved in β4-mediated invasion of breast carcinoma cells identified SPARC, or secreted protein acidic and rich in cysteine. This glycoprotein has been shown to play an important role in matrix remodeling and invasion. Our analysis revealed that manipulation of β4 integrin expression and signaling impacted SPARC expression and that SPARC facilitates β4-mediated invasion. Expression of β4 in β4-deficient cells reduced the expression of a specific microRNA (miR-29a) that targets SPARC and impedes invasion. In cells that express endogenous β4, miR-29a expression is low and β4 ligation facilitates the translation of SPARC through a TOR-dependent mechanism. The results obtained in this study demonstrate that β4 can regulate SPARC expression and that SPARC is an effector of β4-mediated invasion. They also highlight a potential role for specific miRNAs in executing the functions of integrins.

  9. Redox-Relevant Aspects of the Extracellular Matrix and Its Cellular Contacts via Integrins

    PubMed Central

    de Rezende, Flávia Figueiredo

    2014-01-01

    Abstract Significance: The extracellular matrix (ECM) fulfills essential functions in multicellular organisms. It provides the mechanical scaffold and environmental cues to cells. Upon cell attachment, the ECM signals into the cells. In this process, reactive oxygen species (ROS) are physiologically used as signalizing molecules. Recent Advances: ECM attachment influences the ROS-production of cells. In turn, ROS affect the production, assembly and turnover of the ECM during wound healing and matrix remodeling. Pathological changes of ROS levels lead to excess ECM production and increased tissue contraction in fibrotic disorders and desmoplastic tumors. Integrins are cell adhesion molecules which mediate cell adhesion and force transmission between cells and the ECM. They have been identified as a target of redox-regulation by ROS. Cysteine-based redox-modifications, together with structural data, highlighted particular regions within integrin heterodimers that may be subject to redox-dependent conformational changes along with an alteration of integrin binding activity. Critical Issues: In a molecular model, a long-range disulfide-bridge within the integrin β-subunit and disulfide bridges within the genu and calf-2 domains of the integrin α-subunit may control the transition between the bent/inactive and upright/active conformation of the integrin ectodomain. These thiol-based intramolecular cross-linkages occur in the stalk domain of both integrin subunits, whereas the ligand-binding integrin headpiece is apparently unaffected by redox-regulation. Future Directions: Redox-regulation of the integrin activation state may explain the effect of ROS in physiological processes. A deeper understanding of the underlying mechanism may open new prospects for the treatment of fibrotic disorders. Antioxid. Redox Signal. 20, 1977–1993. PMID:24040997

  10. In vivo regulation of integrin turnover by outside-in activation.

    PubMed

    López-Ceballos, Pablo; Herrera-Reyes, Alejandra Donají; Coombs, Daniel; Tanentzapf, Guy

    2016-08-01

    The development of three-dimensional tissue architecture requires precise control over the attachment of cells to the extracellular matrix (ECM). Integrins, the main ECM-binding receptors in animals, are regulated in multiple ways to modulate cell-ECM adhesion. One example is the conformational activation of integrins by extracellular signals ('outside-in activation') or by intracellular signals ('inside-out activation'), whereas another is the modulation of integrin turnover. We demonstrate that outside-in activation regulates integrin turnover to stabilize tissue architecture in vivo Treating Drosophila embryos with Mg(2+) and Mn(2+), known to induce outside-in activation, resulted in decreased integrin turnover. Mathematical modeling combined with mutational analysis provides mechanistic insight into the stabilization of integrins at the membrane. We show that as tissues mature, outside-in activation is crucial for regulating the stabilization of integrin-mediated adhesions. This data identifies a new in vivo role for outside-in activation and sheds light on the key transition between tissue morphogenesis and maintenance.

  11. Algorithmic Strategies in Combinatorial Chemistry

    SciTech Connect

    GOLDMAN,DEBORAH; ISTRAIL,SORIN; LANCIA,GIUSEPPE; PICCOLBONI,ANTONIO; WALENZ,BRIAN

    2000-08-01

    Combinatorial Chemistry is a powerful new technology in drug design and molecular recognition. It is a wet-laboratory methodology aimed at ``massively parallel'' screening of chemical compounds for the discovery of compounds that have a certain biological activity. The power of the method comes from the interaction between experimental design and computational modeling. Principles of ``rational'' drug design are used in the construction of combinatorial libraries to speed up the discovery of lead compounds with the desired biological activity. This paper presents algorithms, software development and computational complexity analysis for problems arising in the design of combinatorial libraries for drug discovery. The authors provide exact polynomial time algorithms and intractability results for several Inverse Problems-formulated as (chemical) graph reconstruction problems-related to the design of combinatorial libraries. These are the first rigorous algorithmic results in the literature. The authors also present results provided by the combinatorial chemistry software package OCOTILLO for combinatorial peptide design using real data libraries. The package provides exact solutions for general inverse problems based on shortest-path topological indices. The results are superior both in accuracy and computing time to the best software reports published in the literature. For 5-peptoid design, the computation is rigorously reduced to an exhaustive search of about 2% of the search space; the exact solutions are found in a few minutes.

  12. A novel integrin {alpha}5{beta}1 antagonistic peptide, A5-1, screened by Protein Chip system as a potent angiogenesis inhibitor

    SciTech Connect

    Kim, Eung-Yoon; Bang, Ji Young; Chang, Soo-Ik; Kang, In-Cheol

    2008-12-26

    Integrin {alpha}5{beta}1 immobilized on a ProteoChip was used to screen new antagonistic peptides from multiple hexapeptide sub-libraries of the positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin {alpha}5{beta}1-Fibronectin interaction was demonstrated on the chip. A novel peptide ligand, A5-1 (VILVLF), with high affinity to integrin {alpha}5{beta}1 was identified from the hexapeptide libraries with this chip-based screening method on the basis of a competitive inhibition assay. A5-1 inhibits the integrin-fibronectin interaction in a dose-dependent manner (IC{sub 50}; 1.56 {+-} 0.28 {mu}M. In addition, it inhibits human umbilical vein endothelial cell proliferation, migration, adhesion, tubular network formation, and bFGF-induced neovascularization in a chick chorioallantoic membrane. These results suggest that A5-1 will be a potent inhibitor of neovascularization.

  13. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3

    PubMed Central

    Osicka, Radim; Osickova, Adriana; Hasan, Shakir; Bumba, Ladislav; Cerny, Jiri; Sebo, Peter

    2015-01-01

    Integrins are heterodimeric cell surface adhesion and signaling receptors that are essential for metazoan existence. Some integrins contain an I-domain that is a major ligand binding site. The ligands preferentially engage the active forms of the integrins and trigger signaling cascades that alter numerous cell functions. Here we found that the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis, preferentially binds an inactive form of the integrin complement receptor 3 (CR3), using a site outside of its I-domain. CyaA binding did not trigger downstream signaling of CR3 in human monocytes and CyaA-catalyzed elevation of cAMP effectively blocked CR3 signaling initiated by a natural ligand. This unprecedented type of integrin-ligand interaction distinguishes CyaA from all other known ligands of the I-domain-containing integrins and provides a mechanistic insight into the previously observed central role of CyaA in the pathogenesis of B. pertussis. DOI: http://dx.doi.org/10.7554/eLife.10766.001 PMID:26650353

  14. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3.

    PubMed

    Osicka, Radim; Osickova, Adriana; Hasan, Shakir; Bumba, Ladislav; Cerny, Jiri; Sebo, Peter

    2015-01-01

    Integrins are heterodimeric cell surface adhesion and signaling receptors that are essential for metazoan existence. Some integrins contain an I-domain that is a major ligand binding site. The ligands preferentially engage the active forms of the integrins and trigger signaling cascades that alter numerous cell functions. Here we found that the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis, preferentially binds an inactive form of the integrin complement receptor 3 (CR3), using a site outside of its I-domain. CyaA binding did not trigger downstream signaling of CR3 in human monocytes and CyaA-catalyzed elevation of cAMP effectively blocked CR3 signaling initiated by a natural ligand. This unprecedented type of integrin-ligand interaction distinguishes CyaA from all other known ligands of the I-domain-containing integrins and provides a mechanistic insight into the previously observed central role of CyaA in the pathogenesis of B. pertussis. PMID:26650353

  15. On schemes of combinatorial transcription logic

    NASA Astrophysics Data System (ADS)

    Buchler, Nicolas E.; Gerland, Ulrich; Hwa, Terence

    2003-04-01

    Cells receive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate specific genetic responses. Here we explore theoretically the potentials and limitations of combinatorial signal integration at the level of cis-regulatory transcription control. Our analysis suggests that many complex transcription-control functions of the type encountered in higher eukaryotes are already implementable within the much simpler bacterial transcription system. Using a quantitative model of bacterial transcription and invoking only specific protein-DNA interaction and weak glue-like interaction between regulatory proteins, we show explicit schemes to implement regulatory logic functions of increasing complexity by appropriately selecting the strengths and arranging the relative positions of the relevant protein-binding DNA sequences in the cis-regulatory region. The architectures that emerge are naturally modular and evolvable. Our results suggest that the transcription regulatory apparatus is a "programmable" computing machine, belonging formally to the class of Boltzmann machines. Crucial to our results is the ability to regulate gene expression at a distance. In bacteria, this can be achieved for isolated genes via DNA looping controlled by the dimerization of DNA-bound proteins. However, if adopted extensively in the genome, long-distance interaction can cause unintentional intergenic cross talk, a detrimental side effect difficult to overcome by the known bacterial transcription-regulation systems. This may be a key factor limiting the genome-wide adoption of complex transcription control in bacteria. Implications of our findings for combinatorial transcription control in eukaryotes are discussed. Abbreviations: TF, transcription factor RNAP, RNA polymerase DNF, disjunctive normal form CNF, conjunctive normal form

  16. Method of increasing radiation sensitivity by inhibition of beta one integrin

    DOEpatents

    Park, Catherine; Bissell, Mina J.

    2009-11-17

    A method for increasing or monitoring apoptosis in tumor cells by the co-administration of ionizing radiation and an anti-integrin antibody. Increasing apoptosis reduces tumor growth in vivo and in a cell culture model. The antibody is directed against the beta-1 integrin subunit and is inhibitory of beta-1 integrin signaling. Other molecules having an inhibitory effect on beta-1 integrin, either in signaling or in binding to its cognate extracellular receptors may also be used. The present method is particularly of interest in treatment of tumor cells associated with breast cancer, wherein radiation is currently used alone. The present method further contemplates a monoclonal antibody suitable for human administration that may further comprise a radioisotope attached thereto.

  17. Specific β-containing Integrins Exert Differential Control on Proliferation and Two-dimensional Collective Cell Migration in Mammary Epithelial Cells*

    PubMed Central

    Jeanes, Alexa I.; Wang, Pengbo; Moreno-Layseca, Paulina; Paul, Nikki; Cheung, Julia; Tsang, Ricky; Akhtar, Nasreen; Foster, Fiona M.; Brennan, Keith; Streuli, Charles H.

    2012-01-01

    Understanding how cell cycle is regulated in normal mammary epithelia is essential for deciphering defects of breast cancer and therefore for developing new therapies. Signals provided by both the extracellular matrix and growth factors are essential for epithelial cell proliferation. However, the mechanisms by which adhesion controls cell cycle in normal epithelia are poorly established. In this study, we describe the consequences of removing the β1-integrin gene from primary cultures of mammary epithelial cells in situ, using CreER. Upon β1-integrin gene deletion, the cells were unable to progress efficiently through S-phase, but were still able to undergo collective two-dimensional migration. These responses are explained by the presence of β3-integrin in β1-integrin-null cells, indicating that integrins containing different β-subunits exert differential control on mammary epithelial proliferation and migration. β1-Integrin deletion did not inhibit growth factor signaling to Erk or prevent the recruitment of core adhesome components to focal adhesions. Instead the S-phase arrest resulted from defective Rac activation and Erk translocation to the nucleus. Rac inhibition prevented Erk translocation and blocked proliferation. Activated Rac1 rescued the proliferation defect in β1-integrin-depleted cells, indicating that this GTPase is essential in propagating proliferative β1-integrin signals. These results show that β1-integrins promote cell cycle in mammary epithelial cells, whereas β3-integrins are involved in migration. PMID:22511753

  18. Transforming Growth Factor-β1 Induces Smad3-Dependent β1 Integrin Gene Expression in Epithelial-to-Mesenchymal Transition during Chronic Tubulointerstitial Fibrosis

    PubMed Central

    Yeh, Yi-Chun; Wei, Wei-Chun; Wang, Yang-Kao; Lin, Shih-Chieh; Sung, Junne-Ming; Tang, Ming-Jer

    2010-01-01

    Transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) contributes to the pathophysiological development of kidney fibrosis. Although it was reported that TGF-β1 enhances β1 integrin levels in NMuMG cells, the detailed molecular mechanisms underlying TGF-β1-induced β1 integrin gene expression and the role of β1 integrin during EMT in the renal system are still unclear. In this study, we examined the role of β1 integrin in TGF-β1-induced EMT both in vitro and in vivo. TGF-β1-induced augmentation of β1 integrin expression was required for EMT in several epithelial cell lines, and knockdown of Smad3 inhibited TGF-β1-induced augmentation of β1 integrin. TGF-β1 triggered β1 integrin gene promoter activity as assessed by luciferase activity assay. Both knockdown of Smad3 and mutation of the Smad-binding element to block binding to the β1 integrin promoter markedly reduced TGF-β1-induced β1 integrin promoter activity. Chromatin immunoprecipitation assay showed that TGF-β1 enhanced Smad3 binding to the β1 integrin promoter. Furthermore, induction of unilateral ureteral obstruction triggered increases of β1 integrin in both renal epithelial and interstitial cells. In human kidney with chronic tubulointerstitial fibrosis, we also found a concomitant increase of β1 integrin and α-smooth muscle actin in tubule epithelia. Blockade of β1 integrin signaling dampened the progression of fibrosis. Taken together, β1 integrin mediates EMT and subsequent tubulointerstitutial fibrosis, suggesting that inhibition of β1 integrin is a possible therapeutic target for prevention of renal fibrosis. PMID:20709799

  19. Constitutive Association of Tie1 and Tie2 with Endothelial Integrins is Functionally Modulated by Angiopoietin-1 and Fibronectin

    PubMed Central

    Dalton, Annamarie C.; Shlamkovitch, Tomer; Papo, Niv; Barton, William A.

    2016-01-01

    Functional cross-talk between Tie2 and Integrin signaling pathways is essential to coordinate endothelial cell adhesion and migration in response to the extracellular matrix, yet the mechanisms behind this phenomenon are unclear. Here, we examine the possibility that receptor cross-talk is driven through uncharacterized Tie-integrin interactions on the endothelial surface. Using a live cell FRET-based proximity assay, we monitor Tie-integrin receptor recognition and demonstrate that both Tie1 and Tie2 readily associate with integrins α5ß1 and αVß3 through their respective ectodomains. Although not required, Tie2-integrin association is significantly enhanced in the presence of the extracellular component and integrin ligand fibronectin. In vitro binding assays with purified components reveal that Tie-integrin recognition is direct, and further demonstrate that the receptor binding domain of the Tie2 ligand Ang-1, but not the receptor binding domain of Ang-2, can independently associate with α5ß1 or αVß3. Finally, we reveal that cooperative Tie/integrin interactions selectively stimulate ERK/MAPK signaling in the presence of both Ang-1 and fibronectin, suggesting a molecular mechanism to sensitize Tie2 to extracellular matrix. We provide a mechanistic model highlighting the role of receptor localization and association in regulating distinct signaling cascades and in turn, the angiogenic switch. PMID:27695111

  20. An EGFR/Src-dependent β4 integrin/FAK complex contributes to malignancy of breast cancer

    PubMed Central

    Tai, Yu-Ling; Chu, Pei-Yu; Lai, I-Rue; Wang, Ming-Yang; Tseng, Hui-Yuan; Guan, Jun-Lin; Liou, Jun-Yang; Shen, Tang-Long

    2015-01-01

    β4 integrin and focal adhesion kinase (FAK) are often associated with a poor prognosis in cancer patients, and their signaling events have recently been linked to malignant outcomes. Here, we demonstrate, for the first time, physical and functional interactions between β4 integrin and FAK that influence breast cancer malignancy. An amino-terminal linker within FAK is essential for its binding with the cytodomain of β4 integrin. Moreover, EGFR/Src-signaling triggers the tyrosine phosphorylation of β4 integrin, which, in turn, recruits FAK to β4 integrin and leads to FAK activation and signaling. Upon disruption of the β4 integrin/FAK complex, tumorigenesis and metastasis in triple-negative breast cancer were markedly reduced. Importantly, the concomitant overexpression of β4 integrin and FAK significantly correlates with malignant potential in patients with triple-negative breast cancer. This study describes a pro-metastatic EGFR/Src-dependent β4 integrin/FAK complex that is involved in breast cancer malignancy and is a novel therapeutic target for triple-negative breast cancer. PMID:26549523

  1. Loss of the Rap1 effector RIAM results in leukocyte adhesion deficiency due to impaired β2 integrin function in mice.

    PubMed

    Klapproth, Sarah; Sperandio, Markus; Pinheiro, Elaine M; Prünster, Monika; Soehnlein, Oliver; Gertler, Frank B; Fässler, Reinhard; Moser, Markus

    2015-12-17

    Talin is an integrin adaptor, which controls integrin activity in all hematopoietic cells. How intracellular signals promote talin binding to the integrin tail leading to integrin activation is still poorly understood, especially in leukocytes. In vitro studies identified an integrin activation complex whose formation is initiated by the interaction of active, guanosine triphosphate (GTP)-bound Ras-related protein 1 (Rap1) with the adapter protein Rap1-GTP-interacting adapter molecule (RIAM) followed by the recruitment of talin to the plasma membrane. Unexpectedly, loss-of-function studies in mice have shown that the talin-activating role of RIAM is neither required for development nor for integrin activation in platelets. In this study, we show that leukocyte integrin activation critically depends on RIAM both in vitro and in vivo. RIAM deficiency results in a loss of β2 integrin activation in multiple leukocyte populations, impaired leukocyte adhesion to inflamed vessels, and accumulation in the circulation. Surprisingly, however, the major leukocyte β1 integrin family member, α4β1, was only partially affected by RIAM deficiency in leukocytes. Thus, although talin is an essential, shared regulator of all integrin classes expressed by leukocytes, we report that β2 and α4 integrins use different RIAM-dependent and -independent pathways to undergo activation by talin.

  2. Alpha 5 Integrin Mediates Osteoarthritic Changes in Mouse Knee Joints

    PubMed Central

    Candela, Maria Elena; Wang, Chao; Gunawardena, Aruni T.; Zhang, Kairui; Cantley, Leslie; Yasuhara, Rika; Usami, Yu; Francois, Noelle; Iwamoto, Masahiro; van der Flier, Arjan; Zhang, Yejia; Qin, Ling; Han, Lin; Enomoto-Iwamoto, Motomi

    2016-01-01

    Osteoarthritis (OA) is one of most common skeletal disorders and can affect synovial joints such as knee and ankle joints. α5 integrin, a major fibronectin receptor, is expressed in articular cartilage and has been demonstrated to play roles in synovial joint development and in the regulation of chondrocyte survival and matrix degradation in articular cartilage. We hypothesized that α5 integrin signaling is involved in pathogenesis of OA. To test this, we generated compound mice that conditionally ablate α5 integrin in the synovial joints using the Gdf5Cre system. The compound mice were born normally and had an overall appearance similar to the control mice. However, when the mutant mice received the OA surgery, they showed stronger resistance to osteoarthritic changes than the control. Specifically the mutant knee joints presented lower levels of cartilage matrix and structure loss and synovial changes and showed stronger biomechanical properties than the control knee joints. These findings indicate that α5 integrin may not be essential for synovial joint development but play a causative role in induction of osteoarthritic changes. PMID:27280771

  3. Alpha 5 Integrin Mediates Osteoarthritic Changes in Mouse Knee Joints.

    PubMed

    Candela, Maria Elena; Wang, Chao; Gunawardena, Aruni T; Zhang, Kairui; Cantley, Leslie; Yasuhara, Rika; Usami, Yu; Francois, Noelle; Iwamoto, Masahiro; van der Flier, Arjan; Zhang, Yejia; Qin, Ling; Han, Lin; Enomoto-Iwamoto, Motomi

    2016-01-01

    Osteoarthritis (OA) is one of most common skeletal disorders and can affect synovial joints such as knee and ankle joints. α5 integrin, a major fibronectin receptor, is expressed in articular cartilage and has been demonstrated to play roles in synovial joint development and in the regulation of chondrocyte survival and matrix degradation in articular cartilage. We hypothesized that α5 integrin signaling is involved in pathogenesis of OA. To test this, we generated compound mice that conditionally ablate α5 integrin in the synovial joints using the Gdf5Cre system. The compound mice were born normally and had an overall appearance similar to the control mice. However, when the mutant mice received the OA surgery, they showed stronger resistance to osteoarthritic changes than the control. Specifically the mutant knee joints presented lower levels of cartilage matrix and structure loss and synovial changes and showed stronger biomechanical properties than the control knee joints. These findings indicate that α5 integrin may not be essential for synovial joint development but play a causative role in induction of osteoarthritic changes. PMID:27280771

  4. NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration.

    PubMed

    Hedrick, Erik; Lee, Syng-Ook; Doddapaneni, Ravi; Singh, Mandip; Safe, Stephen

    2016-05-01

    Overexpression of the nuclear receptor 4A1 (NR4A1) in breast cancer patients is a prognostic factor for decreased survival and increased metastasis, and this has been linked to NR4A1-dependent regulation of transforming growth factor β (TGF-β) signaling. Results of RNA interference studies demonstrate that basal migration of aggressive SKBR3 and MDA-MB-231 breast cancer cells is TGF-β independent and dependent on regulation of β1-integrin gene expression by NR4A1 which can be inhibited by the NR4A1 antagonists 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) and a related p-carboxymethylphenyl [1,1-bis(3'-indolyl)-1-(p-carboxymethylphenyl)methane (DIM-C-pPhCO2Me)] analog. The NR4A1 antagonists also inhibited TGF-β-induced migration of MDA-MB-231 cells by blocking nuclear export of NR4A1, which is an essential step in TGF-β-induced cell migration. We also observed that NR4A1 regulates expression of both β1- and β3-integrins, and unlike other β1-integrin inhibitors which induce prometastatic β3-integrin, NR4A1 antagonists inhibit expression of both β1- and β3-integrin, demonstrating a novel mechanism-based approach for targeting integrins and integrin-dependent breast cancer metastasis. PMID:26929200

  5. Use of surface-enhanced Raman spectroscopy for the detection of human integrins

    NASA Astrophysics Data System (ADS)

    Chowdhury, Mustafa H.; Gant, V. Alexander; Trache, Andreea; Baldwin, Angela M.; Meininger, Gerald A.; Coté, Gerard L.

    2006-03-01

    Current research has revealed the importance of a class of cell surface proteins called integrins in various vital physiological functions such as blood clotting, regulation of blood pressure, tissue blood flow, and vascular remodeling. The key to integrin functionality is its ability to mediate force transmission by interacting with the extracellular matrix and cytoskeleton. In addition, they play a role in signal transduction via their connection with the proteins in focal adhesion (FA) points. To understand the complex mechanism of cell-cell and cell-extracellular matrix (ECM) adhesion that is responsible for these diverse biochemical interactions, it is necessary to identify the integrins on cells and monitor their interaction with various ligands. To this end, for the first time, we employ surface-enhanced Raman spectroscopy (SERS) to detect integrins. The results show the capability using SERS to detect the integrins to the nanomolar concentration regime and to distinguish between two different kinds of integrins, αVβ3 and α5β1, that are present in vascular smooth muscle cells (VSMCs). It is anticipated that the SERS approach will potentially help elucidate the mechanism of integrin-ligand interactions in a variety of phenomena of physiological importance.

  6. Emerging Putative Biomarkers: The Role of Alpha 2 and 6 Integrins in Susceptibility, Treatment, and Prognosis

    PubMed Central

    Marthick, James R.; Dickinson, Joanne L.

    2012-01-01

    The genetic architecture underpinning prostate cancer is complex, polygenic and despite recent significant advances many questions remain. Advances in genetic technologies have greatly improved our ability to identify genetic variants associated with complex disease including prostate cancer. Genome-wide association studies (GWASs) and microarray gene expression studies have identified genetic associations with prostate cancer susceptibility and tumour development. The integrins feature prominently in both studies examining the underlying genetic susceptibility and mechanisms driving prostate tumour development. Integrins are cell adhesion molecules involved in extracellular and intracellular signalling and are imperative for tumour development, migration, and angiogenesis. Although several integrins have been implicated in tumour development, the roles of integrin α2 and integrin α6 are the focus of this paper as evidence is now emerging that these integrins are implicit in prostate cancer susceptibility, cancer stem cell biology, angiogenesis, cell migration, and metastases to bone and represent potential biomarkers and therapeutic targets. There currently exists an urgent need to develop tools that differentiate indolent from aggressive prostate cancers and predict how patients will respond to treatment. This paper outlines the evidence supporting the use of α2 and α6 integrins in clinical applications for tailored patient treatment. PMID:22900191

  7. Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions

    NASA Technical Reports Server (NTRS)

    Chicurel, M. E.; Singer, R. H.; Meyer, C. J.; Ingber, D. E.

    1998-01-01

    The extracellular matrix (ECM) activates signalling pathways that control cell behaviour by binding to cell-surface integrin receptors and inducing the formation of focal adhesion complexes (FACs). In addition to clustered integrins, FACs contain proteins that mechanically couple the integrins to the cytoskeleton and to immobilized signal-transducing molecules. Cell adhesion to the ECM also induces a rapid increase in the translation of preexisting messenger RNAs. Gene expression can be controlled locally by targeting mRNAs to specialized cytoskeletal domains. Here we investigate whether cell binding to the ECM promotes formation of a cytoskeletal microcompartment specialized for translational control at the site of integrin binding. High-resolution in situ hybridization revealed that mRNA and ribosomes rapidly and specifically localized to FACs that form when cells bind to ECM-coated microbeads. Relocation of these protein synthesis components to the FAC depended on the ability of integrins to mechanically couple the ECM to the contractile cytoskeleton and on associated tension-moulding of the actin lattice. Our results suggest a new type of gene regulation by integrins and by mechanical stress which may involve translation of mRNAs into proteins near the sites of signal reception.

  8. Integrin Molecular Tension within Motile Focal Adhesions.

    PubMed

    Wang, Xuefeng; Sun, Jie; Xu, Qian; Chowdhury, Farhan; Roein-Peikar, Mehdi; Wang, Yingxiao; Ha, Taekjip

    2015-12-01

    Forces transmitted by integrins regulate many important cellular functions. Previously, we developed tension gauge tether (TGT) as a molecular force sensor and determined the threshold tension across a single integrin-ligand bond, termed integrin tension, required for initial cell adhesion. Here, we used fluorescently labeled TGTs to study the magnitude and spatial distribution of integrin tension on the cell-substratum interface. We observed two distinct levels of integrin tension. A >54 pN molecular tension is transmitted by clustered integrins in motile focal adhesions (FAs) and such force is generated by actomyosin, whereas the previously reported ∼40 pN integrin tension is transmitted by integrins before FA formation and is independent of actomyosin. We then studied FA motility using a TGT-coated surface as a fluorescent canvas, which records the history of integrin force activity. Our data suggest that the region of the strongest integrin force overlaps with the center of a motile FA within 0.2 μm resolution. We also found that FAs move in pairs and that the asymmetry in the motility of an FA pair is dependent on the initial FA locations on the cell-substratum interface.

  9. NMR methods in combinatorial chemistry.

    PubMed

    Shapiro, M J; Wareing, J R

    1998-06-01

    The use of NMR spectroscopy in combinatorial chemistry has provided a versatile tool for monitoring combinatorial chemistry reactions and for assessing ligand-receptor interactions. The application of magic angle spinning NMR is widespread and has allowed structure determination to be performed on compounds attached to solid supports. A variety of two-dimensional NMR techniques have been applied to enhance the usability of the magic angle spinning NMR data. New developments for solution NMR analysis include high performance liquid chromatography, NMR, mass spectroscopy and flow NMR. NMR based methods currently being investigated may prove valuable as compound screening tools.

  10. Structure of Adenovirus Complexed with Its Internalization Receptor, αvβ5 Integrin

    PubMed Central

    Chiu, Charles Y.; Mathias, Patricia; Nemerow, Glen R.; Stewart, Phoebe L.

    1999-01-01

    The three-dimensional structure of soluble recombinant integrin αvβ5 bound to human adenovirus types 2 and 12 (Ad2 and -12) has been determined at ∼21-Å resolution by cryoelectron microscopy (cryo-EM). The αvβ5 integrin is known to promote Ad cell entry. Cryo-EM has shown that the integrin-binding RGD (Arg-Gly-Asp) protrusion of the Ad2 penton base protein is highly mobile (P. L. Stewart, C. Y. Chiu, S. Huang, T. Muir, Y. Zhao, B. Chait, P. Mathias, and G. R. Nemerow, EMBO J. 16:1189–1198, 1997). Sequence analysis indicated that the Ad12 RGD surface loop is shorter than that of Ad2 and probably less flexible, hence more suitable for structural characterization of the Ad-integrin complex. The cryo-EM structures of the two virus-receptor complexes revealed a ring of integrin density above the penton base of each virus serotype. As expected, the integrin density in the Ad2 complex was diffuse while that in the Ad12 complex was better defined. The integrin consists of two discrete subdomains, a globular domain with an RGD-binding cleft ∼20 Å in diameter and a distal domain with extended, flexible tails. Kinetic analysis of Ad2 interactions with αvβ5 indicated ∼4.2 integrin molecules bound per penton base at close to saturation. These results suggest that the precise spatial arrangement of five RGD protrusions on the penton base promotes integrin clustering and the signaling events required for virus internalization. PMID:10400774

  11. Extracellular matrix stiffness dictates Wnt expression through integrin pathway.

    PubMed

    Du, Jing; Zu, Yan; Li, Jing; Du, Shuyuan; Xu, Yipu; Zhang, Lang; Jiang, Li; Wang, Zhao; Chien, Shu; Yang, Chun

    2016-02-08

    It is well established that extracellular matrix (ECM) stiffness plays a significant role in regulating the phenotypes and behaviors of many cell types. However, the mechanism underlying the sensing of mechanical cues and subsequent elasticity-triggered pathways remains largely unknown. We observed that stiff ECM significantly enhanced the expression level of several members of the Wnt/β-catenin pathway in both bone marrow mesenchymal stem cells and primary chondrocytes. The activation of β-catenin by stiff ECM is not dependent on Wnt signals but is elevated by the activation of integrin/ focal adhesion kinase (FAK) pathway. The accumulated β-catenin then bound to the wnt1 promoter region to up-regulate the gene transcription, thus constituting a positive feedback of the Wnt/β-catenin pathway. With the amplifying effect of positive feedback, this integrin-activated β-catenin/Wnt pathway plays significant roles in mediating the enhancement of Wnt signal on stiff ECM and contributes to the regulation of mesenchymal stem cell differentiation and primary chondrocyte phenotype maintenance. The present integrin-regulated Wnt1 expression and signaling contributes to the understanding of the molecular mechanisms underlying the regulation of cell behaviors by ECM elasticity.

  12. Extracellular matrix stiffness dictates Wnt expression through integrin pathway

    PubMed Central

    Du, Jing; Zu, Yan; Li, Jing; Du, Shuyuan; Xu, Yipu; Zhang, Lang; Jiang, Li; Wang, Zhao; Chien, Shu; Yang, Chun

    2016-01-01

    It is well established that extracellular matrix (ECM) stiffness plays a significant role in regulating the phenotypes and behaviors of many cell types. However, the mechanism underlying the sensing of mechanical cues and subsequent elasticity-triggered pathways remains largely unknown. We observed that stiff ECM significantly enhanced the expression level of several members of the Wnt/β-catenin pathway in both bone marrow mesenchymal stem cells and primary chondrocytes. The activation of β-catenin by stiff ECM is not dependent on Wnt signals but is elevated by the activation of integrin/ focal adhesion kinase (FAK) pathway. The accumulated β-catenin then bound to the wnt1 promoter region to up-regulate the gene transcription, thus constituting a positive feedback of the Wnt/β-catenin pathway. With the amplifying effect of positive feedback, this integrin-activated β-catenin/Wnt pathway plays significant roles in mediating the enhancement of Wnt signal on stiff ECM and contributes to the regulation of mesenchymal stem cell differentiation and primary chondrocyte phenotype maintenance. The present integrin-regulated Wnt1 expression and signaling contributes to the understanding of the molecular mechanisms underlying the regulation of cell behaviors by ECM elasticity. PMID:26854061

  13. Integrins as architects of cell behavior.

    PubMed

    Streuli, Charles H

    2016-10-01

    Integrins are cell surface receptors that bind cells to their physical external environment, linking the extracellular matrix to cell function. They are essential in the biology of all animals. In the late 1980s, we discovered that integrins are required for the ability of breast epithelia to do what they are programmed to do, which is to differentiate and make milk. Since then, integrins have been shown to control most other aspects of phenotype: to stay alive, to divide, and to move about. Integrins also provide part of the mechanism that allows cells to form tissues. Here I discuss how we discovered that integrins control mammary gland differentiation and explore the role of integrins as central architects of other aspects of cell behavior. PMID:27687254

  14. CCN2: a mechanosignaling sensor modulating integrin-dependent connective tissue remodeling in fibroblasts?

    PubMed

    Leask, Andrew

    2013-08-01

    Tensegrity (tensional integrity) is an emerging concept governing the structure of the body. Integrin-mediated mechanical tension is essential for connective tissue function in vivo. For example, in adult skin fibroblasts, the integrin β1 subunit mediates adhesion to collagen and fibronectin. Moreover, integrin β1, through its abilities to activate latent TGFβ1 and promote collagen production through focal adhesion kinase/rac1/nicotinamide adenine dinucleotide phosphate oxidase (NOX)/reactive oxygen species (ROS), is essential for dermal homeostasis, repair and fibrosis. The integrin β1-interacting protein CCN2, a member of the CCN family of proteins, is induced by TGFβ1; yet, CCN2 is not a simple downstream mediator of TGFβ1, but instead synergistically promote TGFβ1-induced adhesive signaling and fibrosis. Due to its selective ability to sense mechanical forces in the microenvironment, CCN2 may represent an exquisitely precise target for therapeutic intervention. PMID:23729366

  15. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

    1999-01-01

    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  16. Integrin-extracellular matrix interactions in connective tissue remodeling and osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; Moursi, A.; Zimmerman, D.; Lull, J.; Damsky, C.

    1995-01-01

    The differentiaton of bone cells is a complex multistep process. Bone is somewhat unusual in that it is very actively and continually remodeled in the adult and that maintenance of its mass in the mature organism is exquisitely sensitive to mechanical as well as chemical signals. Bone is also unique because it consists of a very large amount of extracellular matrix (ECM) that is mineralized. The integrin family of ECM receptors has been shown to play an important role in tissue morphogenesis in several systems. Our studies on the regulation of matrix remodeling enzymes by integrins in rabbit synovial fibroblasts show that two b1 integrin fibronectin (FN) receptor complexes (alpha 5 beta 1 and alpha 4 beta 1) cooperate in detecting subtle changes in the composition of the ECM. As a result of signal transduction by these integrins, the levels of mRNA and protein for several members of the metalloproteinase family are regulated in these cells. We have also used antibody and RGD peptide perturbation studies to determine the significance of cell/ECM interactions to normal osteogenesis. We found that interactions between the cell binding domain of FN and integrins are required for both normal morphogenesis and gene expression in cultured osteoblasts that differentiate to form bone-like tissue in culture. These data lead us to propose that beta 1 integrins play an important role in osteoblast differentiation as well as in bone remodeling.

  17. The cancer glycocalyx mechanically primes integrin-mediated growth and survival.

    PubMed

    Paszek, Matthew J; DuFort, Christopher C; Rossier, Olivier; Bainer, Russell; Mouw, Janna K; Godula, Kamil; Hudak, Jason E; Lakins, Jonathon N; Wijekoon, Amanda C; Cassereau, Luke; Rubashkin, Matthew G; Magbanua, Mark J; Thorn, Kurt S; Davidson, Michael W; Rugo, Hope S; Park, John W; Hammer, Daniel A; Giannone, Grégory; Bertozzi, Carolyn R; Weaver, Valerie M

    2014-07-17

    Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.

  18. The cancer glycocalyx mechanically primes integrin-mediated growth and survival

    PubMed Central

    Paszek, Matthew J.; DuFort, Christopher C.; Rossier, Olivier; Bainer, Russell; Mouw, Janna K.; Godula, Kamil; Hudak, Jason E.; Lakins, Jonathon N.; Wijekoon, Amanda C.; Cassereau, Luke; Rubashkin, Matthew G.; Magbanua, Mark J.; Thorn, Kurt S.; Davidson, Michael W.; Rugo, Hope S.; Park, John W.; Hammer, Daniel A.; Giannone, Grégory; Bertozzi, Carolyn R.; Weaver, Valerie M.

    2015-01-01

    Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function. PMID:25030168

  19. Combinatorial synthesis of ceramic materials

    DOEpatents

    Lauf, Robert J [Oak Ridge, TN; Walls, Claudia A [Oak Ridge, TN; Boatner, Lynn A [Oak Ridge, TN

    2010-02-23

    A combinatorial library includes a gelcast substrate defining a plurality of cavities in at least one surface thereof; and a plurality of gelcast test materials in the cavities, at least two of the test materials differing from the substrate in at least one compositional characteristic, the two test materials differing from each other in at least one compositional characteristic.

  20. Combinatorial synthesis of ceramic materials

    DOEpatents

    Lauf, Robert J.; Walls, Claudia A.; Boatner, Lynn A.

    2006-11-14

    A combinatorial library includes a gelcast substrate defining a plurality of cavities in at least one surface thereof; and a plurality of gelcast test materials in the cavities, at least two of the test materials differing from the substrate in at least one compositional characteristic, the two test materials differing from each other in at least one compositional characteristic.

  1. Mice Lacking beta2-Integrin Function Remain Glucose Tolerant in Spite of Insulin Resistance, Neutrophil Infiltration and Inflammation.

    PubMed

    Meakin, Paul J; Morrison, Vicky L; Sneddon, Claire C; Savinko, Terhi; Uotila, Liisa; Jalicy, Susan M; Gabriel, Jennie L; Kang, Li; Ashford, Michael L J; Fagerholm, Susanna C

    2015-01-01

    Beta2-integrins are important in leukocyte trafficking and function, and are regulated through the binding of cytoplasmic proteins, such as kindlin-3, to their intracellular domain. Here, we investigate the involvement of beta2-integrins in the regulation of metabolic disease using mice where the kindlin-3 binding site in the beta2-integrin cytoplasmic tail has been mutated (TTT/AAA-beta2-integrin knock-in (KI) mice), leading to expressed but dysfunctional beta2-integrins and significant neutrophilia in vivo. Beta2-integrin KI mice fed on a high fat diet showed normal weight gain, and normal accumulation of macrophages and lymphocytes in white adipose tissue (WAT) and liver, but increased neutrophil numbers especially in WAT. In addition, beta2-integrin KI mice fed on a high fat diet showed significantly increased peripheral insulin resistance in response to high-fat feeding. However, this was associated with improved glucose disposal following glucose load. Interestingly, beta2-integrin KI neutrophils produced more elastase in vitro, in response to stimulation. Beta2-integrin KI mice displayed variability of tissue inflammatory status, with liver and WAT exhibiting little or no difference in inflammation compared to high fat fed controls, whereas skeletal muscle demonstrated a raised inflammatory profile in association with higher elastase levels and diminished signalling through the IRS1-PKB pathway. In conclusion, although expression of dysfunctional beta2-integrins increased neutrophil production and infiltration into tissue, skeletal muscle was the most affected tissue exhibiting evidence of higher neutrophil activity and insulin resistance. Thus, beta2-integrins modulate glucose homeostasis during high fat feeding predominantly through actions on skeletal muscle to affect metabolic phenotype in vivo. PMID:26405763

  2. Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling.

    PubMed

    Zhao, Yingxin; Valbuena, Gustavo; Walker, David H; Gazi, Michal; Hidalgo, Marylin; DeSousa, Rita; Oteo, Jose Antonio; Goez, Yenny; Brasier, Allan R

    2016-01-01

    Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from

  3. Talin1 phosphorylation activates β1 integrins: a novel mechanism to promote prostate cancer bone metastasis.

    PubMed

    Jin, J-K; Tien, P-C; Cheng, C-J; Song, J H; Huang, C; Lin, S-H; Gallick, G E

    2015-04-01

    Talins are adaptor proteins that regulate focal adhesion signaling by conjugating integrins to the cytoskeleton. Talins directly bind integrins and are essential for integrin activation. We previously showed that β1 integrins are activated in metastatic prostate cancer (PCa) cells, increasing PCa metastasis to lymph nodes and bone. However, how β1 integrins are activated in PCa cells is unknown. In this study, we identified a novel mechanism of β1 integrin activation. Using knockdown experiments, we first demonstrated that talin1, but not talin2, is important in β1 integrin activation. We next showed that talin1 S425 phosphorylation, but not total talin1 expression, correlates with metastatic potential of PCa cells. Expressing a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 and C4-2B4 PCa cells, decreased activation of β1 integrins, integrin-mediated adhesion, motility and increased the sensitivity of the cells to anoikis. In contrast, reexpression of the phosphorylation-mimicking mutant talin1(S425D) led to increased β1 integrin activation and generated biologic effects opposite to talin1(S425A) expression. In the highly metastatic PC3-MM2 cells, expression of a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 cells, abolished their ability to colonize in the bone following intracardiac injection, while reexpression of phosphorylation-mimicking mutant talin1(S425D) restored their ability to metastasize to bone. Immunohistochemical staining demonstrated that talin S425 phosphorylation is significantly increased in human bone metastases when compared with normal tissues, primary tumors or lymph node metastases. We further showed that p35 expression, an activator of Cdk5, and Cdk5 activity were increased in metastatic tumor cells, and that Cdk5 kinase activity is responsible for talin1 phosphorylation and subsequent β1 integrin activation. Together, our study reveals Cdk5-mediated phosphorylation of talin1 leading

  4. Role of integrins, tetraspanins, and ADAM proteins during the development of apoptotic bodies by spermatogenic cells.

    PubMed

    Kierszenbaum, Abraham L; Rosselot, Carolina; Rivkin, Eugene; Tres, Laura L

    2006-07-01

    cell apoptosis, integrin beta1, alpha3, and alpha6 subunits, and tetraspanins CD9 and C81 become displaced away from the developing apoptotic bodies. In contrast, the intermediate filament protein Sak57, a keratin 5 ortholog, concentrates in the developing apoptotic bodies. We propose that the redistribution of integrin-tetraspanin complexes during spermatogenic cell apoptosis may be evidence of a signaling cascade initiated by Sertoli cell geometric changes. As a result, Sertoli cell reduction in surface area may be a limiting factor of spermatogenic cell survival and in the developmental regulation of spermatogenic cell progenies in the intact seminiferous epithelium.

  5. Wisp2/CCN5 up-regulated in the central nervous system of GM3-only mice facilitates neurite formation in Neuro2a cells via integrin-Akt signaling

    SciTech Connect

    Ohkawa, Yuki; Ohmi, Yuhsuke; Tajima, Orie; Yamauchi, Yoshio; Furukawa, Keiko; Furukawa, Koichi

    2011-08-05

    Highlights: {yields} Wisp2/CCN5 was up-regulated in nervous tissues of GM3-only mutant mice. {yields} Wisp2/CCN5 was found in neurons more strongly in the mutant mice. {yields} Wisp2/CCN5 induces Akt phosphorylation via integrins and facilitates neurite formation. {yields} Wisp2/CCN5 conferred resistance to H{sub 2}O{sub 2}-induced apoptosis. {yields} Up-regulation of Wisp2/CCN5 in GM3-only mice seemed for protection of brains from neurodegeneration. -- Abstract: Wisp2/CCN5 belongs to CCN family proteins which are involved in cell proliferation, angiogenesis, tumorigenesis and wound healing. Although a number of studies on the roles of Wisp2/CCN5 in cancers have been reported, no study on the expression and function of Wisp2/CCN5 in the central nervous system has been reported. In this study, we focused on Wisp2/CCN5 that was up-regulated in nervous tissues in GM3-only mice. Over-expression of Wisp2/CCN5 enhanced neurite outgrowth potently after serum withdrawal with increased phosphorylation levels of Akt and ERKs. When cells were cultured with recombinant Wisp2/CCN5 proteins, more and longer neurites were formed than in the controls. Thus, we demonstrated for the first time that Wisp2/CCN5 facilitates neurite formation in a mouse neuroblastoma cell line, Neuro2a. Akt phosphorylation induced by recombinant Wisp2/CCN5 was suppressed after knockdown of integrin {beta}1. Moreover, Wisp2/CCN5-over-expressing cells were resistant to apoptosis induced by H{sub 2}O{sub 2}. These results suggested that secreted Wisp2/CCN5 induces Akt and ERK phosphorylation via integrins, and consequently facilitates neurite formation and conferred resistance to apoptosis. Up-regulation of Wisp2/CCN5 in GM3-only mice should be, therefore, a reaction to protect nervous tissues from neurodegeneration caused by ganglioside deficiency.

  6. Regional Regulation of Purkinje Cell Dendritic Spines by Integrins and Eph/Ephrins.

    PubMed

    Heintz, Tristan G; Eva, Richard; Fawcett, James W

    2016-01-01

    Climbing fibres and parallel fibres compete for dendritic space on Purkinje cells in the cerebellum. Normally, climbing fibres populate the proximal dendrites, where they suppress the multiple small spines typical of parallel fibres, leading to their replacement by the few large spines that contact climbing fibres. Previous work has shown that ephrins acting via EphA4 are a signal for this change in spine type and density. We have used an in vitro culture model in which to investigate the ephrin effect on Purkinje cell dendritic spines and the role of integrins in these changes. We found that integrins α3, α5 and β4 are present in many of the dendritic spines of cultured Purkinje cells. pFAK, the main downstream signalling molecule from integrins, has a similar distribution, although the intenstity of pFAK staining and the percentage of pFAK+ spines was consistently higher in the proximal dendrites. Activating integrins with Mg2+ led to an increase in the intensity of pFAK staining and an increase in the proportion of pFAK+ spines in both the proximal and distal dendrites, but no change in spine length, density or morphology. Blocking integrin binding with an RGD-containing peptide led to a reduction in spine length, with more stubby spines on both proximal and distal dendrites. Treatment of the cultures with ephrinA3-Fc chimera suppressed dendritic spines specifically on the proximal dendrites and there was also a decrease of pFAK in spines on this domain. This effect was blocked by simultaneous activation of integrins with Mn2+. We conclude that Eph/ephrin signaling regulates proximal dendritic spines in Purkinje cells by inactivating integrin downstream signalling. PMID:27518800

  7. Regional Regulation of Purkinje Cell Dendritic Spines by Integrins and Eph/Ephrins

    PubMed Central

    Heintz, Tristan G.; Eva, Richard; Fawcett, James W.

    2016-01-01

    Climbing fibres and parallel fibres compete for dendritic space on Purkinje cells in the cerebellum. Normally, climbing fibres populate the proximal dendrites, where they suppress the multiple small spines typical of parallel fibres, leading to their replacement by the few large spines that contact climbing fibres. Previous work has shown that ephrins acting via EphA4 are a signal for this change in spine type and density. We have used an in vitro culture model in which to investigate the ephrin effect on Purkinje cell dendritic spines and the role of integrins in these changes. We found that integrins α3, α5 and β4 are present in many of the dendritic spines of cultured Purkinje cells. pFAK, the main downstream signalling molecule from integrins, has a similar distribution, although the intenstity of pFAK staining and the percentage of pFAK+ spines was consistently higher in the proximal dendrites. Activating integrins with Mg2+ led to an increase in the intensity of pFAK staining and an increase in the proportion of pFAK+ spines in both the proximal and distal dendrites, but no change in spine length, density or morphology. Blocking integrin binding with an RGD-containing peptide led to a reduction in spine length, with more stubby spines on both proximal and distal dendrites. Treatment of the cultures with ephrinA3-Fc chimera suppressed dendritic spines specifically on the proximal dendrites and there was also a decrease of pFAK in spines on this domain. This effect was blocked by simultaneous activation of integrins with Mn2+. We conclude that Eph/ephrin signaling regulates proximal dendritic spines in Purkinje cells by inactivating integrin downstream signalling. PMID:27518800

  8. Glioma cell integrin expression and their interactions with integrin antagonists

    PubMed Central

    Mattern, Ralph-Heiko; Read, Susana B.; Pierschbacher, Michael D.; Sze, Chun-I; Eliceiri, Brian P.; Kruse, Carol A.

    2005-01-01

    Summary A panel of human glioma cell explants was screened for integrin expression by flow cytometry using ανβ-specific antibodies. A lower percentage of the glioma cells were positive for the ανβ3 (mean % positive = 20.8%) integrin, whereas higher percentages were positive for the ανβ5 (mean % positive = 72.7%), VLA5α (mean % positive = 87%) and VLAβ1 (mean % positive = 41.7%) integrins. A series of RGD peptides was designed, synthesized and tested for binding to integrin receptors. Based on the results of the binding to the isolated integrin receptors and the expression of integrins on glioma cell lines, a peptide that binds potently to the ανβ3, ανβ5 and α5β1 was selected for further investigations with regards to its effect on glioma cells. The peptide, Ac-c[(Pen)-Tyr(Me)-Ala-Arg-Gly-Asp-Asn-Tic-Cys]NH2 (RGD peptide), exhibited high potential for use in clinical intracranial administration since it had good stability in rat brain cell homogenates placed into artificial cerebrospinal fluid. Using an HPLC method for quantification of peptides in rat brain cell homogenates, we could demonstrate the half-life of the RGD peptide approximated 20 hr. Relative to a scrambled peptide control (non-RGD sequence, same amino acids), the experimental RGD peptide significantly decreased glioma cell proliferation of the entire panel of rat and human glioma cells tested. Adhesion of recently passaged glioma cells to glioma-derived extracellular matrix protein-coated plates was inhibited significantly by the RGD peptide. The peptide also reversed attachment of plated glioma cells. The RGD peptide caused some, but not substantial, glioma cell injury, as evidenced by a quantitative in vitro nuclear DNA morphologic assay and by a flow cytometric assay employing 7-amino actinomycin D (7AAD). We histologically monitored for toxicity caused by various doses of the RGD peptide infused repeatedly into normal cannulated rat brain. At safe doses, the experimental RGD

  9. αvβ3 Integrin Limits the Contribution of Neuropilin-1 to Vascular Endothelial Growth Factor-induced Angiogenesis*

    PubMed Central

    Robinson, Stephen D.; Reynolds, Louise E.; Kostourou, Vassiliki; Reynolds, Andrew R.; da Silva, Rita Graça; Tavora, Bernardo; Baker, Marianne; Marshall, John F.; Hodivala-Dilke, Kairbaan M.

    2009-01-01

    Both vascular endothelial growth factor receptors (VEGFR) and integrins are major regulators of VEGF-induced angiogenesis. Previous work has shown that β3 integrin can regulate negatively VEGFR2 expression. Here we show that β3 integrin can regulate negatively VEGF-mediated angiogenesis by limiting the interaction of the co-receptor NRP1 (neuropilin-1) with VEGFR2. In the presence of αvβ3 integrin, NRP1 contributed minimally to VEGF-induced angiogenic processes in vivo, ex vivo, and in vitro. Conversely, when β3 integrin expression is absent or low or its function is blocked with RGD-mimetic inhibitors, VEGF-mediated responses became NRP1-dependent. Indeed, combined inhibition of β3 integrin and NRP1 decreased VEGF-mediated angiogenic responses further than individual inhibition of these receptors. We also show that αvβ3 integrin can associate with NRP1 in a VEGF-dependent fashion. Our data suggest that β3 integrin may, in part, negatively regulate VEGF signaling by sequestering NRP1 and preventing it from interacting with VEGFR2. PMID:19837659

  10. Vimentin-mediated regulation of cell motility through modulation of beta4 integrin protein levels in oral tumor derived cells.

    PubMed

    Dmello, Crismita; Sawant, Sharada; Alam, Hunain; Gangadaran, Prakash; Tiwari, Richa; Dongre, Harsh; Rana, Neha; Barve, Sai; Costea, Daniela Elena; Chaukar, Davendra; Kane, Shubhada; Pant, Harish; Vaidya, Milind

    2016-01-01

    Vimentin expression correlates well with migratory and invasive potential of the carcinoma cells. The molecular mechanism by which vimentin regulates cell motility is not yet clear. Here, we addressed this issue by depleting vimentin in oral squamous cell carcinoma derived cell line. Vimentin knockdown cells showed enhanced adhesion and spreading to laminin-5. However, we found that they were less invasive as compared to the vector control cells. In addition, signaling associated with adhesion behavior of the cell was increased in vimentin knockdown clones. These findings suggest that the normal function of β4 integrin as mechanical adhesive device is enhanced upon vimentin downregulation. As a proof of principle, the compromised invasive potential of vimentin depleted cells could be rescued upon blocking with β4 integrin adhesion-blocking (ASC-8) antibody or downregulation of β4 integrin in vimentin knockdown background. Interestingly, plectin which associates with α6β4 integrin in the hemidesmosomes, was also found to be upregulated in vimentin knockdown clones. Furthermore, experiments on lysosome and proteasome inhibition revealed that perhaps vimentin regulates the turnover of β4 integrin and plectin. Moreover, an inverse association was observed between vimentin expression and β4 integrin in oral squamous cell carcinoma (OSCC). Collectively, our results show a novel role of vimentin in modulating cell motility by destabilizing β4 integrin-mediated adhesive interactions. Further, vimentin-β4 integrin together may prove to be useful markers for prognostication of human oral cancer.

  11. Tumor suppressor KAI1 affects integrin {alpha}v{beta}3-mediated ovarian cancer cell adhesion, motility, and proliferation

    SciTech Connect

    Ruseva, Zlatna; Geiger, Pamina Xenia Charlotte; Hutzler, Peter; Kotzsch, Matthias; Luber, Birgit; Schmitt, Manfred; Gross, Eva; Reuning, Ute

    2009-06-10

    The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin {alpha}v{beta}3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin {alpha}v{beta}3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with {beta}1-integrins, also colocalizes with integrin {alpha}v{beta}3. Functionally, elevated KAI1 levels drastically increased integrin {alpha}v{beta}3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin {alpha}v{beta}3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin {alpha}v{beta}3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.

  12. Integrin beta 1 enhances the epithelial-mesenchymal transition in association with gefitinib resistance of non-small cell lung cancer.

    PubMed

    Ju, Lixia; Zhou, Caicun

    2013-01-01

    We have previously shown that integrinβ1 associates with gefitinib resistance. As epithelial-mesenchymal transition (EMT) also induces gefitinib resistance in vitro, we wished to determine the relation of them in gefitinib resistance. In this study, we show that integrinβ1 induced epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance in xenograft tumors and gefitinib-resistant NSCLC tumors acquired EMT phenotype. Furthermore, inhibition of integrinβ1 reverses EMT, meanwhile overexpression and activation of integrinβ1 aggravates EMT. Lastly, we further identified that integrinβ1 enhanced EMT via FAK-AKT signaling pathway. These findings highlight a novel relation of integrinβ1 and EMT in EGFR TKI resistant NSCLC. PMID:24440972

  13. Integrin Ligation Results in Nephrin Tyrosine Phosphorylation In Vitro

    PubMed Central

    Verma, Rakesh; Venkatareddy, Madhusudan; Kalinowski, Anne; Patel, Sanjeevkumar R.; Garg, Puneet

    2016-01-01

    Nephrin is expressed at the basolateral aspect of podocytes and is an important signaling protein at the glomerular slit diaphragm. In vitro studies have demonstrated that Nephrin phosphorylation-dependent signaling is able to assemble a protein complex that is able to polymerize actin. However, proximal signaling events that result in nephrin tyrosine phosphorylation are not well understood. Nephrin deletion in mice and human nephrin mutations result in developmental failure of the podocyte intercellular junction resutling in proteinuria. This has been presumed to be due to a failure to respond to an external polarized cue in the absence of nephrin or a failure to transduce an outside-in signal in patients with nephrin mutations. The nephrin extracellular domain binds to itself or neph1 across the foot process intercellular junction. Nephrin is tyrosine phosphorylation-silent in healthy glomeruli when presumably the nephrin extracellular domain is in an engaged state. These observations raise the possibility of an alternate proximal signaling mechanism that might be responsible for nephrin tyrosine phosphorylation. Here we present data showing that integrin engagement at the basal aspect of cultured podocytes results in nephrin tyrosine phosphorylation. This is abrogated by incubating podocytes with an antibody that prevents integrin β1 ligation and activation in response to binding to extracellular matrix. Furthermore, nephrin tyrosine phosphorylation was observed in podocytes expressing a membrane-targeted nephrin construct that lacks the extracellular domain. We propose, integrin-activation based signaling might be responsible for nephrin phosphorylation rather than engagment of the nephrin extracellular domain by a ligand. PMID:26848974

  14. The modulation of MSC integrin expression by RGD presentation

    PubMed Central

    Lam, Jonathan; Segura, Tatiana

    2013-01-01

    Biomaterials designed to mimic the intricate native extracellular matrix (ECM) can use a variety of techniques to control the behavior of encapsulated cells. Common methods include controlling the mechanical properties of the material, incorporating bioactive signals, spatially patterning bioactive signals, and controlling the time-release of bioactive signals. Further design parameters like bioactive signal distribution can be used to manipulate cell behavior. Efforts on clustering adhesion peptides have focused on seeding cells on top of a biomaterial. Here we report the effect of clustering the adhesion peptide RGD on mouse mesenchymal stem cells encapsulated inside three-dimensional hyaluronic acid hydrogels. The clustered bioactive signals resulted in significant differences in both cell spreading and integrin expression. These results indicate that signal RGD peptide clustering is an additional hydrogel design parameter can be used to influence and guide the behavior of encapsulated cells. PMID:23465825

  15. Structure of an integrin with an [alpha]I domain, complement receptor type 4

    SciTech Connect

    Xie, Can; Zhu, Jianghai; Chen, Xing; Mi, Lizhi; Nishida, Noritaka; Springer, Timothy A.

    2010-08-13

    We report the structure of an integrin with an {alpha}I domain, {alpha}{sub X}{beta}{sub 2}, the complement receptor type 4. It was earlier expected that a fixed orientation between the {alpha}I domain and the {beta}-propeller domain in which it is inserted would be required for allosteric signal transmission. However, the {alpha}I domain is highly flexible, enabling two {beta}I domain conformational states to couple to three {alpha}I domain states, and greater accessibility for ligand recognition. Although {alpha}{sub X}{beta}{sub 2} is bent similarly to integrins that lack {alpha}I domains, the terminal domains of the {alpha}- and {beta}-legs, calf-2 and {beta}-tail, are oriented differently than in {alpha}I-less integrins. Linkers extending to the transmembrane domains are unstructured. Previous mutations in the {beta}2-tail domain support the importance of extension, rather than a deadbolt, in integrin activation. The locations of further activating mutations and antibody epitopes show the critical role of extension, and conversion from the closed to the open headpiece conformation, in integrin activation. Differences among 10 molecules in crystal lattices provide unprecedented information on interdomain flexibility important for modelling integrin extension and activation.

  16. Cardiomyocyte contractile status is associated with differences in fibronectin and integrin interactions

    PubMed Central

    Wu, Xin; Sun, Zhe; Foskett, Andrea; Trzeciakowski, Jerome P.; Meininger, Gerald A.

    2010-01-01

    Integrins link the extracellular matrix (ECM) with the intracellular cytoskeleton and other cell adhesion-associated signaling proteins to function as mechanotransducers. However, direct quantitative measurements of the cardiomyocyte mechanical state and its relationship to the interactions between specific ECM proteins and integrins are lacking. The purpose of this study was to characterize the interactions between the ECM protein fibronectin (FN) and integrins in cardiomyocytes and to test the hypothesis that these interactions would vary during contraction and relaxation states in cardiomyocytes. Using atomic force microscopy, we quantified the unbinding force (adhesion force) and adhesion probability between integrins and FN and correlated these measurements with the contractile state as indexed by cell stiffness on freshly isolated mouse cardiomyocytes. Experiments were performed in normal physiological (control), high-K+ (tonically contracted), or low-Ca2+ (fully relaxed) solutions. Under control conditions, the initial peak of adhesion force between FN and myocyte α3β1- and/or α5β1-integrins was 39.6 ± 1.3 pN. The binding specificity between FN and α3β1- and α5β1-integrins was verified by using monoclonal antibodies against α3-, α5-, α3 + α5-, or β1-integrin subunits, which inhibited binding by 48%, 65%, 70%, or 75%, respectively. Cytochalasin D or 2,3-butanedione monoxime (BDM), to disrupt the actin cytoskeleton or block myofilament function, respectively, significantly decreased the cell stiffness; however, the adhesion force and binding probability were not altered. Tonic contraction with high-K+ solution increased total cell adhesion (1.2-fold) and cell stiffness (27.5-fold) compared with fully relaxed cells with low-Ca2+ solution. However, it could be partially prevented by high-K+ bath solution containing BDM, which suppresses contraction by inhibiting the actin-myosin interactions. Thus, our results demonstrate that integrin binding to

  17. Combinatorial control of heterogeneous cell populations

    NASA Astrophysics Data System (ADS)

    Piermarocchi, C.; Duxbury, P.; Paternostro, G.; Feala, J.; Tiziani, S.; Axelrod, J.; Chaudhury, A.; Choi, J.; McCulloch, A.; Cortes, J.

    2010-03-01

    In medicine, a recent pharmacological approach involves systematic discovery of combinatorial therapies, in which different drugs are simultaneously used to control different pathways associated with a cellular function. This control must occur with minimal response in other non-target cells exposed to treatment, i.e. it has to be selective. We have investigated the statistics of selective control of the human apoptosis (cell death) signaling network. We have built a model for a heterogeneous population of cells, characterized by a signaling network with identical topology, but having different link strengths. The control of the life/death signal is realized by acting with external perturbations, modeling the effect of drugs, on the nodes and on the signaling flow. Concepts from statistical physics and information theory, including entropy, frustration, and non-linearity have been used to characterize the general properties of selective control. This knowledge was used as a guide in designing algorithms for identifying selective perturbations. Some of these algorithms have been implemented in vitro in high throughput experiments on real cell lines where a large number of combinations of different drugs can be tested.

  18. Localized LoxL3-Dependent Fibronectin Oxidation Regulates Myofiber Stretch and Integrin-Mediated Adhesion.

    PubMed

    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

    2016-03-01

    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. PMID:26954549

  19. Pull-down assay for analysis of integrin-mediated activation of Rap proteins in adherent platelets.

    PubMed

    Guidetti, Gianni Francesco; Torti, Mauro

    2014-01-01

    Rap1 GTPases operate as molecular switches by cycling between a GDP-bound inactive state and a GTP-bound active state and regulate several cellular pathways in response to different stimuli. Circulating blood platelets express high levels of Rap1 proteins, mainly Rap1b, which plays a critical role in platelet adhesion and activation. Rap1 is a key element in the inside-out signaling pathway leading to the conversion of integrins into the high-affinity state for their ligands. In platelets, Rap1b regulates inside-out activation of both integrin αIIbβ3 and α2β1. In addition, Rap1b is also involved in integrin outside-in signaling. Integrin-mediated platelet adhesion leads to accumulation of GTP-bound Rap1b, which promotes integrin-mediated processes such as spreading and clot retraction. Rap1b is thus a bidirectional regulator of platelet integrin function. Here we describe a method to analyze Rap1b activation induced by platelet adhesion via integrin α2β1.

  20. Combinatorial 3D Mechanical Metamaterials

    NASA Astrophysics Data System (ADS)

    Coulais, Corentin; Teomy, Eial; de Reus, Koen; Shokef, Yair; van Hecke, Martin

    2015-03-01

    We present a class of elastic structures which exhibit 3D-folding motion. Our structures consist of cubic lattices of anisotropic unit cells that can be tiled in a complex combinatorial fashion. We design and 3d-print this complex ordered mechanism, in which we combine elastic hinges and defects to tailor the mechanics of the material. Finally, we use this large design space to encode smart functionalities such as surface patterning and multistability.

  1. YCRD: Yeast Combinatorial Regulation Database

    PubMed Central

    Wu, Wei-Sheng; Hsieh, Yen-Chen; Lai, Fu-Jou

    2016-01-01

    In eukaryotes, the precise transcriptional control of gene expression is typically achieved through combinatorial regulation using cooperative transcription factors (TFs). Therefore, a database which provides regulatory associations between cooperative TFs and their target genes is helpful for biologists to study the molecular mechanisms of transcriptional regulation of gene expression. Because there is no such kind of databases in the public domain, this prompts us to construct a database, called Yeast Combinatorial Regulation Database (YCRD), which deposits 434,197 regulatory associations between 2535 cooperative TF pairs and 6243 genes. The comprehensive collection of more than 2500 cooperative TF pairs was retrieved from 17 existing algorithms in the literature. The target genes of a cooperative TF pair (e.g. TF1-TF2) are defined as the common target genes of TF1 and TF2, where a TF’s experimentally validated target genes were downloaded from YEASTRACT database. In YCRD, users can (i) search the target genes of a cooperative TF pair of interest, (ii) search the cooperative TF pairs which regulate a gene of interest and (iii) identify important cooperative TF pairs which regulate a given set of genes. We believe that YCRD will be a valuable resource for yeast biologists to study combinatorial regulation of gene expression. YCRD is available at http://cosbi.ee.ncku.edu.tw/YCRD/ or http://cosbi2.ee.ncku.edu.tw/YCRD/. PMID:27392072

  2. YCRD: Yeast Combinatorial Regulation Database.

    PubMed

    Wu, Wei-Sheng; Hsieh, Yen-Chen; Lai, Fu-Jou

    2016-01-01

    In eukaryotes, the precise transcriptional control of gene expression is typically achieved through combinatorial regulation using cooperative transcription factors (TFs). Therefore, a database which provides regulatory associations between cooperative TFs and their target genes is helpful for biologists to study the molecular mechanisms of transcriptional regulation of gene expression. Because there is no such kind of databases in the public domain, this prompts us to construct a database, called Yeast Combinatorial Regulation Database (YCRD), which deposits 434,197 regulatory associations between 2535 cooperative TF pairs and 6243 genes. The comprehensive collection of more than 2500 cooperative TF pairs was retrieved from 17 existing algorithms in the literature. The target genes of a cooperative TF pair (e.g. TF1-TF2) are defined as the common target genes of TF1 and TF2, where a TF's experimentally validated target genes were downloaded from YEASTRACT database. In YCRD, users can (i) search the target genes of a cooperative TF pair of interest, (ii) search the cooperative TF pairs which regulate a gene of interest and (iii) identify important cooperative TF pairs which regulate a given set of genes. We believe that YCRD will be a valuable resource for yeast biologists to study combinatorial regulation of gene expression. YCRD is available at http://cosbi.ee.ncku.edu.tw/YCRD/ or http://cosbi2.ee.ncku.edu.tw/YCRD/. PMID:27392072

  3. miR-31 is a broad regulator of β1-integrin expression and function in cancer cells

    PubMed Central

    Augoff, Katarzyna; Das, Mitali; Bialkowska, Katarzyna; McCue, Brian; Plow, Edward F.; Sossey-Alaoui, Khalid

    2011-01-01

    Integrins are adhesion receptors involved in bidirectional signaling that are crucial for various cellular responses during normal homeostasis and pathological conditions such as cancer progression and metastasis. Aberrant expression of noncoding microRNAs has been implicated in the deregulation of integrin expression and activity, leading to the development and progression of cancer tumors, including their acquisition of the metastatic phenotype. miR-31 is a key regulator of several critical genes involved in the invasion-metastasis cascade in cancer. Using diverse cell based, genetic, biochemical, flow cytometry and functional analyses, we report that miR-31 is a master regulator of integrins as it targets multiple α subunit partners (α2, α5 and αV) of β1 integrins and also β3 integrins. We found that expression of miR-31 in cancer cells resulted in a significant repression of these integrin subunits both at the mRNA and protein levels. Loss of expression of α2, α5, αV and β3 was a direct consequence of miR-31 targeting conserved seed sequences in the 3’UTR of these integrin subunits leading to their posttranscriptional repression, which was reflected in their diminished surface expression in live cells. The biological consequence of decreased the cell surface of these integrins was a significant inhibition of cell spreading in a ligand-dependent manner. While different reports have shown that a single integrin can be regulated by several microRNAs, here we show that a single microRNA, miR-31, is able to specifically target several integrin subunits to regulate key aspects of cancer cell invasion and metastasis. PMID:21875932

  4. α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

    SciTech Connect

    Ozeki, Nobuaki; Kawai, Rie; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-02-01

    We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blot and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells. - Highlights: • Odontoblast differentiation requires activation of α2 integrin, Emmprin and MMP-3. • α2 integrin, Emmprin and MMP-3 form a sequential signaling cascade. • β1 integrin acts a specific trigger for odontoblast differentiation. • The role of these effectors is highly novel and unanticipated.

  5. 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.

    PubMed

    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.

  6. Monitoring integrin activation by fluorescence resonance energy transfer.

    PubMed

    Lefort, Craig T; Hyun, Young-Min; Kim, Minsoo

    2012-01-01

    Aberrant integrin activation is associated with several immune pathologies. In leukocyte adhesion deficiency (LAD), the absence or inability of β(2) integrins to undergo affinity upregulation contributes to recurrent infectious episodes and impaired wound healing, while excessive integrin activity leads to an exaggerated inflammatory response with associated tissue damage. Therefore, integrin activation is an attractive target for immunotherapies, and monitoring the effect of agents on integrin activation is necessary during preclinical drug development. The activation of integrins involves the structural rearrangement of both the extracellular and cytoplasmic domains. Here, we describe methods for monitoring integrin conformational activation using fluorescence resonance energy transfer (FRET).

  7. Do Integrins Mediate the Skeletal Response to Altered Loading?

    NASA Technical Reports Server (NTRS)

    vanderMeulen, Marjolein C. H.

    2004-01-01

    In vivo experiments were performed to examine the role of B1 integrin in skeletal adaptation to reduced and increased loading. Transgenic mice were generated with a dominant negative form of the B1 integrin cytoplasmic domain with expression driven by the osteocalcin promoter (pOCb1DN). This fragment consists of the transmembrane and intracellular domains and interferes with endogenous integrin signalling in vitro. This promoter targets expression of the transgene to mature bone cells. Expression of the transgene was confirmed by immunoprecipitation and western blotting. Reduced loading was generated by hindlimb suspension and increased loading the resumption of normal loading following hindlimb suspension. Two groups of female 35-day old mice were examined: poCb1DN transgenic mice (TG) and wild-type littermate controls (WT). Animals were hindlimb suspended for 1 week (HU, n = l0/gp) or 4 weeks (HU, n = 4 - 7/gp) or suspended for 4 weeks followed by reloading by normal ambulation for 4 weeks (RL, n = l0/gp). Age-matched controls (CT) were pairfed based on the HU food intake. The protocols were approved by the NASA Ames Research Center IACUC. Upon completion of the experimental protocol, body mass was recorded and tissues of interest removed and analyzed following standard procedures. Femoral whole bone structural behavior was measured in torsion to failure to obtain whole bone strength (failure torque) and torsional rigidity. Ash content (ash) and fraction (% ash) were determined for the tibia. Total ash is indicative of bone size whereas %ash is a material property. Tibial curvature was measured from microradiographs. For each experiment, the effects of genotype (TG, WT) and treatment (CT, HU/RL) were assessed by two-factor ANOVA followed by the Tukey-Kramer posthoc to identify significant differences at an alpha level of 0.05. Our goal was to understand differences resulting from altered integrin function in the adaptation to altered loading.

  8. Observing force-regulated conformational changes and ligand dissociation from a single integrin on cells

    PubMed Central

    Chen, Wei; Lou, Jizhong; Evans, Evan A.

    2012-01-01

    As adhesion molecules, integrins connect a cell to its environment and transduce signals across the membrane. Their different functional states correspond to distinct conformations. Using a biomembrane force probe, we observed real-time reversible switches between bent and extended conformations of a single integrin, αLβ2, on the surface of a living cell by measuring its nanometer-scale headpiece displacements, bending and unbending frequencies, and molecular stiffness changes. We determined the stabilities of these conformations, their dynamic equilibrium, speeds and rates of conformational changes, and the impact of divalent cations and tensile forces. We quantified how initial and subsequent conformations of αLβ2 regulate the force-dependent kinetics of dissociation from intercellular adhesion molecule 1. Our findings provide new insights into how integrins function as nanomachines to precisely control cell adhesion and signaling. PMID:23109670

  9. Effects of integrin ανβ3 on differentiation and collagen synthesis induced by connective tissue growth factor in human hypertrophic scar fibroblasts.

    PubMed

    Hu, Xiaolong; Li, Na; Tao, Ke; Fang, Xiaobing; Liu, Jiaqi; Wang, Yaojun; Wang, Hongtao; Shi, Jihong; Wang, Yunchuan; Ji, Peng; Cai, Weixia; Bai, Xiaozhi; Zhu, Xiongxiang; Han, Juntao; Hu, Dahai

    2014-11-01

    CCN2 is a matricellular protein that appears to be important in scar formation. CCN2 mediates the pro-fibrotic effects in hypertrophic scars (HTSs) through an unknown mechanism. However, many activities of CCN2 protein are known to be mediated by direct binding to integrin receptors. In this study, we investigated the role of integrin α(ν)β(3) in the differentiation of hypertrophic scar fibroblasts (HTSFs) induced by CCN2. The levels of integrin α(ν)β(3) between normal skin and hypertrophic scar (HTS) tissues were compared, and integrin α(ν)β(3) was found to be upregulated in HTS. CCN2 was shown to induce HTSF differentiation and collagen (COL) synthesis at the mRNA and protein levels. Based on these results, the expression of integrin α(ν)β(3) was upregulated by CCN2 stimulation during HTSF differentiation. Blockade of integrin α(ν)β(3) prevented CCN2-induced HTSF differentiation and COL synthesis. Furthermore, the CCN2-induced increase in contractility of the HTSF in COL lattices was inhibited by integrin α(ν)β(3) blocking antibodies. HTSs were established in a rabbit ear model, and the inhibitor of integrin α(ν)β(3) significantly improved the architecture of the rabbit ear scar. Results of the present study showed that integrin α(ν)β(3) contributes to pro-fibrotic CCN2 signaling. Blocking this pathway may therefore be beneficial for the treatment of HTS.

  10. B-Raf Regulation of Integrin α4β1-mediated Resistance to Shear Stress through Changes in Cell Spreading and Cytoskeletal Association in T Cells*

    PubMed Central

    Brown, Wells S.; Khalili, Jahan S.; Rodriguez-Cruz, Tania G.; Lizee, Greg; McIntyre, Bradley W.

    2014-01-01

    The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to extracellular matrix or other cells as well as initiating intracellular signaling cascades promoting survival or activation. The MAPK pathway is known to be downstream from integrins and to regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show that chemical inhibition of RAF by sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4β1 integrin ligands vascular cell adhesion molecule 1 (VCAM-1) and fibronectin, whereas inhibition of MEK/ERK by U0126 had no effect. Microscopy showed that RAF inhibition leads to significant inhibition of T cell spreading on VCAM-1. The association of α4β1 integrin with the actin cytoskeleton was shown to be dependent on B-Raf activity or expression, whereas α4β1 integrin affinity for soluble VCAM-1 was not. These effects were shown to be specific for α4β1 integrin and not other integrins, such as α5β1 or LFA-1, or a variety of membrane proteins. We demonstrate a novel role for B-Raf in the selective regulation of α4β1 integrin-mediated adhesion. PMID:24936068

  11. Integrin Activation Through the Hematopoietic Adapter Molecule ADAP Regulates Dendritic Development of Hippocampal Neurons

    PubMed Central

    Thiere, Marlen; Kliche, Stefanie; Müller, Bettina; Teuber, Jan; Nold, Isabell; Stork, Oliver

    2016-01-01

    Integrin-mediated cell adhesion and signaling is of critical importance for neuronal differentiation. Recent evidence suggests that an “inside-out” activation of β1-integrin, similar to that observed in hematopoietic cells, contributes to the growth and branching of dendrites. In this study, we investigated the role of the hematopoietic adaptor protein adhesion and degranulation promoting adapter protein (ADAP) in these processes. We demonstrate the expression of ADAP in the developing and adult nervous hippocampus, and in outgrowing dendrites of primary hippocampal neurons. We further show that ADAP occurs in a complex with another adaptor protein signal-transducing kinase-associated phosphoprotein-homolog (SKAP-HOM), with the Rap1 effector protein RAPL and the Hippo kinase macrophage-stimulating 1 (MST1), resembling an ADAP/SKAP module that has been previously described in T-cells and is critically involved in “inside-out” activation of integrins. Knock down of ADAP resulted in reduced expression of activated β1-integrin on dendrites. It furthermore reduced the differentiation of developing neurons, as indicated by reduced dendrite growth and decreased expression of the dendritic marker microtubule-associated protein 2 (MAP2). Our data suggest that an ADAP-dependent integrin-activation similar to that described in hematopoietic cells contributes to the differentiation of neuronal cells. PMID:27746719

  12. The cytoplasmic extension of the integrin β6 subunit regulates epithelial-to-mesenchymal transition.

    PubMed

    Lee, Carlin; Lee, Casey; Lee, Stacey; Siu, Amanda; Ramos, Daniel M

    2014-02-01

    Prognosis for oral cancer patients has not improved in over 60 years due to invasion and recurrence. To understand the invasive behavior of this tumor, we evaluated the role of the αvβ6 integrin. Invasive oral SCC cells express the αvβ6 integrin, which contains an 11-amino-acid extension on its β-subunit unique to the integrin family. We determined that this β6 cytoplasmic extension regulates the composition of the intermediate filament network and the organization of signaling structures called focal contacts. The auto-phosphorylation of FAK, which is localized to focal contacts, was also regulated by the β6-cytoplasmic tail, as were the transcription factors Notch and STAT3. Lastly, we also determined that activation of MAPK required the full-length β6 integrin. Together these results indicate that the signaling critical to epithelial-to-mesenchymal transition (EMT) is regulated by the β6 integrin cytoplasmic domain. PMID:24510996

  13. Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin

    PubMed Central

    Theodosiou, Marina; Widmaier, Moritz; Böttcher, Ralph T; Rognoni, Emanuel; Veelders, Maik; Bharadwaj, Mitasha; Lambacher, Armin; Austen, Katharina; Müller, Daniel J; Zent, Roy; Fässler, Reinhard

    2016-01-01

    Integrins require an activation step prior to ligand binding and signaling. How talin and kindlin contribute to these events in non-hematopoietic cells is poorly understood. Here we report that fibroblasts lacking either talin or kindlin failed to activate β1 integrins, adhere to fibronectin (FN) or maintain their integrins in a high affinity conformation induced by Mn2+. Despite compromised integrin activation and adhesion, Mn2+ enabled talin- but not kindlin-deficient cells to initiate spreading on FN. This isotropic spreading was induced by the ability of kindlin to directly bind paxillin, which in turn bound focal adhesion kinase (FAK) resulting in FAK activation and the formation of lamellipodia. Our findings show that talin and kindlin cooperatively activate integrins leading to FN binding and adhesion, and that kindlin subsequently assembles an essential signaling node at newly formed adhesion sites in a talin-independent manner. DOI: http://dx.doi.org/10.7554/eLife.10130.001 PMID:26821125

  14. Low density lipoprotein receptor-related protein 1 mediated endocytosis of β1-integrin influences cell adhesion and cell migration.

    PubMed

    Rabiej, Verena K; Pflanzner, Thorsten; Wagner, Timo; Goetze, Kristina; Storck, Steffen E; Eble, Johannes A; Weggen, Sascha; Mueller-Klieser, Wolfgang; Pietrzik, Claus U

    2016-01-01

    The low density lipoprotein receptor-related protein 1 (LRP1) has been shown to interact with β1-integrin and regulate its surface expression. LRP1 knock-out cells exhibit altered cytoskeleton organization and decreased cell migration. Here we demonstrate coupled endocytosis of LRP1 and β1-integrin and the involvement of the intracellular NPxY2 motif of LRP1 in this process. Mouse embryonic fibroblasts harboring a knock in replacement of the NPxY2 motif of LRP1 by a multiple alanine cassette (AAxA) showed elevated surface expression of β1-integrin and decreased β1-integrin internalization rates. As a consequence, cell spreading was altered and adhesion rates were increased in our cell model. Cells formed more focal adhesion complexes, whereby in vitro cell migration rates were decreased. Similar results could be observed in a corresponding mouse model, the C57Bl6 LRP1 NPxYxxL knock in mice, therefore, the biochemistry of cellular adhesion was altered in primary cortical neurons. In vivo cell migration experiments demonstrated a disturbance of neuroblast cell migration along the rostral migratory stream. In summary, our results indicate that LRP1 interacts with β1-integrin mediating integrin internalization and thus correlates with downstream signaling of β1-integrin such as focal adhesion dynamics. Consequently, the disturbance of this interaction resulted in a dysfunction in in vivo and in vitro cell adhesion and cell migration.

  15. Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2.

    PubMed

    De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

    2016-02-01

    Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-μ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions.

  16. Analysis of Combinatorial Epigenomic States.

    PubMed

    Soloway, Paul D

    2016-03-18

    Hundreds of distinct chemical modifications to DNA and histone amino acids have been described. Regulation exerted by these so-called epigenetic marks is vital to normal development, stability of cell identity through mitosis, and nongenetic transmission of traits between generations through meiosis. Loss of this regulation contributes to many diseases. Evidence indicates epigenetic marks function in combinations, whereby a given modification has distinct effects on local genome control, depending on which additional modifications are locally present. This review summarizes emerging methods for assessing combinatorial epigenomic states, as well as challenges and opportunities for their refinement.

  17. Patenting inventions in combinatorial chemistry.

    PubMed

    Borson, D B

    2000-02-01

    I have intended to provide an overview of some patent strategies for protecting intellectual property in the combinatorial chemistry arts, along with examples taken from recently issued patents. The opinions in this paper are those of the author, and are not intended to be relied upon as legal advice. Specific questions about any particular patent or invention should be discussed with competent counsel before action is taken. If you would like to look at the patents discussed in this article, they are available on-line at http:¿www.patents.ibm.com/boolquery and at other locations.

  18. Evolution of dynamic combinatorial chemistry.

    PubMed

    Cougnon, Fabien B L; Sanders, Jeremy K M

    2012-12-18

    Since its inception in the mid-1990s, dynamic combinatorial chemistry (DCC), the chemistry of complex systems under thermodynamic control, has proved valuable in identifying unexpected molecules with remarkable binding properties and in providing effective synthetic routes to complex species. Essentially, in this approach, one designs the experiment rather than the molecule. DCC has also provided us with insights into how some chemical systems respond to external stimuli. Using examples from the work of our laboratory and others, this Account shows how the concept of DCC, inspired by the evolution of living systems, has found an increasing range of applications in diverse areas and has evolved conceptually and experimentally. A dynamic combinatorial library (DCL) is a thermodynamically controlled mixture of interconverting species that can respond to various stimuli. The Cambridge version of dynamic combinatorial chemistry was initially inspired by the mammalian immune system and was conceived as a way to create and identify new unpredictable receptors. For example, an added template can select and stabilize a strongly binding member of the library which is then amplified at the expense of the unsuccessful library members, minimizing the free energy of the system. But researchers have exploited DCC in a variety of other ways: over the past two decades, this technique has contributed to the evolution of chemistry and to applications in the diverse fields of catalysis, fragrance release, and responsive materials. Among these applications, researchers have built intricate and well-defined architectures such as catenanes or hydrogen-bonded nanotubes, using the ability of complex chemical systems to reach a high level of organization. In addition, DCC has proved a powerful tool for the study of complex molecular networks and systems. The use of DCC is improving our understanding of chemical and biological systems. The study of folding or self-replicating macrocycles in

  19. Single-molecule decoding of combinatorially modified nucleosomes.

    PubMed

    Shema, Efrat; Jones, Daniel; Shoresh, Noam; Donohue, Laura; Ram, Oren; Bernstein, Bradley E

    2016-05-01

    Different combinations of histone modifications have been proposed to signal distinct gene regulatory functions, but this area is poorly addressed by existing technologies. We applied high-throughput single-molecule imaging to decode combinatorial modifications on millions of individual nucleosomes from pluripotent stem cells and lineage-committed cells. We identified definitively bivalent nucleosomes with concomitant repressive and activating marks, as well as other combinatorial modification states whose prevalence varies with developmental potency. We showed that genetic and chemical perturbations of chromatin enzymes preferentially affect nucleosomes harboring specific modification states. Last, we combined this proteomic platform with single-molecule DNA sequencing technology to simultaneously determine the modification states and genomic positions of individual nucleosomes. This single-molecule technology has the potential to address fundamental questions in chromatin biology and epigenetic regulation. PMID:27151869

  20. Plasmonic activation of gold nanorods for remote stimulation of calcium signaling and protein expression in HEK 293T cells.

    PubMed

    Sanchez-Rodriguez, Sandra P; Sauer, Jeremy P; Stanley, Sarah A; Qian, Xi; Gottesdiener, Andrew; Friedman, Jeffrey M; Dordick, Jonathan S

    2016-10-01

    Remote activation of specific cells of a heterogeneous population can provide a useful research tool for clinical and therapeutic applications. Here, we demonstrate that photostimulation of gold nanorods (AuNRs) using a tunable near-infrared (NIR) laser at specific longitudinal surface plasmon resonance wavelengths can induce the selective and temporal internalization of calcium in HEK 293T cells. Biotin-PEG-Au nanorods coated with streptavidin Alexa Fluor-633 and biotinylated anti-His antibodies were used to decorate cells genetically modified with His-tagged TRPV1 temperature-sensitive ion channel and AuNRs conjugated to biotinylated RGD peptide were used to decorate integrins in unmodified cells. Plasmonic activation can be stimulated at weak laser power (0.7-4.0 W/cm(2) ) without causing cell damage. Selective activation of TRPV1 channels could be controlled by laser power between 1.0 and 1.5 W/cm(2) . Integrin targeting robustly stimulated calcium signaling due to a dense cellular distribution of nanoparticles. Such an approach represents a functional tool for combinatorial activation of cell signaling in heterogeneous cell populations. Our results suggest that it is possible to induce cell activation via NIR-induced gold nanorod heating through the selective targeting of membrane proteins in unmodified cells to produce calcium signaling and downstream expression of specific genes with significant relevance for both in vitro and therapeutic applications. Biotechnol. Bioeng. 2016;113: 2228-2240. © 2016 Wiley Periodicals, Inc. PMID:27563853

  1. Plasmonic activation of gold nanorods for remote stimulation of calcium signaling and protein expression in HEK 293T cells.

    PubMed

    Sanchez-Rodriguez, Sandra P; Sauer, Jeremy P; Stanley, Sarah A; Qian, Xi; Gottesdiener, Andrew; Friedman, Jeffrey M; Dordick, Jonathan S

    2016-10-01

    Remote activation of specific cells of a heterogeneous population can provide a useful research tool for clinical and therapeutic applications. Here, we demonstrate that photostimulation of gold nanorods (AuNRs) using a tunable near-infrared (NIR) laser at specific longitudinal surface plasmon resonance wavelengths can induce the selective and temporal internalization of calcium in HEK 293T cells. Biotin-PEG-Au nanorods coated with streptavidin Alexa Fluor-633 and biotinylated anti-His antibodies were used to decorate cells genetically modified with His-tagged TRPV1 temperature-sensitive ion channel and AuNRs conjugated to biotinylated RGD peptide were used to decorate integrins in unmodified cells. Plasmonic activation can be stimulated at weak laser power (0.7-4.0 W/cm(2) ) without causing cell damage. Selective activation of TRPV1 channels could be controlled by laser power between 1.0 and 1.5 W/cm(2) . Integrin targeting robustly stimulated calcium signaling due to a dense cellular distribution of nanoparticles. Such an approach represents a functional tool for combinatorial activation of cell signaling in heterogeneous cell populations. Our results suggest that it is possible to induce cell activation via NIR-induced gold nanorod heating through the selective targeting of membrane proteins in unmodified cells to produce calcium signaling and downstream expression of specific genes with significant relevance for both in vitro and therapeutic applications. Biotechnol. Bioeng. 2016;113: 2228-2240. © 2016 Wiley Periodicals, Inc.

  2. Expressions of integrin subunits in osteoblasts during weightlessness simulation using clionstat

    NASA Astrophysics Data System (ADS)

    Zhang, S.; Wang, B.; Zhao, D.; Nie, J.; Li, Y.

    Space flight experiments and studies carried out in altered gravity environments have revealed that exposure to altered gravity conditions results in (mal)adaptation of cellular function. In the present study, we used a clinostat to generate a vector-averaged gravity to simulate weightlessness environment. We then observed the responses of rat calvarial osteoblasts subsequent to rotation at 30 revolutions per minute (rpm) for 72 h. We found that the gene expressions of three integrin subunits started to change from 24 h of rotation in clinostat but not in stationary cultures. The decreased percent changes of integrin a5 mRNA at 24, 48 and 72 h were 11.3 +/- 2.6%, 18.7 +/- 4.2% and 9.8 +/ - 2.1%, respectively. The same trend was saw in the expression of integrin av mRNA as 23.0 +/- 4.7%, 12.3 +/- 1.6% and 16.7 +/- 3.2%, respectively. Moreover, the expressions of integrin ß1 mRNA in different periods were also declined with the percent changes of 15.3 +/- 1.3%, 11.4 +/- 1.2% and 26.4 +/- 5.5%, respectively. All cells contain membrane-anchored attachment proteins able to recognize specific chemical motifs in the extracellular macromolecules forming the supporting scaffold, made of various types of collagen, adhesive glycoproteins, elastin, proteoglycans, etc. These cell-matrix interactions are mainly mediated by receptors of the integrins family, heterodimeric molecules made of an extracellular domain connected through a transmembrane sequence to an intracytoplasmic tail. Our results suggest that vector-averaged gravity causes alterations of signal transduction and integrin-mediated cell adhesion in osteoblasts by altering the gene expressions of several crucial integrin subunits. These alterations might contribute to the pathogenesis of osteoporotic bone loss as observed in actual space flights.

  3. Combinatorial effects of odorants on mouse behavior

    PubMed Central

    Saraiva, Luis R.; Kondoh, Kunio; Ye, Xiaolan; Yoon, Kyoung-hye; Hernandez, Marcus; Buck, Linda B.

    2016-01-01

    The mechanisms by which odors induce instinctive behaviors are largely unknown. Odor detection in the mouse nose is mediated by >1, 000 different odorant receptors (ORs) and trace amine-associated receptors (TAARs). Odor perceptions are encoded combinatorially by ORs and can be altered by slight changes in the combination of activated receptors. However, the stereotyped nature of instinctive odor responses suggests the involvement of specific receptors and genetically programmed neural circuits relatively immune to extraneous odor stimuli and receptor inputs. Here, we report that, contrary to expectation, innate odor-induced behaviors can be context-dependent. First, different ligands for a given TAAR can vary in behavioral effect. Second, when combined, some attractive and aversive odorants neutralize one another’s behavioral effects. Both a TAAR ligand and a common odorant block aversion to a predator odor, indicating that this ability is not unique to TAARs and can extend to an aversive response of potential importance to survival. In vitro testing of single receptors with binary odorant mixtures indicates that behavioral blocking can occur without receptor antagonism in the nose. Moreover, genetic ablation of a single receptor prevents its cognate ligand from blocking predator odor aversion, indicating that the blocking requires sensory input from the receptor. Together, these findings indicate that innate odor-induced behaviors can depend on context, that signals from a single receptor can block innate odor aversion, and that instinctive behavioral responses to odors can be modulated by interactions in the brain among signals derived from different receptors. PMID:27208093

  4. Integrin Targeting for Tumor Optical Imaging

    PubMed Central

    Ye, Yunpeng; Chen, Xiaoyuan

    2011-01-01

    Optical imaging has emerged as a powerful modality for studying molecular recognitions and molecular imaging in a noninvasive, sensitive, and real-time way. Some advantages of optical imaging include cost-effectiveness, convenience, and non-ionization safety as well as complementation with other imaging modalities such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). Over the past decade, considerable advances have been made in tumor optical imaging by targeting integrin receptors in preclinical studies. This review has emphasized the construction and evaluation of diverse integrin targeting agents for optical imaging of tumors in mouse models. They mainly include some near-infrared fluorescent dye-RGD peptide conjugates, their multivalent analogs, and nanoparticle conjugates for targeting integrin αvβ3. Some compounds targeting other integrin subtypes such as α4β1 and α3 for tumor optical imaging have also been included. Both in vitro and in vivo studies have revealed some promising integrin-targeting optical agents which have further enhanced our understanding of integrin expression and targeting in cancer biology as well as related anticancer drug discovery. Especially, some integrin-targeted multifunctional optical agents including nanoparticle-based optical agents can multiplex optical imaging with other imaging modalities and targeted therapy, serving as an attractive type of theranostics for simultaneous imaging and targeted therapy. Continued efforts to discover and develop novel, innovative integrin-based optical agents with improved targeting specificity and imaging sensitivity hold great promises for improving cancer early detection, diagnosis, and targeted therapy in clinic. PMID:21546996

  5. Integrins as “Functional Hubs” in the Regulation of Pathological Angiogenesis

    PubMed Central

    Contois, Liangru; Akalu, Abebe; Brooks, Peter C.

    2009-01-01

    It is well accepted that complex biological processes such as angiogenesis are not controlled by a single family of molecules or individually isolated signaling pathways. In this regard, new insight into the interconnected mechanisms that regulate angiogenesis might be gained by examining this process from a more global network perspective. The coordination of signaling cues from both outside and inside many different cell types is required for the successful completion of angiogenesis. Evidence is accumulating that the multifunctional integrin family of cell adhesion receptors represent an important group of molecules that play active roles in sensing, integrating, and distributing a diverse set of signals that regulate many cellular events required for angiogenesis. Given the ability of integrins to bind numerous extracellular ligands and transmit signals in a bi-directional fashion, we will discuss the multiple ways by which integrins may serve as a functional hub during pathological angiogenesis. In addition, we will highlight potential imaging and therapeutic strategies based on the expanding new insight into integrin function. PMID:19482089

  6. Role of Integrin Subunits in Mesenchymal Stem Cell Differentiation and Osteoblast Maturation on Graphitic Carbon-coated Microstructured Surfaces

    PubMed Central

    Olivares-Navarrete, Rene; Rodil, Sandra E.; Hyzy, Sharon L.; Dunn, Ginger R.; Almaguer-Flores, Argelia; Schwartz, Zvi; Boyan, Barbara D.

    2015-01-01

    Surface roughness, topography, chemistry, and energy promote osteoblast differentiation and increase osteogenic local factor production in vitro and bone-to-implant contact in vivo, but the mechanisms involved are not well understood. Knockdown of integrin heterodimer alpha2beta1 (α2β1) blocks the osteogenic effects of the surface, suggesting signaling by this integrin homodimer is required. The purpose of the present study was to separate effects of surface chemistry and surface structure on integrin expression by coating smooth or rough titanium (Ti) substrates with graphitic carbon, retaining surface morphology but altering surface chemistry. Ti surfaces (smooth [Ra<0.4μm], rough [Ra≥3.4μm]) were sputter-coated using a magnetron sputtering system with an ultrapure graphite target, producing a graphitic carbon thin film. Human mesenchymal stem cells and MG63 osteoblast-like cells had higher mRNA for integrin subunits α1, α2, αv, and β1 on rough surfaces in comparison to smooth, and integrin αv on graphitic-carbon-coated rough surfaces in comparison to Ti. Osteogenic differentiation was greater on rough surfaces in comparison to smooth, regardless of chemistry. Silencing integrins β1, α1, or α2 decreased osteoblast maturation on rough surfaces independent of surface chemistry. Silencing integrin αv decreased maturation only on graphitic carbon-coated surfaces, not on Ti. These results suggest a major role of the integrin β1 subunit in roughness recognition, and that integrin alpha subunits play a major role in surface chemistry recognition. PMID:25770999

  7. The integrin-binding motif RGDS induces protein tyrosine phosphorylation without activation in Bufo arenarum (Amphibia) oocytes.

    PubMed

    Mouguelar, Valeria S; Cabada, Marcelo O; Coux, Gabriela

    2011-05-01

    Integrins are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction. Nevertheless, their function in mammalian fertilization is still controversial, as different species behave differently. In amphibians, their role is mainly supported by Xenopus laevis studies, where RGDS peptide induces oocyte activation. We recently provided evidence suggesting the presence and involvement of integrins in the interaction of the oocyte plasma membrane (PM) with sperm in the amphibian Bufo arenarum. In order to understand the role of integrin homologs in oocytes and their possible contribution to egg activation mechanisms, we examined the presence of integrin subunits and the effect of RGDS peptide on oocytes and during fertilization. Western blot studies detected integrin subunits α5, αV and β1 in oocytes. In sperm, we could detect only the αV integrin subunit. We found that RGDS peptide was unable to elicit egg activation or MAPK dephosphorylation, but can induce reversible inhibition of fertilization. A similar partial inhibition was produced by an anti-β1 integrin antibody. Using an anti-phosphotyrosine antibody we found major changes in phosphotyrosine-containing proteins in egg extracts minutes after fertilization. Cytosol and PMs isolated from oocytes and fertilized eggs showed additional fertilization-induced phosphorylated proteins. Some of these were also present in cytosol and PMs from RGDS-treated oocytes (partially mimicking fertilization). These findings suggest that B. arenarum fertilization involves integrins (e.g. β1 subunit) as adhesion proteins. Our data support the view that RGDS-binding receptors may function as signaling receptors in B. arenarum oocytes, but integrin engagement by RGDS is not sufficient for oocyte activation. PMID:21339287

  8. Tumour exosome integrins determine organotropic metastasis.

    PubMed

    Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; Rodrigues, Goncalo; Hashimoto, Ayako; Tesic Mark, Milica; Molina, Henrik; Kohsaka, Shinji; Di Giannatale, Angela; Ceder, Sophia; Singh, Swarnima; Williams, Caitlin; Soplop, Nadine; Uryu, Kunihiro; Pharmer, Lindsay; King, Tari; Bojmar, Linda; Davies, Alexander E; Ararso, Yonathan; Zhang, Tuo; Zhang, Haiying; Hernandez, Jonathan; Weiss, Joshua M; Dumont-Cole, Vanessa D; Kramer, Kimberly; Wexler, Leonard H; Narendran, Aru; Schwartz, Gary K; Healey, John H; Sandstrom, Per; Labori, Knut Jørgen; Kure, Elin H; Grandgenett, Paul M; Hollingsworth, Michael A; de Sousa, Maria; Kaur, Sukhwinder; Jain, Maneesh; Mallya, Kavita; Batra, Surinder K; Jarnagin, William R; Brady, Mary S; Fodstad, Oystein; Muller, Volkmar; Pantel, Klaus; Minn, Andy J; Bissell, Mina J; Garcia, Benjamin A; Kang, Yibin; Rajasekhar, Vinagolu K; Ghajar, Cyrus M; Matei, Irina; Peinado, Hector; Bromberg, Jacqueline; Lyden, David

    2015-11-19

    Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

  9. Tumour exosome integrins determine organotropic metastasis

    PubMed Central

    Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; Rodrigues, Goncalo; Hashimoto, Ayako; Mark, Milica Tesic; Molina, Henrik; Kohsaka, Shinji; Di Giannatale, Angela; Ceder, Sophia; Singh, Swarnima; Williams, Caitlin; Soplop, Nadine; Uryu, Kunihiro; Pharmer, Lindsay; King, Tari; Bojmar, Linda; Davies, Alexander E.; Ararso, Yonathan; Zhang, Tuo; Zhang, Haiying; Hernandez, Jonathan; Weiss, Joshua M.; Dumont-Cole, Vanessa D.; Kramer, Kimberly; Wexler, Leonard H.; Narendran, Aru; Schwartz, Gary K.; Healey, John H.; Sandstrom, Per; Labori, Knut Jørgen; Kure, Elin H.; Grandgenett, Paul M.; Hollingsworth, Michael A.; de Sousa, Maria; Kaur, Sukhwinder; Jain, Maneesh; Mallya, Kavita; Batra, Surinder K.; Jarnagin, William R.; Brady, Mary S.; Fodstad, Oystein; Muller, Volkmar; Pantel, Klaus; Minn, Andy J.; Bissell, Mina J.; Garcia, Benjamin A.; Kang, Yibin; Rajasekhar, Vinagolu K.; Ghajar, Cyrus M.; Matei, Irina; Peinado, Hector; Bromberg, Jacqueline; Lyden, David

    2015-01-01

    Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis. PMID:26524530

  10. Asymmetric endocytosis and remodeling of β1-integrin adhesions during growth cone chemorepulsion by MAG

    PubMed Central

    Hines, Jacob H.; Abu-Rub, Mohammad; Henley, John R.

    2011-01-01

    Gradients of chemorepellent factors released from myelin may impair axon pathfinding and neuro-regeneration after injury. Analogous to the process of chemotaxis in invasive tumor cells, we found that axonal growth cones of Xenopus spinal neurons modulate the functional distribution of integrin receptors during chemorepulsion induced by myelin-associated glycoprotein (MAG). A focal MAG gradient induced polarized endocytosis and concomitant asymmetric loss of β1-integrin and vinculin-containing adhesions on the repellent side during repulsive turning. Loss of symmetrical β1-integrin function was both necessary and sufficient for chemorepulsion, which required internalization by clathrin-mediated endocytosis. Induction of repulsive Ca2+ signals was necessary and sufficient for the stimulated rapid endocytosis of β1-integrin. Altogether, these findings identify β1-integrin as an important functional cargo during Ca2+-dependent rapid endocytosis stimulated by a diffusible guidance cue. Such dynamic redistribution allows the growth cone to rapidly adjust adhesiveness across its axis, an essential feature for initiating chemotactic turning. PMID:20512137

  11. Changes in membrane sphingolipid composition modulate dynamics and adhesion of integrin nanoclusters.

    PubMed

    Eich, Christina; Manzo, Carlo; de Keijzer, Sandra; Bakker, Gert-Jan; Reinieren-Beeren, Inge; García-Parajo, Maria F; Cambi, Alessandra

    2016-01-01

    Sphingolipids are essential constituents of the plasma membrane (PM) and play an important role in signal transduction by modulating clustering and dynamics of membrane receptors. Changes in lipid composition are therefore likely to influence receptor organisation and function, but how this precisely occurs is difficult to address given the intricacy of the PM lipid-network. Here, we combined biochemical assays and single molecule dynamic approaches to demonstrate that the local lipid environment regulates adhesion of integrin receptors by impacting on their lateral mobility. Induction of sphingomyelinase (SMase) activity reduced sphingomyelin (SM) levels by conversion to ceramide (Cer), resulting in impaired integrin adhesion and reduced integrin mobility. Dual-colour imaging of cortical actin in combination with single molecule tracking of integrins showed that this reduced mobility results from increased coupling to the actin cytoskeleton brought about by Cer formation. As such, our data emphasizes a critical role for the PM local lipid composition in regulating the lateral mobility of integrins and their ability to dynamically increase receptor density for efficient ligand binding in the process of cell adhesion. PMID:26869100

  12. Changes in membrane sphingolipid composition modulate dynamics and adhesion of integrin nanoclusters

    PubMed Central

    Eich, Christina; Manzo, Carlo; Keijzer, Sandra de; Bakker, Gert-Jan; Reinieren-Beeren, Inge; García-Parajo, Maria F.; Cambi, Alessandra

    2016-01-01

    Sphingolipids are essential constituents of the plasma membrane (PM) and play an important role in signal transduction by modulating clustering and dynamics of membrane receptors. Changes in lipid composition are therefore likely to influence receptor organisation and function, but how this precisely occurs is difficult to address given the intricacy of the PM lipid-network. Here, we combined biochemical assays and single molecule dynamic approaches to demonstrate that the local lipid environment regulates adhesion of integrin receptors by impacting on their lateral mobility. Induction of sphingomyelinase (SMase) activity reduced sphingomyelin (SM) levels by conversion to ceramide (Cer), resulting in impaired integrin adhesion and reduced integrin mobility. Dual-colour imaging of cortical actin in combination with single molecule tracking of integrins showed that this reduced mobility results from increased coupling to the actin cytoskeleton brought about by Cer formation. As such, our data emphasizes a critical role for the PM local lipid composition in regulating the lateral mobility of integrins and their ability to dynamically increase receptor density for efficient ligand binding in the process of cell adhesion. PMID:26869100

  13. Maternal anti-platelet β3 integrins impair angiogenesis and cause intracranial hemorrhage.

    PubMed

    Yougbaré, Issaka; Lang, Sean; Yang, Hong; Chen, Pingguo; Zhao, Xu; Tai, Wei-She; Zdravic, Darko; Vadasz, Brian; Li, Conglei; Piran, Siavash; Marshall, Alexandra; Zhu, Guangheng; Tiller, Heidi; Killie, Mette Kjaer; Boyd, Shelley; Leong-Poi, Howard; Wen, Xiao-Yan; Skogen, Bjorn; Adamson, S Lee; Freedman, John; Ni, Heyu

    2015-04-01

    Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against β3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-β3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-β3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti-β3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti-β3 integrin antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.

  14. Expression of fibronectin, fibronectin isoforms and integrin receptors in melanocytic lesions.

    PubMed Central

    Natali, P. G.; Nicotra, M. R.; Di Filippo, F.; Bigotti, A.

    1995-01-01

    In vitro studies have demonstrated that fibronectin (FN) can deliver a mitogenic signal to quiescent human melanoma cells and that the alpha 5/beta 1-integrin receptor mediates this stimulus. In view of this finding we have analysed the in vivo expression of FN, and of ED-A and ED-B FN isoforms, in benign and malignant lesions of melanocyte origin. In the same specimens the expression of fibronectin integrin receptors was evaluated. The results demonstrate that, while detection of FN does not correlate with transformation and tumour progression, the expression of the two isoforms is associated with transformation and that only the ED-A variant is found in metastases. Integrin phenotyping disclosed that alpha 3/beta 1 expression is associated with tumour progression, alpha v/beta 3 is a marker of transformation, alpha 4 is rarely expressed and alpha 5 is expressed by about 50% and 30% of the primary and metastatic lesions respectively. Taken together, the results of this study demonstrate that transformation and tumour progression of the melanocyte lineage are associated with modulation of expression of FN isoforms and FN integrin receptors. Furthermore, the expression of alpha 5-integrin in a considerable percentage of primary and metastatic lesions indicates that FN may deliver a proliferative stimulus to melanoma cells in vivo. Images Figure 1 PMID:7779718

  15. Mesodermal expression of integrin α5β1 regulates neural crest development and cardiovascular morphogenesis

    PubMed Central

    Liang, Dong; Wang, Xia; Mittal, Ashok; Dhiman, Sonam; Hou, Shuan-Yu; Degenhardt, Karl; Astrof, Sophie

    2014-01-01

    Integrin α5-null embryos die in mid-gestation from severe defects in cardiovascular morphogenesis, which stem from defective development of the neural crest, heart and vasculature. To investigate the role of integrin α5β1 in cardiovascular development, we used the Mesp1Cre knock-in strain of mice to ablate integrin α5 in the anterior mesoderm, which gives rise to all of the cardiac and many of the vascular and muscle lineages in the anterior portion of the embryo. Surprisingly, we found that mutant embryos displayed numerous defects related to the abnormal development of the neural crest such as cleft palate, ventricular septal defect, abnormal development of hypoglossal nerves, and defective remodeling of the aortic arch arteries. We found that defects in arch artery remodeling stem from the role of mesodermal integrin α5β1 in neural crest proliferation and differentiation into vascular smooth muscle cells, while proliferation of pharyngeal mesoderm and differentiation of mesodermal derivatives into vascular smooth muscle cells was not defective. Taken together our studies demonstrate a requisite role for mesodermal integrin α5β1 in signaling between the mesoderm and the neural crest, thereby regulating neural crest-dependent morphogenesis of essential embryonic structures. PMID:25242040

  16. Combinatorial code governing cellular responses to complex stimuli

    PubMed Central

    Cappuccio, Antonio; Zollinger, Raphaël; Schenk, Mirjam; Walczak, Aleksandra; Servant, Nicolas; Barillot, Emmanuel; Hupé, Philippe; Modlin, Robert L.; Soumelis, Vassili

    2015-01-01

    Cells adapt to their environment through the integration of complex signals. Multiple signals can induce synergistic or antagonistic interactions, currently considered as homogenous behaviours. Here, we use a systematic theoretical approach to enumerate the possible interaction profiles for outputs measured in the conditions 0 (control), signals X, Y, X+Y. Combinatorial analysis reveals 82 possible interaction profiles, which we biologically and mathematically grouped into five positive and five negative interaction modes. To experimentally validate their use in living cells, we apply an original computational workflow to transcriptomics data of innate immune cells integrating physiopathological signal combinations. Up to 9 of the 10 defined modes coexisted in context-dependent proportions. Each interaction mode was preferentially used in specific biological pathways, suggesting a functional role in the adaptation to multiple signals. Our work defines an exhaustive map of interaction modes for cells integrating pairs of physiopathological and pharmacological stimuli. PMID:25896517

  17. Measurement of Cationic and Intracellular Modulation of Integrin Binding Affinity by AFM-Based Nanorobot

    PubMed Central

    Patterson, Kevin C.; Yang, Ruiguo; Zeng, Bixi; Song, Bo; Wang, Shouye; Xi, Ning; Basson, Marc D.

    2013-01-01

    Integrins are dynamic transmembrane cation-dependent heterodimers that both anchor cells in position and transduce signals into and out of cells. We used an atomic force microscope (AFM)-based nanorobotic system to measure integrin-binding forces in intact human intestinal epithelial Caco-2 cells. The AFM-based nanorobot enables human-directed, high-accuracy probe positioning and site-specific investigations. Functionalizing the AFM probe with an arginine-glycine-aspartate (RGD)-containing sequence (consensus binding sequence for integrins) allowed us to detect a series of peptide-cell membrane interactions with a median binding force of 115.1 ± 4.9 pN that were not detected in control interactions. Chelating divalent cations from the culture medium abolished these interactions, as did inhibiting intracellular focal adhesion kinase (FAK) using Y15. Adding 1 mM Mg2+ to the medium caused a rightward shift in the force-binding curve. Adding 1 mM Ca2+ virtually abolished the RGD-membrane specific interactions and blocked the Mg2+ effects. Cell adhesion assays demonstrated parallel effects of divalent cations and the FAK inhibitor on cell adhesion. These results demonstrate direct modulation of integrin-binding affinity by both divalent cations and intracellular signal inhibition. Additionally, three binding states (nonspecific, specific inactivated, and specific activated) were delineated from affinity measurements. Although other research has assumed that this process of integrin conformational change causes altered ligand binding, in this work we directly measured these three states in individual integrins in a physiologically based study. PMID:23823222

  18. Rapid combinatorial screening by synchrotron X-ray imaging

    NASA Astrophysics Data System (ADS)

    Eba, Hiromi; Sakurai, Kenji

    2006-01-01

    An X-ray imaging system, which does not require any scans of the sample or an X-ray beam and which, therefore, dramatically reduces the amount of time required, was employed to evaluate combinatorial libraries efficiently. Two-dimensional X-ray fluorescence (XRF) images of an 8 mm × 8 mm area were observed for combinatorial substrates of manganese-cobalt spinel MnCo 2O 4 and lithium ferrite LiFeO 2 via an exposure time of 1-3 s using synchrotron X-rays. Thus, XRF signals from a whole substrate could be observed at once in a short space of time. In order to observe the chemical environment simultaneously for all materials arranged on the substrate, the fluorescent X-ray absorption fine structure (XAFS) was measured by repeating the imaging during the monochromator scans across the absorption edge for metals. This is extremely efficient because XAFS spectra for all materials placed on the common substrate are obtained from only a single energy scan. One can determine the valence numbers, as well as other aspects of the chemical environment of the metal included in each material, from the differences in spectral features and the energy shifts. Hence, combinatorial libraries can be screened very rapidly, and therefore efficiently, using the X-ray imaging system.

  19. Combinatorial Chemistry for Optical Sensing Applications

    NASA Astrophysics Data System (ADS)

    Díaz-García, M. E.; Luis, G. Pina; Rivero-Espejel, I. A.

    The recent interest in combinatorial chemistry for the synthesis of selective recognition materials for optical sensing applications is presented. The preparation, screening, and applications of libraries of ligands and chemosensors against molecular species and metal ions are first considered. Included in this chapter are also the developments involving applications of combinatorial approaches to the discovery of sol-gel and acrylic-based imprinted materials for optical sensing of antibiotics and pesticides, as well as libraries of doped sol-gels for high-throughput optical sensing of oxygen. The potential of combinatorial chemistry applied to the discovery of new sensing materials is highlighted.

  20. Ameloblastin modulates osteoclastogenesis through the integrin/ERK pathway

    PubMed Central

    Atsawasuwan, Phimon; Dangaria, Smit; Yan, Xiulin; Wu, Tuojiang; Evans, Carla A.; Luan, Xianghong

    2014-01-01

    Proteins of the extracellular matrix often have multiple functions to facilitate complex tasks ranging from signaling to structural support. Here we have focused on the function of one of the matrix proteins expressed in bones and teeth, the matrix adhesion protein ameloblastin (AMBN). Transgenic mice with 5-fold elevated AMBN levels in mandibles suffered from root cementum resorption, delamination, and reduced alveolar bone thickness. AMBN gain of function also resulted in a significant reduction in trabecular bone volume and bone mass dentistry in 42 days postnatal mouse jaws. In an in vitro model of osteoclastogenesis, AMBN modulated osteoclast differentiation from bone marrow derived monocytes (BMMCs), and dramatically increased osteoclast numbers and resorption pits. Furthermore, AMBN more than doubled BMMC adhesion, accelerated cell spreading, and promoted podosome belt and actin ring formation. These effects were associated with elevated ERK1/2 and AKT phosphorylation as well as higher expression of osteoclast activation related genes. Blocking integrin α2β1 and ERK 1/2 pathways alleviated the effects of AMBN on osteoclast differentiation. Together, our data indicate that AMBN increases osteoclast number and differentiation as well as mineralized tissue resorption by regulating cell adhesion and actin cytoskeleton polymerization, initiating integrin-dependent extracellular matrix signaling cascades and enhancing osteoclastogenesis. PMID:23385480

  1. Mechanotransduction molecules in the plant gravisensory response: amyloplast/statolith membranes contain a beta 1 integrin-like protein

    NASA Technical Reports Server (NTRS)

    Lynch, T. M.; Lintilhac, P. M.; Domozych, D.

    1998-01-01

    It has been hypothesized that the sedimentation of amyloplasts within root cap cells is the primary event in the plant gravisensory-signal transduction cascade. Statolith sedimentation, with its ability to generate weighty mechanical signals, is a legitimate means for organisms to discriminate the direction of the gravity vector. However, it has been demonstrated that starchless mutants with reduced statolith densities maintain some ability to sense gravity, calling into question the statolith sedimentation hypothesis. Here we report on the presence of a beta 1 integrin-like protein localized inside amyloplasts of tobacco NT-1 suspension culture, callus cells, and whole-root caps. Two different antibodies to the beta 1 integrin, one to the cytoplasmic domain and one to the extracellular domain, localize in the vicinity of the starch grains within amyloplasts of NT-1. Biochemical data reveals a 110-kDa protein immunoprecipitated from membrane fractions of NT-1 suspension culture indicating size homology to known beta 1 integrin in animals. This study provides the first direct evidence for the possibility of integrin-mediated signal transduction in the perception of gravity by higher plants. An integrin-mediated pathway, initiated by starch grain sedimentation within the amyloplast, may provide the signal amplification necessary to explain the gravitropic response in starch-depleted cultivars.

  2. Integrin αv promotes proliferation by activating ERK 1/2 in the human lung cancer cell line A549.

    PubMed

    Fu, Shijie; Fan, Limin; Pan, Xufeng; Sun, Yifeng; Zhao, Heng

    2015-02-01

    Lung cancer is a leading cause of cancer-related death worldwide, and non-small cell lung cancer (NSCLC) constitutes ~85% of lung cancers. However, the mechanisms underlying the progression of NSCLC remain unclear. In this study, we found the mRNA and protein expression levels of integrin αv are both increased in NSCLC tissues compared to healthy ones, which indicates that integrin αv may play an important role in NSCLC progression. To further investigate the roles of integrin αv in NSCLC, we overexpressed the integrin αv gene in the NSCLC cell line A549, and found that the cell proliferative ability increased. The apoptosis of A549 cells was inhibited with overexpression of integrin αv. To elucidate the molecular mechanism underlying the role of integrin αv in promoting NSCLC progression, we studied the expression of proteins from a number of important pathways associated with tumorigenesis, and found that the extracellular signal regulated protein kinase (ERK)1/2 signaling pathway may be involved in the mediation of the observed integrin αv effects. component of an important pathway for tumorigenesis, the ERK 1/2. Following inhibition of ERK 1/2 signaling, the proliferation of A549 cells induced by integrin αv was reduced, while the inhibition of apoptosis was attenuated. Our findings demonstrate that integrin αv promotes the proliferation of the human lung cancer cell line A549 by activating the ERK 1/2 signaling pathway, which suggests that this pathway may be a promising target for the treatment of human lung cancer.

  3. Endocytosis of Integrin-Binding Human Picornaviruses

    PubMed Central

    Merilahti, Pirjo; Koskinen, Satu; Heikkilä, Outi; Karelehto, Eveliina; Susi, Petri

    2012-01-01

    Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9), echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1) has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses. PMID:23227048

  4. Endocytosis of integrin-binding human picornaviruses.

    PubMed

    Merilahti, Pirjo; Koskinen, Satu; Heikkilä, Outi; Karelehto, Eveliina; Susi, Petri

    2012-01-01

    Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9), echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1) has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  5. Extracellular K(+) and opening of voltage-gated potassium channels activate T cell integrin function: physical and functional association between Kv1.3 channels and beta1 integrins.

    PubMed

    Levite, M; Cahalon, L; Peretz, A; Hershkoviz, R; Sobko, A; Ariel, A; Desai, R; Attali, B; Lider, O

    2000-04-01

    Elevated extracellular K(+) ([K(+)](o)), in the absence of "classical" immunological stimulatory signals, was found to itself be a sufficient stimulus to activate T cell beta1 integrin moieties, and to induce integrin-mediated adhesion and migration. Gating of T cell voltage-gated K(+) channels (Kv1.3) appears to be the crucial "decision-making" step, through which various physiological factors, including elevated [K(+)](o) levels, affect the T cell beta1 integrin function: opening of the channel leads to function, whereas its blockage prevents it. In support of this notion, we found that the proadhesive effects of the chemokine macrophage-inflammatory protein 1beta, the neuropeptide calcitonin gene-related peptide (CGRP), as well as elevated [K(+)](o) levels, are blocked by specific Kv1.3 channel blockers, and that the unique physiological ability of substance P to inhibit T cell adhesion correlates with Kv1.3 inhibition. Interestingly, the Kv1.3 channels and the beta1 integrins coimmunoprecipitate, suggesting that their physical association underlies their functional cooperation on the T cell surface. This study shows that T cells can be activated and driven to integrin function by a pathway that does not involve any of its specific receptors (i.e., by elevated [K(+)](o)). In addition, our results suggest that undesired T cell integrin function in a series of pathological conditions can be arrested by molecules that block the Kv1.3 channels. PMID:10748234

  6. Intrinsic information carriers in combinatorial dynamical systems.

    PubMed

    Harmer, Russ; Danos, Vincent; Feret, Jérôme; Krivine, Jean; Fontana, Walter

    2010-09-01

    Many proteins are composed of structural and chemical features--"sites" for short--characterized by definite interaction capabilities, such as noncovalent binding or covalent modification of other proteins. This modularity allows for varying degrees of independence, as the behavior of a site might be controlled by the state of some but not all sites of the ambient protein. Independence quickly generates a startling combinatorial complexity that shapes most biological networks, such as mammalian signaling systems, and effectively prevents their study in terms of kinetic equations-unless the complexity is radically trimmed. Yet, if combinatorial complexity is key to the system's behavior, eliminating it will prevent, not facilitate, understanding. A more adequate representation of a combinatorial system is provided by a graph-based framework of rewrite rules where each rule specifies only the information that an interaction mechanism depends on. Unlike reactions, which deal with molecular species, rules deal with patterns, i.e., multisets of molecular species. Although the stochastic dynamics induced by a collection of rules on a mixture of molecules can be simulated, it appears useful to capture the system's average or deterministic behavior by means of differential equations. However, expansion of the rules into kinetic equations at the level of molecular species is not only impractical, but conceptually indefensible. If rules describe bona fide patterns of interaction, molecular species are unlikely to constitute appropriate units of dynamics. Rather, we must seek aggregate variables reflective of the causal structure laid down by the rules. We call these variables "fragments" and the process of identifying them "fragmentation." Ideally, fragments are aspects of the system's microscopic population that the set of rules can actually distinguish on average; in practice, it may only be feasible to identify an approximation to this. Most importantly, fragments are

  7. Combinatorial quorum sensing allows bacteria to resolve their social and physical environment.

    PubMed

    Cornforth, Daniel M; Popat, Roman; McNally, Luke; Gurney, James; Scott-Phillips, Thomas C; Ivens, Alasdair; Diggle, Stephen P; Brown, Sam P

    2014-03-18

    Quorum sensing (QS) is a cell-cell communication system that controls gene expression in many bacterial species, mediated by diffusible signal molecules. Although the intracellular regulatory mechanisms of QS are often well-understood, the functional roles of QS remain controversial. In particular, the use of multiple signals by many bacterial species poses a serious challenge to current functional theories. Here, we address this challenge by showing that bacteria can use multiple QS signals to infer both their social (density) and physical (mass-transfer) environment. Analytical and evolutionary simulation models show that the detection of, and response to, complex social/physical contrasts requires multiple signals with distinct half-lives and combinatorial (nonadditive) responses to signal concentrations. We test these predictions using the opportunistic pathogen Pseudomonas aeruginosa and demonstrate significant differences in signal decay between its two primary signal molecules, as well as diverse combinatorial responses to dual-signal inputs. QS is associated with the control of secreted factors, and we show that secretome genes are preferentially controlled by synergistic "AND-gate" responses to multiple signal inputs, ensuring the effective expression of secreted factors in high-density and low mass-transfer environments. Our results support a new functional hypothesis for the use of multiple signals and, more generally, show that bacteria are capable of combinatorial communication.

  8. Transcription factor hlh-2/E/Daughterless drives expression of α integrin ina-1 during DTC migration in C. elegans.

    PubMed

    Meighan, Christopher M; Kann, Allison P; Egress, Emily R

    2015-09-01

    Integrins are involved in a vast number of cell behaviors due to their roles in adhesion and signaling. The regulation of integrin expression is of particular interest as a mechanism to drive developmental events and for the role of altered integrin expression profiles in cancer. Dynamic regulation of the expression of integrin receptors is required for the migration of the distal tip cell (DTC) during gonadogenesis in Caenorhabditis elegans. α integrin ina-1 is required for DTC motility, yet is up-regulated by an unknown mechanism. Analysis of the promoter for α integrin ina-1 identified two E-box sequences that are required for ina-1 expression in the DTC. Knockdown of transcription factor hlh-2, an established E-box binding partner and ortholog of E/Daughterless, prevented expression of a transcriptional fusion of the ina-1 promoter to RFP and blocked DTC migration. Similarly, knockdown of hlh-2 also prevented expression of a translational fusion of the genomic ina-1 gene to GFP while blocking DTC migration. Knockdown of HLH-2 binding partner MIG-24 also reduced ina-1 expression and DTC migration. Overall, these results show that the transcription factor hlh-2 is required for up-regulation of ina-1 at the onset of DTC migration.

  9. Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding

    PubMed Central

    Iturri, Jagoba; García-Fernández, Luis; Reuning, Ute; García, Andrés J.; Campo, Aránzazu del; Salierno, Marcelo J.

    2015-01-01

    The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies. PMID:25825012

  10. Inducible and combinatorial gene manipulation in mouse brain

    PubMed Central

    Dogbevia, Godwin K.; Marticorena-Alvarez, Ricardo; Bausen, Melanie; Sprengel, Rolf; Hasan, Mazahir T.

    2015-01-01

    We have deployed recombinant adeno-associated viruses equipped with tetracycline-controlled genetic switches to manipulate gene expression in mouse brain. Here, we show a combinatorial genetic approach for inducible, cell type-specific gene expression and Cre/loxP mediated gene recombination in different brain regions. Our chemical-genetic approach will help to investigate ‘when’, ‘where’, and ‘how’ gene(s) control neuronal circuit dynamics, and organize, for example, sensory signal processing, learning and memory, and behavior. PMID:25954155

  11. Plakophilin 2 Affects Cell Migration by Modulating Focal Adhesion Dynamics and Integrin Protein Expression

    PubMed Central

    Koetsier, Jennifer L.; Amargo, Evangeline V.; Todorović, Viktor; Green, Kathleen J.; Godsel, Lisa M.

    2014-01-01

    Plakophilin 2 (PKP2), a desmosome component, modulates the activity and localization of the small GTPase RhoA at sites of cell–cell contact. PKP2 regulates cortical actin rearrangement during junction formation, and its loss is accompanied by an increase in actin stress fibers. We hypothesized that PKP2 may regulate focal adhesion dynamics and cell migration. Here we show that PKP2-deficient cells bind efficiently to the extracellular matrix, but upon spreading display total cell areas ~30% smaller than control cells. Focal adhesions in PKP2-deficient cells are ~2× larger and more stable than in control cells, and vinculin displays an increased time for fluorescence recovery after photobleaching. Furthermore, β4 and β1 integrin protein and mRNA expression is elevated in PKP2-silenced cells. Normal focal adhesion phenotypes can be restored in PKP2-null cells by dampening the RhoA pathway or silencing β1 integrin. However, integrin expression levels are not restored by RhoA signaling inhibition. These data uncover a potential role for PKP2 upstream of β1 integrin and RhoA in integrating cell–cell and cell–substrate contact signaling in basal keratinocytes necessary for the morphogenesis, homeostasis, and reepithelialization of the stratified epidermis. PMID:23884246

  12. Multivalent integrin-specific ligands enhance tissue healing and biomaterial integration.

    PubMed

    Petrie, Timothy A; Raynor, Jenny E; Dumbauld, David W; Lee, Ted T; Jagtap, Subodh; Templeman, Kellie L; Collard, David M; García, Andrés J

    2010-08-18

    Engineered biointerfaces covered with biomimetic motifs, including short bioadhesive ligands, are a promising material-based strategy for tissue repair in regenerative medicine. Potentially useful coating molecules are ligands for the integrins, major extracellular matrix receptors that require both ligand binding and nanoscale clustering for maximal signaling efficiency. We prepared coatings consisting of well-defined multimer constructs with a precise number of recombinant fragments of fibronectin (monomer, dimer, tetramer, and pentamer) to assess how nanoscale ligand clustering affects integrin binding, stem cell responses, tissue healing, and biomaterial integration. Clinical-grade titanium was grafted with polymer brushes that presented monomers, dimers, trimers, or pentamers of the alpha(5)beta(1) integrin-specific fibronectin III (7 to 10) domain (FNIII(7-10)). Coatings consisting of trimers and pentamers enhanced integrin-mediated adhesion in vitro, osteogenic signaling, and differentiation in human mesenchymal stem cells more than did surfaces presenting monomers and dimers. Furthermore, ligand clustering promoted bone formation and functional integration of the implant into bone in rat tibiae. This study establishes that a material-based strategy in which implants are coated with clustered bioadhesive ligands can promote robust implant-tissue integration. PMID:20720217

  13. Reprogramming cardiomyocyte mechanosensing by crosstalk between integrins and hyaluronic acid receptors.

    PubMed

    Chopra, Anant; Lin, Victor; McCollough, Amanda; Atzet, Sarah; Prestwich, Glenn D; Wechsler, Andrew S; Murray, Maria E; Oake, Shaina A; Kresh, J Yasha; Janmey, Paul A

    2012-03-15

    The elastic modulus of bioengineered materials has a strong influence on the phenotype of many cells including cardiomyocytes. On polyacrylamide (PAA) gels that are laminated with ligands for integrins, cardiac myocytes develop well organized sarcomeres only when cultured on substrates with elastic moduli in the range 10 kPa-30 kPa, near those of the healthy tissue. On stiffer substrates (>60 kPa) approximating the damaged heart, myocytes form stress fiber-like filament bundles but lack organized sarcomeres or an elongated shape. On soft (<1 kPa) PAA gels myocytes exhibit disorganized actin networks and sarcomeres. However, when the polyacrylamide matrix is replaced by hyaluronic acid (HA) as the gel network to which integrin ligands are attached, robust development of functional neonatal rat ventricular myocytes occurs on gels with elastic moduli of 200 Pa, a stiffness far below that of the neonatal heart and on which myocytes would be amorphous and dysfunctional when cultured on polyacrylamide-based gels. The HA matrix by itself is not adhesive for myocytes, and the myocyte phenotype depends on the type of integrin ligand that is incorporated within the HA gel, with fibronectin, gelatin, or fibrinogen being more effective than collagen I. These results show that HA alters the integrin-dependent stiffness response of cells in vitro and suggests that expression of HA within the extracellular matrix (ECM) in vivo might similarly alter the response of cells that bind the ECM through integrins. The integration of HA with integrin-specific ECM signaling proteins provides a rationale for engineering a new class of soft hybrid hydrogels that can be used in therapeutic strategies to reverse the remodeling of the injured myocardium.

  14. Reprogramming cardiomyocyte mechanosensing by crosstalk between integrins and hyaluronic acid receptors

    PubMed Central

    Chopra, Anant; Lin, Victor; McCollough, Amanda; Atzet, Sarah; Prestwich, Glenn D.; Wechsler, Andrew S.; Murray, Maria E.; Oake, Shaina A.; Kresh, J. Yasha; Janmey, Paul A.

    2012-01-01

    The elastic modulus of bioengineered materials has a strong influence on the phenotype of many cells including cardiomyocytes. On polyacrylamide (PAA) gels that are laminated with ligands for integrins, cardiac myocytes develop well organized sarcomeres only when cultured on substrates with elastic moduli in the range of 10 kPa to 30 kPa, near those of the healthy tissue. On stiffer substrates (>60 kPa) approximating the damaged heart, myocytes form stress fiber-like filament bundles but lack organized sarcomeres or an elongated shape. On soft (<1 kPa) PAA gels myocytes exhibit disorganized actin networks and sarcomeres. However, when the polyacrylamide matrix is replaced by hyaluronic acid (HA) as the gel network to which integrin ligands are attached, robust development of functional neonatal rat ventricular myocytes occurs on gels with elastic moduli of 200 Pa, a stiffness far below that of the neonatal heart and on which myocytes would be amorphous and dysfunctional when cultured on polyacrylamide-based gels. The HA matrix by itself is not adhesive for myocytes, and the myocyte phenotype depends on the type of integrin ligand that is incorporated within the HA gel, with fibronectin, gelatin, or fibrinogen being more effective than collagen 1. These results show that HA alters the integrin-dependent stiffness response of cells in vitro and suggests that expression of HA within the extracellular matrix (ECM) in vivo might similarly alter the response of cells that bind the ECM through integrins. The integration of HA with integrin-specific ECM signaling proteins provides a rationale for engineering a new class of soft hybrid hydrogels that can be used in therapeutic strategies to reverse the remodeling of the injured myocardium. PMID:22196970

  15. Early recognition of lung cancer by integrin targeted imaging in K-ras mouse model.

    PubMed

    Ermolayev, Vladimir; Mohajerani, Pouyan; Ale, Angelique; Sarantopoulos, Athanasios; Aichler, Michaela; Kayser, Gian; Walch, Axel; Ntziachristos, Vasilis

    2015-09-01

    Non-small cell lung cancer is characterized by slow progression and high heterogeneity of tumors. Integrins play an important role in lung cancer development and metastasis and were suggested as a tumor marker; however their role in anticancer therapy remains controversial. In this work, we demonstrate the potential of integrin-targeted imaging to recognize early lesions in transgenic mouse model of lung cancer based on spontaneous introduction of mutated human gene bearing K-ras mutation. We conducted ex vivo and fluorescence molecular tomography-X-ray computed tomography (FMT-XCT) in vivo imaging and analysis for specific targeting of early lung lesions and tumors in rodent preclinical model for lung cancer. The lesions and tumors were characterized by histology, immunofluorescence and immunohistochemistry using a panel of cancer markers. Ex vivo, the integrin-targeted fluorescent signal significantly differed between wild type lung tissue and K-ras pulmonary lesions (PL) at all ages studied. The panel of immunofluorescence experiments demonstrated that PL, which only partially show cancer cell features were detected by αvβ3-integrin targeted imaging. Human patient material analysis confirmed the specificity of target localization in different lung cancer types. Most importantly, small tumors in the lungs of 4-week-old animals could be noninvasively detected in vivo on the fluorescence channel of FMT-XCT. Our findings demonstrated αvβ3-integrin targeted fluorescent imaging to specifically detect premalignant pleural lesions in K-ras mice. Integrin targeted imaging may find application areas in preclinical research and clinical practice, such as early lung cancer diagnostics, intraoperative assistance or therapy monitoring.

  16. Reprogramming cardiomyocyte mechanosensing by crosstalk between integrins and hyaluronic acid receptors.

    PubMed

    Chopra, Anant; Lin, Victor; McCollough, Amanda; Atzet, Sarah; Prestwich, Glenn D; Wechsler, Andrew S; Murray, Maria E; Oake, Shaina A; Kresh, J Yasha; Janmey, Paul A

    2012-03-15

    The elastic modulus of bioengineered materials has a strong influence on the phenotype of many cells including cardiomyocytes. On polyacrylamide (PAA) gels that are laminated with ligands for integrins, cardiac myocytes develop well organized sarcomeres only when cultured on substrates with elastic moduli in the range 10 kPa-30 kPa, near those of the healthy tissue. On stiffer substrates (>60 kPa) approximating the damaged heart, myocytes form stress fiber-like filament bundles but lack organized sarcomeres or an elongated shape. On soft (<1 kPa) PAA gels myocytes exhibit disorganized actin networks and sarcomeres. However, when the polyacrylamide matrix is replaced by hyaluronic acid (HA) as the gel network to which integrin ligands are attached, robust development of functional neonatal rat ventricular myocytes occurs on gels with elastic moduli of 200 Pa, a stiffness far below that of the neonatal heart and on which myocytes would be amorphous and dysfunctional when cultured on polyacrylamide-based gels. The HA matrix by itself is not adhesive for myocytes, and the myocyte phenotype depends on the type of integrin ligand that is incorporated within the HA gel, with fibronectin, gelatin, or fibrinogen being more effective than collagen I. These results show that HA alters the integrin-dependent stiffness response of cells in vitro and suggests that expression of HA within the extracellular matrix (ECM) in vivo might similarly alter the response of cells that bind the ECM through integrins. The integration of HA with integrin-specific ECM signaling proteins provides a rationale for engineering a new class of soft hybrid hydrogels that can be used in therapeutic strategies to reverse the remodeling of the injured myocardium. PMID:22196970

  17. Integrin Involvement in Freeze Resistance of Androgen-Insensitive Prostate Cancer

    PubMed Central

    Baust, John G.; Klossner, Daniel P.; VanBuskirk, Robert G.; Gage, Andrew A.; Mouraviev, Vladimir; Polascik, Thomas J.; Baust, John M.

    2010-01-01

    Cryoablation has emerged as a primary therapy to treat prostate cancer. While effective, the assumption that freezing serves as a ubiquitous lethal stress is challenged by clinical experience and experimental evidence demonstrating time-temperature related cell death dependence. The age-related transformation from an androgen-sensitive (AS) to an androgen-insensitive (AI) phenotype is a major challenge in the management of prostate cancer. AI cells exhibit morphological changes and treatment resistance to many therapies. Since this resistance has been linked with α6β4 integrin overexpression as a result of androgen receptor (AR) loss, we investigated whether α6β4 integrin expression, as a result AR loss, contributes to the reported increased freeze tolerance of AI prostate cancer. A series of studies using AS (LNCaP LP and PC-3 AR) and AI (LNCaP HP and PC-3) cell lines were designed to investigate the cellular mechanisms contributing to variations in freezing response. Investigation into α6β4 integrin expression revealed that AI cell lines overexpressed this protein, thereby altering morphological characteristics and increasing adhesion characteristics. Molecular investigations revealed a significant decrease in caspase 8, 9, and 3 levels AI cells following freezing. Inhibition of α6β4 integrin resulted in increased caspase activity following freezing (similar to AS cells) and enhanced cell death. These data demonstrate that AI cells show an increase in post-freeze susceptibility following inhibition of α6β4 integrin function. Further understanding the role of androgen-receptor related α6β4 integrin expression in prostate cancer cells responses to freezing might lead to novel options for neo-adjunctive treatments targeting the AR signaling pathway. PMID:20066006

  18. β1 and β3 Integrins Cooperate to Induce Syndecan-4-Containing Cross-linked Actin Networks in Human Trabecular Meshwork Cells

    PubMed Central

    Filla, Mark S.; Woods, Anne; Kaufman, Paul L.; Peters, Donna M.

    2006-01-01

    Purpose To characterize the molecular composition of cross-linked actin networks (CLANs) and the regulation of their formation by integrins in normal human trabecular meshwork (TM) cells. CLANs have been observed in steroid-treated and glaucomatous TM cells and have been suggested to contribute to decreased outflow facility by altering the contractility of the TM. Methods Immunofluorescence microscopy was used to identify molecular components of CLANs and quantitate CLAN formation in HTM cells plated on coverslips coated with various extracellular matrix (ECM) proteins (fibronectin, types I and IV collagen, and vitronectin), vascular cell adhesion molecule (VCAM)-1, or activating antibodies against β1, β3, or α2β1 integrins. These integrin antibodies were also used as soluble ligands. Results CLAN vertices contained the actin-binding proteins α-actinin and filamin and the signaling molecules syndecan-4 and PIP2. CLANs lacked Arp3 and cortactin. CLAN formation was dependent on the ECM substrate and was significantly higher on fibronectin and VCAM-1 compared with vitronectin, types I or IV collagen. Adsorbed β1 integrin antibodies also induced CLANs, whereas adsorbed β3 or α2β1 integrin antibodies did not. Soluble β3 integrin antibodies, however, induced CLANs and actually enhanced CLAN formation in cells spread on fibronectin, VCAM-1, type I or type IV collagen, or β1 integrin antibodies. Conclusions CLANs are unique actin-branched networks whose formation can be regulated by β1 and β3 integrin signaling pathways. Thus, integrin-mediated signaling events can modulate the organization of the actin cytoskeleton in TM cells and hence could participate in regulating cytoskeletal events previously demonstrated to be involved in controlling outflow facility. PMID:16639003

  19. N-Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4

    PubMed Central

    Hang, Qinglei; Isaji, Tomoya; Hou, Sicong; Zhou, Ying; Fukuda, Tomohiko; Gu, Jianguo

    2016-01-01

    N-Glycosylation of integrin α5β1 is involved in multiple cell behaviors. We previously reported that the N-glycosylations of the calf domain on integrin α5 (S3–5,10–14) are essential for its inhibitory effect on EGFR signaling in regulating cell proliferation. However, the importance of the individual N-glycosylation and the underlying mechanisms of inhibition remain unclear. Here, we characterize the S3–5,10–14 mutants in detail and found that the N-glycosylation of site-11 (Asn712) is key for cell growth. The restoration of site-11, unlike the other individual sites, significantly suppressed cell growth and EGFR signaling in a manner that was similar to that of wild-type (WT). Mechanistically, this N-glycosylation inhibited the response abilities upon EGF stimulation and EGFR dimerization. Interestingly, we found this N-glycosylation controlled the EGFR complex formation with integrin α5β1 or α6β4; i.e., the loss of site-11 switched EGFR-α5β1 to EGFR-α6β4, which is well known to promote cellular signaling for cell growth. Moreover, the site-11 N-glycan exhibited a more branching structure compared with other sites, which may be required for EGFR-α5β1 formation. Taken together, these data clearly demonstrate that the site-11 N-glycosylation on α5 is most important for its inhibitory effect on EGFR signaling, which may provide a novel regulatory mechanism for crosstalks between integrins and EGFR. PMID:27641064

  20. Integrin activation and focal complex formation in cardiac hypertrophy

    NASA Technical Reports Server (NTRS)

    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.

  1. Regulation of adherence and virulence by the Entamoeba histolytica lectin cytoplasmic domain, which contains a beta2 integrin motif.

    PubMed

    Vines, R R; Ramakrishnan, G; Rogers, J B; Lockhart, L A; Mann, B J; Petri, W A

    1998-08-01

    Killing of human cells by the parasite Entamoeba histolytica requires adherence via an amebic cell surface lectin. Lectin activity in the parasite is regulated by inside-out signaling. The lectin cytoplasmic domain has sequence identity with a region of the beta2 integrin cytoplasmic tail implicated in regulation of integrin-mediated adhesion. Intracellular expression of a fusion protein containing the cytoplasmic domain of the lectin has a dominant negative effect on extracellular lectin-mediated cell adherence. Mutation of the integrin-like sequence abrogates the dominant negative effect. Amebae expressing the dominant negative mutant are less virulent in an animal model of amebiasis. These results suggest that inside-out signaling via the lectin cytoplasmic domain may control the extracellular adhesive activity of the amebic lectin and provide in vivo demonstration of the lectin's role in virulence.

  2. Integrin inhibitors as a therapeutic agent for ovarian cancer.

    PubMed

    Sawada, Kenjiro; Ohyagi-Hara, Chifumi; Kimura, Tadashi; Morishige, Ken-Ichirou

    2012-01-01

    Ovarian cancer is a deadly disease, with a cure rate of only 30%. Despite aggressive treatments, relapse remains almost inevitable in patients with advanced-stage disease. In recent years, great progress has been made towards targeting integrins in cancer treatment, and clinical studies with various integrin inhibitors have demonstrated their effectiveness in blocking cancer progression. Given that the initial critical step of ovarian cancer metastasis is the attachment of cancer cells onto the peritoneum or omentum, in addition to the proven positive clinical results of anti-angiogenic therapy, targeting integrins is likely to be one of the most feasible approaches. This paper summarizes the current understanding of the integrin biology in ovarian cancer metastasis and the various therapeutic approaches attempted with integrin inhibitors. Although no integrin inhibitors have shown favorable results so far, integrin-targeted therapies continue to be a promising approach to be explored for further clinical investigation.

  3. Integrin Inhibitors as a Therapeutic Agent for Ovarian Cancer

    PubMed Central

    Sawada, Kenjiro; Ohyagi-Hara, Chifumi; Kimura, Tadashi; Morishige, Ken-ichirou

    2012-01-01

    Ovarian cancer is a deadly disease, with a cure rate of only 30%. Despite aggressive treatments, relapse remains almost inevitable in patients with advanced-stage disease. In recent years, great progress has been made towards targeting integrins in cancer treatment, and clinical studies with various integrin inhibitors have demonstrated their effectiveness in blocking cancer progression. Given that the initial critical step of ovarian cancer metastasis is the attachment of cancer cells onto the peritoneum or omentum, in addition to the proven positive clinical results of anti-angiogenic therapy, targeting integrins is likely to be one of the most feasible approaches. This paper summarizes the current understanding of the integrin biology in ovarian cancer metastasis and the various therapeutic approaches attempted with integrin inhibitors. Although no integrin inhibitors have shown favorable results so far, integrin-targeted therapies continue to be a promising approach to be explored for further clinical investigation. PMID:22235205

  4. Rigidity sensing and adaptation through regulation of integrin types

    PubMed Central

    Elosegui-Artola, Alberto; Bazellières, Elsa; Allen, Michael D.; Andreu, Ion; Oria, Roger; Sunyer, Raimon; Gomm, Jennifer J.; Marshall, John F.; Jones, J. Louise; Trepat, Xavier; Roca-Cusachs, Pere

    2014-01-01

    Tissue rigidity regulates processes in development, cancer and wound healing. However, how cells detect rigidity, and thereby modulate their behaviour, remains unknown. Here, we show that sensing and adaptation to matrix rigidity in breast myoepithelial cells is determined by the bond dynamics of different integrin types. Cell binding to fibronectin through either α5β1 integrins (constitutively expressed) or αvβ6 integrins (selectively expressed in cancer and development) adapts force generation, actin flow, and integrin recruitment to rigidities associated with healthy or malignant tissue, respectively. In vitro experiments and theoretical modelling further demonstrate that this behaviour is explained by the different binding and unbinding rates of both integrin types to fibronectin. Moreover, rigidity sensing through differences in integrin bond dynamics applies both when integrins bind separately and when they compete for binding to fibronectin. PMID:24793358

  5. Rigidity sensing and adaptation through regulation of integrin types.

    PubMed

    Elosegui-Artola, Alberto; Bazellières, Elsa; Allen, Michael D; Andreu, Ion; Oria, Roger; Sunyer, Raimon; Gomm, Jennifer J; Marshall, John F; Jones, J Louise; Trepat, Xavier; Roca-Cusachs, Pere

    2014-06-01

    Tissue rigidity regulates processes in development, cancer and wound healing. However, how cells detect rigidity, and thereby modulate their behaviour, remains unknown. Here, we show that sensing and adaptation to matrix rigidity in breast myoepithelial cells is determined by the bond dynamics of different integrin types. Cell binding to fibronectin through either α5β1 integrins (constitutively expressed) or αvβ6 integrins (selectively expressed in cancer and development) adapts force generation, actin flow and integrin recruitment to rigidities associated with healthy or malignant tissue, respectively. In vitro experiments and theoretical modelling further demonstrate that this behaviour is explained by the different binding and unbinding rates of both integrin types to fibronectin. Moreover, rigidity sensing through differences in integrin bond dynamics applies both when integrins bind separately and when they compete for binding to fibronectin. PMID:24793358

  6. α6 integrin subunit regulates cerebellar development

    PubMed Central

    Marchetti, Giovanni; De Arcangelis, Adèle; Pfister, Véronique; Georges-Labouesse, Elisabeth

    2013-01-01

    Mutations in genes encoding several basal lamina components as well as their cellular receptors disrupt normal deposition and remodeling of the cortical basement membrane resulting in a disorganized cerebral and cerebellar cortex. The α6 integrin was the first α subunit associated with cortical lamination defects and formation of neural ectopias. In order to understand the precise role of α6 integrin in the central nervous system (CNS), we have generated mutant mice carrying specific deletion of α6 integrin in neuronal and glia precursors by crossing α6 conditional knockout mice with Nestin-Cre line. Cerebral cortex development occurred properly in the resulting α6fl/fl;nestin-Cre mutant animals. Interestingly, however, cerebellum displayed foliation pattern defects although granule cell (GC) proliferation and migration were not affected. Intriguingly, analysis of Bergmann glial (BG) scaffold revealed abnormalities in fibers morphology associated with reduced processes outgrowth and altered actin cytoskeleton. Overall, these data show that α6 integrin receptors are required in BG cells to provide a proper fissure formation during cerebellum morphogenesis. PMID:23722246

  7. Effects of β4 integrin expression on microRNA patterns in breast cancer.

    PubMed

    Gerson, Kristin D; Maddula, V S R Krishna; Seligmann, Bruce E; Shearstone, Jeffrey R; Khan, Ashraf; Mercurio, Arthur M

    2012-07-15

    The integrin α6β4 is defined as an adhesion receptor for laminins. Referred to as 'β4', this integrin plays a key role in the progression of various carcinomas through its ability to orchestrate key signal transduction events and promote cell motility. To identify novel downstream effectors of β4 function in breast cancer, microRNAs (miRNAs) were examined because of their extensive links to tumorigenesis and their ability to regulate gene expression globally. Two breast carcinoma cell lines and a collection of invasive breast carcinomas with varying β4 expression were used to assess the effect of this integrin on miRNA expression. A novel miRNA microarray analysis termed quantitative Nuclease Protection Assay (qNPA) revealed that β4 expression can significantly alter miRNA expression and identified two miRNA families, miR-25/32/92abc/363/363-3p/367 and miR-99ab/100, that are consistently downregulated by expression of this integrin. Analysis of published Affymetrix GeneChip data identified 54 common targets of miR-92ab and miR-99ab/100 within the subset of β4-regulated mRNAs, revealing several genes known to be key components of β4-regulated signaling cascades and effectors of cell motility. Gene ontology classification identified an enrichment in genes associated with cell migration within this population. Finally, gene set enrichment analysis of all β4-regulated mRNAs revealed an enrichment in targets belonging to distinct miRNA families, including miR-92ab and others identified by our initial array analyses. The results obtained in this study provide the first example of an integrin globally impacting miRNA expression and provide evidence that select miRNA families collectively target genes important in executing β4-mediated cell motility.

  8. β1 Integrin Maintains Integrity of the Embryonic Neocortical Stem Cell Niche

    PubMed Central

    Marthiens, Veronique; Relucio, Jenne; Mughal, Mohamed R.; Tang, Sung-Chun; Coksaygan, Turhan; Hall, Peter E.; Chigurupati, Srinivasulu; Patton, Bruce; Colognato, Holly; Rao, Mahendra S.; Mattson, Mark P.

    2009-01-01

    During embryogenesis, the neural stem cells (NSC) of the developing cerebral cortex are located in the ventricular zone (VZ) lining the cerebral ventricles. They exhibit apical and basal processes that contact the ventricular surface and the pial basement membrane, respectively. This unique architecture is important for VZ physical integrity and fate determination of NSC daughter cells. In addition, the shorter apical process is critical for interkinetic nuclear migration (INM), which enables VZ cell mitoses at the ventricular surface. Despite their importance, the mechanisms required for NSC adhesion to the ventricle are poorly understood. We have shown previously that one class of candidate adhesion molecules, laminins, are present in the ventricular region and that their integrin receptors are expressed by NSC. However, prior studies only demonstrate a role for their interaction in the attachment of the basal process to the overlying pial basement membrane. Here we use antibody-blocking and genetic experiments to reveal an additional and novel requirement for laminin/integrin interactions in apical process adhesion and NSC regulation. Transient abrogation of integrin binding and signalling using blocking antibodies to specifically target the ventricular region in utero results in abnormal INM and alterations in the orientation of NSC divisions. We found that these defects were also observed in laminin α2 deficient mice. More detailed analyses using a multidisciplinary approach to analyse stem cell behaviour by expression of fluorescent transgenes and multiphoton time-lapse imaging revealed that the transient embryonic disruption of laminin/integrin signalling at the VZ surface resulted in apical process detachment from the ventricular surface, dystrophic radial glia fibers, and substantial layering defects in the postnatal neocortex. Collectively, these data reveal novel roles for the laminin/integrin interaction in anchoring embryonic NSCs to the ventricular

  9. Binding of a fibrinogen mimetic stabilizes integrin αIIbβ3's open conformation

    PubMed Central

    Hantgan, Roy R.; Rocco, Mattia; Nagaswami, Chandrasekaran; Weisel, John W.

    2001-01-01

    The platelet integrin αIIbβ3 is representative of a class of heterodimeric receptors that upon activation bind extracellular macromolecular ligands and form signaling clusters. This study examined how occupancy of αIIbβ3's fibrinogen binding site affected the receptor's solution structure and stability. Eptifibatide, an integrin antagonist developed to treat cardiovascular disease, served as a high-affinity, monovalent model ligand with fibrinogen-like selectivity for αIIbβ3. Eptifibatide binding promptly and reversibly perturbed the conformation of the αIIbβ3 complex. Ligand-specific decreases in its diffusion and sedimentation coefficient were observed at near-stoichiometric eptifibatide concentrations, in contrast to the receptor-perturbing effects of RGD ligands that we previously observed only at a 70-fold molar excess. Eptifibatide promoted αIIbβ3 dimerization 10-fold more effectively than less selective RGD ligands, as determined by sedimentation equilibrium. Eptifibatide-bound integrin receptors displayed an ectodomain separation and enhanced assembly of dimers and larger oligomers linked through their stalk regions, as seen by transmission electron microscopy. Ligation with eptifibatide protected αIIbβ3 from SDS-induced subunit dissociation, an effect on electrophoretic mobility not seen with RGD ligands. Despite its distinct cleft, the open conformer resisted guanidine unfolding as effectively as the ligand-free integrin. Thus, we provide the first demonstration that binding a monovalent ligand to αIIbβ3's extracellular fibrinogen-recognition site stabilizes the receptor's open conformation and enhances self-association through its distant transmembrane and/or cytoplasmic domains. By showing how eptifibatide and RGD peptides, ligands with distinct binding sites, each affects αIIbβ3's conformation, our findings provide new mechanistic insights into ligand-linked integrin activation, clustering and signaling. PMID:11468358

  10. Myristoylated Alanine Rich C Kinase Substrate (MARCKS) is essential to β2-integrin dependent responses of equine neutrophils

    PubMed Central

    Sheats, Mary K.; Pescosolido, Kimberly C.; Hefner, Ethan M.; Sung, Eui Jae; Adler, Kenneth B.; Jones, Samuel L.

    2014-01-01

    Neutrophil infiltration is a prominent feature in a number of pathologic conditions affecting horses including recurrent airway obstruction, ischemia-reperfusion injury, and laminitis. Cell signaling components involved in neutrophil migration represent targets for novel anti-inflammatory therapies. In order to migrate into tissue, neutrophils must respond to chemoattractant signals in their external environment through activation of adhesion receptors (i.e. integrins) and reorganization of the actin cytoskeleton. Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS), a highly conserved actin-binding protein, has a well demonstrated role in cytoskeletal dependent cellular functions (i.e. adhesion, spreading, and migration), but the details of MARCKS involvement in these processes remain vague. We hypothesized that MARCKS serves as a link between the actin cytoskeleton and integrin function in neutrophils. Using a MARCKS-specific inhibitor peptide known as MANS on equine neutrophils in vitro, we demonstrate that inhibition of MARCKS function significantly attenuates β2-integrin-dependent neutrophil functions including migration, adhesion, and immune complex-mediated respiratory burst. The MANS peptide did not, however, inhibit the β2-integrin-independent PMA mediated respiratory burst. These results attest to the essential role of MARCKS function in regulating neutrophil responses, and strongly implicate MARCKS as a potential regulator of β2-integrins in neutrophils. PMID:24857637

  11. β4 Integrin and Epidermal Growth Factor Coordinately Regulate Electric Field-mediated Directional Migration via Rac1

    PubMed Central

    Pullar, Christine E.; Baier, Brian S.; Kariya, Yoshinobu; Russell, Alan J.; Horst, Basil A.J.; Marinkovich, M. Peter

    2006-01-01

    Endogenous DC electric fields (EF) are present during embryogenesis and are generated in vivo upon wounding, providing guidance cues for directional cell migration (galvanotaxis) required in these processes. To understand the role of beta (β)4 integrin in directional migration, the migratory paths of either primary human keratinocytes (NHK), β4 integrin-null human keratinocytes (β4−), or those in which β4 integrin was reexpressed (β4+), were tracked during exposure to EFs of physiological magnitude (100 mV/mm). Although the expression of β4 integrin had no effect on the rate of cell movement, it was essential for directional (cathodal) migration in the absence of epidermal growth factor (EGF). The addition of EGF potentiated the directional response, suggesting that at least two distinct but synergistic signaling pathways coordinate galvanotaxis. Expression of either a ligand binding–defective β4 (β4+AD) or β4 with a truncated cytoplasmic tail (β4+CT) resulted in loss of directionality in the absence of EGF, whereas inhibition of Rac1 blinded the cells to the EF even in the presence of EGF. In summary, both the β4 integrin ligand–binding and cytoplasmic domains together with EGF were required for the synergistic activation of a Rac-dependent signaling pathway that was essential for keratinocyte directional migration in response to a galvanotactic stimulus. PMID:16914518

  12. Myristoylated Alanine Rich C Kinase Substrate (MARCKS) is essential to β2-integrin dependent responses of equine neutrophils.

    PubMed

    Sheats, Mary K; Pescosolido, Kimberly C; Hefner, Ethan M; Sung, Eui Jae; Adler, Kenneth B; Jones, Samuel L

    2014-08-15

    Neutrophil infiltration is a prominent feature in a number of pathologic conditions affecting horses including recurrent airway obstruction, ischemia-reperfusion injury, and laminitis. Cell signaling components involved in neutrophil migration represent targets for novel anti-inflammatory therapies. In order to migrate into tissue, neutrophils must respond to chemoattractant signals in their external environment through activation of adhesion receptors (i.e. integrins) and reorganization of the actin cytoskeleton. Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS), a highly conserved actin-binding protein, has a well demonstrated role in cytoskeletal dependent cellular functions (i.e. adhesion, spreading, and migration), but the details of MARCKS involvement in these processes remain vague. We hypothesized that MARCKS serves as a link between the actin cytoskeleton and integrin function in neutrophils. Using a MARCKS-specific inhibitor peptide known as MANS on equine neutrophils in vitro, we demonstrate that inhibition of MARCKS function significantly attenuates β2-integrin-dependent neutrophil functions including migration, adhesion, and immune complex-mediated respiratory burst. The MANS peptide did not, however, inhibit the β2-integrin-independent PMA mediated respiratory burst. These results attest to the essential role of MARCKS function in regulating neutrophil responses, and strongly implicate MARCKS as a potential regulator of β2-integrins in neutrophils. PMID:24857637

  13. Combinatorial gene regulation by modulation of relative pulse timing

    PubMed Central

    Lin, Yihan; Sohn, Chang Ho; Dalal, Chiraj K.; Cai, Long; Elowitz, Michael B.

    2015-01-01

    Studies of individual living cells have revealed that many transcription factors activate in dynamic, and often stochastic, pulses within the same cell. However, it has remained unclear whether cells might modulate the relative timing of these pulses to control gene expression. Here, using quantitative single-cell time-lapse imaging of Saccharomyces cerevisiae, we show that the pulsatile transcription factors Msn2 and Mig1 combinatorially regulate their target genes through modulation of their relative pulse timing. The activator Msn2 and repressor Mig1 pulsed in either a temporally overlapping or non-overlapping manner during their transient response to different inputs, with only the non-overlapping dynamics efficiently activating target gene expression. Similarly, under constant environmental conditions, where Msn2 and Mig1 exhibit sporadic pulsing, glucose concentration modulated the temporal overlap between pulses of the two factors. Together, these results reveal a time-based mode of combinatorial gene regulation. Regulation through relative signal timing is common in engineering and neurobiology, and these results suggest that it could also function broadly within the signaling and regulatory systems of the cell. PMID:26466562

  14. Proinflammatory secreted phospholipase A2 type IIA (sPLA-IIA) induces integrin activation through direct binding to a newly identified binding site (site 2) in integrins αvβ3, α4β1, and α5β1.

    PubMed

    Fujita, Masaaki; Zhu, Kan; Fujita, Chitose K; Zhao, Min; Lam, Kit S; Kurth, Mark J; Takada, Yoko K; Takada, Yoshikazu

    2015-01-01

    Integrins are activated by signaling from inside the cell (inside-out signaling) through global conformational changes of integrins. We recently discovered that fractalkine activates integrins in the absence of CX3CR1 through the direct binding of fractalkine to a ligand-binding site in the integrin headpiece (site 2) that is distinct from the classical RGD-binding site (site 1). We propose that fractalkine binding to the newly identified site 2 induces activation of site 1 though conformational changes (in an allosteric mechanism). We reasoned that site 2-mediated activation of integrins is not limited to fractalkine. Human secreted phospholipase A2 type IIA (sPLA2-IIA), a proinflammatory protein, binds to integrins αvβ3 and α4β1 (site 1), and this interaction initiates a signaling pathway that leads to cell proliferation and inflammation. Human sPLA2-IIA does not bind to M-type receptor very well. Here we describe that sPLA2-IIA directly activated purified soluble integrin αvβ3 and transmembrane αvβ3 on the cell surface. This activation did not require catalytic activity or M-type receptor. Docking simulation predicted that sPLA2-IIA binds to site 2 in the closed-headpiece of αvβ3. A peptide from site 2 of integrin β1 specifically bound to sPLA2-IIA and suppressed sPLA2-IIA-induced integrin activation. This suggests that sPLA2-IIA activates αvβ3 through binding to site 2. sPLA2-IIA also activated integrins α4β1 and α5β1 in a site 2-mediated manner. We recently identified small compounds that bind to sPLA2-IIA and suppress integrin-sPLA2-IIA interaction (e.g. compound 21 (Cmpd21)). Cmpd21 effectively suppressed sPLA2-IIA-induced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation. PMID:25398877

  15. Nonlinear dynamics in combinatorial games: Renormalizing Chomp

    NASA Astrophysics Data System (ADS)

    Friedman, Eric J.; Landsberg, Adam Scott

    2007-06-01

    We develop a new approach to combinatorial games that reveals connections between such games and some of the central ideas of nonlinear dynamics: scaling behaviors, complex dynamics and chaos, universality, and aggregation processes. We take as our model system the combinatorial game Chomp, which is one of the simplest in a class of "unsolved" combinatorial games that includes Chess, Checkers, and Go. We discover that the game possesses an underlying geometric structure that "grows" (reminiscent of crystal growth), and show how this growth can be analyzed using a renormalization procedure adapted from physics. In effect, this methodology allows one to transform a combinatorial game like Chomp into a type of dynamical system. Not only does this provide powerful insights into the game of Chomp (yielding a complete probabilistic description of optimal play in Chomp and an answer to a longstanding question about the nature of the winning opening move), but more generally, it offers a mathematical framework for exploring this unexpected relationship between combinatorial games and modern dynamical systems theory.

  16. Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly.

    PubMed

    Horton, Edward R; Byron, Adam; Askari, Janet A; Ng, Daniel H J; Millon-Frémillon, Angélique; Robertson, Joseph; Koper, Ewa J; Paul, Nikki R; Warwood, Stacey; Knight, David; Humphries, Jonathan D; Humphries, Martin J

    2015-12-01

    Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this data set reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome is likely to represent a core cell adhesion machinery, centred around four axes comprising ILK-PINCH-kindlin, FAK-paxillin, talin-vinculin and α-actinin-zyxin-VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community. PMID:26479319

  17. Regulation of Glioblastoma Tumor-Propagating Cells by the Integrin Partner Tetraspanin CD15112

    PubMed Central

    Tilghman, Jessica; Schiapparelli, Paula; Lal, Bachuchu; Ying, Mingyao; Quinones-Hinojosa, Alfredo; Xia, Shuli; Laterra, John

    2016-01-01

    Glioblastoma (GBM) stem cells (GSCs) represent tumor-propagating cells with stem-like characteristics (stemness) that contribute disproportionately to GBM drug resistance and tumor recurrence. Understanding the mechanisms supporting GSC stemness is important for developing therapeutic strategies for targeting GSC-dependent oncogenic mechanisms. Using GBM-derived neurospheres, we identified the cell surface tetraspanin family member CD151 as a novel regulator of glioma cell stemness, GSC self-renewal capacity, migration, and tumor growth. CD151 was found to be overexpressed in GBM tumors and GBM neurospheres enriched in GSCs. Silencing CD151 inhibited neurosphere forming capacity, neurosphere cell proliferation, and migration and attenuated the expression of markers and transcriptional drivers of the GSC phenotype. Conversely, forced CD151 expression promoted neurosphere self-renewal, cell migration, and expression of stemness-associated transcription factors. CD151 was found to complex with integrins α3, α6, and β1 in neurosphere cells, and blocking CD151 interactions with integrins α3 and α6 inhibited AKT phosphorylation, a downstream effector of integrin signaling, and impaired sphere formation and neurosphere cell migration. Additionally, targeting CD151 in vivo inhibited the growth of GBM neurosphere-derived xenografts. These findings identify CD151 and its interactions with integrins α3 and α6 as potential therapeutic targets for inhibiting stemness-driving mechanisms and stem cell populations in GBM. PMID:26992919

  18. The Calcium-Sensing Receptor and Integrins in Cellular Differentiation and Migration

    PubMed Central

    Tharmalingam, Sujeenthar; Hampson, David R.

    2016-01-01

    The calcium-sensing receptor (CaSR) is a widely expressed homodimeric G-protein coupled receptor structurally related to the metabotropic glutamate receptors and GPRC6A. In addition to its well characterized role in maintaining calcium homeostasis and regulating parathyroid hormone release, evidence has accumulated linking the CaSR with cellular differentiation and migration, brain development, stem cell engraftment, wound healing, and tumor growth and metastasis. Elevated expression of the CaSR in aggressive metastatic tumors has been suggested as a potential novel prognostic marker for predicting metastasis, especially to bone tissue where extracellular calcium concentrations may be sufficiently high to activate the receptor. Recent evidence supports a model whereby CaSR-mediated activation of integrins promotes cellular migration. Integrins are single transmembrane spanning heterodimeric adhesion receptors that mediate cell migration by binding to extracellular matrix proteins. The CaSR has been shown to form signaling complexes with the integrins to facilitate both the movement and differentiation of cells, such as neurons during normal brain development and tumor cells under pathological circumstances. Thus, CaSR/integrin complexes may function as a universal cell migration or homing complex. Manipulation of this complex may be of potential interest for treating metastatic cancers, and for developmental disorders pertaining to aberrant neuronal migration. PMID:27303307

  19. Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly

    PubMed Central

    Askari, Janet A.; Ng, Daniel H. J.; Millon-Frémillon, Angélique; Robertson, Joseph; Koper, Ewa J.; Paul, Nikki R.; Warwood, Stacey; Knight, David; Humphries, Jonathan D.; Humphries, Martin J.

    2015-01-01

    Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is currently lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this dataset reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome likely represents a core cell adhesion machinery, centred around four axes comprising ILK-PINCH-kindlin, FAK-paxillin, talin-vinculin and α-actinin-zyxin-VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community. PMID:26479319

  20. Integrin-α9 is required for fibronectin matrix assembly during lymphatic valve morphogenesis

    PubMed Central

    Bazigou, Eleni; Xie, Sherry; Chen, Chun; Weston, Anne; Miura, Naoyuki; Sorokin, Lydia; Adams, Ralf; Muro, Andrés F.; Sheppard, Dean; Makinen, Taija

    2009-01-01

    Summary Dysfunction of lymphatic valves underlies human lymphedema, yet the process of valve morphogenesis is poorly understood. Here, we show that during embryogenesis lymphatic valve leaflet formation is initiated by upregulation of integrin-α9 expression and deposition of its ligand, fibronectin-EIIIA (FN-EIIIA), in the extracellular matrix. Endothelial cell specific deletion of Itga9 (encoding integrin-α9) in mouse embryos results in the development of rudimentary valve leaflets, characterized by disorganized FN matrix, short cusps and retrograde lymphatic flow. Similar morphological and functional defects are observed in mice lacking the EIIIA domain of FN. Mechanistically, we demonstrate that in primary human lymphatic endothelial cells the integrin-α9-EIIIA interaction directly regulates FN fibril assembly, which is essential for the formation of the extracellular matrix core of valve leaflets. Our findings reveal an important role for integrin-α9 signaling during lymphatic valve morphogenesis and implicate it as a candidate gene for primary lymphedema caused by valve defects. PMID:19686679

  1. Interleukin-8 promotes cell migration through integrin αvβ6 upregulation in colorectal cancer.

    PubMed

    Sun, Qi; Sun, Fengkai; Wang, Ben; Liu, Song; Niu, Weibo; Liu, Enyu; Peng, Cheng; Wang, Jiayong; Gao, Huijie; Liang, Benjia; Niu, Zhengchuan; Zou, Xueqing; Niu, Jun

    2014-11-28

    Colorectal cancer (CRC), which is notorious for high morbidity and mortality around the world, shows a predilection for metastasis to liver. Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, has been reported to promote CRC cell migration and is associated with poor prognosis of CRC. However, the underlying molecular mechanism of IL-8-mediated migration remains obscure. In this study, we first demonstrated the cross talk between IL-8 and integrin αvβ6. We analyzed 139 human CRC samples, and found that the immunohistochemical expression of αvβ6 was significantly correlated with expression of IL-8. Furthermore, IL-8 increased the migration through integrin αvβ6 in human CRC cells, and both CXCR1 and CXCR2 were primarily involved during the process. IL-8 upregulated αvβ6 expression in a dose-dependent manner through activation of ERK and Ets-1 signaling pathway. Taken together, our results indicated that IL-8 enhances the migration of CRC cells by increasing αvβ6 integrin expression through the ERK/Ets-1 pathway. Targeting integrin αvβ6 in IL-8 expressing tumors might be a potential therapeutic strategy for CRC patients.

  2. Bit-1 mediates integrin-dependent cell survival through activation of the NFkappaB pathway.

    PubMed

    Griffiths, Genevieve S; Grundl, Melanie; Leychenko, Anna; Reiter, Silke; Young-Robbins, Shirley S; Sulzmaier, Florian J; Caliva, Maisel J; Ramos, Joe W; Matter, Michelle L

    2011-04-22

    Loss of properly regulated cell death and cell survival pathways can contribute to the development of cancer and cancer metastasis. Cell survival signals are modulated by many different receptors, including integrins. Bit-1 is an effector of anoikis (cell death due to loss of attachment) in suspended cells. The anoikis function of Bit-1 can be counteracted by integrin-mediated cell attachment. Here, we explored integrin regulation of Bit-1 in adherent cells. We show that knockdown of endogenous Bit-1 in adherent cells decreased cell survival and re-expression of Bit-1 abrogated this effect. Furthermore, reduction of Bit-1 promoted both staurosporine and serum-deprivation induced apoptosis. Indeed knockdown of Bit-1 in these cells led to increased apoptosis as determined by caspase-3 activation and positive TUNEL staining. Bit-1 expression protected cells from apoptosis by increasing phospho-IκB levels and subsequently bcl-2 gene transcription. Protection from apoptosis under serum-free conditions correlated with bcl-2 transcription and Bcl-2 protein expression. Finally, Bit-1-mediated regulation of bcl-2 was dependent on focal adhesion kinase, PI3K, and AKT. Thus, we have elucidated an integrin-controlled pathway in which Bit-1 is, in part, responsible for the survival effects of cell-ECM interactions.

  3. Interplay of Endosomal pH and Ligand Occupancy in Integrin α5β1 Ubiquitination, Endocytic Sorting, and Cell Migration.

    PubMed

    Kharitidi, Dmitri; Apaja, Pirjo M; Manteghi, Sanaz; Suzuki, Kei; Malitskaya, Elena; Roldan, Ariel; Gingras, Marie-Claude; Takagi, Junichi; Lukacs, Gergely L; Pause, Arnim

    2015-10-20

    Membrane trafficking of integrins plays a pivotal role in cell proliferation and migration. How endocytosed integrins are targeted either for recycling or lysosomal delivery is not fully understood. Here, we show that fibronectin (FN) binding to α5β1 integrin triggers ubiquitination and internalization of the receptor complex. Acidification facilitates FN dissociation from integrin α5β1 in vitro and in early endosomes, promoting receptor complex deubiquitination by the USP9x and recycling to the cell surface. Depending on residual ligand occupancy of receptors, some α5β1 integrins remain ubiquitinated and are captured by ESCRT-0/I, containing histidine domain-containing protein tyrosine phosphatase (HD-PTP) and ubiquitin-associated protein 1 (UBAP1), and are directed for lysosomal proteolysis, limiting receptor downstream signaling and cell migration. Thus, HD-PTP or UBAP1 depletion confers a pro-invasive phenotype. Thus, pH-dependent FN-integrin dissociation and deubiquitination of the activated integrin α5β1 are required for receptor resensitization and cell migration, representing potential targets to modulate tumor invasiveness. PMID:26456826

  4. The role of well-defined patterned substrata on the regeneration of DRG neuron pathfinding and integrin expression dynamics using chondroitin sulfate proteoglycans

    PubMed Central

    Hodgkinson, Gerald N.; Tresco, Patrick A.; Hlady, Vladimir

    2012-01-01

    Injured neurons intrinsically adapt to and partially overcome inhibitory proteoglycan expression in the central nervous system by upregulating integrin expression. It remains unclear however, to what extent varying proteoglycan concentrations influence the strength of this response, how rapidly neurons adapt to proteoglycans, and how pathfinding dynamics are altered over time as integrin expression is modulated in response to proteglycan signals. To investigate these quandaries, we created well-defined substrata in which postnatal DRG neuron pathfinding dynamics and growth cone integrin expression were interrogated as a function of proteoglycan substrata density. DRGs responded by upregulating integrin expression in a proteoglycan dose dependent fashion and exhibited robust outgrowth over all proteoglycan densities at initial time frames. However, after prolonged proteoglycan exposure, neurons exhibited decreasing velocities associated with increasing proteoglycan densities, while neurons growing on low proteoglycan levels exhibited robust outgrowth at all time points. Additionally, DRG outgrowth over proteoglycan density step boundaries, and a brief β1 integrin functional block proved that regeneration was integrin dependent and that DRGs exhibit delayed slowing and loss in persistence after even transient encounters with dense proteoglycan boundaries. These findings demonstrate the complexity of proteoglycan regulation on integrin expression and regenerative pathfinding. PMID:22436802

  5. αvβ8 integrin interacts with RhoGDI1 to regulate Rac1 and Cdc42 activation and drive glioblastoma cell invasion

    PubMed Central

    Reyes, Steve B.; Narayanan, Anjana S.; Lee, Hye Shin; Tchaicha, Jeremy H.; Aldape, Kenneth D.; Lang, Frederick F.; Tolias, Kimberly F.; McCarty, Joseph H.

    2013-01-01

    The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are regulated by extracellular cues within the neural microenvironment. The adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we use human GBM cell lines, primary patient samples, and preclinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin coimmunoprecipitates with Rho-GDP dissociation inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin–RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, regulates activation of Rho proteins to promote GBM cell invasiveness. Hence targeting the αvβ8 integrin–RhoGDI1 signaling axis might be an effective strategy for blocking GBM cell invasion. PMID:23283986

  6. Identification of cancer specific ligands from one-bead one compound combinatorial libraries to develop theranostics agents against oral squamous cell carcinoma

    NASA Astrophysics Data System (ADS)

    Yang, Frances Fan

    Background: Oral squamous cell carcinoma (OSCC) is one of the most prevalent disease worldwide. One-bead one-compound (OBOC) combinatorial technology is a powerful method to identify peptidomimetic ligands against a variety of receptors on cell surfaces. We therefore hypothesized that cancer specific ligands against OSCC might be identified and can be conjugated to optical dyes or nanocarriers to develop theranostic agents against OSCC. Material and methods: Different OSCC cell lines were incubated with OBOC libraries and beads with cell binding were sorted and then screened with normal human cells to identify peptide-beads binding to different OSCC cell lines but not binding to normal human cells. The molecular probes of OSCC were developed by biotinylating the carboxyl end of the ligands. OSCC theranostic agents were developed by decorating LLY13 with NPs and evaluated by using orthotopic bioluminescent oral cancer model. Results: Six OSCC specific ligands were discovered. Initial peptide-histochemistry study indicated that LLY12 and LLY13 were able to specifically detect OSCC cells grown on chamber slides at the concentration of 1 muM. In addition, LLY13 was found to penetrate into the OSCC cells and accumulate in the cytoplasm, and nucleus. After screened with a panel of integrin antibodies, only anti-alpha3 antibody was able to block most of OSCC cells binding to the LLY13 beads. OSCC theranostic agents developed using targeting LLY13 micelles (25+/- 4nm in diameter) were more efficient in binding to HSC-3 cancer cells compared to non-targeting micelles. Ex vivo images demonstrated that xenografts from the mice with targeting micelles appeared to have higher signals than the non-targeting groups. Conclusion: LLY13 has promising in vitro and in vivo targeting activity against OSCC. In addition, LLY13 is also able to penetrate into cancer cells via endocytosis. Initial study indicated that alpha3 integrin might partially be the corresponding receptor involved

  7. Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2.

    PubMed

    Rose, David M; Liu, Shouchun; Woodside, Darren G; Han, Jaewon; Schlaepfer, David D; Ginsberg, Mark H

    2003-06-15

    Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1). PMID:12794117

  8. α6 Integrin Transactivates Insulin-like Growth Factor Receptor-1 (IGF-1R) to Regulate Caspase-3-mediated Lens Epithelial Cell Differentiation Initiation*

    PubMed Central

    Basu, Subhasree; Rajakaruna, Suren; De Arcangelis, Adèle; Zhang, Liping; Georges-Labouesse, Elisabeth; Menko, A. Sue

    2014-01-01

    The canonical mitochondrial death pathway was first discovered for its role in signaling apoptosis. It has since been found to have a requisite function in differentiation initiation in many cell types including the lens through low level activation of the caspase-3 protease. The ability of this pathway to function as a molecular switch in lens differentiation depends on the concurrent induction of survival molecules in the Bcl-2 and IAP families, induced downstream of an IGF-1R/NFκB coordinate survival signal, to regulate caspase-3 activity. Here we investigated whether α6 integrin signals upstream to this IGF-1R-mediated survival-linked differentiation signal. Our findings show that IGF-1R is recruited to and activated specifically in α6 integrin receptor signaling complexes in the lens equatorial region, where lens epithelial cells initiate their differentiation program. In studies with both α6 integrin knock-out mice lenses and primary lens cell cultures following α6 integrin siRNA knockdown, we show that IGF-1R activation is dependent on α6 integrin and that this transactivation requires Src kinase activity. In addition, without α6 integrin, activation and expression of NFκB was diminished, and expression of Bcl-2 and IAP family members were down-regulated, resulting in high levels of caspase-3 activation. As a result, a number of hallmarks of lens differentiation failed to be induced; including nuclear translocation of Prox1 in the differentiation initiation zone and apoptosis was promoted. We conclude that α6 integrin is an essential upstream regulator of the IGF-1R survival pathway that regulates the activity level of caspase-3 for it to signal differentiation initiation of lens epithelial cells. PMID:24381169

  9. Combinatorial Biosynthesis of Polyketides – A Perspective

    PubMed Central

    Wong, Fong T.; Khosla, Chaitan

    2012-01-01

    Since their discovery, polyketide synthases have been attractive targets of biosynthetic engineering to make “unnatural” natural products. Although combinatorial biosynthesis has made encouraging advances over the past two decades, the field remains in its infancy. In this enzyme-centric perspective, we discuss the scientific and technological challenges that could accelerate the adoption of combinatorial biosynthesis as a method of choice for the preparation of encoded libraries of bioactive small molecules. Borrowing a page from the protein structure prediction community, we propose a periodic challenge program to vet the most promising methods in the field, and to foster the collective development of useful tools and algorithms. PMID:22342766

  10. Combinatorial biosynthesis of polyketides--a perspective.

    PubMed

    Wong, Fong T; Khosla, Chaitan

    2012-04-01

    Since their discovery, polyketide synthases have been attractive targets of biosynthetic engineering to make 'unnatural' natural products. Although combinatorial biosynthesis has made encouraging advances over the past two decades, the field remains in its infancy. In this enzyme-centric perspective, we discuss the scientific and technological challenges that could accelerate the adoption of combinatorial biosynthesis as a method of choice for the preparation of encoded libraries of bioactive small molecules. Borrowing a page from the protein structure prediction community, we propose a periodic challenge program to vet the most promising methods in the field, and to foster the collective development of useful tools and algorithms.

  11. beta 1 integrin inhibition dramatically enhances radiotherapy efficacy in human breast cancer xenografts

    SciTech Connect

    Park, Catherine C.; Park, Catherine C.; Zhang, Hui J.; Yao, Evelyn S.; Park, Chong J.; Bissell, Mina J.

    2008-06-02

    {beta}1 integrin signaling has been shown to mediate cellular resistance to apoptosis after exposure to ionizing radiation (IR). Other signaling molecules that increase resistance include Akt, which promotes cell survival downstream of {beta}1 integrin signaling. We showed previously that {beta}1 integrin inhibitory antibodies, AIIB2, enhance apoptosis and decrease growth in human breast cancer cells in 3 dimensional laminin-rich extracellular matrix (3D lrECM) cultures and in vivo. Here we asked whether AIIB2 could synergize with IR to modify Akt-mediated IR resistance. We used 3D lrECM cultures to test the optimal combination of AIIB2 with IR treatment of two breast cancer cell lines, MCF-7 and HMT3522-T4-2, as well as T4-2 myr-Akt breast cancer colonies or HMT3522-S-1, which form normal organotypic structures in 3D lrECM. Colonies were assayed for apoptosis and {beta}1 integrin/Akt signaling pathways were evaluated using western blot. In addition, mice bearing MCF-7 xenografts were used to validate the findings in 3D lrECM. We report that AIIB2 increased apoptosis optimally post-IR by down regulating Akt in breast cancer colonies in 3D lrECM. In vivo, addition of AIIB2 after IR significantly enhanced tumor growth inhibition and apoptosis compared to either treatment alone. Remarkably, the degree of tumor growth inhibition using AIIB2 plus 2 Gy radiation was similar to that of 8 Gy alone. We showed previously that AIIB2 had no discernible toxicity in mice; here, its addition allowed for a significant reduction in the IR dose that was necessary to achieve comparable growth inhibition and apoptosis in breast cancer xenografts in vivo.

  12. Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis

    PubMed Central

    Garciadiego-Cázares, David; Aguirre-Sánchez, Hilda I.; Abarca-Buis, René F.; Kouri, Juan B.; Velasquillo, Cristina; Ibarra, Clemente

    2015-01-01

    The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy

  13. A New Approach for Proving or Generating Combinatorial Identities

    ERIC Educational Resources Information Center

    Gonzalez, Luis

    2010-01-01

    A new method for proving, in an immediate way, many combinatorial identities is presented. The method is based on a simple recursive combinatorial formula involving n + 1 arbitrary real parameters. Moreover, this formula enables one not only to prove, but also generate many different combinatorial identities (not being required to know them "a…

  14. Hypergraph-Based Combinatorial Optimization of Matrix-Vector Multiplication

    ERIC Educational Resources Information Center

    Wolf, Michael Maclean

    2009-01-01

    Combinatorial scientific computing plays an important enabling role in computational science, particularly in high performance scientific computing. In this thesis, we will describe our work on optimizing matrix-vector multiplication using combinatorial techniques. Our research has focused on two different problems in combinatorial scientific…

  15. Effect of surface chemistry on the integrin induced pathway in regulating vascular endothelial cells migration.

    PubMed

    Shen, Yang; Gao, Min; Ma, Yunlong; Yu, Hongchi; Cui, Fu-zhai; Gregersen, Hans; Yu, Qingsong; Wang, Guixue; Liu, Xiaoheng

    2015-02-01

    The migration of vascular endothelial cells (ECs) is essential for reendothelialization after implantation of cardiovascular biomaterials. Reendothelialization is largely determined by surface properties of implants. In this study, surfaces modified with various chemical functional groups (CH3, NH2, COOH, OH) prepared by self-assembled monolayers (SAMs) were used as model system. Expressions and distributions of critical proteins in the integrin-induced signaling pathway were examined to explore the mechanisms of surface chemistry regulating EC migration. The results showed that SAMs modulated cell migration were in the order CH3>NH2>OH>COOH, determined by differences in the expressions of focal adhesion components and Rho GTPases. Multiple integrin subunits showed difference in a surface chemistry-dependent manner, which induced a stepwise activation of signaling cascades associated with EC migration. This work provides a broad overview of surface chemistry regulated endothelial cell migration and establishes association among the surface chemistry, cell migration behavior and associated integrin signaling events. Understanding the relationship between these factors will help us to understand the surface/interface behavior between biomaterials and cells, reveal molecular mechanism of cells sensing surface characterization, and guide surface modification of cardiovascular implanted materials. PMID:25575348

  16. PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

    PubMed Central

    González Wusener, Ana E.; González, Ángela; Nakamura, Fumihiko; Arregui, Carlos O.

    2016-01-01

    ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration. PMID:26700725

  17. Targeting the expression of integrin receptors in tumors

    NASA Astrophysics Data System (ADS)

    Bloch, Sharon; Liang, Kexian; Dorshow, Richard B.; Ye, Yunpeng; Achilefu, Samuel I.

    2004-06-01

    Expression of integrin αvβ3 is upregulated in a number of cancers including colon, pancreas, lung and breast. Additionally, αvβ3 integrin expression has been linked to tumor metastasis and targeting this cell surface protein could provide a viable approach to image and evaluate the metastatic potential of tumors. Accordingly, we evaluated the selective retention of some near infrared (NIR) fluorescent probes in nude mice bearing A549 lung cancer xenograft that express αvβ3 integrin. Our preliminary results indicate that a novel NIR probe designed to target this integrin selectively accumulated in A549 tumor while other non-integrin specific probes were not retained in the tumor. Blocking studies show that tumor uptake of the probe is mediated by αvβ3 integrin receptor.

  18. Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels

    PubMed Central

    1993-01-01

    Electrical signals elicited by integrin interaction with ECM components and their role in neurite outgrowth were studied in two clones (N1 and N7) isolated from 41A3 murine neuroblastoma cell line. Although the two clones similarly adhered to fibronectin (FN) and vitronectin (VN), this adhesion induced neurite outgrowth in N1 but not in N7 cells. Patch clamp recordings in whole cell configuration showed that, upon adhesion to FN or VN but not to platelet factor 4 (PF4), N1 cells undergo a marked (approximately equal to 20 mV) hyperpolarization of the resting potential (Vrest) that occurred within the first 20 min after cell contact with ECM, and persisted for approximately 1 h before reverting to the time zero values. This hyperpolarization was totally absent in N7 cells. A detailed analysis of the molecular mechanisms involved in N1 and N7 cell adhesion to ECM substrata was performed by using antibodies raised against the FN receptor and synthetic peptides variously competing with the FN or VN binding to integrin receptor (GRGDSP and GRGESP). Antibodies, as well as GRGDSP, abolished adhesion of N1 and N7 clones to FN and VN, revealing a similar implication of integrins in the adhesion of these clones to the ECM proteins. However, these anti-adhesive treatments, while ineffective on Vrest of N7 cells, abolished in N1 cells the FN- or VN-induced hyperpolarization and neurite outgrowth, that appeared therefore strictly associated and integrin-mediated phenomena. The nature of this association was deepened through a comparative analysis of the integrin profiles and the ion channels of N1 and N7 cells. The integrin immunoprecipitation profile resulted very similarly in the two clones, with only minor differences concerning the alpha V containing complexes. Both clones possessed Ca2+ and K+ delayed rectifier (KDR) channels, while only N1 cells were endowed with inward rectifier K+ (KIR) channels. The latter governed the Vrest, and, unlike KDR channels, were blocked by

  19. EGFRvIII/integrin β3 interaction in hypoxic and vitronectinenriching microenvironment promote GBM progression and metastasis

    PubMed Central

    Li, Yongsheng; Zhao, Manli; Xie, Hui; Ju, Huanyu; Wang, He; Zhao, Yu; Zheng, Qifan; Wang, Qixue; Su, Jun; Fang, Chuan; Fu, Songbin; Jiang, Tao; Liu, Jiaren; Li, Xia; Kang, Chunsheng; Ren, Huan

    2016-01-01

    Glioblastoma (GBM) is one of the most lethal brain tumors with a short survival time. EGFR amplification and mutation is the most significant genetic signature in GBM. About half of the GBMs with EGFR amplification express a constitutively autophosphorylated variant of EGFR, known as EGFRvIII. Our in vitro data demonstrated further enhanced EGFRvIII activity and tumor cell invasion in the tumor microenvironment of hypoxia plus extracellular matrix (ECM) vitronectin, in which EGFRvIII and integrin β3 tended to form complexes. The treatment with ITGB3 siRNA or the integrin antagonist cilengetide preferentially interrupted the EGFRvIII/integrin β3 complex, effectively reduced tumor cell invasion and activation of downstream signaling effectors. Cilengitide is recently failed in Phase III CENTRIC trial in unselected patients with GBM. However, we found that cilengitide demonstrated efficacious tumor regression via inhibition of tumor growth and angiogenesis in EGFRvIII orthotopic xenografts. Bioinformatics analysis emphasized key roles of integrin β3, hypoxia and vitronectin and their strong correlations with EGFRvIII expression in malignant glioma patient samples in vivo. In conclusion, we demonstrate that EGFRvIII/integrin β3 complexes promote GBM progression and metastasis in the environment of hypoxia and vitronectin-enrichment, and cilengitide may serve as a promising therapeutics for EGFRvIII-positive GBMs. PMID:26717039

  20. "Anticlumping" and Other Combinatorial Effects on Clumped Isotopes: Implications for Tracing Biogeochemical Cycling

    NASA Astrophysics Data System (ADS)

    Yeung, L.

    2015-12-01

    I present a mode of isotopic ordering that has purely combinatorial origins. It can be important when identical rare isotopes are paired by coincidence (e.g., they are neighbors on the same molecule), or when extrinsic factors govern the isotopic composition of the two atoms that share a chemical bond. By itself, combinatorial isotope pairing yields products with isotopes either randomly distributed or with a deficit relative to a random distribution of isotopes. These systematics arise because of an unconventional coupling between the formation of singly- and multiply-substituted isotopic moieties. In a random distribution, rare isotopes are symmetrically distributed: Single isotopic substitutions (e.g., H‒D and D‒H in H2) occur with equal probability, and double isotopic substitutions (e.g., D2) occur according to random chance. The absence of symmetry in a bond-making complex can yield unequal numbers of singly-substituted molecules (e.g., more H‒D than D‒H in H2), which is recorded in the product molecule as a deficit in doubly-substituted moieties and an "anticlumped" isotope distribution (i.e., Δn < 0). Enzymatic isotope pairing reactions, which can have site-specific isotopic fractionation factors and atom reservoirs, should express this class of combinatorial isotope effect. Chemical-kinetic isotope effects, which are related to the bond-forming transition state, arise independently and express second-order combinatorial effects. In general, both combinatorial and chemical factors are important for calculating and interpreting clumped-isotope signatures of individual reactions. In many reactions relevant to geochemical oxygen, carbon, and nitrogen cycling, combinatorial isotope pairing likely plays a strong role in the clumped isotope distribution of the products. These isotopic signatures, manifest as either directly bound isotope clumps or as features of a molecule's isotopic anatomy, could be exploited as tracers of biogeochemistry that can

  1. β3 integrin promotes chemoresistance to epirubicin in MDA-MB-231 through repression of the pro-apoptotic protein, BAD.

    PubMed

    Nair, Madhumathy G; Desai, Krisha; Prabhu, Jyothi S; Hari, P S; Remacle, Jose; Sridhar, T S

    2016-08-01

    Resistance to anthracycline based chemotherapy is a major limitation in the treatment of breast cancer, particularly of the triple negative sub-type that lacks targeted therapies. Resistance that arises from tumor-stromal interaction facilitated by integrins provides the possibility of targeted disruption. In the present study, we demonstrate that integrin β3 signaling inhibits apoptosis induced by a DNA-damaging chemotherapeutic agent, epirubicin, in MDA-MB-231 breast cancer cells. Drug efflux based mechanisms do not contribute to this effect. We show that integrin β3 employs the PI3K-Akt and the MAPK pathway for enabling cell survival and proliferation. Further, our results indicate that integrin β3 helps inhibit epirubicin induced cytotoxicity by repression of the pro-apoptotic protein BAD, thus promoting an anti-apoptotic response. Myristoylated RGT peptide and a monoclonal antibody against integrin β3 brought about a reversal of this effect and chemosensitized the cells. These results identify β3 integrin signaling via repression of BAD as an important survival pathway used by breast cancer cells to evade chemotherapy induced stress.

  2. More Combinatorial Proofs via Flagpole Arrangements

    ERIC Educational Resources Information Center

    DeTemple, Duane; Reynolds, H. David, II

    2006-01-01

    Combinatorial identities are proved by counting the number of arrangements of a flagpole and guy wires on a row of blocks that satisfy a set of conditions. An identity is proved by first deriving and then equating two expressions that each count the number of permissible arrangements. Identities for binomial coefficients and recursion relations…

  3. Students' Verification Strategies for Combinatorial Problems

    ERIC Educational Resources Information Center

    Mashiach Eizenberg, Michal; Zaslavsky, Orit

    2004-01-01

    We focus on a major difficulty in solving combinatorial problems, namely, on the verification of a solution. Our study aimed at identifying undergraduate students' tendencies to verify their solutions, and the verification strategies that they employ when solving these problems. In addition, an attempt was made to evaluate the level of efficiency…

  4. Quantum Resonance Approach to Combinatorial Optimization

    NASA Technical Reports Server (NTRS)

    Zak, Michail

    1997-01-01

    It is shown that quantum resonance can be used for combinatorial optimization. The advantage of the approach is in independence of the computing time upon the dimensionality of the problem. As an example, the solution to a constraint satisfaction problem of exponential complexity is demonstrated.

  5. Ternary resin-bound Dynamic Combinatorial Chemistry.

    PubMed

    Gromova, Anna V; Ciszewski, Joseph M; Miller, Benjamin L

    2012-02-18

    The ability to carry out simultaneous orthogonal exchange chemistries has opened new opportunities for increasing the numerical and structural diversity accessible to Dynamic Combinatorial Chemistry. We present proof-of-concept experiments demonstrating this concept is transferrable to resin-bound DCC, facilitating the generation and analysis of libraries with greater structural diversity.

  6. A Model of Students' Combinatorial Thinking

    ERIC Educational Resources Information Center

    Lockwood, Elise

    2013-01-01

    Combinatorial topics have become increasingly prevalent in K-12 and undergraduate curricula, yet research on combinatorics education indicates that students face difficulties when solving counting problems. The research community has not yet addressed students' ways of thinking at a level that facilitates deeper understanding of how students…

  7. Loss of β1-integrin from urothelium results in overactive bladder and incontinence in mice: a mechanosensory rather than structural phenotype

    PubMed Central

    Kanasaki, Keizo; Yu, Weiqun; von Bodungen, Maximilian; Larigakis, John D.; Kanasaki, Megumi; Ayala de la Pena, Francisco; Kalluri, Raghu; Hill, Warren G.

    2013-01-01

    Bladder urothelium senses and communicates information about bladder fullness. However, the mechanoreceptors that respond to tissue stretch are poorly defined. Integrins are mechanotransducers in other tissues. Therefore, we eliminated β1-integrin selectively in urothelium of mice using Cre-LoxP targeted gene deletion. β1-Integrin localized to basal/intermediate urothelial cells by confocal microscopy. β1-Integrin conditional-knockout (β1-cKO) mice lacking urothelial β1-integrin exhibited down-regulation and mislocalization of α3- and α5-integrins by immunohistochemistry but, surprisingly, had normal morphology, permeability, and transepithelial resistance when compared with Cre-negative littermate controls. β1-cKO mice were incontinent, as judged by random urine leakage on filter paper (4-fold higher spotting, P<0.01; 2.5-fold higher urine area percentage, P<0.05). Urodynamic function assessed by cystometry revealed bladder overfilling with 80% longer intercontractile intervals (P<0.05) and detrusor hyperactivity (3-fold more prevoid contractions, P<0.05), but smooth muscle contractility remained intact. ATP secretion into the lumen was elevated (49 vs. 22 nM, P<0.05), indicating abnormal filling-induced purinergic signaling, and short-circuit currents (measured in Ussing chambers) revealed 2-fold higher stretch-activated ion channel conductances in response to hydrostatic pressure of 1 cmH2O (P<0.05). We conclude that loss of integrin signaling from urothelium results in incontinence and overactive bladder due to abnormal mechanotransduction; more broadly, our findings indicate that urothelium itself directly modulates voiding.—Kanasaki, K., Yu, W., von Bodungen, M., Larigakis, J. D., Kanasaki, M., Ayala de la Pena, F., Kalluri, R., Hill, W.G. Loss of β1-integrin from urothelium results in overactive bladder and incontinence in mice: a mechanosensory rather than structural phenotype. PMID:23395910

  8. Leukocyte arrest: Biomechanics and molecular mechanisms of β2 integrin activation.

    PubMed

    Fan, Zhichao; Ley, Klaus

    2015-01-01

    Integrins are a group of heterodimeric transmembrane receptors that play essential roles in cell-cell and cell-matrix interaction. Integrins are important in many physiological processes and diseases. Integrins acquire affinity to their ligand by undergoing molecular conformational changes called activation. Here we review the molecular biomechanics during conformational changes of integrins, integrin functions in leukocyte biorheology (adhesive functions during rolling and arrest) and molecules involved in integrin activation. PMID:26684674

  9. Integrating virtual screening and combinatorial chemistry for accelerated drug discovery.

    PubMed

    López-Vallejo, Fabian; Caulfield, Thomas; Martínez-Mayorga, Karina; Giulianotti, Marc A; Nefzi, Adel; Houghten, Richard A; Medina-Franco, Jose L

    2011-07-01

    Virtual screening is increasingly being used in drug discovery programs with a growing number of successful applications. Experimental methodologies developed to speed up the drug discovery processes include high-throughput screening and combinatorial chemistry. The complementarities between computational and experimental screenings have been recognized and reviewed in the literature. Computational methods have also been used in the combinatorial chemistry field, in particular in library design. However, the integration of computational and combinatorial chemistry screenings has been attempted only recently. Combinatorial libraries (experimental or virtual) represent a notable source of chemically related compounds. Advances in combinatorial chemistry and deconvolution strategies, have enabled the rapid exploration of novel and dense regions in the chemical space. The present review is focused on the integration of virtual and experimental screening of combinatorial libraries. Applications of virtual screening to discover novel anticancer agents and our ongoing efforts towards the integration of virtual screening and combinatorial chemistry are also discussed.

  10. Use of protein-engineered fabrics to identify design rules for integrin ligand clustering in biomaterials.

    PubMed

    Benitez, Patrick L; Mascharak, Shamik; Proctor, Amy C; Heilshorn, Sarah C

    2016-01-01

    While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand's effective dissociation constant (KD,eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122 000 ligands μm(-2)) and global (0 to 71 000 ligand μm(-2)) densities of an arginine-glycine-aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to αVβ3 integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's KD,eff of 12 000 RGD μm(-2). Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122 000 RGD μm(-2), cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer

  11. Regulation of β1 integrin-Klf2-mediated angiogenesis by CCM proteins.

    PubMed

    Renz, Marc; Otten, Cécile; Faurobert, Eva; Rudolph, Franziska; Zhu, Yuan; Boulday, Gwénola; Duchene, Johan; Mickoleit, Michaela; Dietrich, Ann-Christin; Ramspacher, Caroline; Steed, Emily; Manet-Dupé, Sandra; Benz, Alexander; Hassel, David; Vermot, Julien; Huisken, Jan; Tournier-Lasserve, Elisabeth; Felbor, Ute; Sure, Ulrich; Albiges-Rizo, Corinne; Abdelilah-Seyfried, Salim

    2015-01-26

    Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor β1 integrin and occurs in the absence of blood flow. Downregulation of β1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a β1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells. PMID:25625207

  12. An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens.

    PubMed

    Meliopoulos, Victoria A; Van de Velde, Lee-Ann; Van de Velde, Nicholas C; Karlsson, Erik A; Neale, Geoff; Vogel, Peter; Guy, Cliff; Sharma, Shalini; Duan, Susu; Surman, Sherri L; Jones, Bart G; Johnson, Michael D L; Bosio, Catharine; Jolly, Lisa; Jenkins, R Gisli; Hurwitz, Julia L; Rosch, Jason W; Sheppard, Dean; Thomas, Paul G; Murray, Peter J; Schultz-Cherry, Stacey

    2016-08-01

    The healthy lung maintains a steady state of immune readiness to rapidly respond to injury from invaders. Integrins are important for setting the parameters of this resting state, particularly the epithelial-restricted αVβ6 integrin, which is upregulated during injury. Once expressed, αVβ6 moderates acute lung injury (ALI) through as yet undefined molecular mechanisms. We show that the upregulation of β6 during influenza infection is involved in disease pathogenesis. β6-deficient mice (β6 KO) have increased survival during influenza infection likely due to the limited viral spread into the alveolar spaces leading to reduced ALI. Although the β6 KO have morphologically normal lungs, they harbor constitutively activated lung CD11b+ alveolar macrophages (AM) and elevated type I IFN signaling activity, which we traced to the loss of β6-activated transforming growth factor-β (TGF-β). Administration of exogenous TGF-β to β6 KO mice leads to reduced numbers of CD11b+ AMs, decreased type I IFN signaling activity and loss of the protective phenotype during influenza infection. Protection extended to other respiratory pathogens such as Sendai virus and bacterial pneumonia. Our studies demonstrate that the loss of one epithelial protein, αVβ6 integrin, can alter the lung microenvironment during both homeostasis and respiratory infection leading to reduced lung injury and improved survival.

  13. An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens

    PubMed Central

    Van de Velde, Nicholas C.; Karlsson, Erik A.; Neale, Geoff; Vogel, Peter; Sharma, Shalini; Duan, Susu; Surman, Sherri L.; Jones, Bart G.; Johnson, Michael D. L.; Bosio, Catharine; Jolly, Lisa; Jenkins, R. Gisli; Hurwitz, Julia L.; Rosch, Jason W.; Sheppard, Dean; Thomas, Paul G.; Murray, Peter J.; Schultz-Cherry, Stacey

    2016-01-01

    The healthy lung maintains a steady state of immune readiness to rapidly respond to injury from invaders. Integrins are important for setting the parameters of this resting state, particularly the epithelial-restricted αVβ6 integrin, which is upregulated during injury. Once expressed, αVβ6 moderates acute lung injury (ALI) through as yet undefined molecular mechanisms. We show that the upregulation of β6 during influenza infection is involved in disease pathogenesis. β6-deficient mice (β6 KO) have increased survival during influenza infection likely due to the limited viral spread into the alveolar spaces leading to reduced ALI. Although the β6 KO have morphologically normal lungs, they harbor constitutively activated lung CD11b+ alveolar macrophages (AM) and elevated type I IFN signaling activity, which we traced to the loss of β6-activated transforming growth factor-β (TGF-β). Administration of exogenous TGF-β to β6 KO mice leads to reduced numbers of CD11b+ AMs, decreased type I IFN signaling activity and loss of the protective phenotype during influenza infection. Protection extended to other respiratory pathogens such as Sendai virus and bacterial pneumonia. Our studies demonstrate that the loss of one epithelial protein, αVβ6 integrin, can alter the lung microenvironment during both homeostasis and respiratory infection leading to reduced lung injury and improved survival. PMID:27505057

  14. JAK tyrosine kinases promote hierarchical activation of Rho and Rap modules of integrin activation.

    PubMed

    Montresor, Alessio; Bolomini-Vittori, Matteo; Toffali, Lara; Rossi, Barbara; Constantin, Gabriela; Laudanna, Carlo

    2013-12-23

    Lymphocyte recruitment is regulated by signaling modules based on the activity of Rho and Rap small guanosine triphosphatases that control integrin activation by chemokines. We show that Janus kinase (JAK) protein tyrosine kinases control chemokine-induced LFA-1- and VLA-4-mediated adhesion as well as human T lymphocyte homing to secondary lymphoid organs. JAK2 and JAK3 isoforms, but not JAK1, mediate CXCL12-induced LFA-1 triggering to a high affinity state. Signal transduction analysis showed that chemokine-induced activation of the Rho module of LFA-1 affinity triggering is dependent on JAK activity, with VAV1 mediating Rho activation by JAKs in a Gαi-independent manner. Furthermore, activation of Rap1A by chemokines is also dependent on JAK2 and JAK3 activity. Importantly, activation of Rap1A by JAKs is mediated by RhoA and PLD1, thus establishing Rap1A as a downstream effector of the Rho module. Thus, JAK tyrosine kinases control integrin activation and dependent lymphocyte trafficking by bridging chemokine receptors to the concurrent and hierarchical activation of the Rho and Rap modules of integrin activation.

  15. An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens.

    PubMed

    Meliopoulos, Victoria A; Van de Velde, Lee-Ann; Van de Velde, Nicholas C; Karlsson, Erik A; Neale, Geoff; Vogel, Peter; Guy, Cliff; Sharma, Shalini; Duan, Susu; Surman, Sherri L; Jones, Bart G; Johnson, Michael D L; Bosio, Catharine; Jolly, Lisa; Jenkins, R Gisli; Hurwitz, Julia L; Rosch, Jason W; Sheppard, Dean; Thomas, Paul G; Murray, Peter J; Schultz-Cherry, Stacey

    2016-08-01

    The healthy lung maintains a steady state of immune readiness to rapidly respond to injury from invaders. Integrins are important for setting the parameters of this resting state, particularly the epithelial-restricted αVβ6 integrin, which is upregulated during injury. Once expressed, αVβ6 moderates acute lung injury (ALI) through as yet undefined molecular mechanisms. We show that the upregulation of β6 during influenza infection is involved in disease pathogenesis. β6-deficient mice (β6 KO) have increased survival during influenza infection likely due to the limited viral spread into the alveolar spaces leading to reduced ALI. Although the β6 KO have morphologically normal lungs, they harbor constitutively activated lung CD11b+ alveolar macrophages (AM) and elevated type I IFN signaling activity, which we traced to the loss of β6-activated transforming growth factor-β (TGF-β). Administration of exogenous TGF-β to β6 KO mice leads to reduced numbers of CD11b+ AMs, decreased type I IFN signaling activity and loss of the protective phenotype during influenza infection. Protection extended to other respiratory pathogens such as Sendai virus and bacterial pneumonia. Our studies demonstrate that the loss of one epithelial protein, αVβ6 integrin, can alter the lung microenvironment during both homeostasis and respiratory infection leading to reduced lung injury and improved survival. PMID:27505057

  16. A novel member of the integrin receptor family mediates Arg-Gly-Asp- stimulated neutrophil phagocytosis

    PubMed Central

    1989-01-01

    Human neutrophils (PMN) express a heterodimeric receptor that has ligand binding specificity for the Arg-Gly-Asp (RGD) sequence within many adhesive proteins. A monoclonal antibody, B6H12, which binds to this receptor, inhibits both RGD-mediated ligand binding and stimulation of IgG-mediated phagocytosis by fibronectin, fibrinogen, vitronectin, von Willebrand's factor, and collagen type IV. By several criteria this receptor is neither a known very late antigen, a known cytoadhesin (gp IIb/IIIa-vitronectin receptor), nor a member of the LFA- 1, Mac-1, p150,95 group of integrin receptors. Ligand binding via this receptor is rapidly inactivated by products of the myeloperoxidase- hydrogen peroxide-halide system of PMN. We conclude that this receptor, for which we propose the name leukocyte response integrin, is a signal- transducing molecule on PMN which may have a significant early role in modulation of PMN function at inflammatory sites. PMID:2785522

  17. How Osteoblasts Sense their Environment: Integrin-Extracellular Matrix Interactions and Mechanical Loading of Bone

    NASA Technical Reports Server (NTRS)

    Globus, Ruth K.; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    Osteoblasts are the cells responsible for forming and replacing bone throughout life. We know that mechanical stimulation through weight-bearing at I gravity on Earth is needed to maintain healthy bone, and that osteoblasts play a critical role in that process. Over the last 9 years in my laboratory at NASA ARC, we have studied the regulation of osteoblast function by interactions between the extracellular matrix and die cell. Using a cell culture approach, we defined the repertoire of adhesion receptors, called integrins, which are expressed on the osteoblast surface, as well as specific extracellular matrix proteins, which are needed for cellular differentiation and survival. We are now extending these observations to determine if integrin signaling is involved in the skeletal responses to disuse and recovery from disuse using the rodent model of hindlimb unloading by tail suspension. Together, our cell culture and animal studies are providing new insight into the regulation of osteoblast function in bone.

  18. Human-restricted bacterial pathogens block shedding of epithelial cells by stimulating integrin activation.

    PubMed

    Muenzner, Petra; Bachmann, Verena; Zimmermann, Wolfgang; Hentschel, Jochen; Hauck, Christof R

    2010-09-01

    Colonization of mucosal surfaces is the key initial step in most bacterial infections. One mechanism protecting the mucosa is the rapid shedding of epithelial cells, also termed exfoliation, but it is unclear how pathogens counteract this process. We found that carcinoembryonic antigen (CEA)-binding bacteria colonized the urogenital tract of CEA transgenic mice, but not of wild-type mice, by suppressing exfoliation of mucosal cells. CEA binding triggered de novo expression of the transforming growth factor receptor CD105, changing focal adhesion composition and activating beta1 integrins. This manipulation of integrin inside-out signaling promotes efficient mucosal colonization and represents a potential target to prevent or cure bacterial infections. PMID:20813953

  19. FOREWORD: Focus on Combinatorial Materials Science Focus on Combinatorial Materials Science

    NASA Astrophysics Data System (ADS)

    Chikyo, Toyohiro

    2011-10-01

    About 15 years have passed since the introduction of modern combinatorial synthesis and high-throughput techniques for the development of novel inorganic materials; however, similar methods existed before. The most famous was reported in 1970 by Hanak who prepared composition-spread films of metal alloys by sputtering mixed-material targets. Although this method was innovative, it was rarely used because of the large amount of data to be processed. This problem is solved in the modern combinatorial material research, which is strongly related to computer data analysis and robotics. This field is still at the developing stage and may be enriched by new methods. Nevertheless, given the progress in measurement equipment and procedures, we believe the combinatorial approach will become a major and standard tool of materials screening and development. The first article of this journal, published in 2000, was titled 'Combinatorial solid state materials science and technology', and this focus issue aims to reintroduce this topic to the Science and Technology of Advanced Materials audience. It covers recent progress in combinatorial materials research describing new results in catalysis, phosphors, polymers and metal alloys for shape memory materials. Sophisticated high-throughput characterization schemes and innovative synthesis tools are also presented, such as spray deposition using nanoparticles or ion plating. On a technical note, data handling systems are introduced to familiarize researchers with the combinatorial methodology. We hope that through this focus issue a wide audience of materials scientists can learn about recent and future trends in combinatorial materials science and high-throughput experimentation.

  20. A combinatorial code for pattern formation in Drosophila oogenesis

    PubMed Central

    Yakoby, N.; Bristow, C.A.; Gong, D.; Schafer, X.; Lembong, J.; Zartman, J.J.; Halfon, M.S.; Schüpbach, T.; Shvartsman, S.Y.

    2010-01-01

    Summary Two-dimensional patterning of the follicular epithelium in Drosophila oogenesis is required for the formation of three-dimensional eggshell structures. Our analysis of a large number of published gene expression patterns in the follicle cells suggested that they follow a simple combinatorial code, based on six spatial building blocks and the operations of union, difference, intersection, and addition. The building blocks are related to the distribution of the inductive signals, provided by the highly conserved EGFR and DPP pathways. We demonstrated the validity of the code by testing it against a set of newly identified expression patterns, obtained in a large-scale transcriptional profiling experiment. Using the proposed code, we distinguished 36 distinct patterns for 81 genes expressed in the follicular epithelium and characterized their joint dynamics over four stages of oogenesis. This work provides the first systematic analysis of the diversity and dynamics of two-dimensional gene expression patterns in a developing tissue. PMID:19000837

  1. Structures and Interaction Analyses of Integrin αMβ2 Cytoplasmic Tails*

    PubMed Central

    Chua, Geok-Lin; Tang, Xiao-Yan; Amalraj, Monalisa; Tan, Suet-Mien; Bhattacharjya, Surajit

    2011-01-01

    Integrins are heterodimeric (α and β subunits) signal transducer proteins involved in cell adhesions and migrations. The cytosolic tails of integrins are essential for transmitting bidirectional signaling and also implicated in maintaining the resting states of the receptors. In addition, cytosolic tails of integrins often undergo post-translation modifications like phosphorylation. However, the consequences of phosphorylation on the structures and interactions are not clear. The leukocyte-specific integrin αMβ2 is essential for myeloid cell adhesion, phagocytosis, and degranulation. In this work, we determined solution structures of the myristoylated cytosolic tail of αM and a Ser phosphorylated variant in dodecylphosphocholine micelles by NMR spectroscopy. Furthermore, the interactions between non-phosphorylated and phosphorylated αM tails with β2 tail were investigated by NMR and fluorescence resonance energy transfer (FRET). The three-dimensional structures of the 24-residue cytosolic tail of αM or phosphorylated αM are characterized by an N-terminal amphipathic helix and a loop at the C terminus. The residues at the loop are involved in packing interactions with the hydrophobic face of the helix. 15N-1H heteronuclear single quantum coherence experiments identified residues of αM and β2 tails that may be involved in the formation of a tail-tail heterocomplex. We further examined interactions between myristoylated β2 tail in dodecylphosphocholine micelles with dansylated αM tail peptides by FRET. These studies revealed enhanced interactions between αM or phosphorylated αM tails with β2 tail with Kd values ∼5.2 ± 0.6 and ∼4.4 ± 0.7 μm, respectively. Docked structures of tail-tail complexes delineated that the αM/β2 interface at the cytosolic region could be sustained by a network of polar interactions, ionic interactions, and/or hydrogen bonds. PMID:22052909

  2. Differential combinatorial coding of pheromones in two olfactory subsystems of the honey bee brain.

    PubMed

    Carcaud, Julie; Giurfa, Martin; Sandoz, Jean-Christophe

    2015-03-11

    Neural coding of pheromones has been intensively studied in insects with a particular focus on sex pheromones. These studies favored the view that pheromone compounds are processed within specific antennal lobe glomeruli following a specialized labeled-line system. However, pheromones play crucial roles in an insect's life beyond sexual attraction, and some species use many different pheromones making such a labeled-line organization unrealistic. A combinatorial coding scheme, in which each component activates a set of broadly tuned units, appears more adapted in this case. However, this idea has not been tested thoroughly. We focused here on the honey bee Apis mellifera, a social insect that relies on a wide range of pheromones to ensure colony cohesion. Interestingly, the honey bee olfactory system harbors two central parallel pathways, whose functions remain largely unknown. Using optophysiological recordings of projection neurons, we compared the responses of these two pathways to 27 known honey bee pheromonal compounds emitted by the brood, the workers, and the queen. We show that while queen mandibular pheromone is processed by l-ALT (lateral antennal lobe tract) neurons and brood pheromone is mainly processed by m-ALT (median antennal lobe tract) neurons, worker pheromones induce redundant activity in both pathways. Moreover, all tested pheromonal compounds induce combinatorial activity from several AL glomeruli. These findings support the combinatorial coding scheme and suggest that higher-order brain centers reading out these combinatorial activity patterns may eventually classify olfactory signals according to their biological meaning. PMID:25762663

  3. Insulin-like growth factor-binding protein 2-driven glioma progression is prevented by blocking a clinically significant integrin, integrin-linked kinase, and NF-κB network

    PubMed Central

    Holmes, Kristen M.; Annala, Matti; Chua, Corrine Y. X.; Dunlap, Sarah M.; Liu, Yuexin; Hugen, Niek; Moore, Lynette M.; Cogdell, David; Hu, Limei; Nykter, Matti; Hess, Kenneth; Fuller, Gregory N.; Zhang, Wei

    2012-01-01

    Insulin-like growth factor-binding protein 2 (IGFBP2) is increasingly recognized as a glioma oncogene, emerging as a target for therapeutic intervention. In this study, we used an integrative approach to characterizing the IGFBP2 network, combining transcriptional profiling of human glioma with validation in glial cells and the replication-competent ASLV long terminal repeat with a splice acceptor/tv-a glioma mouse system. We demonstrated that IGFBP2 expression is closely linked to genes in the integrin and integrin-linked kinase (ILK) pathways and that these genes are associated with prognosis. We further showed that IGFBP2 activates integrin β1 and downstream invasion pathways, requires ILK to induce cell motility, and activates NF-κB. Most significantly, the IGFBP2/integrin/ILK/NF-κB network functions as a physiologically active signaling pathway in vivo by driving glioma progression; interfering with any point in the pathway markedly inhibits progression. The results of this study reveal a signaling pathway that is both targetable and highly relevant to improving the survival of glioma patients. PMID:22345562

  4. Receptor activator of NF-κB ligand induces cell adhesion and integrin α2 expression via NF-κB in head and neck cancers

    PubMed Central

    Yamada, Tamaki; Tsuda, Masumi; Wagatsuma, Takanori; Fujioka, Yoichiro; Fujioka, Mari; Satoh, Aya O.; Horiuchi, Kosui; Nishide, Shinya; Nanbo, Asuka; Totsuka, Yasunori; Haga, Hisashi; Tanaka, Shinya; Shindoh, Masanobu; Ohba, Yusuke

    2016-01-01

    Cellular interactions with the extracellular matrix play critical roles in tumor progression. We previously reported that receptor activator of NF-κB ligand (RANKL) specifically facilitates head and neck squamous cell carcinoma (HNSCC) progression in vivo. Here, we report a novel role for RANKL in the regulation of cell adhesion. Among the major type I collagen receptors, integrin α2 was significantly upregulated in RANKL-expressing cells, and its knockdown suppressed cell adhesion. The mRNA abundance of integrin α2 positively correlated with that of RANKL in human HNSCC tissues. We also revealed that RANK-NF-κB signaling mediated integrin α2 expression in an autocrine/paracrine manner. Interestingly, the amount of active integrin β1 on the cell surface was increased in RANKL-expressing cells through the upregulation of integrin α2 and endocytosis. Moreover, the RANK-integrin α2 pathway contributed to RANKL-dependent enhanced survival in a collagen gel and inhibited apoptosis in a xenograft model, demonstrating an important role for RANKL-mediated cell adhesion in three-dimensional environments. PMID:27009236

  5. High throughput combinatorial screening of semiconductor materials

    NASA Astrophysics Data System (ADS)

    Mao, Samuel S.

    2011-11-01

    This article provides an overview of an advanced combinatorial material discovery platform developed recently for screening semiconductor materials with properties that may have applications ranging from radiation detectors to solar cells. Semiconductor thin-film libraries, each consisting of 256 materials of different composition arranged into a 16×16 matrix, were fabricated using laser-assisted evaporation process along with a combinatorial mechanism to achieve variations. The composition and microstructure of individual materials on each thin-film library were characterized with an integrated scanning micro-beam x-ray fluorescence and diffraction system, while the band gaps were determined by scanning optical reflection and transmission of the libraries. An ultrafast ultraviolet photon-induced charge probe was devised to measure the mobility and lifetime of individual thin-film materials on semiconductor libraries. Selected results on the discovery of semiconductors with desired band gaps and transport properties are illustrated.

  6. Assessment of structural diversity in combinatorial synthesis.

    PubMed

    Fergus, Suzanne; Bender, Andreas; Spring, David R

    2005-06-01

    This article covers the combinatorial synthesis of small molecules with maximal structural diversity to generate a collection of pure compounds that are attractive for lead generation in a phenotypic, high-throughput screening approach. Nature synthesises diverse small molecules, but there are disadvantages with using natural product sources. The efficient chemical synthesis of structural diversity (and complexity) is the aim of diversity-oriented synthesis, and recent progress is reviewed. Specific highlights include a discussion of strategies to obtain structural diversity and an analysis of molecular descriptors used to classify compounds. The assessment of how successful one synthesis is versus another is subjective, therefore we test-drive software to assess structural diversity in combinatorial synthesis, which is freely available via a web interface.

  7. [The research progress of dynamic combinatorial chemistry].

    PubMed

    He, Wei; She, Peng-Wei; Fang, Zheng; Guo, Kai

    2013-06-01

    As a novel branch of combinational chemistry, dynamic combinatorial chemistry (DCC) can be viewed as a technique which combines library synthesis and screening in one pot. By addition of molecular target, ligangds, which show binding affinity or strong interaction with the molecular target, can be amplified an young but rapidly growing branch of combinatorial chemistry, has been widely used in organic chemistry, biochemistry, material fields. Ligands in the library can be amplified, since synthesis of the library is screened by a molecular target. Therefore, these structures could be identified easily. Consequently DCC has been widely used in the lead discovery, material chemistry and other fields. On the basis of the principle and method of DCC, this review emphasizes the three factors of DCC, including molecular targets (bio-enzyme, lectin, nucleic acid, organic molecule, inorganic molecule); reaction (disulphide chemistry, ammoniation reduction reaction, hydrazone chemistry, etc.) and analytical method. Meanwhile, limitation, current situation and future development of DCC were also discussed in this paper.

  8. Combinatorial biosynthesis--potential and problems.

    PubMed

    Floss, Heinz G

    2006-06-25

    Because of their ecological functions, natural products have been optimized in evolution for interaction with biological systems and receptors. However, they have not necessarily been optimized for other desirable drug properties and thus can often be improved by structural modification. Using examples from the literature, this paper reviews the opportunities for increasing structural diversity among natural products by combinatorial biosynthesis, i.e., the genetic manipulation of biosynthetic pathways. It distinguishes between combinatorial biosynthesis in a narrower sense to generate libraries of modified structures, and metabolic engineering for the targeted formation of specific structural analogs. Some of the problems and limitations encountered with these approaches are also discussed. Work from the author's laboratory on ansamycin antibiotics is presented which illustrates some of the opportunities and limitations.

  9. Exploiting Quantum Resonance to Solve Combinatorial Problems

    NASA Technical Reports Server (NTRS)

    Zak, Michail; Fijany, Amir

    2006-01-01

    Quantum resonance would be exploited in a proposed quantum-computing approach to the solution of combinatorial optimization problems. In quantum computing in general, one takes advantage of the fact that an algorithm cannot be decoupled from the physical effects available to implement it. Prior approaches to quantum computing have involved exploitation of only a subset of known quantum physical effects, notably including parallelism and entanglement, but not including resonance. In the proposed approach, one would utilize the combinatorial properties of tensor-product decomposability of unitary evolution of many-particle quantum systems for physically simulating solutions to NP-complete problems (a class of problems that are intractable with respect to classical methods of computation). In this approach, reinforcement and selection of a desired solution would be executed by means of quantum resonance. Classes of NP-complete problems that are important in practice and could be solved by the proposed approach include planning, scheduling, search, and optimal design.

  10. Breast Cancer Cells in Three-dimensional Culture Display an Enhanced Radioresponse after Coordinate Targeting of Integrin ?5?1 and Fibronectin

    SciTech Connect

    Nam, Jin-Min; Onodera, Yasuhito; Bissell, Mina J; Park, Catherine C

    2010-04-07

    Tactics to selectively enhance cancer radioresponse are of great interest. Cancer cells actively elaborate and remodel their extracellular matrix (ECM) to aid in survival and progression. Previous work has shown that {beta}1-integrin inhibitory antibodies can enhance the growth-inhibitory and apoptotic responses of human breast cancer cell lines to ionizing radiation, either when cells are cultured in three-dimensional laminin-rich ECM (3D lrECM) or grown as xenografts in mice. Here, we show that a specific {alpha} heterodimer of {beta}1-integrin preferentially mediates a prosurvival signal in human breast cancer cells that can be specifically targeted for therapy. 3D lrECM culture conditions were used to compare {alpha}-integrin heterodimer expression in malignant and nonmalignant cell lines. Under these conditions, we found that expression of {alpha}5{beta}1-integrin was upregulated in malignant cells compared with nonmalignant breast cells. Similarly, we found that normal and oncofetal splice variants of fibronectin, the primary ECM ligand of {alpha}5{beta}1-integrin, were also strikingly upregulated in malignant cell lines compared with nonmalignant acini. Cell treatment with a peptide that disrupts the interactions of {alpha}5{beta}1-integrin with fibronectin promoted apoptosis in malignant cells and further heightened the apoptotic effects of radiation. In support of these results, an analysis of gene expression array data from breast cancer patients revealed an association of high levels of {alpha}5-integrin expression with decreased survival. Our findings offer preclinical validation of fibronectin and {alpha}5{beta}1-integrin as targets for breast cancer therapy.

  11. The Combinatorial Trace Method in Action

    ERIC Educational Resources Information Center

    Krebs, Mike; Martinez, Natalie C.

    2013-01-01

    On any finite graph, the number of closed walks of length k is equal to the sum of the kth powers of the eigenvalues of any adjacency matrix. This simple observation is the basis for the combinatorial trace method, wherein we attempt to count (or bound) the number of closed walks of a given length so as to obtain information about the graph's…

  12. Combinatorial and algorithm aspects of hyperbolic polynomials

    SciTech Connect

    Gurvits, Leonid I.

    2004-01-01

    Univariate polynomials with real roots appear quite often in modern combinatorics, especially in the context of integer polytopes. We discovered in this paper rather unexpected and very likely far-reaching connections between hyperbolic polynomials and many classical combinatorial and algorithmic problems. There are still several open problems. The most interesting is a hyperbolic generalization of the van der Waerden conjecture for permanents of doubly stochastic matrices.

  13. Simulated Microgravity Alters Actin Cytoskeleton and Integrin-Mediated Focal Adhesions of Cultured Human Mesenchymal Stromal Cells

    NASA Astrophysics Data System (ADS)

    Gershovich, P. M.; Gershovic, J. G.; Buravkova, L. B.

    2008-06-01

    Cytoskeletal alterations occur in several cell types including lymphocytes, glial cells, and osteoblasts, during spaceflight and under simulated microgravity (SMG) (3, 4). One potential mechanism for cytoskeletal gravisensitivity is disruption of extracellular matrix (ECM) and integrin interactions. Focal adhesions are specialized sites of cell-matrix interaction composed of integrins and the diversity of focal adhesion-associated cytoplasmic proteins including vinculin, talin, α-actinin, and actin filaments (4, 5). Integrins produce signals essential for proper cellular function, survival and differentiation. Therefore, we investigated the effects of SMG on F-actin cytoskeleton structure, vinculin focal adhesions, expression of some integrin subtypes and cellular adhesion molecules (CAMs) in mesenchymal stem cells derived from human bone marrow (hMSCs). Simulated microgravity was produced by 3D-clinostat (Dutch Space, Netherlands). Staining of actin fibers with TRITC-phalloidin showed reorganization even after 30 minutes of simulated microgravity. The increasing of cells number with abnormal F-actin was observed after subsequent terms of 3D-clinorotation (6, 24, 48, 120 hours). Randomization of gravity vector altered dimensional structure of stress fibers and resulted in remodeling of actin fibers inside the cells. In addition, we observed vinculin redistribution inside the cells after 6 hours and prolonged terms of clinorotation. Tubulin fibers in a contrast with F-actin and vinculin didn't show any reorganization even after long 3Dclinorotation (120 hours). The expression of integrin α2 increased 1,5-6-fold in clinorotated hMSCs. Also we observed decrease in number of VCAM-1-positive cells and changes in expression of ICAM-1. Taken together, our findings indicate that SMG leads to microfilament and adhesion alterations of hMSCs most probably associated with involvement of some integrin subtypes.

  14. Immunoexpression of α2-integrin and Hsp47 in hereditary gingival fibromatosis and gingival fibromatosis-associated dental abnormalities

    PubMed Central

    de Oliveira-Santos, Carolina; Della-Coletta, Ricardo; Cristianismo-Costa, Daiane; Paranaíba, Lívia M.-R.; Martelli-Júnior, Hercílio

    2013-01-01

    Objective: The purpose of the present study was to investigate the expression of the α2-integrin subunit and heat shock protein 47 (Hsp47) in two families with isolated gingival fibromatosis (GF) form and one family with GF associated with dental abnormalities and normal gingiva (NG). Study Design: Immunohistochemistry was performed with antibodies against α2-integrin and Hsp47 in specimens from two unrelated families with hereditary gingival fibromatosis (Families 1 and 2) and from one family with a gingival fibromatosis-associated dental abnormality (Family 3); NG samples were used for comparison. The results were analysed statistically. Results: Immunoreactivity for α2-integrin and Hsp47 was observed in the nucleus of epithelial cells of both the basal and suprabasal layer and a more discreet signal