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Sample records for beta5 integrin activation

  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. Vitronectin enhances internalization of crocidolite asbestos by rabbit pleural mesothelial cells via the integrin alpha v beta 5.

    PubMed Central

    Boylan, A M; Sanan, D A; Sheppard, D; Broaddus, V C

    1995-01-01

    The mechanism by which pleural mesothelial cells, the likely progenitor cells of asbestos-induced mesothelioma, recognize and internalize crocidolite asbestos is unknown. Because incubation of asbestos fibers with serum increases their association with cells, we asked whether a protein coat on asbestos increased internalization of fibers via specific cellular receptors. Coating crocidolite with citronectin, but not with fibronectin or other proteins, increased fiber internalization by rabbit pleural mesothelial cells, as measured by a new technique using fluorescence confocal microscopy. Receptors for vitronectin, alpha v beta 3 and alpha v beta 5, were identified on mesothelial cells. Inhibiting vitronectin receptors by plating cells on a vitronectin substrate or incubating cells with excess soluble vitronectin reduced internalization of vitronectin-coated crocidolite. Inhibition of alpha v beta 5, but not alpha v beta 3, with blocking antibodies similarly reduced internalization. In addition, alpha v beta 5, but not alpha v beta 3, showed immunocytochemical colocalization with fibers. Of biologic relevance, coating crocidolite with serum also increased internalization via alpha v beta 5, an effect dependent on the vitronectin in serum. We conclude that pleural mesothelial cells recognize and internalize vitronectin- and serum-coated asbestos via the integrin alpha v beta 5. Since integrins initiate some of the same signaling pathways as does asbestos, our findings may provide insights into the mechanisms of asbestos-induced biologic effects. Images PMID:7560092

  3. cDNA cloning reveals two mouse beta5 integrin transcripts distinct in cytoplasmic domains as a result of alternative splicing.

    PubMed Central

    Zhang, H; Tan, S M; Lu, J

    1998-01-01

    The integrin beta5 subunit has only been found to form a heterodimer with subunit alphav which acts as a vitronectin receptor. Integrin alphavbeta5 has been implicated in cell migration and growth factor-induced angiogenesis. In the present study, a mouse liver cDNA library was screened using a human beta5 cDNA fragment obtained by reverse transcriptase PCR (RT-PCR). Three of the clones (MB5, MB15 and MB17) overlapped to give an open reading frame, called beta5A, which is homologous to the human beta5 subunit. The sequence of another clone (MB26), called beta5B, was identical with beta5A, except for a deletion of 29 bp near the 3' end of the open reading frame. The 29 bp deletion resulted in an open-reading-frame shift and a completely different C-terminal sequence in beta5B. beta5A and beta5B were shown, by RT-PCR, to be co-expressed in most mouse tissues tested, although beta5B mRNA was detected at much lower levels than beta5A. beta5A and beta5B mRNAs were also detected in the mouse monocytic cell line, J774, and in isolated mouse peritoneal macrophages. Adhesion of peritoneal macrophages has been shown to up-regulate the expression of both beta5A and beta5B mRNAs. The 29 bp sequence begins with a putative intron-splicing donor site (GTGAT...). A 3' fragment of the mouse integrin beta5 gene was cloned by PCR and sequenced showing that the 29 bp sequence was also immediately followed by an intron. Therefore, the 29 bp sequence was apparently expressed as part of the beta5A mRNA but was spliced out as part of the downstream intron in beta5B. Since the cytoplasmic domains of the integrin beta subunits are important in cytoskeleton attachment and signalling, the two alternatively spliced beta5 isoforms may have distinct roles in cell adhesion and other cellular functions. PMID:9531507

  4. Two-stage genome-wide association study identifies integrin beta 5 as having potential role in bull fertility

    PubMed Central

    Feugang, Jean M; Kaya, Abdullah; Page, Grier P; Chen, Lang; Mehta, Tapan; Hirani, Kashif; Nazareth, Lynne; Topper, Einko; Gibbs, Richard; Memili, Erdogan

    2009-01-01

    Background Fertility is one of the most critical factors controlling biological and financial performance of animal production systems and genetic improvement of lines. The objective of this study was to identify molecular defects in the sperm that are responsible for uncompensable fertility in Holstein bulls. We performed a comprehensive genome wide analysis of single nucleotide polymorphisms (SNP) for bull fertility followed by a second-stage replication in additional bulls for a restricted set of markers. Results In the Phase I association study, we genotyped the genomic sperm DNA of 10 low-fertility and 10 high-fertility bulls using Bovine SNP Gene Chips containing approximately 10,000 random SNP markers. In these animals, 8,207 markers were found to be polymorphic, 97 of which were significantly associated with fertility (p < 0.01). In the Phase II study, we tested the four most significant SNP from the Phase I study in 101 low-fertility and 100 high-fertility bulls, with two SNPs (rs29024867 and rs41257187) significantly replicated. Rs29024867 corresponds to a nucleotide change of C → G 2,190 bp 3' of the collagen type I alpha 2 gene on chromosome 4, while the rs41257187 (C → T) is in the coding region of integrin beta 5 gene on chromosome 1. The SNP rs41257187 induces a synonymous (Proline → Proline), suggesting disequilibrium with the true causative locus (i), but we found that the incubation of bull spermatozoa with integrin beta 5 antibodies significantly decreased the ability to fertilize oocytes. Our findings suggest that the bovine sperm integrin beta 5 protein plays a role during fertilization and could serve as a positional or functional marker of bull fertility. Conclusion We have identified molecular markers associated with bull fertility and established that at least one of the genes harboring such variation has a role in fertility. The findings are important in understanding mechanisms of uncompensatory infertility in bulls, and in other

  5. Nonessential role of beta3 and beta5 integrin subunits for efficient clearance of cellular debris after light-induced photoreceptor degeneration.

    PubMed

    Joly, Sandrine; Samardzija, Marijana; Wenzel, Andreas; Thiersch, Markus; Grimm, Christian

    2009-03-01

    During light-induced photoreceptor degeneration, large amounts of cellular debris are formed that must be cleared from the subretinal space. The integrins alphavbeta5 and alphavbeta3 are involved in the normal physiological process of phagocytosis in the retina. This study was conducted to investigate the question of whether the lack of beta5 and/or beta3 integrin subunits might influence the course of retinal degeneration and/or clearance of photoreceptor debris induced by acute exposure to light. Wild-type, beta5(-/-) and beta3(-/-) single-knockout, and beta3(-/-)/beta5(-/-) Ccl2(-/-)/beta5(-/-) double-knockout mice were exposed to 13,000 lux of white light for 2 hours to induce severe photoreceptor degeneration. Real-time PCR and Western blot analysis were used to analyze gene and protein expression, light- and electron microscopy to judge retinal morphology, and immunofluorescence to study retinal distribution of proteins. Individual or combined deletion of beta3 and beta5 integrin subunits did not affect the pattern of photoreceptor cell loss or the clearance of photoreceptor debris in mice compared with that in wild-type mice. Invading macrophages may contribute to efficient phagocytosis. However, ablation of the MCP-1 gene did not prevent macrophage recruitment. Several chemokines in addition to MCP-1 were induced after light-induced damage that may have compensated for the deletion of MCP-1. Acute clearance of a large amount of cellular debris from the subretinal space involves invading macrophages and does not depend on beta3 and beta5 integrins.

  6. Integrin activation and structural rearrangement.

    PubMed

    Takagi, Junichi; Springer, Timothy A

    2002-08-01

    Among adhesion receptor families, integrins are particularly important in biological processes that require rapid modulation of adhesion and de-adhesion. Activation on a timescale of < 1 s of beta2 integrins on leukocytes and beta3 integrins on platelets enables deposition of these cells at sites of inflammation or vessel wall injury. Recent crystal, nuclear magnetic resonance (NMR), and electron microscope (EM) structures of integrins and their domains lead to a unifying mechanism of activation for both integrins that contain and those that lack an inserted (I) domain. The I domain adopts two alternative conformations, termed open and closed. In striking similarity to signaling G-proteins, rearrangement of a Mg2+-binding site is linked to large conformational movements in distant backbone regions. Mutations that stabilize a particular conformation show that the open conformation has high affinity for ligand, whereas the closed conformation has low affinity. Movement of the C-terminal alpha-helix 10 A down the side of the domain in the open conformation is sufficient to increase affinity at the distal ligand-binding site 9,000-fold. This C-terminal "bell-rope" provides a mechanism for linkage to conformational movements in other domains. Recent structures and functional studies reveal interactions between beta-propeller, I, and I-like domains in the integrin headpiece, and a critical role for integrin epidermal growth factor (EGF) domains in the stalk region. The headpiece of the integrin faces down towards the membrane in the inactive conformation, and extends upward in a "switchblade"-like opening upon activation. These long-range structural rearrangements of the entire integrin molecule involving interdomain contacts appear closely linked to conformational changes within the I and I-like domains, which result in increased affinity and competence for ligand binding.

  7. Transforming growth factor-beta 1 modulates beta 1 and beta 5 integrin receptors and induces the de novo expression of the alpha v beta 6 heterodimer in normal human keratinocytes: implications for wound healing

    PubMed Central

    1995-01-01

    The molecular mechanism underlying the promotion of wound healing by TGF-beta 1 is incompletely understood. We report that TGF-beta 1 regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-beta 1: (a) strongly upregulates the expression of the fibronectin receptor alpha 5 beta 1, the vitronectin receptor alpha v beta 5, and the collagen receptor alpha 2 beta 1 by differentially modulating the synthesis of their alpha and beta subunits; (b) downregulates the multifunctional alpha 3 beta 1 heterodimer; (c) induces the de novo expression and surface exposure of the alpha v beta 6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both beta 1 and beta 4 dimers; and (f) causes a pericellular redistribution of alpha v beta 5. These data suggest that alpha 5 beta 1, alpha v beta 6, and alpha v beta 5, not routinely used by keratinocytes resting on an intact basement membrane, act as "emergency" receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-beta 1 reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-beta 1-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells. PMID:7537276

  8. SHARPIN is an endogenous inhibitor of beta1-integrin activation

    PubMed Central

    Rantala, Juha K.; Pouwels, Jeroen; Pellinen, Teijo; Veltel, Stefan; Laasola, Petra; Potter, Christopher S.; Duffy, Ted; Sundberg, John P.; Kallioniemi, Olli; Askari, Janet A.; Humphries, Martin; Parsons, Maddy; Salmi, Marko; Ivaska, Johanna

    2012-01-01

    Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and Kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. Here we identified SHARPIN as an important inactivator of β1-integrins in an RNAi-screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity which was fully rescued by re-expression of SHARPIN. SHARPIN directly bound to a conserved cytoplasmic region of integrin α-subunits and inhibited recruitment of Talin and Kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations. PMID:21947080

  9. Distinct Recycling of Active and Inactive β1 Integrins

    PubMed Central

    Arjonen, Antti; Alanko, Jonna; Veltel, Stefan; Ivaska, Johanna

    2012-01-01

    Integrin trafficking plays an important role in cellular motility and cytokinesis. Integrins undergo constant endo/exocytic shuttling to facilitate the dynamic regulation of cell adhesion. Integrin activity toward the components of the extracellular matrix is regulated by the ability of these receptors to switch between active and inactive conformations. Several cellular signalling pathways have been described in the regulation of integrin traffic under different conditions. However, the interrelationship between integrin activity conformations and their endocytic fate have remained incompletely understood. Here, we have investigated the endocytic trafficking of active and inactive β1 integrins in cancer cells. Both conformers are endocytosed in a clathrin- and dynamin-dependent manner. The net endocytosis rate of the active β1 integrins is higher, whereas endocytosis of the inactive β1 integrin is counteracted by rapid recycling back to the plasma membrane via an ARF 6- and early endosome antigen 1-positive compartment in an Rab 4a- and actin-dependent manner. Owing to these distinct trafficking routes, the two receptor pools display divergent subcellular localization. At steady state, the inactive β1 integrin is mainly on the plasma membrane, whereas the active receptor is predominantly intracellular. These data provide new insights into the endocytic traffic of integrins and imply the possibility of a previously unappreciated crosstalk between pathways regulating integrin activity and traffic. PMID:22222055

  10. The P2Y2 nucleotide receptor requires interaction with alpha v integrins to access and activate G12.

    PubMed

    Liao, Zhongji; Seye, Cheikh I; Weisman, Gary A; Erb, Laurie

    2007-05-01

    The P2Y2 nucleotide receptor (P2Y2R) interacts with alpha v integrins to activate G(o) and induce chemotaxis in human 1321N1 astrocytoma cells. In this study, it was determined that the P2Y2R also requires interaction with alpha v integrins to activate G12 and associated signaling pathways that control chemotaxis in 1321N1 cells. Mutation of the Arg-Gly-Asp (RGD) integrin-binding sequence in the first extracellular loop of the human P2Y2R to Arg-Gly-Glu (RGE), which prevents integrin interaction, did not inhibit G(q) or ERK1/2 signaling by the P2Y2R agonist UTP but completely inhibited activation of G12 and G12-mediated events, including Rho activation, cofilin and myosin light chain-2 phosphorylation, stress fiber formation and chemotaxis towards UTP. The involvement of G12 in all these events was verified by using a dominant negative G alpha12 construct. G12 activation by the P2Y2R also was inhibited by anti-alpha v beta5 integrin antibodies and alpha v integrin antisense oligonucleotides, suggesting that alpha v integrin activity and expression are required for the P2Y2R to activate G12. Co-immunoprecipitation experiments confirmed that G alpha12 protein associates with the wild-type P2Y2R and with alpha v integrins but not with the RGE mutant P2Y2R or with alpha3 integrins. Collectively, these results suggest that alpha v integrin complexes provide the P2Y2R with access to G12, thereby allowing activation of this heterotrimeric G protein that controls actin cytoskeletal rearrangements required for chemotaxis.

  11. Focal Adhesion Kinase Modulates Cell Adhesion Strengthening via Integrin Activation

    PubMed Central

    Michael, Kristin E.; Dumbauld, David W.; Burns, Kellie L.; Hanks, Steven K.

    2009-01-01

    Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces. PMID:19297531

  12. Tests of the Extension and Deadbolt Models of Integrin Activation*

    PubMed Central

    Zhu, Jieqing; Boylan, Brian; Luo, Bing-Hao; Newman, Peter J.; Springer, Timothy A.

    2007-01-01

    Despite extensive evidence that integrin conformational changes between bent and extended conformations regulate affinity for ligands, an alternative hypothesis has been proposed in which a “deadbolt” can regulate affinity for ligand in the absence of extension. Here, we tested both the deadbolt and the extension models. According to the deadbolt model, a hairpin loop in the β3 tail domain could act as a deadbolt to restrain the displacement of the β3 I domain β6-α7 loop and maintain integrin in the low affinity state. We found that mutating or deleting the β3 tail domain loop has no effect on ligand binding by either αIIbβ3 or αVβ3 integrins. In contrast, we found that mutations that lock integrins in the bent conformation with disulfide bonds resist inside-out activation induced by cytoplasmic domain mutation. Furthermore, we demonstrated that extension is required for accessibility to fibronectin but not smaller fragments. The data demonstrate that integrin extension is required for ligand binding during integrin inside-out signaling and that the deadbolt does not regulate integrin activation. PMID:17301049

  13. Integrin activation controls metastasis in human breast cancer

    NASA Astrophysics Data System (ADS)

    Felding-Habermann, Brunhilde; O'Toole, Timothy E.; Smith, Jeffrey W.; Fransvea, Emilia; Ruggeri, Zaverio M.; Ginsberg, Mark H.; Hughes, Paul E.; Pampori, Nisar; Shattil, Sanford J.; Saven, Alan; Mueller, Barbara M.

    2001-02-01

    Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin v3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin v3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated αvβ3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant αvβ3D723R, but not αvβ3WT, in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin αvβ3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.

  14. Integrin activation controls metastasis in human breast cancer

    PubMed Central

    Felding-Habermann, Brunhilde; O'Toole, Timothy E.; Smith, Jeffrey W.; Fransvea, Emilia; Ruggeri, Zaverio M.; Ginsberg, Mark H.; Hughes, Paul E.; Pampori, Nisar; Shattil, Sanford J.; Saven, Alan; Mueller, Barbara M.

    2001-01-01

    Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin αvβ3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin αvβ3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated αvβ3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant αvβ3D723R, but not αvβ3WT, in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin αvβ3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer. PMID:11172040

  15. 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

  16. Integrin activation by a cold atmospheric plasma jet

    NASA Astrophysics Data System (ADS)

    Volotskova, Olga; Stepp, Mary Ann; Keidar, Michael

    2012-05-01

    Current breakthrough research on cold atmospheric plasma (CAP) demonstrates that CAP has great potential in various areas, including medicine and biology, thus providing a new tool for living tissue treatment. In this paper, we explore potential mechanisms by which CAP alters cell migration and influences cell adhesion. We focus on the study of CAP interaction with fibroblasts and corneal epithelial cells. The data show that fibroblasts and corneal epithelial cells have different thresholds (treatment times) required to achieve maximum inhibition of cell migration. Both cell types reduced their migration rates by ˜30-40% after CAP compared to control cells. Also, the impact of CAP treatment on cell migration and persistence of fibroblasts after integrin activation by MnCl2, serum starvation or replating cells onto surfaces coated with integrin ligands is assessed; the results show that activation by MnCl2 or starvation attenuates cells’ responses to plasma. Studies carried out to assess the impact of CAP treatment on the activation state of β1 integrin and focal adhesion size by using immunofluorescence show that fibroblasts have more active β1 integrin on their surface and large focal adhesions after CAP treatment. Based on these data, a thermodynamic model is presented to explain how CAP leads to integrin activation and focal adhesion assembly.

  17. Synthesis and anti-glioma activity of 25(R)-spirostan-3beta,5alpha,6beta,19-tetrol.

    PubMed

    Leng, TianDong; Zhang, JingXia; Xie, Jun; Zhou, ShuJia; Huang, YiJun; Zhou, YueHan; Zhu, WenBo; Yan, GuangMei

    2010-03-01

    Malignant gliomas are common and aggressive brain tumours in adults. The rapid proliferation and diffuse brain migration are the main obstacles to successful treatment. Here, we show 25(R)-spirostan-3beta,5alpha,6beta,19-tetrol, a polyhydroxy steroid, is capable of suppressing proliferation and migration of C6 malignant glioma cells in a concentration-dependent manner. The compound 25(R)-spirostan-3beta,5alpha,6beta,19-tetrol was synthesised by seven steps starting from diosgenin in 8.55% overall yield. The structures of the synthetic compounds were characterised by infrared (IR), (1)H nuclear magnetic resonance (NMR), (13)C NMR spectra and EA.

  18. Lamellipodial tension, not integrin/ligand binding, is the crucial factor to realise integrin activation and cell migration.

    PubMed

    Schulte, Carsten; Ferraris, Gian Maria Sarra; Oldani, Amanda; Galluzzi, Massimiliano; Podestà, Alessandro; Puricelli, Luca; de Lorenzi, Valentina; Lenardi, Cristina; Milani, Paolo; Sidenius, Nicolai

    2016-01-01

    The molecular clutch (MC) model proposes that actomyosin-driven force transmission permits integrin-dependent cell migration. To investigate the MC, we introduced diverse talin (TLN) and integrin variants into Flp-In™ T-Rex™ HEK293 cells stably expressing uPAR. Vitronectin variants served as substrate providing uPAR-mediated cell adhesion and optionally integrin binding. This particular system allowed us to selectively analyse key MC proteins and interactions, effectively from the extracellular matrix substrate to intracellular f-actin, and to therewith study mechanobiological aspects of MC engagement also uncoupled from integrin/ligand binding. With this experimental approach, we found that for the initial PIP2-dependent membrane/TLN/f-actin linkage and persistent lamellipodia formation the C-terminal TLN actin binding site (ABS) is dispensable. The establishment of an adequate MC-mediated lamellipodial tension instead depends predominantly on the coupling of this C-terminal TLN ABS to the actomyosin-driven retrograde actin flow force. This lamellipodial tension is crucial for full integrin activation eventually determining integrin-dependent cell migration. In the integrin/ligand-independent condition the frictional membrane resistance participates to these processes. Integrin/ligand binding can also contribute but is not necessarily required.

  19. The Anticancer Activity of Organotelluranes: Potential Role in Integrin Inactivation.

    PubMed

    Silberman, Alon; Kalechman, Yona; Hirsch, Shira; Erlich, Ziv; Sredni, Benjamin; Albeck, Amnon

    2016-05-17

    Organic Te(IV) compounds (organotelluranes) differing in their labile ligands exhibited anti-integrin activities in vitro and anti-metastatic properties in vivo. They underwent ligand substitution with l-cysteine, as a thiol model compound. Unlike inorganic Te(IV) compounds, the organotelluranes did not form a stable complex with cysteine, but rather immediately oxidized it. The organotelluranes inhibited integrin functions, such as adhesion, migration, and metalloproteinase secretion mediation in B16F10 murine melanoma cells. In comparison, a reduced derivative with no labile ligand inhibited adhesion of B16F10 cells to a significantly lower extent, thus pointing to the importance of the labile ligands of the Te(IV) atom. One of the organotelluranes inhibited circulating cancer cells in vivo, possibly by integrin inhibition. Our results extend the current knowledge on the reactivity and mechanism of organotelluranes with different labile ligands and highlight their clinical potential.

  20. Endothelial destabilization by angiopoietin-2 via integrin β1 activation

    PubMed Central

    Hakanpaa, Laura; Sipila, Tuomas; Leppanen, Veli-Matti; Gautam, Prson; Nurmi, Harri; Jacquemet, Guillaume; Eklund, Lauri; Ivaska, Johanna; Alitalo, Kari; Saharinen, Pipsa

    2015-01-01

    Angiopoietins regulate vascular homeostasis via the endothelial Tie receptor tyrosine kinases. Angiopoietin-1 (Ang1) supports endothelial stabilization via Tie2 activation. Angiopoietin-2 (Ang2) functions as a context-dependent Tie2 agonist/antagonist promoting pathological angiogenesis, vascular permeability and inflammation. Elucidating Ang2-dependent mechanisms of vascular destablization is critical for rational design of angiopoietin antagonists that have demonstrated therapeutic efficacy in cancer trials. Here, we report that Ang2, but not Ang1, activates β1-integrin, leading to endothelial destablization. Autocrine Ang2 signalling upon Tie2 silencing, or in Ang2 transgenic mice, promotes β1-integrin-positive elongated matrix adhesions and actin stress fibres, regulating vascular endothelial-cadherin-containing cell–cell junctions. The Tie2-silenced monolayer integrity is rescued by β1-integrin, phosphoinositide-3 kinase or Rho kinase inhibition, and by re-expression of a membrane-bound Tie2 ectodomain. Furthermore, Tie2 silencing increases, whereas Ang2 blocking inhibits transendothelial tumour cell migration in vitro. These results establish Ang2-mediated β1-integrin activation as a promoter of endothelial destablization, explaining the controversial vascular functions of Ang1 and Ang2. PMID:25635707

  1. Platelet Activation, P-Selectin, and Eosinophil β1-Integrin Activation in Asthma

    PubMed Central

    Gunderson, Kristin A.; Busse, William W.; Jarjour, Nizar N.; Mosher, Deane F.

    2012-01-01

    Rationale: Eosinophil β1-integrin activation correlates inversely with FEV1 and directly with eosinophil-bound P-selectin in subjects with nonsevere allergic asthma. Objectives: Determine the relationships between β1-integrin activation and pulmonary function or eosinophil-bound P-selectin in subjects with asthma of varying severity and discern the source of eosinophil-bound P-selectin. Methods: Blood was assayed by flow cytometry for P-selectin and activated β1-integrin on eosinophils and platelets. Plasma was analyzed with ELISA for soluble P-selectin, platelet factor 4, and thrombospondin-1. Measurements and Main Results: Activated β1-integrin correlated with eosinophil-bound P-selectin among all subjects with asthma even though activated β1-integrin was higher in subjects with nonsevere asthma than severe asthma. Activated β1-integrin correlated inversely with FEV1 corrected for FVC only in younger subjects with nonsevere asthma. Paradoxically, platelet surface P-selectin, a platelet activation marker, was low in subjects with severe asthma, whereas plasma platelet factor 4, a second platelet activation marker, was high. Correlations indicated that P-selectin–positive platelets complexed to eosinophils are the major source of the eosinophil-bound P-selectin associated with β1-integrin activation. After whole-lung antigen challenge of subjects with nonsevere asthma, a model of asthma exacerbation known to cause platelet activation, circulating eosinophils bearing P-selectin and activated β1-integrin disappeared. Conclusions: The relationship between eosinophil β1-integrin activation and pulmonary function was replicated only for younger subjects with nonsevere asthma. However, we infer that platelet activation and binding of activated platelets to eosinophils followed by P-selectin–mediated eosinophil β1-integrin activation occur in both nonsevere and severe asthma with rapid movement of platelet–eosinophil complexes into the lung in more severe

  2. A proteomic approach reveals integrin activation state-dependent control of microtubule cortical targeting

    PubMed Central

    Byron, Adam; Askari, Janet A.; Humphries, Jonathan D.; Jacquemet, Guillaume; Koper, Ewa J.; Warwood, Stacey; Choi, Colin K.; Stroud, Matthew J.; Chen, Christopher S.; Knight, David; Humphries, Martin J.

    2015-01-01

    Integrin activation, which is regulated by allosteric changes in receptor conformation, enables cellular responses to the chemical, mechanical and topological features of the extracellular microenvironment. A global view of how activation state converts the molecular composition of the region proximal to integrins into functional readouts is, however, lacking. Here, using conformation-specific monoclonal antibodies, we report the isolation of integrin activation state-dependent complexes and their characterization by mass spectrometry. Quantitative comparisons, integrating network, clustering, pathway and image analyses, define multiple functional protein modules enriched in a conformation-specific manner. Notably, active integrin complexes are specifically enriched for proteins associated with microtubule-based functions. Visualization of microtubules on micropatterned surfaces and live cell imaging demonstrate that active integrins establish an environment that stabilizes microtubules at the cell periphery. These data provide a resource for the interrogation of the global molecular connections that link integrin activation to adhesion signalling. PMID:25609142

  3. Coordinated integrin activation by actin-dependent force during T-cell migration.

    PubMed

    Nordenfelt, Pontus; Elliott, Hunter L; Springer, Timothy A

    2016-10-10

    For a cell to move forward it must convert chemical energy into mechanical propulsion. Force produced by actin polymerization can generate traction across the plasma membrane by transmission through integrins to their ligands. However, the role this force plays in integrin activation is unknown. Here we show that integrin activity and cytoskeletal dynamics are reciprocally linked, where actin-dependent force itself appears to regulate integrin activity. We generated fluorescent tension-sensing constructs of integrin αLβ2 (LFA-1) to visualize intramolecular tension during cell migration. Using quantitative imaging of migrating T cells, we correlate tension in the αL or β2 subunit with cell and actin dynamics. We find that actin engagement produces tension within the β2 subunit to induce and stabilize an active integrin conformational state and that this requires intact talin and kindlin motifs. This supports a general mechanism where localized actin polymerization can coordinate activation of the complex machinery required for cell migration.

  4. 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.

  5. 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.

  6. Suppression of integrin activation by activated Ras or Raf does not correlate with bulk activation of ERK MAP kinase.

    PubMed

    Hughes, Paul E; Oertli, Beat; Hansen, Malene; Chou, Fan-Li; Willumsen, Berthe M; Ginsberg, Mark H

    2002-07-01

    The rapid modulation of ligand-binding affinity ("activation") is a central property of the integrin family of cell adhesion receptors. The Ras family of small GTP-binding proteins and their downstream effectors are key players in regulating integrin activation. H-Ras can suppress integrin activation in fibroblasts via its downstream effector kinase, Raf-1. In contrast, to H-Ras, a closely related small GTP-binding protein R-Ras has the opposite activity, and promotes integrin activation. To gain insight into the regulation of integrin activation by Ras GTPases, we created a series of H-Ras/R-Ras chimeras. We found that a 35-amino acid stretch of H-Ras was required for full suppressive activity. Furthermore, the suppressive chimeras were weak activators of the ERK1/2 MAP kinase pathway, suggesting that the suppression of integrin activation may be independent of the activation of the bulk of ERK MAP kinase. Additional data demonstrating that the ability of H-Ras or Raf-1 to suppress integrin activation was unaffected by inhibition of bulk ERK1/2 MAP kinase activation supported this hypothesis. Thus, the suppression of integrin activation is a Raf kinase induced regulatory event that can be mediated independently of bulk activation of the ERK MAP-kinase pathway.

  7. Conformational equilibria and intrinsic affinities define integrin activation.

    PubMed

    Li, Jing; Su, Yang; Xia, Wei; Qin, Yan; Humphries, Martin J; Vestweber, Dietmar; Cabañas, Carlos; Lu, Chafen; Springer, Timothy A

    2017-03-01

    We show that the three conformational states of integrin α5β1 have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000-fold higher affinity of the extended-open state than the bent-closed and extended-closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α5β1 On the surface of K562 cells, α5β1 is 99.8% bent-closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N-glycans and leg domains in each subunit that connect the ligand-binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single-span transmembrane domains.

  8. Vinculin activators target integrins from within the cell to increase melanoma sensitivity to chemotherapy

    PubMed Central

    Nelson, Elke S.; Folkmann, Andrew W.; Henry, Michael D.; DeMali, Kris A.

    2011-01-01

    Metastatic melanoma is an aggressive skin disease for which there are no effective therapies. Emerging evidence indicates that melanomas can be sensitized to chemotherapy by increasing integrin function. Current integrin therapies work by targeting the extracellular domain, resulting in complete gains or losses of integrin function that lead to mechanism-based toxicities. An attractive alternative approach is to target proteins, such as vinculin, that associate with the integrin cytoplasmic domains and regulate its ligand binding properties. Here we report that a novel reagent, denoted vinculin activating peptide or VAP, increases integrin activity from within the cell, as measured by elevated: (1) numbers of active integrins, (2) adhesion of cells to extracellular matrix ligands, (3) numbers of cell-matrix adhesions, and (4) downstream signaling. These effects are dependent on both integrins and a key regulatory residue A50 in the vinculin head domain. We further show that VAP dramatically increases the sensitivity of melanomas to chemotherapy in clonal growth assays and in vivo mouse models of melanoma. Finally, we demonstrate that the increase in chemosensitivity results from increases in DNA damage-induced apoptosis in a p53-dependent manner. Collectively these findings demonstrate for the first time that integrin function can be manipulated from within the cell and validate integrins as a new therapeutic target for the treatment of chemoresistant melanomas. PMID:21460181

  9. Integrin Cytoplasmic Tail Interactions

    PubMed Central

    2015-01-01

    Integrins are heterodimeric cell surface adhesion receptors essential for multicellular life. They connect cells to the extracellular environment and transduce chemical and mechanical signals to and from the cell. Intracellular proteins that bind the integrin cytoplasmic tail regulate integrin engagement of extracellular ligands as well as integrin localization and trafficking. Cytoplasmic integrin-binding proteins also function downstream of integrins, mediating links to the cytoskeleton and to signaling cascades that impact cell motility, growth, and survival. Here, we review key integrin-interacting proteins and their roles in regulating integrin activity, localization, and signaling. PMID:24467163

  10. Genetic perturbation of the putative cytoplasmic membrane-proximal salt bridge aberrantly activates α4 integrins

    PubMed Central

    Imai, Yoichi; Park, Eun Jeong; Peer, Dan; Peixoto, António; Cheng, Guiying; von Andrian, Ulrich H.; Carman, Christopher V.

    2008-01-01

    α4 integrins play a pivotal role in leukocyte migration and tissue-specific homing. The ability of integrins to bind ligand is dynamically regulated by activation-dependent conformational changes triggered in the cytoplasmic domain. An NMR solution structure defined a putative membrane-proximal salt bridge between the αIIbβ3 integrin cytoplasmic tails, which restrains integrins in their low-affinity state. However, the physiological importance of this salt bridge in α4 integrin regulation remains to be elucidated. To address this question, we disrupted the salt bridge in murine germ line by mutating the conserved cytoplasmic arginine RGFFKR in α4 integrins. In lymphocytes from knock-in mice (α4-R/AGFFKR), α4β1 and α4β7 integrins exhibited constitutively up-regulated ligand binding. However, transmigration of these cells across VCAM-1 and MAdCAM-1 substrates, or across endothelial monolayers, was reduced. Perturbed detachment of the tail appeared to cause the reduced cell migration of α4-R/AGFFKR lymphocytes. In vivo, α4-R/AGFFKR cells exhibited increased firm adhesion to Peyer patch venules but reduced homing to the gut. Our results demonstrate that the membrane-proximal salt bridge plays a critical role in supporting proper α4 integrin adhesive dynamics. Loss of this interaction destabilizes the nonadhesive conformation, and thereby perturbs the properly balanced cycles of adhesion and deadhesion required for efficient cell migration. PMID:18809756

  11. Integrin-dependent Control of Translation: Engagement of Integrin αIIbβ3 Regulates Synthesis of Proteins in Activated Human Platelets

    PubMed Central

    Pabla, Ravinder; Weyrich, Andrew S.; Dixon, Dan A.; Bray, Paul F.; McIntyre, Thomas M.; Prescott, Stephen M.; Zimmerman, Guy A.

    1999-01-01

    Integrins are widely expressed plasma membrane adhesion molecules that tether cells to matrix proteins and to one another in cell–cell interactions. Integrins also transmit outside-in signals that regulate functional responses of cells, and are known to influence gene expression by regulating transcription. In previous studies we found that platelets, which are naturally occurring anucleate cytoplasts, translate preformed mRNA transcripts when they are activated by outside-in signals. Using strategies that interrupt engagement of integrin αIIbβ3 by fibrinogen and platelets deficient in this integrin, we found that αIIbβ3 regulates the synthesis of B cell lymphoma 3 (Bcl-3) when platelet aggregation is induced by thrombin. We also found that synthesis of Bcl-3, which occurs via a specialized translation control pathway regulated by mammalian target of rapamycin (mTOR), is induced when platelets adhere to immobilized fibrinogen in the absence of thrombin and when integrin αIIbβ3 is engaged by a conformation-altering antibody against integrin αIIbβ3. Thus, outside-in signals delivered by integrin αIIbβ3 are required for translation of Bcl-3 in thrombin-stimulated aggregated platelets and are sufficient to induce translation of this marker protein in the absence of thrombin. Engagement of integrin α2β1 by collagen also triggered synthesis of Bcl-3. Thus, control of translation may be a general mechanism by which surface adhesion molecules regulate gene expression. PMID:9885253

  12. An integrin from oyster Crassostrea gigas mediates the phagocytosis toward Vibrio splendidus through LPS binding activity.

    PubMed

    Jia, Zhihao; Zhang, Tao; Jiang, Shuai; Wang, Mengqiang; Cheng, Qi; Sun, Mingzhe; Wang, Lingling; Song, Linsheng

    2015-11-01

    Integrins are a family of cell adhesion molecules which play important roles in the regulation of cell adhesion, migration, proliferation, apoptosis and phagocytosis. In the present study, the immune function of an integrin from the oyster Crassostrea gigas (designated CgIntegrin) was characterized to understand the regulatory mechanism of hemocyte phagocytosis toward different microbes. The full-length cDNA of CgIntegrin was 2571 bp with an open reading frame (ORF) of 2397 bp, encoding a polypeptide of 799 amino acids. The mRNA transcripts of CgIntegrin were predominantly detected in hemocytes, gonad and adductor muscle, while lowly in hepatopancreas, mantle and gill. The mRNA expression level was up-regulated at 6 h post lipopolysaccharide (LPS) stimulation (p < 0.01), while no significant change was observed after peptidoglycan (PGN) stimulation. The oyster hemocytes with relative high CgIntegrin expression level exhibited different phagocytic abilities towards different microorganism and particles, such as Gram-positive bacteria Vibrio splendidus, Gram-negative bacteria Staphylococcus aureus and latex beads. Moreover, the phagocytic rate towards V. splendidus was significantly decreased after the blockade of CgIntegrin using the polyclonal antibody. The recombinant CgIntegrin (rCgIntegrin) displayed agglutinating activity towards V. splendidus but not S. aureus and Y. lipolytica. It also exhibited a higher binding affinity towards LPS (compared to rTrx group) in a dose-dependent manner with the apparent dissociation constant (Kd) of 5.53 × 10(-6) M. The results indicated that CgIntegrin served as a pattern recognition receptor with LPS binding activity, which could directly bind to V. splendidus and enhance the phagocytosis of oyster hemocytes.

  13. The Neutrophil Btk Signalosome Regulates Integrin Activation during Sterile Inflammation

    PubMed Central

    Volmering, Stephanie; Block, Helena; Boras, Mark; Lowell, Clifford A.; Zarbock, Alexander

    2016-01-01

    SUMMARY Neutrophils are recruited from the blood to sites of sterile inflammation, where they are involved in wound healing but can also cause tissue damage. During sterile inflammation, necrotic cells release pro-inflammatory molecules including formylated peptides. However, the signaling pathway triggered by formylated peptides to integrin activation and leukocyte recruitment is unknown. By using spinning-disk confocal intravital microscopy, we examined the molecular mechanisms of leukocyte recruitment to sites of focal hepatic necrosis in vivo. We demonstrated that the Bruton’s tyrosine kinase (Btk) was required for multiple Mac-1 activation events involved in neutrophil recruitment and functions during sterile inflammation triggered by fMLF. The Src family kinase Hck, Wiskott-Aldrich-syndrome protein, and phospholipase Cγ2 were also involved in this pathway required for fMLF-triggered Mac-1 activation and neutrophil recruitment. Thus, we have identified a neutrophil Btk signalosome that is involved in a signaling pathway triggered by formylated peptides leading to the selective activation of Mac-1 and neutrophil recruitment during sterile inflammation. PMID:26777396

  14. Tiam1 is recruited to β1-integrin complexes by 14-3-3ζ where it mediates integrin-induced Rac1 activation and motility.

    PubMed

    O'Toole, Timothy E; Bialkowska, Katarzyna; Li, Xiaohong; Fox, Joan E B

    2011-11-01

    14-3-3 is an adaptor protein that localizes to the leading edge of spreading cells, returning to the cytoplasm as spreading ceases. Previously, we showed that integrin-induced Rac1 activation and spreading were inhibited by sequestration of 14-3-3ζ and restored by its overexpression. Here, we determined whether 14-3-3 mediates integrin signaling by localizing a guanine nucleotide exchange factor (GEF) to Rac1-activating integrin complexes. We showed that GST-14-3-3ζ recruited the Rac1-GEF, Tiam1, from cell lysates through Tiam1 residues 1-182 (N(1-182) Tiam1). The physiological relevance of this interaction was examined in serum-starved Hela cells plated on fibronectin. Both Tiam1 and N(1-182) Tiam1 were recruited to 14-3-3-containing β1-integrin complexes, as shown by co-localization and co-immunoprecipitation. Integrin-induced Rac1 activation was inhibited when Tiam1 was depleted with siRNA or by overexpression of catalytically inactive N(1-182) Tiam1, which was incorporated into 14-3-3/β1-integrin complexes and inhibited spreading in a manner that was overcome by constitutively active Rac1. Integrin-induced Rac1 activation, spreading, and migration were also inhibited by overexpression of 14-3-3ζ S58D, which was unable to recruit Tiam1 from lysates, co-immunoprecipitate with Tiam1, or mediate its incorporation into β1-integrin complexes. Taken together, these findings suggest a previously unrecognized mechanism of integrin-induced Rac1 activation in which 14-3-3 dimers localize Tiam1 to integrin complexes, where it mediates integrin-dependent Rac1 activation, thus initiating motility-inducing pathways. Moreover, since Tiam1 is recruited to other sites of localized Rac1 activation through its PH-CC-EX domain, the present findings show that a mechanism involving its N-terminal 182 residues is utilized to recruit Tiam1 to motility-inducing integrin complexes. Copyright © 2011 Wiley-Liss, Inc.

  15. Upregulation and activation of eosinophil integrins in blood and airway after segmental lung antigen challenge1

    PubMed Central

    Johansson, Mats W.; Kelly, Elizabeth A. B.; Busse, William W.; Jarjour, Nizar N.; Mosher, Deane F.

    2008-01-01

    We hypothesized that there are clinically relevant differences in eosinophil integrin expression and activation in patients with asthma. To evaluate this, surface densities and activation states of integrins on eosinophils in blood and bronchoalveolar lavage (BAL) of 19 asthmatic subjects were studied before and 48 h after segmental Ag challenge. At 48 h, there was increased expression of αD and the N29 epitope of activated β1 integrins on blood eosinophils and of αM, β2, and the mAb24 epitope of activated β2 integrins on airway eosinophils. Changes correlated with the late-phase fall in forced expiratory volume in 1 s (FEV1) after whole-lung inhalation of the Ag that was subsequently used in segmental challenge and were greater in subjects defined as dual responders. Increased surface densities of αM and β2 and activation of β2 on airway eosinophils correlated with the concentration of IL-5 in BAL fluid. Activation of β1 and β2 on airway eosinophils correlated with eosinophil percentage in BAL. Thus, eosinophils respond to an allergic stimulus by activation of integrins in a sequence that likely promotes eosinophilic inflammation of the airway. Before challenge, β1 and β2 integrins of circulating eosinophils are in low-activation conformations, and αDβ2 surface expression is low. After Ag challenge, circulating eosinophils adopt a phenotype with activated β1 integrins and upregulated αDβ2, changes that are predicted to facilitate eosinophil arrest on VCAM-1 in bronchial vessels. Finally, eosinophils present in IL-5-rich airway fluid have a hyperadhesive phenotype associated with increased surface expression of αMβ2 and activation of β2 integrins. PMID:18490765

  16. Exclusion of Integrins from CNS Axons Is Regulated by Arf6 Activation and the AIS

    PubMed Central

    Franssen, Elske H. P.; Zhao, Rong-Rong; Koseki, Hiroaki; Kanamarlapudi, Venkateswarlu; Hoogenraad, Casper C.

    2015-01-01

    Integrins are adhesion and survival molecules involved in axon growth during CNS development, as well as axon regeneration after injury in the peripheral nervous system (PNS). Adult CNS axons do not regenerate after injury, partly due to a low intrinsic growth capacity. We have previously studied the role of integrins in axon growth in PNS axons; in the present study, we investigate whether integrin mechanisms involved in PNS regeneration may be altered or lacking from mature CNS axons by studying maturing CNS neurons in vitro. In rat cortical neurons, we find that integrins are present in axons during initial growth but later become restricted to the somato-dendritic domain. We investigated how this occurs and whether it can be altered to enhance axonal growth potential. We find a developmental change in integrin trafficking; transport becomes predominantly retrograde throughout axons, but not dendrites, as neurons mature. The directionality of transport is controlled through the activation state of ARF6, with developmental upregulation of the ARF6 GEF ARNO enhancing retrograde transport. Lowering ARF6 activity in mature neurons restores anterograde integrin flow, allows transport into axons, and increases axon growth. In addition, we found that the axon initial segment is partly responsible for exclusion of integrins and removal of this structure allows integrins into axons. Changing posttranslational modifications of tubulin with taxol also allows integrins into the proximal axon. The experiments suggest that the developmental loss of regenerative ability in CNS axons is due to exclusion of growth-related molecules due to changes in trafficking. PMID:26019348

  17. Akt1 Signaling Regulates Integrin Activation, Matrix Recognition, and Fibronectin Assembly*S

    PubMed Central

    Somanath, Payaningal R.; Kandel, Eugene S.; Hay, Nissim; Byzova, Tatiana V.

    2009-01-01

    Akt, a serine-threonine kinase, regulates multiple cellular processes in vascular cells. We have previously documented that Akt activates integrins and Akt1 deficiency results in matrix abnormalities in skin and blood vessels in vivo. Based on these observations, we hypothesized that Akt1 is necessary for integrin activation and matrix assembly by fibroblasts. In this study, using various cell systems, we show that Akt1 is essential for the inside-out activation of integrins in endothelial cells and fibroblasts, which in turn, mediates matrix assembly. Fibronectin is a major extracellular matrix component of the skin and the vascular basement membrane, which possesses binding sites for many integrins and extracellular matrix proteins. Akt1−/− fibroblasts and NIH fibroblasts expressing dominant negative Akt1 (K179M-Akt1) showed impaired fibronectin assembly compared with control fibroblasts. In contrast, expression of constitutively active Akt1 (myrAkt1) resulted in enhanced fibronectin assembly. Although increased fibronectin assembly by myrAkt1-expressing human foreskin fibroblasts was abolished by treatment with anti-integrin β1 blocking antibodies, treatment with β1-stimulating antibodies rescued the impaired fibronectin assembly that was due to lack of Akt activity. Finally, expression of myrAkt1 corrected the phenotype of Akt1−/− fibroblasts thus showing that Akt1 regulates fibronectin assembly through activation of integrin α5β1. PMID:17562714

  18. Applications of snake venom components to modulate integrin activities in cell-matrix interactions

    PubMed Central

    Marcinkiewicz, Cezary

    2013-01-01

    Snake venom proteins are broadly investigated in the different areas of life science. Direct interaction of these compounds with cells may involve a variety of mechanisms that result in diverse cellular responses leading to the activation or blocking of physiological functions of the cell. In this review, the snake venom components interacting with integrins will be characterized in context of their effect on cellular response. Currently, two major families of snake venom proteins are considered as integrin-binding molecules. The most attention has been devoted to the disintegrin family, which binds certain types of integrins through specific motifs recognized as a tri-peptide structurally localized on an integrin-binding loop. Other snake venom integrin-binding proteins belong to the C-type lectin family. Snake venom molecules bind to the cellular integrins resulting in a modulation of cell signaling and in consequence, the regulation of cell proliferation, migration and apoptosis. Therefore, snake venom research on the integrin-binding molecules may have significance in biomedicine and basic cell biology. PMID:23811033

  19. Low affinity binding in cis to P2Y2R mediates force-dependent integrin activation during hantavirus infection.

    PubMed

    Bondu, Virginie; Wu, Chenyu; Cao, Wenpeng; Simons, Peter C; Gillette, Jennifer; Zhu, Jieqing; Erb, Laurie; Zhang, X Frank; Buranda, Tione

    2017-08-23

    Pathogenic hantaviruses bind to the Plexin Semaphorin Integrin (PSI) domain of inactive, β3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation β5/β3 integrins and an RGD sequence in the first extracellular loop of P2Y2R (Erb et al., 2001). With single-molecule atomic force microscopy, we show a cognate interaction between (RGD)P2Y2R and an AFM tip decorated with recombinant αIIbβ3 integrins expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y2R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y2R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing αIIbβ3 integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation specific mAB PAC1. The data also show that blocking of the Gα13 protein from binding to the cytoplasmic domain of the β3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y2R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force, yields physiological integrin activation. © 2017 by The American Society for Cell Biology.

  20. Coordinated integrin activation by actin-dependent force during T-cell migration

    PubMed Central

    Nordenfelt, Pontus; Elliott, Hunter L.; Springer, Timothy A.

    2016-01-01

    For a cell to move forward it must convert chemical energy into mechanical propulsion. Force produced by actin polymerization can generate traction across the plasma membrane by transmission through integrins to their ligands. However, the role this force plays in integrin activation is unknown. Here we show that integrin activity and cytoskeletal dynamics are reciprocally linked, where actin-dependent force itself appears to regulate integrin activity. We generated fluorescent tension-sensing constructs of integrin αLβ2 (LFA-1) to visualize intramolecular tension during cell migration. Using quantitative imaging of migrating T cells, we correlate tension in the αL or β2 subunit with cell and actin dynamics. We find that actin engagement produces tension within the β2 subunit to induce and stabilize an active integrin conformational state and that this requires intact talin and kindlin motifs. This supports a general mechanism where localized actin polymerization can coordinate activation of the complex machinery required for cell migration. PMID:27721490

  1. Acidic Extracellular pH Promotes Activation of Integrin αvβ3

    PubMed Central

    Paradise, Ranjani K.; Lauffenburger, Douglas A.; Van Vliet, Krystyn J.

    2011-01-01

    Acidic extracellular pH is characteristic of the cell microenvironment in several important physiological and pathological contexts. Although it is well established that acidic extracellular pH can have profound effects on processes such as cell adhesion and migration, the underlying molecular mechanisms are largely unknown. Integrin receptors physically connect cells to the extracellular matrix, and are thus likely to modulate cell responses to extracellular conditions. Here, we examine the role of acidic extracellular pH in regulating activation of integrin αvβ3. Through computational molecular dynamics simulations, we find that acidic extracellular pH promotes opening of the αvβ3 headpiece, indicating that acidic pH can thereby facilitate integrin activation. This prediction is consistent with our flow cytometry and atomic force microscope-mediated force spectroscopy assays of integrin αvβ3 on live cells, which both demonstrate that acidic pH promotes activation at the intact cell surface. Finally, quantification of cell morphology and migration measurements shows that acidic extracellular pH affects cell behavior in a manner that is consistent with increased integrin activation. Taken together, these computational and experimental results suggest a new and complementary mechanism of integrin activation regulation, with associated implications for cell adhesion and migration in regions of altered pH that are relevant to wound healing and cancer. PMID:21283814

  2. 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.

  3. Direct interactions with the integrin β1 cytoplasmic tail activate the Abl2/Arg kinase.

    PubMed

    Simpson, Mark A; Bradley, William D; Harburger, David; Parsons, Maddy; Calderwood, David A; Koleske, Anthony J

    2015-03-27

    Integrins are heterodimeric α/β extracellular matrix adhesion receptors that couple physically to the actin cytoskeleton and regulate kinase signaling pathways to control cytoskeletal remodeling and adhesion complex formation and disassembly. β1 integrins signal through the Abl2/Arg (Abl-related gene) nonreceptor tyrosine kinase to control fibroblast cell motility, neuronal dendrite morphogenesis and stability, and cancer cell invasiveness, but the molecular mechanisms by which integrin β1 activates Arg are unknown. We report here that the Arg kinase domain interacts directly with a lysine-rich membrane-proximal segment in the integrin β1 cytoplasmic tail, that Arg phosphorylates the membrane-proximal Tyr-783 in the β1 tail, and that the Arg Src homology domain then engages this phosphorylated region in the tail. We show that these interactions mediate direct binding between integrin β1 and Arg in vitro and in cells and activate Arg kinase activity. These findings provide a model for understanding how β1-containing integrins interact with and activate Abl family kinases.

  4. Activation of integrins in endothelial cells by fluid shear stress mediates Rho-dependent cytoskeletal alignment

    PubMed Central

    Tzima, Eleni; del Pozo, Miguel Angel; Shattil, Sanford J.; Chien, Shu; Schwartz, Martin Alexander

    2001-01-01

    Fluid shear stress is a critical determinant of vascular remodeling and atherogenesis. Both integrins and the small GTPase Rho are implicated in endothelial cell responses to shear but the mechanisms are poorly understood. We now show that shear stress rapidly stimulates conformational activation of integrin αvβ3 in bovine aortic endothelial cells, followed by an increase in its binding to extracellular cell matrix (ECM) proteins. The shear-induced new integrin binding to ECM induces a transient inactivation of Rho similar to that seen when suspended cells are plated on ECM proteins. This transient inhibition is necessary for cytoskeletal alignment in the direction of flow. The results therefore define the role of integrins and Rho in a pathway leading to endothelial cell adaptation to flow. PMID:11532928

  5. P-Selectin Activates Integrin-mediated Colon Carcinoma Cell Adhesion to Fibronectin

    PubMed Central

    Reyes-Reyes, E. Merit; George, Margaret D.; Roberts, John D.; Akiyama, Steven K.

    2006-01-01

    During hematogenous cancer metastasis, tumor cells separate from a primary mass, enter the bloodstream, disperse throughout the body, migrate across vessel walls, and generate distant colonies. The later steps of metastasis superficially resemble leukocyte extravasation, a process initiated by selectin-mediated cell tethering to the blood vessel wall followed by integrin-mediated arrest and transendothelial migration. Some cancer cells express P-selectin ligands and attach to immobilized Pselectin, suggesting that these cells can arrest in blood vessels using sequential selectin- and integrin-mediated adhesion, as do leukocytes. We hypothesize that selectin binding may regulate subsequent integrin-mediated steps in metastasis. Using a model system of cultured Colo 320 human colon adenocarcinoma cells incubated with soluble P-selectin-IgG chimeric protein, we have found that P-selectin can stimulate activation of the α5β1 integrin resulting in a specific increase of adhesion and spreading of these cells on fibronectin substrates. P-selectin binding also induced activation of p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (PI3-K). PI3-K inhibitors blocked P-selectin-mediated integrin activation, cell attachment, and cell spreading. Inhibition of p38 MAPK activation blocked cell spreading, but not cell attachment. P-Selectin binding also resulted in formation of a signaling complex containing PI3-K and p38 MAPK. These results suggest that P-selectin binding to tumor cells can activate α5β1 integrin via PI3-K and p38 MAPK signaling pathways leading to increased cell adhesion. We propose that P-selectin ligands are important tumor cell signaling molecules that modulate integrin-mediated cell adhesion in the metastatic process. PMID:17056038

  6. Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation

    PubMed Central

    Svensson, Lena; Howarth, Kimberley; McDowall, Alison; Patzak, Irene; Evans, Rachel; Ussar, Siegfried; Moser, Markus; Metin, Ayse; Fried, Mike; Tomlinson, Ian; Hogg, Nancy

    2009-01-01

    Integrins are the major adhesion receptors of leukocytes and platelets. β1 and β2 integrin function on leukocytes is crucial for a successful immune response and the platelet integrin αIIbβ3 initiates the process of blood clotting through binding fibrinogen1-3. Integrins on circulating cells bind poorly to their ligands but become active after ‘inside-out’ signaling through other membrane receptors4,5. Subjects with leukocyte adhesion deficiency-1 (LAD-I) do not express β2 integrins because of mutations in the gene specifying the β2 subunit, and they suffer recurrent bacterial infections6,7. Mutations affecting αIIbβ3 integrin cause the bleeding disorder termed Glanzmann’s thrombasthenia3. Subjects with LAD-III show symptoms of both LAD-I and Glanzmann’s thrombasthenia. Their hematopoietically-derived cells express β1, β2 and β3 integrins, but defective inside-out signaling causes immune deficiency and bleeding problems8. The LAD-III lesion has been attributed to a C→A mutation in the gene encoding calcium and diacylglycerol guanine nucleotide exchange factor (CALDAGGEF1; official symbol RASGRP2) specifying the CALDAG-GEF1 protein9, but we show that this change is not responsible for the LAD-III disorder. Instead, we identify mutations in the KINDLIN3 (official symbol FERMT3) gene specifying the KINDLIN-3 protein as the cause of LAD-III in Maltese and Turkish subjects. Two independent mutations result in decreased KINDLIN3 messenger RNA levels and loss of protein expression. Notably, transfection of the subjects’ lymphocytes with KINDLIN3 complementary DNA but not CALDAGGEF1 cDNA reverses the LAD-III defect, restoring integrin-mediated adhesion and migration. PMID:19234463

  7. Hydrogels with precisely controlled integrin activation dictate vascular patterning and permeability.

    PubMed

    Li, Shuoran; Nih, Lina R; Bachman, Haylee; Fei, Peng; Li, Yilei; Nam, Eunwoo; Dimatteo, Robert; Carmichael, S Thomas; Barker, Thomas H; Segura, Tatiana

    2017-09-01

    Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here, we show that through engineering hydrogel materials to promote α3/α5β1 integrin binding, we can promote the formation of a space-filling and mature vasculature compared with hydrogel materials that promote αvβ3 integrin binding. In vitro, α3/α5β1 scaffolds promoted endothelial cells to sprout and branch, forming organized extensive networks that eventually reached and anastomosed with neighbouring branches. In vivo, α3/α5β1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel formation and non-leaky blood vessels by 10 days post-stroke. In contrast, materials that promote αvβ3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF-induced vascular permeability in vivo.

  8. Hydrogels with precisely controlled integrin activation dictate vascular patterning and permeability

    NASA Astrophysics Data System (ADS)

    Li, Shuoran; Nih, Lina R.; Bachman, Haylee; Fei, Peng; Li, Yilei; Nam, Eunwoo; Dimatteo, Robert; Carmichael, S. Thomas; Barker, Thomas H.; Segura, Tatiana

    2017-09-01

    Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here, we show that through engineering hydrogel materials to promote α3/α5β1 integrin binding, we can promote the formation of a space-filling and mature vasculature compared with hydrogel materials that promote αvβ3 integrin binding. In vitro, α3/α5β1 scaffolds promoted endothelial cells to sprout and branch, forming organized extensive networks that eventually reached and anastomosed with neighbouring branches. In vivo, α3/α5β1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel formation and non-leaky blood vessels by 10 days post-stroke. In contrast, materials that promote αvβ3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF-induced vascular permeability in vivo.

  9. 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

  10. Integrin Activation Through the Hematopoietic Adapter Molecule ADAP Regulates Dendritic Development of Hippocampal Neurons.

    PubMed

    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.

  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. Expression of an Activated Integrin Promotes Long-Distance Sensory Axon Regeneration in the Spinal Cord.

    PubMed

    Cheah, Menghon; Andrews, Melissa R; Chew, Daniel J; Moloney, Elizabeth B; Verhaagen, Joost; Fässler, Reinhard; Fawcett, James W

    2016-07-06

    After CNS injury, axon regeneration is blocked by an inhibitory environment consisting of the highly upregulated tenascin-C and chondroitin sulfate proteoglycans (CSPGs). Tenascin-C promotes growth of axons if they express a tenascin-binding integrin, particularly α9β1. Additionally, integrins can be inactivated by CSPGs, and this inhibition can be overcome by the presence of a β1-binding integrin activator, kindlin-1. We examined the synergistic effect of α9 integrin and kindlin-1 on sensory axon regeneration in adult rat spinal cord after dorsal root crush and adeno-associated virus transgene expression in dorsal root ganglia. After 12 weeks, axons from C6-C7 dorsal root ganglia regenerated through the tenascin-C-rich dorsal root entry zone into the dorsal column up to C1 level and above (>25 mm axon length) through a normal pathway. Animals also showed anatomical and electrophysiological evidence of reconnection to the dorsal horn and behavioral recovery in mechanical pressure, thermal pain, and ladder-walking tasks. Expression of α9 integrin or kindlin-1 alone promoted much less regeneration and recovery. The study demonstrates that long-distance sensory axon regeneration over a normal pathway and with sensory and sensory-motor recovery can be achieved. This was achieved by expressing an integrin that recognizes tenascin-C, one of the components of glial scar tissue, and an integrin activator. This enabled extensive long-distance (>25 mm) regeneration of both myelinated and unmyelinated sensory axons with topographically correct connections in the spinal cord. The extent of growth and recovery we have seen would probably be clinically significant. Restoration of sensation to hands, perineum, and genitalia would be a significant improvement for a spinal cord-injured patient. Copyright © 2016 CHEAH et al.

  13. Expression of an Activated Integrin Promotes Long-Distance Sensory Axon Regeneration in the Spinal Cord

    PubMed Central

    Cheah, Menghon; Chew, Daniel J.; Moloney, Elizabeth B.; Verhaagen, Joost; Fässler, Reinhard

    2016-01-01

    After CNS injury, axon regeneration is blocked by an inhibitory environment consisting of the highly upregulated tenascin-C and chondroitin sulfate proteoglycans (CSPGs). Tenascin-C promotes growth of axons if they express a tenascin-binding integrin, particularly α9β1. Additionally, integrins can be inactivated by CSPGs, and this inhibition can be overcome by the presence of a β1-binding integrin activator, kindlin-1. We examined the synergistic effect of α9 integrin and kindlin-1 on sensory axon regeneration in adult rat spinal cord after dorsal root crush and adeno-associated virus transgene expression in dorsal root ganglia. After 12 weeks, axons from C6–C7 dorsal root ganglia regenerated through the tenascin-C-rich dorsal root entry zone into the dorsal column up to C1 level and above (>25 mm axon length) through a normal pathway. Animals also showed anatomical and electrophysiological evidence of reconnection to the dorsal horn and behavioral recovery in mechanical pressure, thermal pain, and ladder-walking tasks. Expression of α9 integrin or kindlin-1 alone promoted much less regeneration and recovery. SIGNIFICANCE STATEMENT The study demonstrates that long-distance sensory axon regeneration over a normal pathway and with sensory and sensory–motor recovery can be achieved. This was achieved by expressing an integrin that recognizes tenascin-C, one of the components of glial scar tissue, and an integrin activator. This enabled extensive long-distance (>25 mm) regeneration of both myelinated and unmyelinated sensory axons with topographically correct connections in the spinal cord. The extent of growth and recovery we have seen would probably be clinically significant. Restoration of sensation to hands, perineum, and genitalia would be a significant improvement for a spinal cord-injured patient. PMID:27383601

  14. Mechanical stress-activated integrin α5β1 induces opening of connexin 43 hemichannels.

    PubMed

    Batra, Nidhi; Burra, Sirisha; Siller-Jackson, Arlene J; Gu, Sumin; Xia, Xuechun; Weber, Gregory F; DeSimone, Douglas; Bonewald, Lynda F; Lafer, Eileen M; Sprague, Eugene; Schwartz, Martin A; Jiang, Jean X

    2012-02-28

    The connexin 43 (Cx43) hemichannel (HC) in the mechanosensory osteocytes is a major portal for the release of factors responsible for the anabolic effects of mechanical loading on bone formation and remodeling. However, little is known about how the Cx43 molecule responds to mechanical stimulation leading to the opening of the HC. Here, we demonstrate that integrin α5β1 interacts directly with Cx43 and that this interaction is required for mechanical stimulation-induced opening of the Cx43 HC. Direct mechanical perturbation via magnetic beads or conformational activation of integrin α5β1 leads to the opening of the Cx43 HC, and this role of the integrin is independent of its association with an extracellular fibronectin substrate. PI3K signaling is responsible for the shear stress-induced conformational activation of integrin α5β1 leading to the opening of the HC. These results identify an unconventional function of integrin that acts as a mechanical tether to induce opening of the HC and provide a mechanism connecting the effect of mechanical forces directly to anabolic function of the bone.

  15. THE TMEFF2 TUMOR SUPPRESSOR MODULATES INTEGRIN EXPRESSION, RHOA ACTIVATION AND MIGRATION OF PROSTATE CANCER CELLS

    PubMed Central

    Chen, Xiaofei; Corbin, Joshua M.; Tipton, Greg J.; Yang, Li V.; Asch, Adam S.; Ruiz-Echevarría, Maria J.

    2014-01-01

    Cell adhesion and migration play important roles in physiological and pathological states, including embryonic development and cancer invasion and metastasis. The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed mainly in brain and prostate and its expression is deregulated in prostate cancer. We have previously shown that TMEFF2 can function as a tumor suppressor by inhibiting cell migration and invasion of prostate cells. However, the molecular mechanisms involved in this inhibition are not clear. In this study we demonstrate that TMEFF2 affects cell adhesion and migration of prostate cancer cells and that this effect correlates with changes in integrin expression and RhoA activation. Deletion of a 13 basic-rich amino acid region in the cytoplasmic domain of TMEFF2 prevented these effects. Overexpression of TMEFF2 reduced cell attachment and migration on vitronectin and caused a concomitant decrease in RhoA activation, stress fiber formation and expression of αv, β1 and β3 integrin subunits. Conversely, TMEFF2 interference in 22Rv1 prostate cancer cells resulted in increased integrin expression. Results obtained with a double TRAMP/TMEFF2 transgenic mouse also indicated that TMEFF2 expression reduced integrin expression in the mouse prostate. In summary, the data presented here indicate an important role of TMEFF2 in regulating cell adhesion and migration that involves integrin signaling and is mediated by its cytoplasmic domain. PMID:24632071

  16. Activation of integrin α5 mediated by flow requires its translocation to membrane lipid rafts in vascular endothelial cells.

    PubMed

    Sun, Xiaoli; Fu, Yi; Gu, Mingxia; Zhang, Lu; Li, Dan; Li, Hongliang; Chien, Shu; Shyy, John Y-J; Zhu, Yi

    2016-01-19

    Local flow patterns determine the uneven distribution of atherosclerotic lesions. Membrane lipid rafts and integrins are crucial for shear stress-regulated endothelial function. In this study, we investigate the role of lipid rafts and integrin α5 in regulating the inflammatory response in endothelial cells (ECs) under atheroprone versus atheroprotective flow. Lipid raft proteins were isolated from ECs exposed to oscillatory shear stress (OS) or pulsatile shear stress, and then analyzed by quantitative proteomics. Among 396 proteins redistributed in lipid rafts, integrin α5 was the most significantly elevated in lipid rafts under OS. In addition, OS increased the level of activated integrin α5 in lipid rafts through the regulation of membrane cholesterol and fluidity. Disruption of F-actin-based cytoskeleton and knockdown of caveolin-1 prevented the OS-induced integrin α5 translocation and activation. In vivo, integrin α5 activation and EC dysfunction were observed in the atheroprone areas of low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice, and knockdown of integrin α5 markedly attenuated EC dysfunction in partially ligated carotid arteries. Consistent with these findings, mice with haploinsufficency of integrin α5 exhibited a reduction of atherosclerotic lesions in the regions under atheroprone flow. The present study has revealed an integrin- and membrane lipid raft-dependent mechanotransduction mechanism by which atheroprone flow causes endothelial dysfunction.

  17. Calreticulin activates β1 integrin via fucosylation by fucosyltransferase 1 in J82 human bladder cancer cells.

    PubMed

    Lu, Yi-Chien; Chen, Chiung-Nien; Chu, Chia-Ying; Lu, Jenher; Wang, Bo-Jeng; Chen, Chia-Hua; Huang, Min-Chuan; Lin, Tsui-Hwa; Pan, Chin-Chen; Chen, Swey-Shen Alex; Hsu, Wen-Ming; Liao, Yung-Feng; Wu, Pei-Yi; Hsia, Hsin-Yi; Chang, Cheng-Chi; Lee, Hsinyu

    2014-05-15

    Fucosylation regulates various pathological events in cells. We reported that different levels of CRT (calreticulin) affect the cell adhesion and metastasis of bladder cancer. However, the precise mechanism of tumour metastasis regulated by CRT remains unclear. Using a DNA array, we identified FUT1 (fucosyltransferase 1) as a gene regulated by CRT expression levels. CRT regulated cell adhesion through α1,2-linked fucosylation of β1 integrin and this modification was catalysed by FUT1. To clarify the roles for FUT1 in bladder cancer, we transfected the human FUT1 gene into CRT-RNAi stable cell lines. FUT1 overexpression in CRT-RNAi cells resulted in increased levels of β1 integrin fucosylation and rescued cell adhesion to type-I collagen. Treatment with UEA-1 (Ulex europaeus agglutinin-1), a lectin that recognizes FUT1-modified glycosylation structures, did not affect cell adhesion. In contrast, a FUT1-specific fucosidase diminished the activation of β1 integrin. These results indicated that α1,2-fucosylation of β1 integrin was not involved in integrin-collagen interaction, but promoted β1 integrin activation. Moreover, we demonstrated that CRT regulated FUT1 mRNA degradation at the 3'-UTR. In conclusion, the results of the present study suggest that CRT stabilized FUT1 mRNA, thereby leading to an increase in fucosylation of β1 integrin. Furthermore, increased fucosylation levels activate β1 integrin, rather than directly modifying the integrin-binding sites.

  18. Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion

    PubMed Central

    Eppler, Felix J.

    2017-01-01

    Leukocyte trafficking is crucial to facilitate efficient immune responses. Here, we report that the large GTPase dynamin2, which is generally considered to have a key role in endocytosis and membrane remodeling, is an essential regulator of integrin-dependent human T lymphocyte adhesion and migration. Chemical inhibition or knockdown of dynamin2 expression significantly reduced integrin-dependent T cell adhesion in vitro. This phenotype was not observed when T cells were treated with various chemical inhibitors which abrogate endocytosis or actin polymerization. We furthermore detected dynamin2 in signaling complexes and propose that it controls T cell adhesion via FAK/Pyk2- and RapGEF1-mediated Rap1 activation. In addition, the dynamin2 inhibitor-induced reduction of lymphocyte adhesion can be rescued by Rap1a overexpression. We demonstrate that the dynamin2 effect on T cell adhesion does not involve integrin affinity regulation but instead relies on its ability to modulate integrin valency. Taken together, we suggest a previously unidentified role of dynamin2 in the regulation of integrin-mediated lymphocyte adhesion via a Rap1 signaling pathway. PMID:28273099

  19. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

    SciTech Connect

    Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

    1998-06-29

    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

  20. Syndecan-1 couples the insulin-like growth factor-1 receptor to inside-out integrin activation

    PubMed Central

    Beauvais, DeannaLee M.; Rapraeger, Alan C.

    2010-01-01

    Syndecan-1 (Sdc1) engages and activates the αvβ3 (and/or αvβ5) integrin when clustered in human carcinoma and endothelial cells. Although the engagement is extracellular, the activation mechanism is cytoplasmic. This talin-dependent, inside-out signaling pathway is activated downstream of the insulin-like growth factor-1 receptor (IGF1R), whose kinase activity is triggered by Sdc1 clustering. In vitro binding assays using purified receptors suggest that association of the Sdc1 ectodomain with the integrin provides a ‘docking face’ for IGF1R. IGF1R docking and activation of the associated integrin is blocked by synstatin (SSTN92–119), a peptide derived from the integrin engagement site in Sdc1. IGF1R colocalizes with αvβ3 integrin and Sdc1 in focal contacts, but fails to associate with or activate the integrin in cells either lacking Sdc1 or expressing Sdc1Δ67–121, a mutant that is unable to form the Sdc1–integrin–IGF1R ternary complex. Integrin activation is also blocked by IGF1R inhibitors or by silencing IGF1R or talin expression with small-interfering RNAs (siRNAs). In both cases, expression of the constitutively active talin F23 head domain rescues integrin activation. We recently reported that SSTN92–119 blocks angiogenesis and impairs tumor growth in mice, therefore this Sdc1-mediated integrin regulatory mechanism might be a crucial regulator of disease processes known to rely on these integrins, including tumor cell metastasis and tumor-induced angiogenesis. PMID:20971705

  1. Activation of β1 Integrins on Blood Eosinophils by P-Selectin

    PubMed Central

    Mosher, Deane F.

    2011-01-01

    Activation of β1 integrins of blood eosinophils, assessed by mAb N29, correlates inversely with FEV1 in two paradigms for studying control of human asthma. We asked whether P-selectin causes eosinophil β1 integrin activation and results in increased adhesivity. By dual-label flow cytometry, eosinophils with high levels of surface-associated P-selectin had higher reactivity with the activation-sensitive anti-β1 mAbs N29, 8E3, and 9EG7 than eosinophils with no or with a low-level of surface-associated P-selectin. Among patients with nonsevere asthma, surface P-selectin correlated with N29, 8E3, and 9EG7 signals. By immunofluorescence microscopy, surface-associated P-selectin was present in patches on eosinophils, some of which stained for the platelet marker thrombospondin-1. Activated β1 and P-selectin partially colocalized on eosinophils. Soluble P-selectin added to whole blood enhanced activation of eosinophil β1, but not β2, integrins. In contrast, IL-5 activated eosinophil β2, but not β1, integrins. Eosinophils that did not attach to vascular cell adhesion molecule-1 (VCAM-1) in a static adhesion assay had a lower N29 signal than the original population. Soluble P-selectin added to whole blood enhanced eosinophil adhesion to VCAM-1. These findings are compatible with a scenario whereby P-selectin, on eosinophil-associated activated platelets or acquired from plasma or from prior interactions with endothelial cells or platelets, activates eosinophil α4β1 integrin and stimulates eosinophils to adhere to VCAM-1 and move to the airway in asthma. PMID:21441381

  2. The P2Y2 nucleotide receptor requires interaction with αv integrins to access and activate G12

    PubMed Central

    Liao, Zhongji; Seye, Cheikh I.; Weisman, Gary A.; Erb, Laurie

    2009-01-01

    Summary The P2Y2 nucleotide receptor (P2Y2R) interacts with αv integrins to activate Go and induce chemotaxis in human 1321N1 astrocytoma cells. In this study, it was determined that the P2Y2R also requires interaction with αv integrins to activate G12 and associated signaling pathways that control chemotaxis in 1321N1 cells. Mutation of the Arg-Gly-Asp (RGD) integrin-binding sequence in the first extracellular loop of the human P2Y2R to Arg-Gly-Glu (RGE), which prevents integrin interaction, did not inhibit Gq or ERK1/2 signaling by the P2Y2R agonist UTP but completely inhibited activation of G12 and G12-mediated events, including Rho activation, cofilin and myosin light chain-2 phosphorylation, stress fiber formation and chemotaxis towards UTP. The involvement of G12 in all these events was verified by using a dominant negative Gα12 construct. G12 activation by the P2Y2R also was inhibited by anti-αvβ5 integrin antibodies and αv integrin antisense oligonucleotides, suggesting that αv integrin activity and expression are required for the P2Y2R to activate G12. Coimmunoprecipitation experiments confirmed that Gα12 protein associates with the wild-type P2Y2R and with αv integrins but not with the RGE mutant P2Y2R or with α3 integrins. Collectively, these results suggest that αv integrin complexes provide the P2Y2R with access to G12, thereby allowing activation of this heterotrimeric G protein that controls actin cytoskeletal rearrangements required for chemotaxis. PMID:17452627

  3. Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1).

    PubMed

    Jung, S M; Moroi, M

    2000-03-17

    Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin alpha(2)beta(1) activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1), but those operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1) induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin alpha(2)beta(1) activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin alpha(2)beta(1) activation, while at the high agonist concentrations, there would be several pathways through which integrin alpha(2)beta(1) activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B(max)) as thrombin-induced platelets, but their affinity (K(d)) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin alpha(2)beta(1) induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1) would have different conformations that determine their ligand affinity.

  4. Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated β1-integrin-dependent cell migration.

    PubMed

    Kapustin, Alexander; Stepanova, Victoria; Aniol, Natalia; Cines, Douglas B; Poliakov, Alexei; Yarovoi, Serge; Lebedeva, Tatiana; Wait, Robin; Ryzhakov, Grigory; Parfyonova, Yelena; Gursky, Yaroslav; Yanagisawa, Hiromi; Minashkin, Mikhail; Beabealashvilli, Robert; Vorotnikov, Alexander; Bobik, Alex; Tkachuk, Vsevolod

    2012-04-15

    uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.

  5. Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated β1-integrin-dependent cell migration

    PubMed Central

    Kapustin, Alexander; Stepanova, Victoria; Aniol, Natalia; Cines, Douglas B.; Poliakov, Alexei; Yarovoi, Serge; Lebedeva, Tatiana; Wait, Robin; Ryzhakov, Grigory; Parfyonova, Yelena; Gursky, Yaroslav; Yanagisawa, Hiromi; Minashkin, Mikhail; Beabealashvilli, Robert; Vorotnikov, Alexander; Bobik, Alex; Tkachuk, Vsevolod

    2015-01-01

    uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5−/−) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (FblnRGE/RGE MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+ -binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility. PMID:22280367

  6. The oligodendrocyte precursor mitogen PDGF stimulates proliferation by activation of αvβ3 integrins

    PubMed Central

    Baron, Wia; Shattil, Sanford J.; ffrench-Constant, Charles

    2002-01-01

    Central nervous system development requires precise and localized regulation of neural precursor behaviour. Here we show how the interaction between growth factor and integrin signalling pathways provides a mechanism for such precision in oligodendrocyte progenitor (OP) proliferation. While physiological concentrations of platelet-derived growth factor (PDGF) were not in themselves sufficient to promote OP proliferation, they did so on extracellular matrix (ECM) substrates that bind αvβ3 integrin. Upon PDGF-AA exposure and αvβ3 engagement, a physical co-association between both receptors was demonstrated, confirming the interaction between these signalling pathways. Furthermore, we found that PDGFαR stimulated a protein kinase C-dependent activation of integrin αvβ3, which in turn induced OP proliferation via a phosphatidylinositol 3-kinase-dependent signalling pathway. These studies establish a mechanism by which OP proliferation is dependent on the availability of both an ECM ligand and a mitogenic growth factor. Growth factor- mediated integrin activation is the critical integrative step in proliferation signalling, and ensures that the response of neural precursor cells to long-range cues can be regulated by their cellular neighbours, allowing precise control of cell behaviour during development. PMID:11953315

  7. SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.

    PubMed

    Barker, Thomas H; Baneyx, Gretchen; Cardó-Vila, Marina; Workman, Gail A; Weaver, Matt; Menon, Priya M; Dedhar, Shoukat; Rempel, Sandra A; Arap, Wadih; Pasqualini, Renata; Vogel, Viola; Sage, E Helene

    2005-10-28

    SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.

  8. In the newborn hippocampus, neurotrophin-dependent survival requires spontaneous activity and integrin signaling

    PubMed Central

    Murase, Sachiko; Owens, David F.; McKay, Ronald D.

    2012-01-01

    The nervous system develops through a program that first produces neurons in excess and then eliminates as many as half in a specific period of early post-natal life. Neurotrophins are widely thought to regulate neuronal survival but this role has not been clearly defined in the central nervous system. Here we show that neurotrophins promote survival of young neurons by promoting spontaneous activity. Survival of hippocampal neurons in neonatal rat requires spontaneous activity that depends on the excitatory action of γ-aminobutyric acid (GABA). Neurotrophins facilitate recruitment of cultured neurons into active networks, and it is this activity, combined with integrin receptor signaling, that controls neuronal survival. In vivo, neurotrophins require integrin signaling to control neuron number. These data are the first to link the early excitatory action of GABA to the developmental death period and to assign an essential role for activity in neurotrophin-mediated survival that establishes appropriate networks. PMID:21613492

  9. Bit-1 Mediates Integrin-dependent Cell Survival through Activation of the NFκB Pathway*

    PubMed Central

    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.

    2011-01-01

    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. PMID:21383007

  10. A computational analysis of the dynamic roles of talin, Dok1, and PIPKI for integrin activation.

    PubMed

    Geier, Florian; Fengos, Georgios; Iber, Dagmar

    2011-01-01

    Integrin signaling regulates cell migration and plays a pivotal role in developmental processes and cancer metastasis. Integrin signaling has been studied extensively and much data is available on pathway components and interactions. Yet the data is fragmented and an integrated model is missing. We use a rule-based modeling approach to integrate available data and test biological hypotheses regarding the role of talin, Dok1 and PIPKI in integrin activation. The detailed biochemical characterization of integrin signaling provides us with measured values for most of the kinetics parameters. However, measurements are not fully accurate and the cellular concentrations of signaling proteins are largely unknown and expected to vary substantially across different cellular conditions. By sampling model behaviors over the physiologically realistic parameter range we find that the model exhibits only two different qualitative behaviors and these depend mainly on the relative protein concentrations, which offers a powerful point of control to the cell. Our study highlights the necessity to characterize model behavior not for a single parameter optimum, but to identify parameter sets that characterize different signaling modes.

  11. Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels

    PubMed Central

    1993-01-01

    Ba2+ and Cs+. By moving patched cells in contact with FN-coated beads, it was shown that KIR channel activation was responsible for the FN-mediated hyperpolarization of Vrest. Treatment with Pertuxis toxin (PTX) abolished this hyperpolarization and neurite outgrowth, indicating that a G protein is interposed between integrins and KIR channels and that the activation of these channels is required for neuritogenesis. In fact, the block of KIR channels by Cs+ abolished both hyperpolarization and neurite outgrowth, provided that the cation was supplied during the first two hours after N1 cell contact with FN.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8354696

  12. Lateral Mobility and Nanoscale Spatial Arrangement of Chemokine-activated α4β1 Integrins on T Cells*

    PubMed Central

    Sosa-Costa, Alberto; Isern de Val, Sol; Sevilla-Movilla, Silvia; Teixidó, Joaquin

    2016-01-01

    Chemokine stimulation of integrin α4β1-dependent T lymphocyte adhesion is a key step during lymphocyte trafficking. A central question regarding α4β1 function is how its lateral mobility and organization influence its affinity and avidity following cell stimulation with chemokines and/or ligands. Using single particle tracking and superresolution imaging approaches, we explored the lateral mobility and spatial arrangement of individual α4β1integrins on T cells exposed to different activating stimuli. We show that CXCL12 stimulation leads to rapid and transient α4β1activation, measured by induction of the activation epitope recognized by the HUTS-21 anti-β1antibody and by increased talin-β1 association. CXCL12-dependent α4β1 activation directly correlated with restricted lateral diffusion and integrin immobilization. Moreover, co-stimulation by CXCL12 together with soluble VCAM-1 potentiated integrin immobilization with a 5-fold increase in immobile integrins compared with unstimulated conditions. Our data indicate that docking by talin of the chemokine-activated α4β1 to the actin cytoskeleton favors integrin immobilization, which likely facilitates ligand interaction and increased adhesiveness. Superresolution imaging showed that the nanoscale organization of high-affinity α4β1 remains unaffected following chemokine and/or ligand addition. Instead, newly activated α4β1 integrins organize on the cell membrane as independent units without joining pre-established integrin sites to contribute to cluster formation. Altogether, our results provide a rationale to understand how the spatiotemporal organization of activated α4β1 integrins regulates T lymphocyte adhesion. PMID:27481944

  13. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

    PubMed Central

    King, W G; Mattaliano, M D; Chan, T O; Tsichlis, P N; Brugge, J S

    1997-01-01

    Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases. PMID:9234699

  14. Cysteine protease cathepsin X modulates immune response via activation of β2 integrins

    PubMed Central

    Obermajer, Nataša; Repnik, Urška; Jevnikar, Zala; Turk, Boris; Kreft, Marko; Kos, Janko

    2008-01-01

    Cathepsin X is a lysosomal, cysteine dependent carboxypeptidase. Its expression is restricted to cells of the immune system, suggesting a function related to the processes of inflammatory and immune responses. It has been shown to stimulate macrophage antigen-1 (Mac-1) receptor-dependent adhesion and phagocytosis via interaction with integrin β2 subunit. Here its potential role in regulating lymphocyte proliferation via Mac-1 and the other β2 integrin receptor, lymphocyte function-associated antigen-1 (LFA-1) has been investigated. Cathepsin X has been shown to suppress proliferation of human peripheral blood mononuclear cells, by activation of Mac-1, known as a suppressive factor for lymphocyte proliferation. On the other hand, co-localization of cathepsin X and LFA-1 supports the role of cathepsin X in regulating LFA-1 activity, which enhances lymphocyte proliferation. As shown by fluorescence resonance energy transfer, using U-937 and Jurkat cells transfected with αL-mCFP and β2-mYFP, recombinant cathepsin X directly activates LFA-1. The activation was confirmed by increased binding of monoclonal antibody 24, recognizing active LFA-1. We demonstrate that cathepsin X is involved in the regulation of two β2 integrin receptors, LFA-1 and Mac-1, which exhibit opposing roles in lymphocyte activation. PMID:18194276

  15. Signalling pathways induced by protease-activated receptors and integrins in T cells.

    PubMed

    Bar-Shavit, Rachel; Maoz, Miriam; Yongjun, Yin; Groysman, Maya; Dekel, Idit; Katzav, Shulamit

    2002-01-01

    Recent characterization of the thrombin receptor indicates that it plays a role in T-cell signalling pathways. However, little is known regarding the signalling events following stimulation of additional members of the protease-activated receptor (PAR) family, i.e. PAR2 and PAR3. Most of the postligand cascades are largely unknown. Here, we illustrate that in Jurkat T-leukaemic cells, activation of PAR1, PAR2 and PAR3 induce tyrosine phosphorylation of Vav1. This response was impaired in Jurkat T cells deficient in p56lck (JCaM1.6). Activation of PARs also led to an increase in tyrosine phosphorylation of ZAP-70 and SLP-76, two key proteins in T-cell receptor (TCR) signalling. We also demonstrated that p56lck is meaningful for integrin signalling. Thus, JCaM1.6 cells exhibited a marked reduction in their adherence to fibronectin-coated plates, as compared to the level of adherence of Jurkat T cells. While the phosphorylation of Vav1 in T cells is augmented following adhesion, no additional increase was noted following treatment of the adhered cells with PARs. Altogether, we have identified key components in the postligand-signalling cascade of PARs and integrins. Furthermore, we have identified Lck as a critical and possibly upstream component of PAR-induced Vav1 phosphorylation, as well as integrin activation, in Jurkat T cells.

  16. Distinct roles of AKT isoforms in regulating β1-integrin activity, migration, and invasion in prostate cancer

    PubMed Central

    Virtakoivu, Reetta; Pellinen, Teijo; Rantala, Juha K.; Perälä, Merja; Ivaska, Johanna

    2012-01-01

    AKT1 and AKT2 kinases have been shown to play opposite roles in breast cancer migration and invasion. In this study, an RNA interference screen for integrin activity inhibitors identified AKT1 as an inhibitor of β1-integrin activity in prostate cancer. Validation experiments investigating all three AKT isoforms demonstrated that, unlike in breast cancer, both AKT1 and AKT2 function as negative regulators of cell migration and invasion in PC3 prostate cancer cells. Down-regulation of AKT1 and AKT2, but not AKT3, induced activation of cell surface β1-integrins and enhanced adhesion, migration, and invasion. Silencing of AKT1 and AKT2 also resulted in increased focal adhesion size. Importantly, the mechanisms involved in integrin activity regulation were distinct for the two AKT isoforms. Silencing of AKT1 relieved feedback suppression of the expression and activity of several receptor tyrosine kinases, including EGFR and MET, with established cross-talk with β1-integrins. Silencing of AKT2, on the other hand, induced up-regulation of the microRNA-200 (miR-200) family, and overexpression of miR-200 was sufficient to induce integrin activity and cell migration in PC3 cells. Taken together, these data define an inhibitory role for both AKT1 and AKT2 in prostate cancer migration and invasion and highlight the cell type–specific actions of AKT kinases in the regulation of cell motility. PMID:22809628

  17. Phospholipase C-gamma1 potentiates integrin-dependent cell spreading and migration through Pyk2/paxillin activation.

    PubMed

    Choi, Jang Hyun; Yang, Yong-Ryoul; Lee, Seul Ki; Kim, Il-Shin; Ha, Sang Hoon; Kim, Eung-Kyun; Bae, Yun Soo; Ryu, Sung Ho; Suh, Pann-Ghill

    2007-08-01

    Phospholipase C-gamma1 (PLC-gamma1), which generates two second messengers, namely, inositol-1, 4, 5-trisphosphate and diacylglycerol, is implicated in growth factor-mediated chemotaxis. However, the exact role of PLC-gamma1 in integrin-mediated cell adhesion and migration remains poorly understood. In this study, we demonstrate that PLC-gamma1 is required for actin cytoskeletal organization and cell motility through the regulation of Pyk2 and paxillin activation. After fibronectin stimulation, PLC-gamma1 directly interacted with the cytoplasmic tail of integrin beta1. In PLC-gamma1-silenced cells, integrin-induced Pyk2 and paxillin phosphorylation were significantly reduced and PLC-gamma1 potentiated the integrin-induced Pyk2/paxillin activation in its enzymatic activity-dependent manner. In addition, specific knock-down of PLC-gamma1 resulted in a failure to form focal adhesions dependent on fibronectin stimulation, which appeared to be caused by the suppression of Pyk2 and paxillin phosphorylation. Interestingly, PLC-gamma1 potentiated the activations of Rac, thus integrin-induced lamellipodia formation was up-regulated. Consequently, the strength of cell-substratum interaction and cell motility were profoundly up-regulated by PLC-gamma1. Taken together, these results suggest that PLC-gamma1 is a key player in integrin-mediated cell spreading and motility achieved by the activation of Pyk2/paxillin/Rac signaling.

  18. Definition of EGF-like, closely interacting modules that bear activation epitopes in integrin beta subunits.

    PubMed

    Takagi, J; Beglova, N; Yalamanchili, P; Blacklow, S C; Springer, T A

    2001-09-25

    Integrin beta subunits contain four cysteine-rich repeats in a long extracellular stalk that connects the headpiece to the membrane. Most mAbs to integrin activation epitopes map to these repeats, and they are important in propagating conformational signals from the membrane/cytosol to the ligand-binding headpiece. Sequence analysis of a protein containing only 10 integrin-like, cysteine-rich repeats suggests that these repeats start one cysteine earlier than previously reported. By using the new repeat boundaries, statistically significant sequence homology to epidermal growth factor-like domains is found, and a disulfide bond connectivity of the eight cysteines is predicted that differs in three of four disulfides from a previous prediction of epidermal growth factor-like modules [Berg, R. W., Leung, E., Gough, S., Morris, C., Yao, W.-P., Wang, S.-x., Ni, J. & Krissansen, G. W. (1999) Genomics 56, 169-178]. N-terminally truncated beta2 integrin stalk fragments were well expressed and secreted from 293 T cells when they began at repeat boundaries but not when they began one cysteine earlier or later. Furthermore, peptides that correspond to module 3 or modules 2 + 3 were expressed in bacteria and refolded. The module 2 + 3 fragment was as reactive with three mAbs to activation epitopes as a beta2 fragment expressed in eukaryotic cells, indicating a native fold. Only one residue intervenes between the last cysteine of one module and the first cysteine of the next. This arrangement is consistent with a tight intermodule connection, a prerequisite for signal propagation from the membrane to the ligand binding headpiece.

  19. Activation induced morphological changes and integrin αIIbβ3 activity of living platelets.

    PubMed

    Posch, Sandra; Neundlinger, Isabel; Leitner, Michael; Siostrzonek, Peter; Panzer, Simon; Hinterdorfer, Peter; Ebner, Andreas

    2013-04-01

    Platelets are essential in hemostasis. Upon activation they undergo a shape-change accompanied with receptor presentation. Atomic force microscopy (AFM) imaging and single molecule force spectroscopy (SMFS) were used as powerful tools for exploring morphological changes as well as receptor activities of platelets. Imaging time series was accomplished with and without fixation steps at the single platelet level. Hereby the response of mechanical stimulation of the platelet by the AFM cantilever tip was directly observed. We demonstrate that living and fixed platelets develop filopodia after a short activation time followed by their disappearance including cellular bleb formation. Thereafter a second filopodia formation (filopodia extrusion) was observed; those filopodia subsequently disappeared again, and finally platelets detached from the support due to cell death. We determined the influence of mechanical stress on the chronology of morphological changes of platelets and demonstrated shear force induced filopodia formation. Through recordings over several hours, topographical AFM images over the full platelet lifetime - from early activation up to apoptosis - are presented. SMFS measurements on living platelets allowed determining the activation state of the most prominent membrane receptor integrin αIIbβ3 at all different phases of activation. αIIbβ3 was fully activated, independent of the morphological state.

  20. Integrin-Dependent Activation of the JNK Signaling Pathway by Mechanical Stress

    PubMed Central

    Kanger, Johannes S.; Subramaniam, Vinod; Martin-Blanco, Enrique

    2011-01-01

    Mechanical force is known to modulate the activity of the Jun N-terminal kinase (JNK) signaling cascade. However, the effect of mechanical stresses on JNK signaling activation has previously only been analyzed by in vitro detection methods. It still remains unknown how living cells activate the JNK signaling cascade in response to mechanical stress and what its functions are in stretched cells. We assessed in real-time the activity of the JNK pathway in Drosophila cells by Fluorescence Lifetime Imaging Microscopy (FLIM), using an intramolecular phosphorylation-dependent dJun-FRET (Fluorescence Resonance Energy Transfer) biosensor. We found that quantitative FRET-FLIM analysis and confocal microscopy revealed sustained dJun-FRET biosensor activation and stable morphology changes in response to mechanical stretch for Drosophila S2R+ cells. Further, these cells plated on different substrates showed distinct levels of JNK activity that associate with differences in cell morphology, integrin expression and focal adhesion organization. These data imply that alterations in the cytoskeleton and matrix attachments may act as regulators of JNK signaling, and that JNK activity might feed back to modulate the cytoskeleton and cell adhesion. We found that this dynamic system is highly plastic; at rest, integrins at focal adhesions and talin are key factors suppressing JNK activity, while multidirectional static stretch leads to integrin-dependent, and probably talin-independent, Jun sensor activation. Further, our data suggest that JNK activity has to coordinate with other signaling elements for the regulation of the cytoskeleton and cell shape remodeling associated with stretch. PMID:22180774

  1. Rac1 is deactivated at integrin activation sites through an IQGAP1–filamin-A–RacGAP1 pathway

    PubMed Central

    Jacquemet, Guillaume; Morgan, Mark R.; Byron, Adam; Humphries, Jonathan D.; Choi, Colin K.; Chen, Christopher S.; Caswell, Patrick T.; Humphries, Martin J.

    2013-01-01

    Summary Cell migration makes a fundamental contribution to both normal physiology and disease pathogenesis. Integrin engagement with extracellular ligands spatially controls, via the cyclical activation and deactivation of the small GTPase Rac1, the dynamic membrane protrusion and cytoskeletal reorganization events that are required for directional migration. Although the pathways that control integrin-mediated Rac1 activation are reasonably well defined, the mechanisms that are responsible for switching off activity are poorly understood. Here, proteomic analysis of activated integrin-associated complexes suggests filamin-A and IQ-motif-containing GTPase-activating protein 1 (IQGAP1) as candidates that link β1 integrin to Rac1. siRNA-mediated knockdown of either filamin-A or IQGAP1 induced high, dysregulated Rac1 activity during cell spreading on fibronectin. Using immunoprecipitation and immunocytochemistry, filamin-A and IQGAP1 were shown to be part of a complex that is recruited to active β1 integrin. Mass spectrometric analysis of individual filamin-A, IQGAP1 and Rac1 pull-downs and biochemical analysis, identified RacGAP1 as a novel IQGAP1 binding partner. Further immunoprecipitation and immunocytochemistry analyses demonstrated that RacGAP1 is recruited to IQGAP1 and active β1 integrin, and that suppression of RacGAP1 expression triggered elevated Rac1 activity during spreading on fibronectin. Consistent with these findings, reduced expression of filamin-A, IQGAP1 or RacGAP1 triggered unconstrained membrane protrusion and disrupted directional cell migration on fibrillar extracellular matrices. These findings suggest a model whereby integrin engagement, followed by filamin-A, IQGAP1 and RacGAP1 recruitment, deactivates Rac1 to constrain its activity spatially and thereby coordinate directional cell migration. PMID:23843620

  2. Macrolide analog F806 suppresses esophageal squamous cell carcinoma (ESCC) by blocking β1 integrin activation.

    PubMed

    Li, Li-Yan; Jiang, Hong; Xie, Yang-Min; Liao, Lian-Di; Cao, Hui-Hui; Xu, Xiu-E; Chen, Bo; Zeng, Fa-Min; Zhang, Ying-Li; Du, Ze-Peng; Chen, Hong; Huang, Wei; Jia, Wei; Zheng, Wei; Xie, Jian-Jun; Li, En-Min; Xu, Li-Yan

    2015-06-30

    The paucity of new drugs for the treatment of esophageal squamous cell carcinoma (ESCC) limits the treatment options. This study characterized the therapeutic efficacy and action mechanism of a novel natural macrolide compound F806 in human ESCC xenograft models and cell lines. F806 inhibited growth of ESCC, most importantly, it displayed fewer undesirable side effects on normal tissues in two human ESCC xenograft models. F806 inhibited proliferation of six ESCC cells lines, with the half maximal inhibitory concentration (IC50) ranging from 9.31 to 16.43 μM. Furthermore, F806 induced apoptosis of ESCC cells, contributing to its growth-inhibitory effect. Also, F806 inhibited cell adhesion resulting in anoikis. Mechanistic studies revealed that F806 inhibited the activation of β1 integrin in part by binding to a novel site Arg610 of β1 integrin, suppressed focal adhesion formation, decreased cell adhesion to extracellular matrix and eventually triggered apoptosis. We concluded that F806 would potentially be a well-tolerated anticancer drug by targeting β1 integrin, resulting in anoikis in ESCC cells.

  3. 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

  4. Intact αIIbβ3 Integrin Is Extended after Activation as Measured by Solution X-ray Scattering and Electron Microscopy*

    PubMed Central

    Eng, Edward T.; Smagghe, Benoit J.; Walz, Thomas; Springer, Timothy A.

    2011-01-01

    Integrins are bidirectional signaling molecules on the cell surface that have fundamental roles in regulating cell behavior and contribute to cell migration and adhesion. Understanding of the mechanism of integrin signaling and activation has been advanced with truncated ectodomain preparations; however, the nature of conformational change in the full-length intact integrin molecule remains an active area of research. Here we used small angle x-ray scattering and electron microscopy to study detergent-solubilized, intact platelet integrin αIIbβ3. In the resting state, the intact αIIbβ3 adopted a compact, bent conformation. Upon activation with Mn2+, the average integrin extension increased. Further activation by addition of ligand led to stabilization of the extended state and opening of the headpiece. The observed extension and conformational rearrangement upon activation are consistent with the extension and headpiece opening model of integrin activation. PMID:21832081

  5. Activity-Induced Synaptic Structural Modifications by an Activator of Integrin Signaling at the Drosophila Neuromuscular Junction.

    PubMed

    Lee, Joo Yeun; Geng, Junhua; Lee, Juhyun; Wang, Andrew R; Chang, Karen T

    2017-03-22

    Activity-induced synaptic structural modification is crucial for neural development and synaptic plasticity, but the molecular players involved in this process are not well defined. Here, we report that a protein named Shriveled (Shv) regulates synaptic growth and activity-dependent synaptic remodeling at the Drosophila neuromuscular junction. Depletion of Shv causes synaptic overgrowth and an accumulation of immature boutons. We find that Shv physically and genetically interacts with βPS integrin. Furthermore, Shv is secreted during intense, but not mild, neuronal activity to acutely activate integrin signaling, induce synaptic bouton enlargement, and increase postsynaptic glutamate receptor abundance. Consequently, loss of Shv prevents activity-induced synapse maturation and abolishes post-tetanic potentiation, a form of synaptic plasticity. Our data identify Shv as a novel trans-synaptic signal secreted upon intense neuronal activity to promote synapse remodeling through integrin receptor signaling.SIGNIFICANCE STATEMENT The ability of neurons to rapidly modify synaptic structure in response to neuronal activity, a process called activity-induced structural remodeling, is crucial for neuronal development and complex brain functions. The molecular players that are important for this fundamental biological process are not well understood. Here we show that the Shriveled (Shv) protein is required during development to maintain normal synaptic growth. We further demonstrate that Shv is selectively released during intense neuronal activity, but not mild neuronal activity, to acutely activate integrin signaling and trigger structural modifications at the Drosophila neuromuscular junction. This work identifies Shv as a key modulator of activity-induced structural remodeling and suggests that neurons use distinct molecular cues to differentially modulate synaptic growth and remodeling to meet synaptic demand. Copyright © 2017 the authors 0270-6474/17/373246-18$15.00/0.

  6. Integrin activation by P-Rex1 is required for selectin-mediated slow leukocyte rolling and intravascular crawling.

    PubMed

    Herter, Jan M; Rossaint, Jan; Block, Helena; Welch, Heidi; Zarbock, Alexander

    2013-03-21

    Integrin activation is essential for the function of leukocytes. Impaired integrin activation on leukocytes is the hallmark of the leukocyte adhesion deficiency syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. In inflammation, leukocytes collect different signals during the contact with the microvasculature, which activate signaling pathways leading to integrin activation and leukocyte recruitment. We report the role of P-Rex1, a Rac-specific guanine nucleotide exchanging factor, in integrin activation and leukocyte recruitment. We find that P-Rex1 is required for inducing selectin-mediated lymphocyte function-associated antigen-1 (LFA-1) extension that corresponds to intermediate affinity and induces slow leukocyte rolling, whereas P-Rex1 is not involved in the induction of the high-affinity conformation of LFA-1 obligatory for leukocyte arrest. Furthermore, we demonstrate that P-Rex1 is involved in Mac-1-dependent intravascular crawling. In vivo, both LFA-1-dependent slow rolling and Mac-1-dependent crawling are defective in P-Rex1(-/-) leukocytes, whereas chemokine-induced arrest and postadhesion strengthening remain intact in P-Rex1-deficient leukocytes. Rac1 is involved in E-selectin-mediated slow rolling and crawling. In vivo, in an ischemia-reperfusion-induced model of acute kidney injury, abolished selectin-mediated integrin activation contributed to decreased neutrophil recruitment and reduced kidney damage in P-Rex1-deficient mice. We conclude that P-Rex1 serves distinct functions in LFA-1 and Mac-1 activation.

  7. Integrin Targeting and Toxicological Assessment of Peptide-Conjugated Liposome Delivery Systems to Activated Endothelial Cells.

    PubMed

    Kermanizadeh, Ali; Villadsen, Klaus; Østrem, Ragnhild G; Jensen, Knud J; Møller, Peter; Loft, Steffen

    2017-04-01

    Utilization of functionalized liposomes as the means of targeted delivery of therapeutics may enhance specific transport of biologically active drugs to target tissues, while avoiding or reducing undesired side effects. In the present investigation, peptide-conjugated cationic liposomes were constructed with the aim of targeting integrins (i.e. vitronectin and/or fibronectin receptors) on activated endothelial cells. The peptide-conjugated liposomes induced only cytotoxicity at the highest concentration in non-activated or activated endothelial cells, as well as in co-culture of endothelial cells and macrophages. There was unaltered secretion of cytokines after exposure of peptide-conjugated liposomes to endothelial cells, indicating that the materials were not inflammogenic. Liposomes with a peptide targeting the fibronectin receptor (integrin α5β1) were more effective in targeting of activated endothelial cells, as compared to a liposome with a peptide that targeted both the fibronectin and vitronectin receptors, as well as liposomes with a control peptide. The liposome targeted to the fibronectin receptor also displayed uptake in endothelial cells in co-culture with activated macrophages. Therefore, this study demonstrates the feasibility of constructing a peptide-conjugated cationic liposome, which displays targeting to activated endothelial cells at concentrations that are not cytotoxic or inflammogenic to the cells.

  8. LW protein: a promiscuous integrin receptor activated by adrenergic signaling.

    PubMed

    Delahunty, M; Zennadi, R; Telen, M J

    2006-01-01

    The LW blood group antigen glycoprotein, although part of the Rh macromolecular complex, is nonetheless a member of the intercellular adhesion molecule (ICAM) family. Thus, while it is only rarely clinically important in the setting of transfusion and pregnancy, LW is likely to contribute to red cell adhesion in a variety of settings, including during hematopoiesis, as well as in vascular disorders. The best documentation of a pathophysiological role for LW in human disease is in sickle cell disease, where it contributes to red cell adhesion to endothelial cells and the development of vaso-occlusion, the hallmark of that disease. LW may also contribute to other intravascular processes, such as both venous and arterial thrombosis, due to its ability to interact with both activated platelets as well as leukocytes. The evidence that LW itself can undergo activation on red cells holds promise that pharmacotherapeutic maneuvers may be found to prevent such pathophysiologic interactions.

  9. Degradation of Internalized αvβ5 Integrin Is Controlled by uPAR Bound uPA: Effect on β1 Integrin Activity and α-SMA Stress Fiber Assembly

    PubMed Central

    Wang, Lingyan; Pedroja, Benjamin S.; Meyers, Erin E.; Garcia, Angelo L.; Twining, Sally S.; Bernstein, Audrey M.

    2012-01-01

    Myofibroblasts (Mfs) that persist in a healing wound promote extracellular matrix (ECM) accumulation and excessive tissue contraction. Increased levels of integrin αvβ5 promote the Mf phenotype and other fibrotic markers. Previously we reported that maintaining uPA (urokinase plasminogen activator) bound to its cell-surface receptor, uPAR prevented TGFβ-induced Mf differentiation. We now demonstrate that uPA/uPAR controls integrin β5 protein levels and in turn, the Mf phenotype. When cell-surface uPA was increased, integrin β5 levels were reduced (61%). In contrast, when uPA/uPAR was silenced, integrin β5 total and cell-surface levels were increased (2–4 fold). Integrin β5 accumulation resulted from a significant decrease in β5 ubiquitination leading to a decrease in the degradation rate of internalized β5. uPA-silencing also induced α-SMA stress fiber organization in cells that were seeded on collagen, increased cell area (1.7 fold), and increased integrin β1 binding to the collagen matrix, with reduced activation of β1. Elevated cell-surface integrin β5 was necessary for these changes after uPA-silencing since blocking αvβ5 function reversed these effects. Our data support a novel mechanism by which downregulation of uPA/uPAR results in increased integrin αvβ5 cell-surface protein levels that regulate the activity of β1 integrins, promoting characteristics of the persistent Mf. PMID:22470492

  10. Activated αvβ3 Integrin Regulates αvβ5 Integrin–Mediated Phagocytosis in Trabecular Meshwork Cells

    PubMed Central

    Gagen, Debjani; Filla, Mark S.; Clark, Ross; Liton, Paloma; Peters, Donna M.

    2013-01-01

    Purpose. To investigate the roles of αvβ3 and αvβ5 integrins in phagocytosis in human trabecular meshwork (HTM) cells. Methods. Immunofluorescence microscopy and FACS analysis were used to determine levels of αvβ3 and αvβ5 integrins in TM tissue and cultures of normal and immortalized TM cells. Phagocytosis was measured using pHrodo-labeled S. aureus bioparticles followed by FACS analysis. The role of αvβ5 integrin in phagocytosis was evaluated by knocking down αvβ5 integrin expression with siRNA against the human β5 gene. Signaling from focal adhesion kinase (FAK) was blocked using FAK inhibitor 14. The role of αvβ3 integrins in phagocytosis was determined by treating HTM cells with dexamethasone (DEX) or ethanol (EtOH) and by generating stable cell lines that overexpressed either wild type (WT) or constitutively active (CA) β3 integrin subunit. Results. Both TM tissue and cell lines expressed αvβ3 and αvβ5 integrins. Knockdown of αvβ5 integrin reduced phagocytosis by ∼60% and FAK inhibition significantly reduced phagocytosis up to 84%, in a dose-dependent manner. DEX treatment increased αvβ3 integrin expression in HTM cells but reduced phagocytosis by ∼50% compared with untreated and EtOH-treated cells. The CA β3 integrin–expressing cell line showed increased αvβ3 integrin levels and decreased phagocytosis by ∼50% compared with the control. Conclusions. The αvβ5 integrin-FAK–mediated pathway regulates phagocytosis in TM cells and this pathway is inhibited by activation of αvβ3 integrins. This suggests that changes in integrin expression and activity may be responsible for alterations in phagocytosis observed in steroid induced glaucoma. PMID:23821196

  11. Highly Potent, Water Soluble Benzimidazole Antagonist for Activated (alpha)4(beta)1 Integrin

    SciTech Connect

    Carpenter, R D; Andrei, M; Lau, E Y; Lightstone, F C; Liu, R; Lam, K S; Kurth, M J

    2007-08-29

    The cell surface receptor {alpha}{sub 4}{beta}{sub 1} integrin, activated constitutively in lymphoma, can be targeted with the bisaryl urea peptidomimetic antagonist 1 (LLP2A). However, concerns on its preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl urea to a 2-arylaminobenzimidazole moiety resulting in improved solubility while maintaining picomolar potency [5 (KLCA4); IC{sub 50} = 305 pM]. With exceptional solubility, this finding has potential for improving PK to help diagnose and treat lymphomas.

  12. Integrins and metastasis

    PubMed Central

    Ganguly, Kirat Kumar; Pal, Sekhar; Moulik, Shuvojit; Chatterjee, Amitava

    2013-01-01

    Metastasis is a combination of biological events that makes the difference between cancer and other diseases. Metastasis requires flow of erroneous but precisely coordinated basic cellular activities like cell migration–invasion, cell survival–apoptosis, cell proliferation, etc. All of these processes require efficient regulation of cell attachment and detachment, which recruit integrin receptors in this flow of events. World literatures show several aspects of interrelation of integrins and metastasis. Integrin molecules are being used as prime target to battle metastasis. In this review we are collating the observations showing importance of integrin biology in regulation of metastasis and the strategies where integrin receptors are being used as targets to regulate metastasis. PMID:23563505

  13. Modulation of tumor cell stiffness and migration by type IV collagen through direct activation of integrin signaling pathway.

    PubMed

    Chen, Sheng-Yi; Lin, Jo-Shi; Yang, Bei-Chang

    2014-08-01

    Excessive collagen deposition plays a critical role in tumor progression and metastasis. To understand how type IV collagen affects mechanical stiffness and migration, low-collagen-IV-expressing transfectants of B16F10, U118MG, and Huh7 (denoted shCol cells) were established by the lentiviral-mediated delivery of small interfering RNA against type IV-α1 collagen (Col4A1). Although having similar growth rates, shCol cells showed a flatter morphology compared to that of the corresponding controls. Notably, knocking down the Col4A1 gene conferred the cells with higher levels of elasticity and lower motility. Exposure to blocking antibodies against human β1 integrin or α2β1 integrin or the pharmacological inhibition of Src and ERK activity by PP1 and U0126, respectively, effectively reduced cell motility and raised cell stiffness. Reduced Src and ERK activities in shCol cells indicate the involvement of a collagen IV/integrin signaling pathway. The forced expression of β1 integrin significantly stimulated Src and ERK phosphorylation, reduced cell stiffness, and accelerated cell motility. In an experimental metastasis assay using C57BL/6 mice, B16F10 shCol cells formed significantly fewer and smaller lung nodules, confirming the contribution of collagen to metastasis. In summary, the integrin signaling pathway activated in a tumor environment with collagen deposition is responsible for low cell elasticity and high metastatic ability.

  14. Bacterial genotoxins promote inside-out integrin β1 activation, formation of focal adhesion complexes and cell spreading.

    PubMed

    Levi, Laura; Toyooka, Tatsushi; Patarroyo, Manuel; Frisan, Teresa

    2015-01-01

    Integrins are membrane bound receptors that regulate several cellular processes, such as cell adhesion, migration, survival and proliferation, and may contribute to tumor initiation/progression in cells exposed to genotoxic stress. The extent of integrin activation and its role in cell survival upon intoxication with bacterial genotoxins are still poorly characterized. These toxins induce DNA strand breaks in the target cells and activate the DNA damage response (DDR), coordinated by the Ataxia Telangectasia Mutated (ATM) kinase. In the present study, we demonstrate that induction of DNA damage by two bacterial genotoxins promotes activation of integrin β1, leading to enhanced assembly of focal adhesions and cell spreading on fibronectin, but not on vitronectin. This phenotype is mediated by an ATM-dependent inside-out integrin signaling, and requires the actin cytoskeleton remodeler NET1. The toxin-mediated cell spreading and anchorage-independent survival further relies on ALIX and TSG101, two components of the endosomal sorting complex required for transport (ESCRT), known to regulate integrin intracellular trafficking. These data reveal a novel aspect of the cellular response to bacterial genotoxins, and provide new tools to understand the carcinogenic potential of these effectors in the context of chronic intoxication and infection.

  15. Blocking α5β1 Integrin Attenuates sCD40L-Mediated Platelet Activation.

    PubMed

    Simic, Damir; Bogdan, Nancy; Teng, Fang; Otieno, Monicah

    2017-09-01

    The soluble form of CD40L (sCD40L) is a platelet-derived mediator that links inflammation, hemostasis, and vascular dysfunction. Indeed, blockade of CD40L by neutralizing antibodies or genetic disruption in mice prevents atherosclerosis and atherothrombosis. Until recently, it was believed that CD40 and αIIbβ3 were the only receptors on platelets responsible for binding sCD40L, leading to platelet activation and initiation of thrombotic events. Recent findings showed α5β1 integrin as a novel platelet sCD40L receptor, with an unknown function. For the first time, using anti-α5β1 blocking antibodies, we show that sCD40L/α5β1 interaction leads to platelet activation as evaluated in the human whole blood. Establishing α5β1 integrin's role in platelet activation, and therefore thrombosis will help further shed light on the etiology of thrombotic disease.

  16. Integrin beta and receptor for activated protein kinase C are involved in the cell entry of Bombyx mori cypovirus.

    PubMed

    Zhang, Yiling; Cao, Guangli; Zhu, Liyuan; Chen, Fei; Zar, Mian Sahib; Wang, Simei; Hu, Xiaolong; Wei, Yuhong; Xue, Renyu; Gong, Chengliang

    2017-02-07

    Receptor-mediated endocytosis using a β1 integrin-dependent internalization was considered as the primary mechanism for the initiation of mammalian reovirus infection. Bombyx mori cypovirus (BmCPV) is a member of Reoviridae family which mainly infects the midgut epithelium of silkworm; the cell entry of BmCPV is poorly explored. In this study, co-immunoprecipitation (Co-IP), virus overlay protein binding assay (VOPBA), and BmCPV-protein interaction on the polyvinylidene difluoride membrane (BmCPV-PI-PVDF) methods were employed to screen the interacting proteins of BmCPV, and several proteins including integrin beta and receptor for activated protein kinase C (RACK1) were identified as the candidate interacting proteins for establishing the infection of BmCPV. The infectivity of BmCPV was investigated in vivo and in vitro by RNA interference (RNAi) and antibody blocking methods, and the results showed that the infectivity of BmCPV was significantly reduced by either small interfering RNA-mediated silencing of integrin beta and RACK1 or antibody blocking of integrin beta and RACK1. The expression level of integrin beta or RACK1 is not the highest in the silkworm midgut which is a principal target tissue of BmCPV, suggesting that the molecules other than integrin beta or RACK1 might play a key role in determining the tissue tropism of BmCPV infection. The establishment of BmCPV infection depends on other factors, and these factors interacted with integrin beta and RACK1 to form receptor complex for the cell entry of BmCPV.

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

    PubMed

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

    2008-12-15

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

  18. 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.

  19. Activation of the platelet collagen receptor integrin alpha(2)beta(1): its mechanism and participation in the physiological functions of platelets.

    PubMed

    Jung, S M; Moroi, M

    2000-10-01

    When platelets are stimulated by agonists, integrin alpha(2)beta(1) (GP Ia/IIa), one of the platelet collagen receptors, is activated to forms with high affinities for its ligand collagen. Here we describe our studies to characterize the binding kinetics of the activated integrin forms and the activation mechanism. Under low agonist concentrations, integrin alpha(2)beta(1) is activated through a mechanism involving ADP/ADP receptors; and under high agonist concentrations, multiple signaling pathways are involved in its activation. Such differences in mechanism at low and high agonist concentrations are also suggested in the activation of integrin alpha(IIb)beta(3), the platelet fibrinogen receptor. We describe our flow adhesion studies, from which evidence was obtained about the involvement of integrin alpha(2)beta(1) activation in the physiological function of platelets, adhesion and thrombus formation.

  20. Integrins in periodontal disease.

    PubMed

    Larjava, Hannu; Koivisto, Leeni; Heino, Jyrki; Häkkinen, Lari

    2014-07-15

    Cell surface integrin receptors mediate cell adhesion, migration and cellular signaling in all nucleated cells. They are activated by binding to extracellular ligands or by intracellular proteins, such as kindlins that engage with their cytoplasmic tails. Cells in the periodontal tissues express several integrins with overlapping ligand-binding capabilities. A distinct phenotype in the periodontium has only been described for knockouts or mutations of three integrin subunits, α11, β6 and β2. Integrin α11β1 appears to have some regulatory function in the periodontal ligament of continuously erupting incisors in mice. Integrin αvβ6 is expressed in the junctional epithelium (JE) of the gingiva. Animals deficient in this receptor develop classical signs of periodontal disease, including inflammation, apical migration of the JE and bone loss, suggesting that it plays a role in the regulation of periodontal inflmmation, likely through activation of transforming growth factor-β1. Lack of integrin activation in the JE is also associated with periodontitis. Patients with kindlin-1 mutations have severe early-onset periodontal disease. Finally, patients with mutations in the leukocyte-specific β2 integrin subunit have severe periodontal problems due to lack of transiting neutrophils in the periodontal tissues.

  1. 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

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

    PubMed

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

    2016-08-23

    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.

  3. The integrins.

    PubMed

    Takada, Yoshikazu; Ye, Xiaojing; Simon, Scott

    2007-01-01

    The integrins are a superfamily of cell adhesion receptors that bind to extracellular matrix ligands, cell-surface ligands, and soluble ligands. They are transmembrane alphabeta heterodimers and at least 18 alpha and eight beta subunits are known in humans, generating 24 heterodimers. Members of this family have been found in mammals, chicken and zebrafish, as well as lower eukaryotes, including sponges, the nematode Caenorhabditis elegans (two alpha and one beta subunits, generating two integrins) and the fruitfly Drosophila melanogaster (five alpha and one beta, generating five integrins). The alpha and beta subunits have distinct domain structures, with extracellular domains from each subunit contributing to the ligand-binding site of the heterodimer. The sequence arginine-glycine-aspartic acid (RGD) was identified as a general integrin-binding motif, but individual integrins are also specific for particular protein ligands. Immunologically important integrin ligands are the intercellular adhesion molecules (ICAMs), immunoglobulin superfamily members present on inflamed endothelium and antigen-presenting cells. On ligand binding, integrins transduce signals into the cell interior; they can also receive intracellular signals that regulate their ligand-binding affinity. Here we provide a brief overview that concentrates mostly on the organization, structure and function of mammalian integrins, which have been more extensively studied than integrins in other organisms.

  4. Ambroxol inhibits neutrophil respiratory burst activated by alpha chain integrin adhesion.

    PubMed

    Peroni, D G; Moser, S; Gallo, G; Pigozzi, R; Tenero, L; Zanoni, L; Boner, A L; Piacentini, G L

    2013-01-01

    The purpose of the present study was to investigate the possible anti-oxidant effect(s) of Ambroxol on neutrophils activated by ligand-binding of the drug with membrane-associated adhesion integrin CD11a and to estimate dose-response changes in oxygen free radical production. The amount of free radical production by anti-CD11a- and anti-CD4-coated neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (FMLP) and challenged with increasing concentration of Ambroxol, was evaluated within a time frame of 90 minutes. A significant dose-dependent effect response of Ambroxol on O2‾ production by cells coated with anti-CD11a antibody was observed. This preliminary study opens a new perspective on the therapeutic role of Ambroxol as an antioxidant drug and for its potential use in controlling oxidative stress, particularly in leukocyte-dependent inflammation.

  5. CD81 expressed on human thymocytes mediates integrin activation and interleukin 2-dependent proliferation.

    PubMed

    Todd, S C; Lipps, S G; Crisa, L; Salomon, D R; Tsoukas, C D

    1996-11-01

    Lymphocyte recognition of antigen by the antigen-specific T cell receptor (TCR) and coreceptor complexes rapidly alters the cell's adhesive properties facilitating high avidity cell-ligand interactions necessary for lymphocyte development and function. Here, we report the expression of CD81 (target of antiproliferative antigen [TAPA]-1) on human thymocytes and the physical association of CD81 with CD4 and CD8 T cell coreceptors. Antibody ligation of CD81 on thymocytes promotes the rapid induction of integrin-mediated cell-cell adhesion via lymphocyte function-associated molecule-1 (LFA-1). Cross-linking CD81 is also shown to be costimulatory with signaling through the TCR/CD3 complex inducing interleukin 2-dependent thymocyte proliferation. These data suggest that a CD81-mediated pathway in thymocytes is involved in the regulation of both cell adhesion and activation.

  6. Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity

    SciTech Connect

    Whelan, Jarrett T.; Wang, Lei; Chen, Jianming; Metts, Meagan E.; Nasser, Taj A.; McGoldrick, Liam J.; Bridges, Lance C.

    2014-11-28

    Highlights: • Transcription and translation are required for retinoid-induced lymphocyte adhesion. • RAR activation is sufficient to induced lymphocyte cell adhesion. • Vitamin D derivatives inhibit RAR-prompted lymphocyte adhesion. • Adhesion occurs through a novel binding site within ADAM disintegrin domains. • RARα is a key nuclear receptor for retinoid-dependent lymphocyte cell adhesion. - Abstract: Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH){sub 2}D{sub 3}, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.

  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).

  8. Linked regulation of motility and integrin function in activated migrating neutrophils revealed by interference in remodelling of the cytoskeleton.

    PubMed

    Anderson, Stephen I; Behrendt, Barbara; Machesky, Laura M; Insall, Robert H; Nash, Gerard B

    2003-02-01

    Neutrophils migrate rapidly by co-ordinating regulation of their beta2-integrin adhesion with turnover of filamentous F-actin. The seven-protein Arp2/3 complex regulates actin polymerisation upon activation by proteins of the WASP-family. To investigate links between actin polymerisation, adhesion, and migration, we used a novel osmotic-shock method to load neutrophils with peptides: (1). WASP-WA and Scar-WA (which incorporate the actin- and Arp2/3-binding regions of WASP and Scar1), to compete with endogenous WASP-family members; (2). proline rich motifs (PRM) from the ActA protein of L. monocytogenes or from vinculin, which bind vasodilator-stimulated phosphoprotein (VASP), a regulator of cytoskeleton assembly. In a flow system, rolling-adherent neutrophils were stimulated with formyl tri-peptide. This caused rapid immobilisation, followed by migration with increasing velocity, supported by activated beta2-integrin CD11b/CD18. Loading ActA PRM (but not vinculin PRM) caused concentration-dependent reduction in migration velocity. At the highest concentration, unstimulated neutrophils had elevated F-actin and were rigid, but could not change their F-actin content or shape upon stimulation. Scar-WA also caused marked reduction in migration rate, but WASP-WA had a lesser effect. Scar-WA did not modify activation-dependent formation of F-actin or change in shape. However, a reduction in rate of downregulation of integrin adhesion appeared to contribute to impaired migration. These studies show that interference in cytoskeletal reorganisation that follows activation in neutrophils, can impair regulation of integrin function as well as motility. They also suggest a role of the Arp2/3 complex and WASP-family in co-ordinating actin polymerisation and integrin function in migrating neutrophils. Copyright 2003 Wiley-Liss, Inc.

  9. VLA-4 integrin concentrates at the peripheral supramolecular activation complex of the immune synapse and drives T helper 1 responses.

    PubMed

    Mittelbrunn, María; Molina, Ana; Escribese, María M; Yáñez-Mó, María; Escudero, Ester; Ursa, Angeles; Tejedor, Reyes; Mampaso, Francisco; Sánchez-Madrid, Francisco

    2004-07-27

    The integrin alpha 4 beta 1 (VLA-4) not only mediates the adhesion and transendothelial migration of leukocytes, but also provides costimulatory signals that contribute to the activation of T lymphocytes. However, the behavior of alpha 4 beta 1 during the formation of the immune synapse is currently unknown. Here, we show that alpha 4 beta 1 is recruited to both human and murine antigen-dependent immune synapses, when the antigen-presenting cell is a B lymphocyte or a dendritic cell, colocalizing with LFA-1 at the peripheral supramolecular activation complex. However, when conjugates are formed in the presence of anti-alpha 4 antibodies, VLA-4 colocalizes with the CD3-zeta chain at the center of the synapse. In addition, antibody engagement of alpha 4 integrin promotes polarization toward a T helper 1 (Th1) response in human in vitro models of CD4(+) T cell differentiation and naïve T cell priming by dendritic cells. The in vivo administration of anti-alpha 4 integrin antibodies also induces an immune deviation to Th1 response that dampens a Th2-driven autoimmune nephritis in Brown Norway rats. These data reveal a regulatory role of alpha 4 integrins on T lymphocyte-antigen presenting cell cognate immune interactions.

  10. VLA-4 integrin concentrates at the peripheral supramolecular activation complex of the immune synapse and drives T helper 1 responses

    NASA Astrophysics Data System (ADS)

    Mittelbrunn, María; Molina, Ana; Escribese, María M.; Yáñez-Mó, María; Escudero, Ester; Ursa, Ángeles; Tejedor, Reyes; Mampaso, Francisco; Sánchez-Madrid, Francisco

    2004-07-01

    The integrin 41 (VLA-4) not only mediates the adhesion and transendothelial migration of leukocytes, but also provides costimulatory signals that contribute to the activation of T lymphocytes. However, the behavior of 41 during the formation of the immune synapse is currently unknown. Here, we show that 41 is recruited to both human and murine antigen-dependent immune synapses, when the antigen-presenting cell is a B lymphocyte or a dendritic cell, colocalizing with LFA-1 at the peripheral supramolecular activation complex. However, when conjugates are formed in the presence of anti-4 antibodies, VLA-4 colocalizes with the CD3- chain at the center of the synapse. In addition, antibody engagement of 4 integrin promotes polarization toward a T helper 1 (Th1) response in human in vitro models of CD4+ T cell differentiation and naïve T cell priming by dendritic cells. The in vivo administration of anti-4 integrin antibodies also induces an immune deviation to Th1 response that dampens a Th2-driven autoimmune nephritis in Brown Norway rats. These data reveal a regulatory role of 4 integrins on T lymphocyte-antigen presenting cell cognate immune interactions.

  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. © 2015. Published by The Company of Biologists Ltd.

  12. Expression/activation of α5β1 integrin is linked to the β-catenin signaling pathway to drive migration in glioma cells

    PubMed Central

    Renner, Guillaume; Noulet, Fanny; Mercier, Marie-Cécile; Choulier, Laurence; Etienne-Selloum, Nelly; Gies, Jean-Pierre; Lehmann, Maxime; Lelong-Rebel, Isabelle; Martin, Sophie; Dontenwill, Monique

    2016-01-01

    The Wnt/beta catenin pathway has been highlighted as an important player of brain tumors aggressiveness and resistance to therapies. Increasing knowledges of the regulation of beta-catenin transactivation point out its hub position in different pathophysiological outcomes in glioma such as survival and migration. Crosstalks between integrins and beta-catenin pathways have been suggested in several tumor tissues. As we demonstrated earlier that α5β1 integrin may be considered as a therapeutic target in high grade glioma through its contribution to glioma cell migration and resistance to chemotherapy, we addressed here the potential relationship between α5β1 integrin and beta-catenin activation in glioma cells. We demonstrated that overexpression and activation by fibronectin of α5β1 integrin allowed the transactivation of beta-catenin gene targets included in an EMT-like program that induced an increase in cell migration. Hampering of beta catenin activation and cell migration could be similarly achieved by a specific integrin antagonist. In addition we showed that α5β1 integrin/AKT axis is mainly involved in these processes. However, blockade of beta-catenin by XAV939 (tankyrase inhibitor leading to beta-catenin degradation) did not synergize with p53 activation aiming to cell apoptosis as was the case with integrin antagonists. We therefore propose a dual implication of α5β1 integrin/AKT axis in glioma cell resistance to therapies and migration each supported by different signaling pathways. Our data thus suggest that α5β1 integrin may be added to the growing list of beta-catenin modulators and provide new evidences to assign this integrin as a valuable target to fight high grade glioma. PMID:27613837

  13. Fibroblast activation protein increases metastatic potential of fibrosarcoma line HT1080 through upregulation of integrin-mediated signaling pathways.

    PubMed

    Baird, Sarah K; Allan, Laura; Renner, Christoph; Scott, Fiona E; Scott, Andrew M

    2015-06-01

    The serine protease fibroblast activation protein (FAP) is selectively expressed on tumour-associated fibroblasts in most human epithelial tumours, as well as on some mesenchymal tumours such as sarcoma. High FAP expression is most often associated with poor outcome and increased metastasis. Here, we compare the in vitro metastatic potential of HT1080 fibrosarcoma cells with and without FAP expression in order to elucidate the mechanism by which FAP may influence metastasis. In the presence of FAP, cells were more adhesive to extracellular matrix proteins and migrated and invaded through Matrigel to a greater degree. The anti-FAP antibody ESC11, which caused internalization of FAP, decreased adhesion and migration, but only when cells expressed FAP. It was also found that blocking activity of integrins β1 and αvβ3 reduced both cell adhesion and migration and this effect was much more marked in FAP-expressing HT1080 cells than mock-transfected HT1080 cells. The expression or activation of intracellular proteins that form part of the downstream signaling of integrins, including integrin-linked kinase, Rac1 and focal adhesion kinase, was also upregulated when FAP was expressed, suggesting that FAP not only upregulates metastatic-like cell behaviours through interaction with integrins, but also influences the intracellular signaling of integrins. This was confirmed using both PI3 kinase and Src kinase inhibitors, which decreased adhesion and migration in FAP-expressing cells, but did not affect mock-transfected HT1080 cells. FAP is therefore a useful target for anti-cancer therapy, as not only is its expression tumour-selective, but its downregulation has the potential to reduce incidence of metastasis.

  14. Integrin α6β4 cooperates with LPA signaling to stimulate Rac through AKAP-Lbc-mediated RhoA activation.

    PubMed

    O'Connor, Kathleen L; Chen, Min; Towers, L Nicole

    2012-02-01

    The α(6)β(4) integrin promotes carcinoma invasion through its ability to promote directed migration and polarization of carcinoma cells. In this study, we explore how the α(6)β(4) integrin cooperates with lysophosphatidic acid (LPA) to activate Rho and Rac small GTPases. Through the use of dominant negative Rho constructs, C3 exotransferase, and Rho kinase inhibitor, we find that Rho is critical for LPA-dependent chemotaxis and lamellae formation. However, utilization of specific Rho isoforms depends on integrin α(6)β(4) expression status. Integrin α(6)β(4)-negative MDA-MB-435 cells utilize only RhoC for motility, whereas integrin α(6)β(4)-expressing cells utilize RhoC but additionally activate and utilize RhoA for LPA-dependent cell motility and lamellae formation. Notably, the activation of RhoA by cooperative LPA and integrin α(6)β(4) signaling requires the Rho guanine nucleotide exchange factor AKAP-Lbc. We also determine that integrin α(6)β(4) cannot activate Rac1 directly but promotes LPA-mediated Rac1 activation that is dependent on RhoA activity and de novo β(1) integrin ligation. Finally, we find that the regulation of Rac1 and RhoA in response to LPA is differentially regulated by phosphodiesterases, PKA, and phosphatidylinositol 3-kinase, thus supporting their spatially distinct compartmentalization. In summary, signaling from integrin α(6)β(4) facilitates LPA-stimulated chemotaxis through preferential activation of RhoA, which, in turn, facilitates activation of Rac1.

  15. The NLRP3 Inflammasome Is a Pathogen Sensor for Invasive Entamoeba histolytica via Activation of α5β1 Integrin at the Macrophage-Amebae Intercellular Junction.

    PubMed

    Mortimer, Leanne; Moreau, France; Cornick, Steve; Chadee, Kris

    2015-05-01

    Entamoeba histolytica (Eh) is an extracellular protozoan parasite of humans that invades the colon to cause life-threatening intestinal and extra-intestinal amebiasis. Colonized Eh is asymptomatic, however, when trophozoites adhere to host cells there is a considerable inflammatory response that is critical in the pathogenesis of amebiasis. The host and/or parasite factors that trigger the inflammatory response to invading Eh are not well understood. We recently identified that Eh adherence to macrophages induces inflammasome activation and in the present study we sought to determine the molecular events upon contact that coordinates this response. Here we report that Eh contact-dependent activation of α5β1 integrin is critical for activation of the NLRP3 inflammasome. Eh-macrophage contact triggered recruitment of α5β1 integrin and NLRP3 into the intercellular junction, where α5β1 integrin underwent activation by an integrin-binding cysteine protease on the parasite surface, termed EhCP5. As a result of its activation, α5β1 integrin induced ATP release into the extracellular space through opening of pannexin-1 channels that signalled through P2X7 receptors to deliver a critical co-stimulatory signal that activated the NLRP3 inflammasome. Both the cysteine protease activity and integrin-binding domain of EhCP5 were required to trigger α5β1 integrin that led to ATP release and NLRP3 inflammasome activation. These findings reveal engagement of α5β1 integrin across the parasite-host junction is a key regulatory step that initiates robust inflammatory responses to Eh. We propose that α5β1 integrin distinguishes Eh direct contact and functions with NLRP3 as pathogenicity sensor for invasive Eh infection.

  16. The NLRP3 Inflammasome Is a Pathogen Sensor for Invasive Entamoeba histolytica via Activation of α5β1 Integrin at the Macrophage-Amebae Intercellular Junction

    PubMed Central

    Mortimer, Leanne; Moreau, France; Cornick, Steve; Chadee, Kris

    2015-01-01

    Entamoeba histolytica (Eh) is an extracellular protozoan parasite of humans that invades the colon to cause life-threatening intestinal and extra-intestinal amebiasis. Colonized Eh is asymptomatic, however, when trophozoites adhere to host cells there is a considerable inflammatory response that is critical in the pathogenesis of amebiasis. The host and/or parasite factors that trigger the inflammatory response to invading Eh are not well understood. We recently identified that Eh adherence to macrophages induces inflammasome activation and in the present study we sought to determine the molecular events upon contact that coordinates this response. Here we report that Eh contact-dependent activation of α5β1 integrin is critical for activation of the NLRP3 inflammasome. Eh-macrophage contact triggered recruitment of α5β1 integrin and NLRP3 into the intercellular junction, where α5β1 integrin underwent activation by an integrin-binding cysteine protease on the parasite surface, termed EhCP5. As a result of its activation, α5β1 integrin induced ATP release into the extracellular space through opening of pannexin-1 channels that signalled through P2X7 receptors to deliver a critical co-stimulatory signal that activated the NLRP3 inflammasome. Both the cysteine protease activity and integrin-binding domain of EhCP5 were required to trigger α5β1 integrin that led to ATP release and NLRP3 inflammasome activation. These findings reveal engagement of α5β1 integrin across the parasite-host junction is a key regulatory step that initiates robust inflammatory responses to Eh. We propose that α5β1 integrin distinguishes Eh direct contact and functions with NLRP3 as pathogenicity sensor for invasive Eh infection. PMID:25955828

  17. Distinct determinants on collagen support alpha 2 beta 1 integrin-mediated platelet adhesion and platelet activation.

    PubMed Central

    Santoro, S A; Walsh, J J; Staatz, W D; Baranski, K J

    1991-01-01

    Recent studies have revealed that the sequence of amino acids asp-gly-glu-ala represents an essential determinant of the site within the alpha 1(I)-CB3 fragment of collagen recognized by the alpha 2 beta 1 integrin cell surface collagen receptor (Staatz et al., 1991). Studies employing chemical modifications of collagen amino acid side chains confirm both the essential nature of the acidic side chains of aspartic acid and glutamic acid residues and the nonessentiality of lysine epsilon-amino groups in supporting adhesion mediated by the alpha 2 beta 1 integrin. The approach also indicates the presence of a distinct determinant on collagen separate from the alpha 2 beta 1 recognition site that contains essential lysine side chains and that is necessary for subsequent interactions with the platelet surface that give rise to collagen-induced platelet activation and secretion. The two-step, two-site model for cellular signaling involving both an integrin and a signal-transducing coreceptor suggested by these data may be common to other integrin-mediated processes. PMID:1809397

  18. Anti-alpha 4 integrin antibody induces apoptosis in murine thymocytes and staphylococcal enterotoxin B-activated lymph node T cells.

    PubMed Central

    Tchilian, E Z; Owen, J J; Jenkinson, E J

    1997-01-01

    We have shown that an antibody (9C10) to the alpha 4 integrin induces apoptosis in murine immature CD4+ CD8+ thymocytes and in activated (but not resting) mature lymph node T cells. In both cases, apoptosis is blocked by the highly selective protein kinase C (PKC) inhibitor Ro31.8425, suggesting that 9C10 induces signalling through the alpha 4 integrin resulting in PKC activation leading to apoptosis. Overall, our results indicate the potential role of the alpha 4 integrin-mediated interactions in apoptosis induction during T-cell development and following mature T-cell activation. PMID:9486103

  19. Induced keratinocyte hyper-proliferation in alpha2beta1 integrin transgenic mice results in systemic immune cell activation.

    PubMed

    Teige, Ingrid; Bäcklund, Alexandra; Svensson, Lars; Kvist, Peter Helding; Petersen, Thomas Kongstad; Kemp, Kåre

    2010-01-01

    alpha2beta1 integrins are normally confined to the proliferating basal layers of the epidermis. However, during wound healing and in psoriasis, these integrins are expressed on keratinocytes in suprabasal layers correlating with a less differentiated phenotype. Transgenic mice expressing alpha2beta1 integrins under the involucrine promoter have previously been demonstrated, to various degrees, spontaneously develop a skin disorder resembling psoriasis. Herein, we show that a mild epidermal wounding induces a uniform acanthosis together with an influx of immune cells. The disease initiates as a normal wound healing process and is completely restored in wildtype mice by day 14. However, in the integrin transgenic mice a chronic inflammation develops, a process that can be compared to the Koebner phenomenon in psoriatic patients. In this study, we have followed the integrin transgenic mice for five weeks, where substantial keratinocyte hyper-proliferation, inflammatory infiltration and high cytokine levels within the skin can still be observed. In addition, draining lymph nodes were dramatically increased in size and contained highly activated T cells, as well as APCs secreting large amounts of pro-inflammatory cytokines. Furthermore, the systemic immune response was affected with increased spleen size, elevated cytokine levels in the serum and altered lymphocyte trafficking patterns, very much resembling what is seen in psoriasis patients. Finally, CD4(+) T cell depletion was not able to affect the onset or progression of skin inflammation. This suggests that altered keratinocyte differentiation and proliferation can drive a skin inflammation and cause chronic immune cell activation both at a local and systemic level.

  20. SNX17 Affects T Cell Activation by Regulating T Cell Receptor and Integrin Recycling

    PubMed Central

    Osborne, Douglas G.; Piotrowski, Joshua T.; Dick, Christopher J.; Zhang, Jin-San; Billadeau, Daniel D.

    2015-01-01

    A key component in T cell activation is the endosomal recycling of receptors to the cell surface, thereby allowing continual integration of signaling and antigen recognition. One protein potentially involved in T cell receptor transport is sorting nexin 17 (SNX17). SNX proteins have been found to bind proteins involved in T cell activation, but specifically the role of SNX17 in receptor recycling and T cell activation is unknown. Using immunofluorescence, we find that SNX17 co-localizes with TCR and localizes to the immune synapse in T-APC conjugates. Significantly, knockdown of the SNX17 resulted in fewer T-APC conjugates, lower CD69, TCR, and LFA-1 surface expression, as well as lower overall TCR recycling compared to control T cells. Lastly, we identified the FERM-domain of SNX17 as being responsible in the binding and trafficking of TCR and LFA-1 to the cell surface. These data suggest that SNX17 plays a role in the maintenance of normal surface levels of activating receptors and integrins to permit optimum T cell activation at the immune synapse. PMID:25825439

  1. Regulation and function of an activation-dependent epitope of the beta 1 integrins in vascular cells after balloon injury in baboon arteries and in vitro.

    PubMed Central

    Koyama, N.; Seki, J.; Vergel, S.; Mattsson, E. J.; Yednock, T.; Kovach, N. L.; Harlan, J. M.; Clowes, A. W.

    1996-01-01

    Migration and proliferation of endothelial cells (ECs) and smooth muscle cells (SMCs) contribute to the response to injury in damaged and atherosclerotic vessels. These events might be regulated by cellular interactions with extracellular matrix through the expression and activation of integrins. To study the functions of beta 1 integrins in the vessel wall, we used monoclonal antibody (MAb) 15/7, which recognizes an activation epitope of beta 1 integrin subunits, and MAb 8A2, which induces a high affinity form of beta 1 integrins recognized by MAb 15/7. Immunohistochemical analyses were done on samples of normal baboon saphenous arteries and from arteries subjected to balloon injury. EC and SMC expressed the activation epitope of beta 1 integrin in uninjured arteries. By contrast, in balloon-injured arteries 6 weeks after injury, regenerating EC did not express the activation epitope, and there was no decrease in the expression of total beta 1 integrin, whereas SMC migrating into the intima exhibited decreased expression of the total and activated beta 1 integrin. Flow cytometer analysis of cultured cells indicated that baboon EC and SMC weakly express the activation epitope of beta 1 integrin. Next, we determined by utilizing MAb 8A2 the effects of increased expression of activation epitope of beta 1 integrin on the functions of SMC and EC. The activation of beta 1 integrins on SMC induced by MAb 8A2 enhanced SMC adhesion and suppressed SMC migration in a Boyden chamber assay. SMC proliferation was inhibited by MAb 8A2 dose-dependently. Similarly, MAb 8A2-induced activation of beta 1 integrins on EC suppressed EC migration into a wound. However, MAb 8A2 did not affect the basic fibroblast growth factor-induced proliferation of EC, although it blocked the decrease in EC number caused by the removal of basic fibroblast growth factor. These results suggest that activation of beta 1 integrins in vascular cells is regulated in a cell-type dependent manner and plays an

  2. Differential regulation of phosphoinositide metabolism by alphaVbeta3 and alphaVbeta5 integrins upon smooth muscle cell migration.

    PubMed

    Paulhe, F; Racaud-Sultan, C; Ragab, A; Albiges-Rizo, C; Chap, H; Iberg, N; Morand, O; Perret, B

    2001-11-09

    Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins.

  3. PTH-related protein upregulates integrin {alpha}6{beta}4 expression and activates Akt in breast cancer cells

    SciTech Connect

    Shen Xiaoli; Falzon, Miriam . E-mail: mfalzon@utmb.edu

    2006-11-15

    Breast cancer is the most common carcinoma that metastasizes to bone. Tumor-produced parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption, is a major mediator of the osteolytic process in breast cancer. We have previously shown that PTHrP increases breast cancer cell proliferation, survival, migration, and pro-invasive integrin {alpha}6{beta}4 expression. To determine the role of integrin {alpha}6{beta}4 in these PTHrP-mediated effects, we utilized two strategies to modulate expression of the {alpha}6 and {beta}4 subunits in parental and PTHrP-overexpressing MDA-MB-231 and MCF-7 cells: overexpression of {alpha}6{beta}4 by transfection with constructs encoding the {alpha}6 and {beta}4 subunits, and suppression of endogenous {alpha}6{beta}4 expression by transfection with siRNAs targeting these subunits. We now show that the effects of PTHrP are mediated via upregulation of integrin {alpha}6{beta}4 expression. We also show that integrin {alpha}6{beta}4 expression is modulated at the mRNA level, indicating a transcriptional and/or post-transcriptional mechanism of action for PTHrP. PTHrP expression also increased the levels of phosphorylated Akt, with a consequent increase in the levels of phosphorylated (inactive) glycogen synthase kinase-3 (GSK-3). The role of PTHrP in breast cancer growth and metastasis may thus be mediated via upregulation of integrin {alpha}6{beta}4 expression and Akt activation, with consequent inactivation of GSK-3.

  4. Paired Ig-Like Type 2 Receptor-Derived Agonist Ligands Ameliorate Inflammatory Reactions by Downregulating β1 Integrin Activity

    PubMed Central

    Lee, Kyoung-Jin; Lim, Dongyoung; Yoo, Yeon Ho; Park, Eun-Ji; Lee, Sun-Hee; Yadav, Birendra Kumar; Lee, Yong-Ki; Park, Jeong Hyun; Kim, Daejoong; Park, Kyeong Han; Hahn, Jang-Hee

    2016-01-01

    The paired immunoglobulin-like type 2 receptor (PILR) family consists of two functionally opposite members, inhibitory PILRα and activating PILRβ receptors. PILRs are widely expressed in various immune cells and interact with their ligands, especially CD99 expressed on activated T cells, to participate in immune responses. Here we investigated whether PILR-derived agonists inhibit β1 integrin activity as ligands for CD99. PILR-derived peptides as well as PILR-Fc fusion proteins prevented cell adhesion to fibronectin through the regulation of β1 integrin activity. Especially, PILRpep3, a representative 3-mer peptide covering the conserved motifs of the PILR extracellular domain, prevented the clustering and activation of β1 integrin by dephosphorylating FAK and vinculin, which are major components of focal adhesion. In addition, PILRpep3 inhibited transendothelial migration of monocytes as well as endothelial cell tube formation. Furthermore, upon intraperitoneal injection of PILRpep3 into mice with collagen-induced arthritis, the inflammatory response of rheumatoid arthritis was strongly suppressed. Taken together, these results suggest that PILR-derived agonist ligands may prevent the inflammatory reactions of rheumatoid arthritis by activating CD99. PMID:27306643

  5. Kindlin-3–mediated integrin adhesion is dispensable for quiescent but essential for activated hematopoietic stem cells

    PubMed Central

    Ruppert, Raphael; Moser, Markus; Sperandio, Markus; Rognoni, Emanuel; Orban, Martin; Liu, Wen-Hsin; Schulz, Ansgar S.; Oostendorp, Robert A.J.; Massberg, Steffen

    2015-01-01

    Hematopoietic stem cells (HSCs) generate highly dividing hematopoietic progenitor cells (HPCs), which produce all blood cell lineages. HSCs are usually quiescent, retained by integrins in specific niches, and become activated when the pools of HPCs decrease. We report that Kindlin-3–mediated integrin activation controls homing of HSCs to the bone marrow (BM) and the retention of activated HSCs and HPCs but not of quiescent HSCs in their BM niches. Consequently, Kindlin-3–deficient HSCs enter quiescence and remain in the BM when cotransplanted with wild-type hematopoietic stem and progenitor cells (HSPCs), whereas they are hyperactivated and lost in the circulation when wild-type HSPCs are absent, leading to their exhaustion and reduced survival of recipients. The accumulation of HSPCs in the circulation of leukocyte adhesion deficiency type III patients, who lack Kindlin-3, underlines the conserved functions of Kindlin-3 in man and the importance of our findings for human disease. PMID:26282877

  6. Polymorphonuclear leukocyte integrin pattern, at baseline and after activation, in type 2 diabetic subjects with macrovascular complications.

    PubMed

    Caimi, G; Montana, M; Ferrara, F; Porretto, F; Musso, M; Canino, B; Lo Presti, R

    2003-03-01

    In vascular atherosclerotic disease and in diabetes mellitus few studies have evaluated the polymorphonuclear leukocyte (PMN) adhesion molecule pattern. In this study we examined the PMN integrin expression at baseline and after activation in controls and type 2 diabetic subjects with macrovascular complications (MVC). We enrolled 21 subjects with type 2 diabetes mellitus and macrovascular complications, localized in peripheral, coronary and cerebral sites. The patients had peripheral occlusive arterial disease, chronic cerebrovascular disease or coronary heart disease. We evaluated the expression of some PMN integrins (CD11a, CD11b, CD11c, CD18), using flow cytofluorimetry, at baseline and after in vitro activation with 4-phorbol-12-myristate-13 acetate. Type 2 diabetic subjects with MVC showed, compared to normals, an increase of CD11a and CD18 and a decrease of CD11b and CD11c. After activation, in PMN(s) of normal subjects, we found an increase in the expression of all adhesion molecules, while in PMNS of type 2 diabetic subjects with MVC we observed an increase of CD11b and CD11c and a decrease of CD11a. In type 2 diabetic patients with MVC the basal upregulation of CD11a and CD18 may be related to the PMN spontaneous activation, while the behavior of CD11b may depend on its self-consumption. After activation the CD11a modification may be due to its cleavage or to an altered integrin phosphorylation/dephosphorylation balance.

  7. Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment

    PubMed Central

    Maza, Paloma K.; Suzuki, Erika

    2016-01-01

    Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-β-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation. PMID:27148251

  8. Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating β1 integrin

    PubMed Central

    Shih, Peggy T.; Elices, Mariano J.; Fang, Zhuang T.; Ugarova, Tatiana P.; Strahl, Dana; Territo, Mary C.; Frank, Joy S.; Kovach, Nicholas L.; Cabanas, Carlos; Berliner, Judith A.; Vora, Devendra K.

    1999-01-01

    We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic α4β1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial α4β1 ligand in MM-LDL-treated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation of β1integrins, particularly those associated with α5 integrins.Activation of β1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated β1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated β1 showed increased staining on the luminal endothelium of human coronary lesions with active monocyte entry. These results suggest the importance of these integrin ligand interactions in human atherosclerosis. J. Clin. Invest. 103:613–625 (1999) PMID:10074478

  9. The Transcription Factor IRF8 Activates Integrin-mediated TGFβ Signaling and Promotes Neuroinflammation

    PubMed Central

    Yoshida, Yuko; Yoshimi, Ryusuke; Yoshii, Hiroaki; Kim, Daniel; Dey, Anup; Xiong, Huabao; Munasinghe, Jeeva; Yazawa, Itaru; O’Donovan, Michael J.; Maximova, Olga A.; Sharma, Suveena; Zhu, Jinfang; Wang, Hongsheng; Morse, Herbert C.; Ozato, Keiko

    2014-01-01

    SUMMARY Recent epidemiological studies have identified interferon regulatory factor 8 (IRF8) as a susceptibility factor for multiple sclerosis (MS). However, how IRF8 influences the neuroinflammatory disease has remained unknown. By studying the role of IRF8 in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we found that Irf8−/− mice are resistant to EAE. Furthermore, expression of IRF8 in antigen presenting cells (APCs, such as macrophages, dendritic cells and microglia), but not in T cells, facilitated disease onset and progression through multiple pathways. IRF8 enhanced αvβ8 integrin expression in APCs and activated TGFβ signaling leading to T helper 17 (Th17) cell differentiation. IRF8 induced a cytokine milieu that favored growth and maintenance of Th1 and Th17 cells, by stimulating interleukin-12 (IL12) and IL23 production, but inhibiting IL27 during EAE. Finally, IRF8 activated microglia and exacerbated neuroinflammation. Together, this work provides mechanistic bases by which IRF8 contributes to the pathogenesis of MS. PMID:24485804

  10. The transcription factor IRF8 activates integrin-mediated TGF-β signaling and promotes neuroinflammation.

    PubMed

    Yoshida, Yuko; Yoshimi, Ryusuke; Yoshii, Hiroaki; Kim, Daniel; Dey, Anup; Xiong, Huabao; Munasinghe, Jeeva; Yazawa, Itaru; O'Donovan, Michael J; Maximova, Olga A; Sharma, Suveena; Zhu, Jinfang; Wang, Hongsheng; Morse, Herbert C; Ozato, Keiko

    2014-02-20

    Recent epidemiological studies have identified interferon regulatory factor 8 (IRF8) as a susceptibility factor for multiple sclerosis (MS). However, how IRF8 influences the neuroinflammatory disease has remained unknown. By studying the role of IRF8 in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we found that Irf8(-/-) mice are resistant to EAE. Furthermore, expression of IRF8 in antigen-presenting cells (APCs, such as macrophages, dendritic cells, and microglia), but not in T cells, facilitated disease onset and progression through multiple pathways. IRF8 enhanced αvβ8 integrin expression in APCs and activated TGF-β signaling leading to T helper 17 (Th17) cell differentiation. IRF8 induced a cytokine milieu that favored growth and maintenance of Th1 and Th17 cells, by stimulating interleukin-12 (IL-12) and IL-23 production, but inhibiting IL-27 during EAE. Finally, IRF8 activated microglia and exacerbated neuroinflammation. Together, this work provides mechanistic bases by which IRF8 contributes to the pathogenesis of MS.

  11. PPFIA1 drives active α5β1 integrin recycling and controls fibronectin fibrillogenesis and vascular morphogenesis

    PubMed Central

    Mana, Giulia; Clapero, Fabiana; Panieri, Emiliano; Panero, Valentina; Böttcher, Ralph T.; Tseng, Hui-Yuan; Saltarin, Federico; Astanina, Elena; Wolanska, Katarzyna I.; Morgan, Mark R.; Humphries, Martin J.; Santoro, Massimo M.; Serini, Guido; Valdembri, Donatella

    2016-01-01

    Basolateral polymerization of cellular fibronectin (FN) into a meshwork drives endothelial cell (EC) polarity and vascular remodelling. However, mechanisms coordinating α5β1 integrin-mediated extracellular FN endocytosis and exocytosis of newly synthesized FN remain elusive. Here we show that, on Rab21-elicited internalization, FN-bound/active α5β1 is recycled to the EC surface. We identify a pathway, comprising the regulators of post-Golgi carrier formation PI4KB and AP-1A, the small GTPase Rab11B, the surface tyrosine phosphatase receptor PTPRF and its adaptor PPFIA1, which we propose acts as a funnel combining FN secretion and recycling of active α5β1 integrin from the trans-Golgi network (TGN) to the EC surface, thus allowing FN fibrillogenesis. In this framework, PPFIA1 interacts with active α5β1 integrin and localizes close to EC adhesions where post-Golgi carriers are targeted. We show that PPFIA1 is required for FN polymerization-dependent vascular morphogenesis, both in vitro and in the developing zebrafish embryo. PMID:27876801

  12. Slingshot-Cofilin activation mediates mitochondrial and synaptic dysfunction via Aβ ligation to β1-integrin conformers

    PubMed Central

    Woo, J A; Zhao, X; Khan, H; Penn, C; Wang, X; Joly-Amado, A; Weeber, E; Morgan, D; Kang, D E

    2015-01-01

    The accumulation of amyloid-β protein (Aβ) is an early event associated with synaptic and mitochondrial damage in Alzheimer's disease (AD). Recent studies have implicated the filamentous actin (F-actin) severing protein, Cofilin, in synaptic remodeling, mitochondrial dysfunction, and AD pathogenesis. However, whether Cofilin is an essential component of the AD pathogenic process and how Aβ impinges its signals to Cofilin from the neuronal surface are unknown. In this study, we found that Aβ42 oligomers (Aβ42O, amyloid-β protein 1–42 oligomers) bind with high affinity to low or intermediate activation conformers of β1-integrin, resulting in the loss of surface β1-integrin and activation of Cofilin via Slingshot homology-1 (SSH1) activation. Specifically, conditional loss of β1-integrin prevented Aβ42O-induced Cofilin activation, and allosteric modulation or activation of β1-integrin significantly reduced Aβ42O binding to neurons while blocking Aβ42O-induced reactive oxygen species (ROS) production, mitochondrial dysfunction, depletion of F-actin/focal Vinculin, and apoptosis. Cofilin, in turn, was required for Aβ42O-induced loss of cell surface β1-integrin, disruption of F-actin/focal Talin–Vinculin, and depletion of F-actin-associated postsynaptic proteins. SSH1 reduction, which mitigated Cofilin activation, prevented Aβ42O-induced mitochondrial Cofilin translocation and apoptosis, while AD brain mitochondria contained significantly increased activated/oxidized Cofilin. In mechanistic support in vivo, AD mouse model (APP (amyloid precursor protein)/PS1) brains contained increased SSH1/Cofilin and decreased SSH1/14-3-3 complexes, indicative of SSH1–Cofilin activation via release of SSH1 from 14-3-3. Finally, genetic reduction in Cofilin rescued APP/Aβ-induced synaptic protein loss and gliosis in vivo as well as deficits in long-term potentiation (LTP) and contextual memory in APP/PS1 mice. These novel findings therefore implicate the essential

  13. AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic

    PubMed Central

    Thavarajah, Thanusi; Medvedev, Sergei; Bowden, Peter; Marshall, John G.; Antonescu, Costin N.

    2015-01-01

    The cell surface proteome controls numerous cellular functions including cell migration and adhesion, intercellular communication and nutrient uptake. Cell surface proteins are controlled by acute changes in protein abundance at the plasma membrane through regulation of endocytosis and recycling (endomembrane traffic). Many cellular signals regulate endomembrane traffic, including metabolic signaling; however, the extent to which the cell surface proteome is controlled by acute regulation of endomembrane traffic under various conditions remains incompletely understood. AMP-activated protein kinase (AMPK) is a key metabolic sensor that is activated upon reduced cellular energy availability. AMPK activation alters the endomembrane traffic of a few specific proteins, as part of an adaptive response to increase energy intake and reduce energy expenditure. How increased AMPK activity during energy stress may globally regulate the cell surface proteome is not well understood. To study how AMPK may regulate the cell surface proteome, we used cell-impermeable biotinylation to selectively purify cell surface proteins under various conditions. Using ESI-MS/MS, we found that acute (90 min) treatment with the AMPK activator A-769662 elicits broad control of the cell surface abundance of diverse proteins. In particular, A-769662 treatment depleted from the cell surface proteins with functions in cell migration and adhesion. To complement our mass spectrometry results, we used other methods to show that A-769662 treatment results in impaired cell migration. Further, A-769662 treatment reduced the cell surface abundance of β1-integrin, a key cell migration protein, and AMPK gene silencing prevented this effect. While the control of the cell surface abundance of various proteins by A-769662 treatment was broad, it was also selective, as this treatment did not change the cell surface abundance of the transferrin receptor. Hence, the cell surface proteome is subject to acute

  14. A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins

    PubMed Central

    Kapp, Tobias G.; Rechenmacher, Florian; Neubauer, Stefanie; Maltsev, Oleg V.; Cavalcanti-Adam, Elisabetta A.; Zarka, Revital; Reuning, Ute; Notni, Johannes; Wester, Hans-Jürgen; Mas-Moruno, Carlos; Spatz, Joachim; Geiger, Benjamin; Kessler, Horst

    2017-01-01

    Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC50-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins αvβ3, αvβ5, αvβ6, αvβ8, α5β1, αIIbβ3, using homogenous ELISA-like solid phase binding assay. PMID:28074920

  15. Janus kinases 1 and 2 regulate chemokine-mediated integrin activation and naïve T-cell homing.

    PubMed

    Pérez-Rivero, Gema; Cascio, Graciela; Soriano, Silvia Fernández; Sanz, Álvaro Gil; de Guinoa, Julia Sáez; Rodríguez-Frade, José Miguel; Gomariz, Rosa P; Holgado, Borja L; Cabañas, Carlos; Carrasco, Yolanda R; Stein, Jens V; Mellado, Mario

    2013-07-01

    Janus kinases (JAKs) are central signaling molecules in cytokine receptor cascades. Although they have also been implicated in chemokine receptor signaling, this function continues to be debated. To address this issue, we established a nucleofection model in primary, nonactivated mouse T lymphocytes to silence JAK expression and to evaluate the ability of these cells to home to lymph nodes. Reduced JAK1 and JAK2 expression impaired naïve T-cell migration in response to gradients of the chemokines CXCL12 and CCL21. In vivo homing of JAK1/JAK2-deficient cells to lymph nodes decreased, whereas intranodal localization and motility were unaffected. JAK1 and JAK2 defects altered CXCL12- and CCL21-triggered ezrin/radixin/moesin (ERM) dephosphorylation and F-actin polymerization, as well as activation of lymphocyte function-associated Ag-1 and very late Ag-4 integrins. As a result, the cells did not adhere firmly to integrin substrates in response to these chemokines. The results demonstrate that JAK1/JAK2 participate in chemokine-induced integrin activation and might be considered a target for modulation of immune cell extravasation and therefore, control of inflammatory reactions. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Constitutive activation of integrin alpha 4 beta 1 defines a unique stage of human thymocyte development

    PubMed Central

    1994-01-01

    Our understanding of thymocyte development and of the positive and negative selection events involved in shaping the repertoire of mature T lymphocytes has been greatly facilitated by the use of transgenic and gene knockout animals. Much less is known about the factors that control the homing and population of the thymus by T cell precursors and the subsequent migration of developing thymocytes through the thymic architecture. As the integrins represent a candidate group of cell surface receptors that may regulate thymocyte development, we have analyzed the expression and function of alpha 4 beta 1 and alpha 5 beta 1 on human thymocytes. A major portion of double positive (CD4+ CD8+) human thymocytes express alpha 4 beta 1 in a constitutively active form and adhere to fibronectin and vascular cell adhesion molecule 1. alpha 4 beta 1 expression is similar on adherent and nonadherent populations, thus, activity reflects the receptor state and not simple expression. The adherent cells are immature, expressing high levels of CD4/CD8 and low levels of CD3 and CD69. In contrast, nonadherent cells possess the phenotype of thymocytes after positive selection, expressing intermediate levels of CD4 and/or CD8 and high levels of CD3 and CD69. The adherent population fails to respond to activation with anti-CD3 and fibronectin, whereas nonadherents exhibit an alpha 5 beta 1- dependent proliferation. Differential regulation of alpha 4 beta 1 and alpha 5 beta 1 receptors may provide a mechanism controlling cellular traffic, differentiation, and positive selection of thymocytes. PMID:8163937

  17. Integrin beta3 regions controlling binding of murine mAb 7E3: implications for the mechanism of integrin alphaIIbbeta3 activation.

    PubMed

    Artoni, Andrea; Li, JiHong; Mitchell, Beau; Ruan, Jian; Takagi, Junichi; Springer, Timothy A; French, Deborah L; Coller, Barry S

    2004-09-07

    Abciximab, a derivative of the murine mAb 7E3, protects against ischemic complications of percutaneous coronary interventions by inhibiting ligand binding to the alphaIIbbeta3 receptor. In this study we identified regions on integrin beta3 that control 7E3 binding. Murine/human amino acid substitutions were created in two regions of the betaA domain that previous studies found to influence 7E3 binding: the C177-C184 loop and K125-N133. The T182N substitution and a K125Q mutation reduced 7E3 binding to human beta3 in complex with alphaIIb. The introduction of both the human C177-C184 region and human W129 into murine beta3 was necessary and sufficient to permit 7E3 binding to the human alphaIIb/murine beta3 complex. Although we cannot exclude allosteric effects, we propose that 7E3 binds between C177-C184 and W129, which are within 15 A of each other in the crystal structure and close to the beta3 metal ion-dependent adhesion site. We previously demonstrated that 7E3 binds more rapidly to activated than unactivated platelets. Because it has been proposed that alphaIIbbeta3 changes from a bent to an extended conformation upon activation, we hypothesized that 7E3 binds less well to the bent than the extended conformation. In support of this hypothesis we found that 7E3 bound less well to an alphaIIbbeta3 construct locked in a bent conformation, and unlocking the conformation restored 7E3 binding. Thus, our data are consistent with alphaIIbbeta3 existing in variably bent conformations in equilibrium with each other on unactivated platelets, and activation resulting in alphaIIbbeta3 adopting a more extended conformation.

  18. Integrin-linked kinase activity regulates Rac- and Cdc42-mediated actin cytoskeleton reorganization via alpha-PIX.

    PubMed

    Filipenko, Nolan R; Attwell, Sarah; Roskelley, Calvin; Dedhar, Shoukat

    2005-09-01

    Cell spreading and migration are regulated in a Rho family GTPase-dependent manner by growth factors and integrin-mediated cell-extracellular matrix (ECM) interactions. The molecular mechanisms involved in the ECM- and growth factor-mediated activation of these small GTPases remain unclear. In the present study, we demonstrate that integrin-linked kinase (ILK), which is a focal adhesion protein activated by both ECM and growth factors, is required for the activation of Rac and Cdc42 in epithelial cells. Ectopic expression of active ILK in mammary epithelial cells induces dramatic reorganization of the actin cytoskeleton and promotes rapid cell spreading on fibronectin. These effects are associated with constitutive activation of both Rac and Cdc42, but not Rho. The use of ILK siRNA or small molecule inhibitors to inhibit ILK expression and kinase activity, respectively, results in diminished cell spreading and actin cytoskeleton reorganization, concomitant with a reduction in Rac and Cdc42 activation. Studies into the mechanism of ILK-mediated Rac activation suggest an important role for the ILK-beta-parvin interaction and the activity of the Rac/Cdc42-specific guanine nucleotide exchange factor alpha-PIX downstream of ILK. Taken together, these data demonstrate an essential role of ILK kinase activity in Rac- and Cdc42-mediated actin cytoskeleton reorganization in epithelial cells, further solidifying a role for ILK in the regulation of cancer cell motility and invasiveness.

  19. Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix.

    PubMed

    Gronthos, S; Simmons, P J; Graves, S E; Robey, P G

    2001-02-01

    To date, the precise interactions between bone marrow stromal cells and the extracellular matrix that govern stromal cell development remain unclear. The integrin super-family of cell-surface adhesion molecules represents a major pathway used by virtually all cell types to interact with different extracellular matrix components. In this study, purified populations of stromal precursor cells were isolated from the STRO-1-positive fraction of normal human marrow, by fluoresence-activated cell sorting, and then assayed for their ability to initiate clonogenic growth in the presence of various integrin ligands. Bone marrow-derived stromal progenitors displayed differential growth to fibronectin, vitronectin, and laminin, over collagen types I and III, but showed a similar affinity for collagen type IV. The integrin heterodimers alpha1beta1, alpha2beta1, alpha5beta1, alpha6beta1, alpha(v)beta3, and alpha(v)beta5 were found to coexpress with the STRO-1 antigen on the cell surface of CFU-F, using dual-color analysis. Furthermore, only a proportion of stromal precursors expressed the integrin alpha4beta1, while no measurable levels of the integrin alpha3beta1 could be detected. Subsequent adhesion studies using functional blocking antibodies to different integrin alpha/beta heterodimers showed that stromal cell growth on collagen, laminin, and fibronectin was mediated by multiple beta1 integrins. In contrast, cloning efficiency in the presence of vitronectin was mediated in part by alpha(v)beta3. When human marrow stromal cells were cultured under osteoinductive conditions, their ability to form a mineralized matrix in vitro was significantly diminished in the presence of a functional blocking monoclonal antibody to the beta1 integrin subunit. The results of this study indicate that beta1 integrins appear to be the predominant adhesion receptor subfamily utilized by stromal precursor cells to adhere and proliferate utilizing matrix glycoproteins commonly found in the bone

  20. The integrin effector PINCH regulates JNK activity and epithelial migration in concert with Ras suppressor 1.

    PubMed

    Kadrmas, Julie L; Smith, Mark A; Clark, Kathleen A; Pronovost, Stephen M; Muster, Nemone; Yates, John R; Beckerle, Mary C

    2004-12-20

    Cell adhesion and migration are dynamic processes requiring the coordinated action of multiple signaling pathways, but the mechanisms underlying signal integration have remained elusive. Drosophila embryonic dorsal closure (DC) requires both integrin function and c-Jun amino-terminal kinase (JNK) signaling for opposed epithelial sheets to migrate, meet, and suture. Here, we show that PINCH, a protein required for integrin-dependent cell adhesion and actin-membrane anchorage, is present at the leading edge of these migrating epithelia and is required for DC. By analysis of native protein complexes, we identify RSU-1, a regulator of Ras signaling in mammalian cells, as a novel PINCH binding partner that contributes to PINCH stability. Mutation of the gene encoding RSU-1 results in wing blistering in Drosophila, demonstrating its role in integrin-dependent cell adhesion. Genetic interaction analyses reveal that both PINCH and RSU-1 antagonize JNK signaling during DC. Our results suggest that PINCH and RSU-1 contribute to the integration of JNK and integrin functions during Drosophila development.

  1. The integrin effector PINCH regulates JNK activity and epithelial migration in concert with Ras suppressor 1

    PubMed Central

    Kadrmas, Julie L.; Smith, Mark A.; Clark, Kathleen A.; Pronovost, Stephen M.; Muster, Nemone; Yates, John R.; Beckerle, Mary C.

    2004-01-01

    Cell adhesion and migration are dynamic processes requiring the coordinated action of multiple signaling pathways, but the mechanisms underlying signal integration have remained elusive. Drosophila embryonic dorsal closure (DC) requires both integrin function and c-Jun amino-terminal kinase (JNK) signaling for opposed epithelial sheets to migrate, meet, and suture. Here, we show that PINCH, a protein required for integrin-dependent cell adhesion and actin–membrane anchorage, is present at the leading edge of these migrating epithelia and is required for DC. By analysis of native protein complexes, we identify RSU-1, a regulator of Ras signaling in mammalian cells, as a novel PINCH binding partner that contributes to PINCH stability. Mutation of the gene encoding RSU-1 results in wing blistering in Drosophila, demonstrating its role in integrin-dependent cell adhesion. Genetic interaction analyses reveal that both PINCH and RSU-1 antagonize JNK signaling during DC. Our results suggest that PINCH and RSU-1 contribute to the integration of JNK and integrin functions during Drosophila development. PMID:15596544

  2. Analysis of the activation state of alpha4beta1 integrin in human B cell lines derived from myeloma, leukemia or lymphoma.

    PubMed

    García-Gila, M; Cabañas, C; García-Pardo, A

    1997-12-01

    Myeloma cells specifically localize in the bone marrow and rarely circulate in blood. To study whether this immobilization could be partially explained by the presence of constitutively activated integrins, particularly alpha4beta1, we used the activation reporter HUTS-21 anti-beta1 mAb. These analyses showed that beta1 integrins on myeloma cells were moderately active and could be upregulated similarly to integrins on lymphoma or leukemia cells. Myeloma cells were also tested for their ability to attach to RGD-containing fibronectin fragments, a property of activated (but not resting) alpha4beta1. Two cell lines adhered to these fragments and this was inhibited by anti-alpha5 but not by anti-alpha4 mAbs. These results show that myeloma cells bear low/moderately active alpha4beta1 and support the notion that multiple interactions contribute to their localization in the bone marrow.

  3. Enhancement of activated beta1-integrin expression by prostaglandin E2 via EP receptors in isolated human coronary arterial endothelial cells: implication for the treatment of Kawasaki disease.

    PubMed

    Kajimoto, M; Ichiyama, T; Ueno, Y; Shiraishi, M; Hasegawa, M; Furukawa, S

    2009-04-01

    Plasma prostaglandin E(2) (PGE(2)) levels are markedly elevated in acute Kawasaki disease (KD). We evaluated the function of the EP receptors in the expression of activated beta(1)-integrin stimulated by PGE(2) in human coronary arterial endothelial cells (HCAEC). We determined the mRNA expression of the PGE(2) receptors, EP receptors (EP(1-4)) in HCAEC by RT-PCR and protein expression by Western blotting. We evaluated the function of the EP receptors in the expression of activated beta(1)-integrin stimulated by PGE(2) in HCAEC, using antagonists and agonists of the EP receptors, by flow cytometry. RT-PCR revealed mRNAs for all four EP receptors in HCAEC. Western blotting demonstrated EP(1), EP(2) and EP(3) expression in HCAEC. The EP(2) and EP(3) agonists enhanced the expression of activated beta(1)-integrin in HCAEC. The potency of the EP(2) agonist was significantly greater than that of the EP(3) agonist. Pretreatment with the EP(1), EP(2) and EP(3) antagonists inhibited the expression of activated beta(1)-integrin induced by PGE(2) in HCAEC. The potency of the EP(2) antagonist was significantly greater than that of the EP(1) and EP(3) antagonists. Our results suggest that PGE(2) mainly induces the activation of beta(1)-integrins via the EP(2) receptor in HCAEC. Our results further suggest that the EP(2) antagonist modulates the inflammatory response during KD vasculitis.

  4. E-cadherin endocytosis regulates the activity of Rap1: a traffic light GTPase at the crossroads between cadherin and integrin function.

    PubMed

    Balzac, Fiorella; Avolio, Maria; Degani, Simona; Kaverina, Irina; Torti, Mauro; Silengo, Lorenzo; Small, J Victor; Retta, Saverio Francesco

    2005-10-15

    The coordinate modulation of cadherin and integrin functions plays an essential role in fundamental physiological and pathological processes, including morphogenesis and cancer. However, the molecular mechanisms underlying the functional crosstalk between cadherins and integrins are still elusive. Here, we demonstrate that the small GTPase Rap1, a crucial regulator of the inside-out activation of integrins, is a target for E-cadherin-mediated outside-in signaling. In particular, we show that a strong activation of Rap1 occurs upon adherens junction disassembly that is triggered by E-cadherin internalization and trafficking along the endocytic pathway. By contrast, Rap1 activity is not influenced by integrin outside-in signaling. Furthermore, we demonstrate that the E-cadherin endocytosis-dependent activation of Rap1 is associated with and controlled by an increased Src kinase activity, and is paralleled by the colocalization of Rap1 and E-cadherin at the perinuclear Rab11-positive recycling endosome compartment, and the association of Rap1 with a subset of E-cadherin-catenin complexes that does not contain p120ctn. Conversely, Rap1 activity is suppressed by the formation of E-cadherin-dependent cell-cell junctions as well as by agents that inhibit either Src activity or E-cadherin internalization and intracellular trafficking. Finally, we demonstrate that the E-cadherin endocytosis-dependent activation of Rap1 is associated with and is required for the formation of integrin-based focal adhesions. Our findings provide the first evidence of an E-cadherin-modulated endosomal signaling pathway involving Rap1, and suggest that cadherins may have a novel modulatory role in integrin adhesive functions by fine-tuning Rap1 activation.

  5. 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

  6. Integrin αIIb-Mediated PI3K/Akt Activation in Platelets

    PubMed Central

    Niu, Haixia; Chen, Xue; Gruppo, Ralph A.; Li, Ding; Wang, Yanhua; Zhang, Lin; Wang, Kemin; Chai, Weiran; Sun, Yueping; Ding, Zhongren; Gartner, T. Kent; Liu, Junling

    2012-01-01

    Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R724KEFAKFEEER734. In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R724KEFAKFEEER734, each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E724AERKFERKFE734, but not in cells expressing wild type αIIbβ3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets. PMID:23082158

  7. Integrin alpha1beta1 controls reactive oxygen species synthesis by negatively regulating epidermal growth factor receptor-mediated Rac activation.

    PubMed

    Chen, Xiwu; Abair, Tristin D; Ibanez, Maria R; Su, Yan; Frey, Mark R; Dise, Rebecca S; Polk, D Brent; Singh, Amar B; Harris, Raymond C; Zent, Roy; Pozzi, Ambra

    2007-05-01

    Integrins control many cell functions, including generation of reactive oxygen species (ROS) and regulation of collagen synthesis. Mesangial cells, found in the glomerulus of the kidney, are able to produce large amounts of ROS via the NADPH oxidase. We previously demonstrated that integrin alpha1-null mice develop worse fibrosis than wild-type mice following glomerular injury and this is due, in part, to excessive ROS production by alpha1-null mesangial cells. In the present studies, we describe the mechanism whereby integrin alpha1-null mesangial cells produce excessive ROS. Integrin alpha1-null mesangial cells have constitutively increased basal levels of activated Rac1, which result in its increased translocation to the cell membrane, excessive ROS production, and consequent collagen IV deposition. Basal Rac1 activation is a direct consequence of ligand-independent increased epidermal growth factor receptor (EGFR) phosphorylation in alpha1-null mesangial cells. Thus, our study demonstrates that integrin alpha1beta1-EGFR cross talk is a key step in negatively regulating Rac1 activation, ROS production, and excessive collagen synthesis, which is a hallmark of diseases characterized by irreversible fibrosis.

  8. Talin1 is critical for force-dependent reinforcement of initial integrin-cytoskeleton bonds but not tyrosine kinase activation.

    PubMed

    Giannone, Grégory; Jiang, Guoying; Sutton, Deborah H; Critchley, David R; Sheetz, Michael P

    2003-10-27

    Cells rapidly transduce forces exerted on extracellular matrix contacts into tyrosine kinase activation and recruitment of cytoskeletal proteins to reinforce integrin-cytoskeleton connections and initiate adhesion site formation. The relationship between these two processes has not been defined, particularly at the submicrometer level. Using talin1-deficient cells, it appears that talin1 is critical for building early mechanical linkages. Deletion of talin1 blocked laser tweezers, force-dependent reinforcement of submicrometer fibronectin-coated beads and early formation of adhesion sites in response to force, even though Src family kinases, focal adhesion kinase, and spreading were activated normally. Recruitment of vinculin and paxillin to sites of force application also required talin1. FilaminA had a secondary role in strengthening fibronectin-integrin-cytoskeleton connections and no role in stretch-dependent adhesion site assembly. Thus, force-dependent activation of tyrosine kinases is independent of early force-dependent structural changes that require talin1 as part of a critical scaffold.

  9. β3 Integrin Promotes Long-Lasting Activation and Polarization of Vascular Endothelial Growth Factor Receptor 2 by Immobilized Ligand

    PubMed Central

    Ravelli, Cosetta; Grillo, Elisabetta; Corsini, Michela; Coltrini, Daniela

    2015-01-01

    Objective— During neovessel formation, angiogenic growth factors associate with the extracellular matrix. These immobilized factors represent a persistent stimulus for the otherwise quiescent endothelial cells (ECs), driving directional EC migration and proliferation and leading to new blood vessel growth. Vascular endothelial growth factor receptor 2 (VEGFR2) is the main mediator of angiogenesis. Although VEGFR2 signaling has been deeply characterized, little is known about its subcellular localization during neovessel formation. Aim of this study was the characterization and molecular determinants of activated VEGFR2 localization in ECs during neovessel formation in response to matrix-immobilized ligand. Approach and Results— Here we demonstrate that ECs stimulated by extracellular matrix–associated gremlin, a noncanonical VEGFR2 ligand, are polarized and relocate the receptor in close contact with the angiogenic factor–enriched matrix both in vitro and in vivo. GM1 (monosialotetrahexosylganglioside)-positive planar lipid rafts, β3 integrin receptors, and the intracellular signaling transducers focal adhesion kinase and RhoA (Ras homolog gene family, member A) cooperate to promote VEGFR2 long-term polarization and activation. Conclusions— A ligand anchored to the extracellular matrix induces VEGFR2 polarization in ECs. Long-lasting VEGFR2 relocation is closely dependent on lipid raft integrity and activation of β3 integrin pathway. The study of the endothelial responses to immobilized growth factors may offer insights into the angiogenic process in physiological and pathological conditions, including cancer, and for a better engineering of synthetic tissue scaffolds to blend with the host vasculature. PMID:26293466

  10. Integrin affinity modulation in angiogenesis

    PubMed Central

    Mahabeleshwar, Ganapati H.; Chen, Juhua; Feng, Weiyi; Somanath, Payaningal R.; Razorenova, Olga V.; Byzova, Tatiana V.

    2008-01-01

    Integrins, transmembrane glycoprotein receptors, play vital roles in pathological angiogenesis, but their precise regulatory functions are not completely understood and remain controversial. This study aims to assess the regulatory functions of individual beta subunits of endothelial integrins in angiogenic responses induced by vascular endothelial growth factor (VEGF). Inhibition of expression of β1, β3 or β5 integrins in endothelial cells resulted in down regulation of EC adhesion and migration on the primary ligand for the corresponding integrin receptor, while no effects on the recognition of other ligands were detected. Although inhibition of expression of each subunit substantially affected capillary growth stimulated by VEGF, the loss of β3 integrin was the most inhibitory. EC stimulation by VEGF induced formation of the high affinity (activated) state of αvβ3 in a monolayer and activated αvβ3 was co-localized with VEGF receptor-2 (VEGFR-2). Inhibition of expression of β1, β3 or β5 did not affect expression levels of VEGFR-2 in EC. However, inhibition of β3, but not β1 or β5, resulted in substantial inhibition of VEGFR-2 phosphorylation stimulated by VEGF. Exogenous stimulation of αvβ3 integrin with activating antibodies augmented VEGF-dependent phosphorylation of VEGFR-2, whereas integrin blockade suppressed this response. Most importantly, activated αvβ3 was detected on endothelial cells of tumor vasculature. Activation of αvβ3 was substantially increased in highly-vascularized tumors as compared to normal tissues. Moreover, activated αvβ3 was co-localized with VEGFR-2 on endothelial cells of proliferating blood vessels. Together, these results show the unique role of αvβ3 integrin in cross-talk with VEGFR-2 in the context of pathological angiogenesis. PMID:18287811

  11. Integrin α4β7 expression increases HIV susceptibility in activated cervical CD4+ T cells via an HIV attachment-independent mechanism

    PubMed Central

    Ding, Jian; Tasker, Carley; Lespinasse, Pierre; Dai, Jihong; Fitzgerald-Bocarsly, Patricia; Lu, Wuyuan; Heller, Debra; Chang, Theresa L.

    2015-01-01

    Background CD4+ T cells, the principal target in acute SIV and HIV infection, are crucial for the establishment and dissemination of HIV infection in mucosal tissues. Studies indicate that α4β7 CD4+ T cells are preferentially infected by HIV in vitro and during acute SIV infection. The integrin α4β7 is thought to promote HIV capture by target cells; however, the role of integrin α4β7 in HIV transmission remains controversial. In this study, we characterized immune phenotypes of human cervical T cells and examined HIV preference in integrin α4β7+ CD4+ T cells. In vitro all-trans retinoic acid differentiated peripheral CD4+ T cells (at-RA differentiated cells) were included as a comparison. Results In both peripheral and cervical cells, the majority of HIV p24+ cells were activated CD4+ T cells expressing integrin α4β7. Among infected at-RA differentiated cells, the frequency of CCR5 expression was higher in HIV p24+ cells than in HIV p24- cells; no such difference was observed in cervical cells. Neither the cyclic hexapeptide CWLDVC nor a monoclonal antibody against integrin α4β7 blocked HIV attachment or gp120 binding to target cells regardless of the presence of CD4, indicating that integrin α4β7 did not facilitate HIV capture by target cells. Conclusion Integrin α4β7 expression increases HIV susceptibility, but the mechanism is not through promoting HIV binding to target cells. PMID:26167616

  12. The Arf6 GTPase-activating Proteins ARAP2 and ACAP1 Define Distinct Endosomal Compartments That Regulate Integrin α5β1 Traffic*

    PubMed Central

    Chen, Pei-Wen; Luo, Ruibai; Jian, Xiaoying; Randazzo, Paul A.

    2014-01-01

    Arf6 and the Arf6 GTPase-activating protein (GAP) ACAP1 are established regulators of integrin traffic important to cell adhesion and migration. However, the function of Arf6 with ACAP1 cannot explain the range of Arf6 effects on integrin-based structures. We propose that Arf6 has different functions determined, in part, by the associated Arf GAP. We tested this idea by comparing the Arf6 GAPs ARAP2 and ACAP1. We found that ARAP2 and ACAP1 had opposing effects on apparent integrin β1 internalization. ARAP2 knockdown slowed, whereas ACAP1 knockdown accelerated, integrin β1 internalization. Integrin β1 association with adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif (APPL)-positive endosomes and EEA1-positive endosomes was affected by ARAP2 knockdown and depended on ARAP2 GAP activity. ARAP2 formed a complex with APPL1 and colocalized with Arf6 and APPL in a compartment distinct from the Arf6/ACAP1 tubular recycling endosome. In addition, although ACAP1 and ARAP2 each colocalized with Arf6, they did not colocalize with each other and had opposing effects on focal adhesions (FAs). ARAP2 overexpression promoted large FAs, but ACAP1 overexpression reduced FAs. Taken together, the data support a model in which Arf6 has at least two sites of opposing action defined by distinct Arf6 GAPs. PMID:25225293

  13. Regulation of E-cadherin expression and beta-catenin/Tcf transcriptional activity by the integrin-linked kinase.

    PubMed

    Oloumi, Arusha; McPhee, Timothy; Dedhar, Shoukat

    2004-04-01

    Integrin-linked kinase (ILK) is a serine/threonine protein kinase which interacts with the cytoplasmic domains of beta1 and beta3 integrins. ILK structure and its localization at the focal adhesion allows it not only to interact with different structural proteins, but also to mediate many different signalling pathways. Extracellular matrices (ECM) and growth factors each stimulate ILK signalling. Constitutive activation of ILK in epithelial cells results in oncogenic phenotypes such as disruption of cell extracellular matrix and cell to cell interactions, suppression of suspension-induced apoptosis, and induction of anchorage independent cell growth and cell cycle progression. More specifically, pathological overexpression of ILK results in down-regulation of E-cadherin expression, and nuclear accumulation of beta-catenin, leading to the subsequent activation of the beta-catenin/Tcf transcription complex, the downstream components of the Wnt signalling pathway. Here we review the data implicating ILK in the regulation of these two signalling pathways, and discuss recent novel insights into the molecular basis and requirement of ILK in the process of epithelial to mesenchymal transformation (EMT).

  14. Novel synthetic cyclic integrin αvβ3 binding peptide ALOS4: antitumor activity in mouse melanoma models

    PubMed Central

    Yacobovich, Shiri; Tuchinsky, Lena; Kirby, Michael; Kardash, Tetiana; Agranyoni, Oryan; Nesher, Elimelech; Redko, Boris; Gellerman, Gary; Tobi, Dror; Gurova, Katerina; Koman, Igor; Fabian, Osnat Ashur; Pinhasov, Albert

    2016-01-01

    ALOS4, a unique synthetic cyclic peptide without resemblance to known integrin ligand sequences, was discovered through repeated biopanning with pIII phage expressing a disulfide-constrained nonapeptide library. Binding assays using a FITC-labeled analogue demonstrated selective binding to immobilized αvβ3 and a lack of significant binding to other common proteins, such as bovine serum albumin and collagen. In B16F10 cell cultures, ALOS4 treatment at 72 h inhibited cell migration (30%) and adhesion (up to 67%). Immunofluorescent imaging an ALOS4-FITC analogue with B16F10 cells demonstrated rapid cell surface binding, and uptake and localization in the cytoplasm. Daily injections of ALOS4 (0.1, 0.3 or 0.5 mg/kg i.p.) to mice inoculated with B16F10 mouse melanoma cells in two different cancer models, metastatic and subcutaneous tumor, resulted in reduction of lung tumor count (metastatic) and tumor mass (subcutaneous) and increased survival of animals monitored to 45 and 60 days, respectively. Examination of cellular activity indicated that ALOS4 produces inhibition of cell migration and adhesion in a concentration-dependent manner. Collectively, these results suggest that ALOS4 is a structurally-unique selective αvβ3 integrin ligand with potential anti-metastatic activity. PMID:27556860

  15. Integrin α2β1 inhibits MST1 kinase phosphorylation and activates Yes-associated protein oncogenic signaling in hepatocellular carcinoma.

    PubMed

    Wong, Kwong-Fai; Liu, Angela M; Hong, Wanjin; Xu, Zhi; Luk, John M

    2016-11-22

    The Hippo pathway regulates the down-stream target Yes-associated protein (YAP) to maintain organ homeostasis, which is commonly inactivated in many types of cancers. However, how cell adhesion dysregulates the Hippo pathway activating YAP oncogene in hepatocellular carcinoma (HCC) remains unclear. Our findings demonstrate that α2β1 integrin (but not other β1 integrins) expressed in HCC cells, after binding to collagen extracellular matrix, could inhibit MST1 kinase phosphorylation and activate YAP pro-oncogenic activities. Knockdown of integrin α2 gene (ITGA2) suppressed YAP targeted gene expression in vitro. α2β1 and collagen binding resulted in suppressing Hippo signaling of mammalian sterile 20-like kinase 1 (MST1) and Large tumor suppressor homolog 1 (LATS1) with concomitant activation of YAP-mediated connective tissue growth factor (CTGF) gene expression. In vitro kinase assay showed that MST1 is an immediate downstream target of integrin α2 with S1180 residue as the critical phosphorylation site. Clinical correlational analysis using a gene expression dataset of 228 HCC tumors revealed that ITGA2 expression was significantly associated with tumor progression, and co-expression with YAP targeted genes (AXL receptor tyrosine kinase, CTGF, cyclin D1, glypican 3, insulin like growth factor 1 receptor, and SRY-box 4) correlated with survivals of HCC patients. In conclusion, α2β1 integrin activation through cellular adhesion impacts the Hippo pathway in solid tumors and modulates MST1-YAP signaling cascade. Targeting integrin α2 holds promises for treating YAP-positive HCC.

  16. Integrin α2β1 inhibits MST1 kinase phosphorylation and activates Yes-associated protein oncogenic signaling in hepatocellular carcinoma

    PubMed Central

    Wong, Kwong-Fai; Liu, Angela M.; Hong, Wanjin; Xu, Zhi; Luk, John M.

    2016-01-01

    The Hippo pathway regulates the down-stream target Yes-associated protein (YAP) to maintain organ homeostasis, which is commonly inactivated in many types of cancers. However, how cell adhesion dysregulates the Hippo pathway activating YAP oncogene in hepatocellular carcinoma (HCC) remains unclear. Our findings demonstrate that α2β1 integrin (but not other β1 integrins) expressed in HCC cells, after binding to collagen extracellular matrix, could inhibit MST1 kinase phosphorylation and activate YAP pro-oncogenic activities. Knockdown of integrin α2 gene (ITGA2) suppressed YAP targeted gene expression in vitro. α2β1 and collagen binding resulted in suppressing Hippo signaling of mammalian sterile 20-like kinase 1 (MST1) and Large tumor suppressor homolog 1 (LATS1) with concomitant activation of YAP-mediated connective tissue growth factor (CTGF) gene expression. In vitro kinase assay showed that MST1 is an immediate downstream target of integrin α2 with S1180 residue as the critical phosphorylation site. Clinical correlational analysis using a gene expression dataset of 228 HCC tumors revealed that ITGA2 expression was significantly associated with tumor progression, and co-expression with YAP targeted genes (AXL receptor tyrosine kinase, CTGF, cyclin D1, glypican 3, insulin like growth factor 1 receptor, and SRY-box 4) correlated with survivals of HCC patients. In conclusion, α2β1 integrin activation through cellular adhesion impacts the Hippo pathway in solid tumors and modulates MST1-YAP signaling cascade. Targeting integrin α2 holds promises for treating YAP-positive HCC. PMID:27765911

  17. Small molecule integrin antagonists that bind to the beta2 subunit I-like domain and activate signals in one direction and block them in the other.

    PubMed

    Shimaoka, Motomu; Salas, Azucena; Yang, Wei; Weitz-Schmidt, Gabriele; Springer, Timothy A

    2003-09-01

    Leukocyte integrins contain an inserted (I) domain in their alpha subunits and an I-like domain in their beta(2) subunit, which directly bind ligand and regulate ligand binding, respectively. We describe a novel mechanistic class of integrin inhibitors that bind to the metal ion-dependent adhesion site of the beta(2) I-like domain and prevent its interaction with and activation of the alpha(L) I domain. The inhibitors do not bind to the alpha(L) I domain but stabilize alpha/beta subunit association and can show selectivity for alpha(L)beta(2) compared to alpha(M)beta(2). The inhibitors reveal a crucial intersection for relaying conformational signals within integrin extracellular domains. While blocking signals in one direction to the I domain, the antagonists induce the active conformation of the I-like domain and stalk domains, and thus transmit conformational signals in the other direction toward the transmembrane domains.

  18. Plasminogen Activator Inhibitor-1 Regulates Integrin αvβ3 Expression and Autocrine Transforming Growth Factor β Signaling*

    PubMed Central

    Pedroja, Benjamin S.; Kang, Leah E.; Imas, Alex O.; Carmeliet, Peter; Bernstein, Audrey M.

    2009-01-01

    Fibrosis is characterized by elevated transforming growth factor β (TGFβ) signaling, resulting in extracellular matrix accumulation and increased PAI-1 (plasminogen activator inhibitor) expression. PAI-1 induces the internalization of urokinase plasminogen activator/receptor and integrin αvβ3 from the cell surface. Since increased αvβ3 expression correlates with increased TGFβ signaling, we hypothesized that aberrant PAI-1-mediated αvβ3 endocytosis could initiate an autocrine loop of TGFβ activity. We found that in PAI-1 knock-out (KO) mouse embryonic fibroblasts), αvβ3 endocytosis was reduced by ∼75%, leaving αvβ3 in enlarged focal adhesions, similar to wild type cells transfected with PAI-1 small interfering RNA. TGFβ signaling was significantly enhanced in PAI-1 KO cells, as demonstrated by a 3-fold increase in SMAD2/3-containing nuclei and a 2.9-fold increase in TGFβ activity that correlated with an increase in αvβ3 and TGFβ receptor II expression. As expected, PAI-1 KO cells had unregulated plasmin activity, which was only partially responsible for TGFβ activation, as evidenced by a mere 25% reduction in TGFβ activity when plasmin was inhibited. Treatment of cells with an αvβ3-specific cyclic RGD peptide (GpenGRGD) led to a more profound (59%) TGFβ inhibition; a nonspecific RGD peptide (GRGDNP) inhibited TGFβ by only 23%. Human primary fibroblasts were used to confirm that PAI-1 inhibition and β3 overexpression led to an increase in TGFβ activity. Consistent with a fibrotic phenotype, PAI-1 KO cells were constitutively myofibroblasts that had a 1.6-fold increase in collagen deposition over wild type cells. These data suggest that PAI-1-mediated regulation of αvβ3 integrin is critical for the control of TGFβ signaling and the prevention of fibrotic disease. PMID:19487690

  19. Integrin Targeted Delivery of Radiotherapeutics

    PubMed Central

    Liu, Zhaofei; Wang, Fan; Chen, Xiaoyuan

    2011-01-01

    Targeted radionuclide therapy, which is based on the selective delivery of a sufficient radiation dose to tumors without significantly affecting normal tissues, is a promising therapeutic approach for the treatment of a wide variety of malignancies. Integrins, a family of cell adhesion molecules, play key roles during tumor angiogenesis and metastasis. Among all the integrins, αvβ3 seems to be the most important in the process of tumor angiogenesis. Integrin αvβ3 is highly expressed on activated endothelial cells, new-born vessels as well as some tumor cells, but is not present in resting endothelial cells and most normal organ systems, making it a suitable target for anti-tumor therapy. In this review, we summarize the current development and applications of antibody-, peptide-, and other ligand-based integrin targeted radiotherapeutics for tumor radiation therapy. PMID:21547160

  20. Angiostatic activity of obtustatin as α1β1 integrin inhibitor in experimental melanoma growth

    PubMed Central

    Brown, Meghan C.; Staniszewska, Izabela; Valle, Luis Del; Tuszynski, George P.; Marcinkiewicz, Cezary

    2008-01-01

    The presented results show the effect of targeting of collagen receptor, α1β1 integrin expressed on the endothelial cells on the development of experimental melanoma and pathological angiogenesis. Obtustatin, a snake venom KTS-disintegrin, was applied as a specific inhibitor of this integrin. This low molecular weight peptide revealed a potent therapeutic effect on melanoma progression in two animal systems, mouse and quail. Its oncostatic effect was related to the inhibition of angiogenesis. Obtustatin inhibited the neovascularization ratio on the CAM embryo of quail, which was pathologically induced by the developing tumor. The i.v. administration of obtustatin completely blocked cancer growth of MV3 human melanoma in nude mice. In B16F10 syngeneic mouse model treatment with the disintegrin revealed a lower effect, although the development of the tumor was significantly reduced for both dosages. The mechanism of obtustatin action is related to the blocking of microvascular endothelial cell proliferation, which undergoes apoptosis in caspase-dependent manner. Summarizing, we present studies of low molecular weight disintegrin, obtustatin as a potential therapeutic compound for treatment of melanoma that contain a high level of vascularization. PMID:18712720

  1. MFG-E8 inhibits neutrophil migration through αvβ3-integrin-dependent MAP kinase activation

    PubMed Central

    AZIZ, MONOWAR; YANG, WENG-LANG; CORBO, LANA M; CHAUNG, WAYNE W; MATSUO, SHINGO; WANG, PING

    2015-01-01

    We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG-E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG-E8 attenuates neutrophil migration. Recombinant human MFG-E8 (rhMFG-E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL-60, was treated with rhMFG-E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin-8 (IL-8) as the chemoattractant. Surface CXCR2 and intracellular G protein-coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen-activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG-E8 resulted in a significant inhibition of dHL-60 cell migration in a dose-dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG-E8-treated dHL-60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL-60 cells, treatment with rhMFG-E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10–30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL-60 cell migration which was significantly inhibited treatment with rhMFG-E8. Furthermore, blocking the MFG-E8 receptors, αvβ3/αvβ5-integrins, by anti-αv-integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG-E8-induced inhibition of dHL-60 cell migration. Finally, treatment of the dHL-60 cells with SB203580 and PD98059 neutralized the rhMFG-E8-induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG-E8 through which it inhibits neutrophil migration through αvβ3-integrin

  2. Matrix Metalloproteinase 2-Integrin αvβ3 Binding Is Required for Mesenchymal Cell Invasive Activity but Not Epithelial Locomotion: A Computational Time-Lapse Study

    PubMed Central

    Rupp, Paul A.; Visconti, Richard P.; Czirók, András; Cheresh, David A.

    2008-01-01

    Cellular invasive behavior through three-dimensional collagen gels was analyzed using computational time-lapse imaging. A subpopulation of endocardial cells, derived from explanted quail cardiac cushions, undergoes an epithelial-to-mesenchymal transition and invades the substance of the collagen gels when placed in culture. In contrast, other endocardial cells remain epithelial and move over the gel surface. Here, we show that integrin αvβ3 and matrix metalloproteinase (MMP)2 are present and active in cushion mesenchymal tissue. More importantly, functional assays show that mesenchymal invasive behavior is dependent on MMP2 activity and integrin αvβ3 binding. Inhibitors of MMP enzymatic activity and molecules that prevent integrin αvβ3 binding to MMP2, via its hemopexin domain, result in significantly reduced cellular protrusive activity and invasive behavior. Computational analyses show diminished intensity and persistence time of motility in treated invasive mesenchymal cells, but no reduction in motility of the epithelial-like cells moving over the gel surface. Thus, quantitative time-lapse data show that mesenchymal cell invasive behavior, but not epithelial cell locomotion over the gel surface, is partially regulated by the MMP2–integrin interactions. PMID:18923152

  3. Anti-integrin therapy for multiple sclerosis.

    PubMed

    Kawamoto, Eiji; Nakahashi, Susumu; Okamoto, Takayuki; Imai, Hiroshi; Shimaoka, Motomu

    2012-01-01

    Integrins are the foremost family of cell adhesion molecules that regulate immune cell trafficking in health and diseases. Integrin alpha4 mediates organ-specific migration of immune cells to the inflamed brain, thereby playing the critical role in the pathogenesis of multiple sclerosis. Anti-alpha4 integrin therapy aiming to block infiltration of autoreactive lymphocytes to the inflamed brain has been validated in several clinical trials for the treatment of multiple sclerosis. This paper provides readers with an overview of the molecular and structural bases of integrin activation as well as rationale for using anti-alpha4 integrin therapy for multiple sclerosis and then chronicles the rise and fall of this treatment strategy using natalizumab, a humanized anti-alpha4 integrin.

  4. Regulation of integrin-mediated adhesions

    PubMed Central

    Iwamoto, Daniel V.; Calderwood, David A.

    2015-01-01

    Integrins are heterodimeric transmembrane adhesion receptors that couple the actin cytoskeleton to the extracellular environment and bidirectionally relay signals across the cell membrane. These processes are critical for cell attachment, migration, differentiation, and survival, and therefore play essential roles in metazoan development, physiology, and pathology. Integrin-mediated adhesions are regulated by diverse factors, including the conformation-specific affinities of integrin receptors for their extracellular ligands, the clustering of integrins and their intracellular binding partners into discrete adhesive structures, mechanical forces exerted on the adhesion, and the intracellular trafficking of integrins themselves. Recent advances shed light onto how the interaction of specific intracellular proteins with the short cytoplasmic tails of integrins controls each of these activities. PMID:26189062

  5. The integrin αL leg region controls the Mg/EGTA mediated activation of LFA-1.

    PubMed

    Guan, Siyu; Cheng, Ming; Law, S K Alex

    2015-03-06

    We have shown that Mg/EGTA (5 mM Mg(2+) and 1.5 mM EGTA) could effectively promote the adhesion of integrin αLβ2 to its ligand ICAM-1 but could not promote that of the αMβ2 to denatured BSA. In order to determine the structural differences between αL and αM that specifically contribute to Mg/EGTA sensitivity, a series of αL/αM chimeras were constructed. Our results showed that αLβ2 with αM calf-1 domain completely lost the response to Mg/EGTA activation. In the reverse experiment, αMβ2 would require the presence of both the αL calf-1 and calf-2 domain to initiate the Mg/EGTA sensitivity. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. The laminin-binding activity of the alpha 7 integrin receptor is defined by developmentally regulated splicing in the extracellular domain.

    PubMed Central

    Ziober, B L; Chen, Y; Kramer, R H

    1997-01-01

    The expression pattern of the laminin-binding alpha 7 beta 1 integrin is developmentally regulated in skeletal, cardiac, and smooth muscle. The X1/X2 alternative splicing in the extracellular domain of alpha 7 is found in the variable region between conserved alpha-chain homology repeat domains III and IV, a site implicated in ligand binding. To assess differences in X1/X2 isoform activity, we generated MCF-7 cell lines transfected with alpha 7-X1/X2 cDNAs. Transfectants expressing the alpha 7-X2 variant adhered rapidly to laminin 1, whereas those expressing alpha 7-X1 failed to attach. That alpha 7-X1 exists in an inactive state was established in assays using an activating beta 1 antibody that induced X1-dependent cell adhesion and spreading. Furthermore, the activation of alpha 7-X1 was cell type specific, and when expressed in HT1080 cells, the integrin was converted into a fully functional receptor capable of promoting adhesion. Thus, the expression of the alpha 7-X1/X2 integrin is a novel mechanism that regulates receptor affinity states in a cell-specific context and may modulate integrin-dependent events during muscle development and repair. Images PMID:9307969

  7. Laminin regulates the osteogenic differentiation of dental follicle cells via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway.

    PubMed

    Viale-Bouroncle, Sandra; Gosau, Martin; Morsczeck, Christian

    2014-07-01

    Dental follicle cells (DFCs) are ideal for studies concerning the differentiation of dental precursor cells into alveolar osteoblasts and cementoblasts. Previous investigations have suggested that the extracellular matrix (ECM) protein laminin and the ECM receptor integrin-α2/-β1 play regulatory roles during the osteogenic differentiation of DFCs. Our present data indicate that laminin impairs alkaline phosphatase (ALP) activity following osteogenic induction while inducing integrin-α2/-β1 expression, osteogenic differentiation marker elevation, and DFC biomineralization. Integrin-α2/-β1 facilitates the laminin-dependent expression of osteogenic differentiation markers and the laminin-dependent inhibition of ALP activity. Moreover, these laminin-dependent effects on the osteogenic differentiation of DFCs can be reversed by the inhibition of the FAK/ERK signaling pathway. Thus, laminin regulates the inhibition of early osteogenic differentiation markers and the induction of late osteogenic differentiation markers via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway.

  8. Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through alphavbeta3 and alpha5beta1 integrins.

    PubMed Central

    Huynh-Do, U; Stein, E; Lane, A A; Liu, H; Cerretti, D P; Daniel, T O

    1999-01-01

    Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. Here, EphB1 regulated cell attachment by discriminating the density at which ephrin-B1 was displayed on a reconstituted surface. EphB1-ephrin-B1 engagement did not promote cell attachment through mechanical tethering, but did activate integrin-mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen was mediated through alphavbeta3 integrin. EphB1 transfection conferred ephrin-B1-responsive activation of alpha5beta1 integrin-mediated cell attachment in human embryonic kidney cells. Activation-competent but signaling-defective EphB1 point mutants failed to stimulate ephrin-B1 dependent attachment. These findings lead us to propose that EphB1 functions as a 'ligand density sensor' to signal integrin-mediated cell-matrix attachment. PMID:10205170

  9. CCN1 induces hepatic ductular reaction through integrin αvβ₅-mediated activation of NF-κB.

    PubMed

    Kim, Ki-Hyun; Chen, Chih-Chiun; Alpini, Gianfranco; Lau, Lester F

    2015-05-01

    Liver cholestatic diseases, which stem from diverse etiologies, result in liver toxicity and fibrosis and may progress to cirrhosis and liver failure. We show that CCN1 (also known as CYR61), a matricellular protein that dampens and resolves liver fibrosis, also mediates cholangiocyte proliferation and ductular reaction, which are repair responses to cholestatic injury. In cholangiocytes, CCN1 activated NF-κB through integrin αvβ5/αvβ3, leading to Jag1 expression, JAG1/NOTCH signaling, and cholangiocyte proliferation. CCN1 also induced Jag1 expression in hepatic stellate cells, whereupon they interacted with hepatic progenitor cells to promote their differentiation into cholangiocytes. Administration of CCN1 protein or soluble JAG1 induced cholangiocyte proliferation in mice, which was blocked by inhibitors of NF-κB or NOTCH signaling. Knock-in mice expressing a CCN1 mutant that is unable to bind αvβ5/αvβ3 were impaired in ductular reaction, leading to massive hepatic necrosis and mortality after bile duct ligation (BDL), whereas treatment of these mice with soluble JAG1 rescued ductular reaction and reduced hepatic necrosis and mortality. Blockade of integrin αvβ5/αvβ3, NF-κB, or NOTCH signaling in WT mice also resulted in defective ductular reaction after BDL. These findings demonstrate that CCN1 induces cholangiocyte proliferation and ductular reaction and identify CCN1/αvβ5/NF-κB/JAG1 as a critical axis for biliary injury repair.

  10. RGD-peptide lunasin inhibits Akt-mediated NF-κB activation in human macrophages through interaction with the αVβ3 integrin.

    PubMed

    Cam, Anthony; de Mejia, Elvira Gonzalez

    2012-10-01

    Cardiovascular disease is the leading cause of mortality in the United States and regulation of aberrant macrophage activity under inflammatory conditions is critical for its prevention. The objective was to determine the effect of lunasin on the inhibition of Akt-mediated activation of nuclear factor-kappa B (NF-κB)-dependent markers of inflammation and to characterize the physical interaction of lunasin with the αVβ3 integrin receptor in lipopolysaccharide (LPS)-induced human THP-1 macrophages. The effect of lunasin was evaluated in vitro in LPS-induced THP-1 human macrophages using immunoassays, co-immunoprecipitation (Co-IP), and fluorescence confocal microscopy. Lunasin (50 μM) reduced cyclooxygenase-2, inducible nitric oxide synthase, and NO levels by 57.9, 64.5, and 76.2%, respectively, and inhibited the activation of phosphorylated Akt and NF-κB p65 by 59.5 and 74.5%, respectively. Lunasin (50 μM) reduced exogenous release of prostaglandin E(2) and tumor necrosis factor-α by 92.5 and 94.9%, respectively. Vitronectin (10 μg/mL), an integrin ligand, increased expression of proinflammatory markers, whereas lunasin (50 μM) attenuated them. Co-IP of lunasin-treated cells confirmed direct interaction with αVβ3 integrin and LC/MS/MS verified its identity. Lunasin was detected within intracellular vesicles and reduced total αVβ3 intensity as observed by fluorescence microscopy. Lunasin inhibited αVβ3 integrin-mediated proinflammatory markers and downregulated Akt-mediated NF-κB pathways through interaction with αVβ3 integrin. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. HGF/scatter factor selectively promotes cell invasion by increasing integrin avidity.

    PubMed

    Trusolino, L; Cavassa, S; Angelini, P; Andó, M; Bertotti, A; Comoglio, P M; Boccaccio, C

    2000-08-01

    Hepatocyte growth factor/scatter factor (HGF/SF) controls a genetic program known as 'invasive growth', which involves as critical steps cell adhesion, migration, and trespassing of basement membranes. We show here that in MDA-MB-231 carcinoma cells, these steps are elicited by HGF/SF but not by epidermal growth factor (EGF). Neither factor substantially alters the production or activity of extracellular matrix proteases. HGF/SF, but not EGF, selectively promotes cell adhesion on laminins 1 and 5, fibronectin, and vitronectin through a PI3-K-dependent mechanism. Increased adhesion is followed by enhanced invasiveness through isolated matrix proteins as well as through reconstituted basement membranes. Inhibition assays using function-blocking antibodies show that this phenomenon is mediated by multiple integrins including beta1, beta3, beta4, and beta5. HGF/SF triggers clustering of all these integrins at actin-rich adhesive sites and lamellipodia but does not quantitatively modify their membrane expression. These data suggest that HGF/SF promotes cell adhesion and invasiveness by increasing the avidity of integrins for their specific ligands.

  12. Divergent viral superantigens delete V beta 5+ T lymphocytes.

    PubMed Central

    Gollob, K J; Palmer, E

    1992-01-01

    Several murine superantigens in association with class II major histocompatibility complex proteins have been shown to cause the deletion of T cells based on the expression of particular beta-chain variable region (V beta) gene segments. We have previously shown that mice expressing the Etc-1 superantigen, encoded by an open reading frame within the 3' long terminal repeat of the endogenous mouse mammary tumor provirus (Mtv), Mtv-9, delete T cells expressing either V beta 5 or V beta 11 gene segments. Comparison of several Mtv 3' long terminal repeat open reading frame sequences has indicated that the carboxyl terminus likely encodes the V beta specificity of these proteins. Our analysis of C57BL/6 x DBA/2 recombinant inbred strains of mouse revealed three Mtv-9-negative strains that nevertheless have a low frequency of V beta 5-expressing T cells. Here we demonstrate that a second endogenous superantigen, responsible for the deletion of V beta 5-bearing T cells, is encoded by a gene mapping to Mtv-6 on chromosome 16. Surprisingly, the carboxyl-terminal sequences of the Mtv-6 and -9 superantigens are extremely divergent, in spite of the fact that they both mediate the deletion of V beta 5+ lymphocytes. Images PMID:1317583

  13. Divergent viral superantigens delete V beta 5+ T lymphocytes.

    PubMed

    Gollob, K J; Palmer, E

    1992-06-01

    Several murine superantigens in association with class II major histocompatibility complex proteins have been shown to cause the deletion of T cells based on the expression of particular beta-chain variable region (V beta) gene segments. We have previously shown that mice expressing the Etc-1 superantigen, encoded by an open reading frame within the 3' long terminal repeat of the endogenous mouse mammary tumor provirus (Mtv), Mtv-9, delete T cells expressing either V beta 5 or V beta 11 gene segments. Comparison of several Mtv 3' long terminal repeat open reading frame sequences has indicated that the carboxyl terminus likely encodes the V beta specificity of these proteins. Our analysis of C57BL/6 x DBA/2 recombinant inbred strains of mouse revealed three Mtv-9-negative strains that nevertheless have a low frequency of V beta 5-expressing T cells. Here we demonstrate that a second endogenous superantigen, responsible for the deletion of V beta 5-bearing T cells, is encoded by a gene mapping to Mtv-6 on chromosome 16. Surprisingly, the carboxyl-terminal sequences of the Mtv-6 and -9 superantigens are extremely divergent, in spite of the fact that they both mediate the deletion of V beta 5+ lymphocytes.

  14. Characterization of a Novel Integrin Binding Protein, VPS33B, Which is Important for Platelet Activation and in vivo Thrombosis and Hemostasis

    PubMed Central

    Xiang, Binggang; Zhang, Guoying; Ye, Shaojing; Zhang, Rui; Huang, Cai; Liu, Jun; Tao, Min; Ruan, Changgeng; Smyth, Susan S.; Whiteheart, Sidney W.; Li, Zhenyu

    2015-01-01

    Background Integrins are heterodimeric (α/β) membrane proteins that play fundamental roles in many biological processes, e.g. cell adhesion and spreading, which are important for platelet function and hemostasis. The molecular mechanism that regulates integrin activation is not completely understood. Methods and Results Here, we show that VPS33B, a member of the Sec1/Munc18 (SM) family and a component of the Vps-C tethering complexes, binds directly to the integrin β subunit. Overexpression of VPS33B in CHO cells potentiated αIIbβ3 outside-in signaling but not inside-out signaling. Platelets, from megakaryocyte- and platelet-specific VPS33B conditional knockout mice, had normal morphology yet their spreading on fibrinogen was impaired and they failed to support clot retraction. Platelet aggregation and ATP secretion in response to low-dose agonists were reduced in the VPS33B knockout mice. αIIbβ3-mediated endocytosis of fibrinogen was also defective. Tail bleeding times and times to occlusion in a FeCl3-induced thrombosis model were prolonged in the VPS33B knockout mice. Furthermore, VPS33B acted upstream of the RhoA-ROCK-MLC and Rac1 dependent pathways that lead to clot retraction and cell spreading, respectively. Conclusions Our work demonstrates that vesicular trafficking complexes, containing VPS33B, are a novel class of modifiers of integrin function. Our data also provide insights into the molecular mechanism and treatment of ARC syndrome. PMID:26399659

  15. Activated tumor cell integrin αvβ3 cooperates with platelets to promote extravasation and metastasis from the blood stream.

    PubMed

    Weber, Martin R; Zuka, Masahiko; Lorger, Mihaela; Tschan, Mario; Torbett, Bruce E; Zijlstra, Andries; Quigley, James P; Staflin, Karin; Eliceiri, Brian P; Krueger, Joseph S; Marchese, Patrizia; Ruggeri, Zaverio M; Felding, Brunhilde H

    2016-04-01

    Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvβ3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvβ3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvβ3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvβ3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease.

  16. Genome-wide association study implicates immune activation of multiple integrin genes in inflammatory bowel disease

    PubMed Central

    Lamb, Christopher A.; Luo, Yang; Kennedy, Nicholas A.; Jostins, Luke; Rice, Daniel L.; Gutierrez-Achury, Javier; Ji, Sun-Gou; Heap, Graham; Nimmo, Elaine R.; Edwards, Cathryn; Henderson, Paul; Mowat, Craig; Sanderson, Jeremy; Satsangi, Jack; Simmons, Alison; Wilson, David C.; Tremelling, Mark; Hart, Ailsa; Mathew, Christopher G.; Newman, William G.; Parkes, Miles; Lees, Charlie W.; Uhlig, Holm; Hawkey, Chris; Prescott, Natalie J.; Ahmad, Tariq; Mansfield, John C.; Anderson, Carl A.; Barrett, Jeffrey C.

    2016-01-01

    Genetic association studies have identified 215 risk loci for inflammatory bowel disease 1–8, which have revealed fundamental aspects of its molecular biology. We performed a genome-wide association study of 25,305 individuals, and meta-analyzed with published summary statistics, yielding a total sample size of 59,957 subjects. We identified 25 new loci, three of which contain integrin genes that encode proteins in pathways identified as important therapeutic targets in inflammatory bowel disease. The associated variants are correlated with expression changes in response to immune stimulus at two of these genes (ITGA4, ITGB8) and at previously implicated loci (ITGAL, ICAM1). In all four cases, the expression increasing allele also increases disease risk. We also identified likely causal missense variants in the primary immune deficiency gene PLCG2 and a negative regulator of inflammation, SLAMF8. Our results demonstrate that new common variant associations continue to identify genes relevant to therapeutic target identification and prioritization. PMID:28067908

  17. Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1

    PubMed Central

    Peer, Dan; Zhu, Pengcheng; Carman, Christopher V.; Lieberman, Judy; Shimaoka, Motomu

    2007-01-01

    Silencing gene expression by RNAi is a powerful method for exploring gene function and validating drug targets and potentially for therapy. Lymphocytes and other primary blood cells are resistant to lipid-based transfection in vitro and are difficult to target in vivo. We show here that antibody-protamine fusion proteins targeting the human integrin lymphocyte function-associated antigen-1 (LFA-1) efficiently deliver siRNAs and specifically induce silencing in primary lymphocytes, monocytes, and dendritic cells. Moreover, a fusion protein constructed from an antibody that preferentially recognizes activation-dependent conformational changes in LFA-1 selectively targets activated leukocytes and can be used to suppress gene expression and cell proliferation only in activated lymphocytes. The siRNA-fusion protein complexes do not cause lymphocyte activation or induce IFN responses. K562 cells expressing latent WT or constitutively activated LFA-1 engrafted in the lungs of SCID mice are selectively targeted by intravenously injected fusion protein–siRNA complexes, demonstrating the potential in vivo applicability of LFA-1-directed siRNA delivery. PMID:17360483

  18. β8 Integrin Binds Rho GDP Dissociation Inhibitor-1 and Activates Rac1 to Inhibit Mesangial Cell Myofibroblast Differentiation*

    PubMed Central

    Lakhe-Reddy, Sujata; Khan, Shenaz; Konieczkowski, Martha; Jarad, George; Wu, Karen L.; Reichardt, Louis F.; Takai, Yoshimi; Bruggeman, Leslie A.; Wang, Bingcheng; Sedor, John R.; Schelling, Jeffrey R.

    2009-01-01

    αvβ8 integrin expression is restricted primarily to kidney, brain, and placenta. Targeted αv or β8 deletion is embryonic lethal due to defective placenta and brain angiogenesis, precluding investigation of kidney αvβ8 function. We find that kidney β8 is localized to glomerular mesangial cells, and expression is decreased in mouse models of glomerulosclerosis, suggesting that β8 regulates normal mesangial cell differentiation. To interrogate β8 signaling pathways, yeast two-hybrid and co-precipitation studies demonstrated β8 interaction with Rho guanine nucleotide dissociation inhibitor-1 (GDI). Selective β8 stimulation enhanced β8-GDI interaction as well as Rac1 (but not RhoA) activation and lamellipodia formation. Mesangial cells from itgb8−/− mice backcrossed to a genetic background that permitted survival, or gdi−/− mice, which develop glomerulosclerosis, demonstrated RhoA (but not Rac1) activity and α-smooth muscle actin assembly, which characterizes mesangial cell myofibroblast transformation in renal disease. To determine whether Rac1 directly modulates RhoA-associated myofibroblast differentiation, mesangial cells were transduced with inhibitory Rac peptide fused to human immunodeficiency virus-Tat, resulting in enhanced α-smooth muscle actin organization. We conclude that the β8 cytosolic tail in mesangial cells organizes a signaling complex that culminates in Rac1 activation to mediate wild-type differentiation, whereas decreased β8 activation shifts mesangial cells toward a RhoA-dependent myofibroblast phenotype. PMID:16690620

  19. Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4β1 integrin interaction, cell adhesion and anti-proliferative activity

    PubMed Central

    Maiorani, Orlando; Pivetta, Eliana; Capuano, Alessandra; Modica, Teresa Maria Elisa; Wassermann, Bruna; Bucciotti, Francesco; Colombatti, Alfonso; Doliana, Roberto; Spessotto, Paola

    2017-01-01

    The extracellular matrix glycoprotein EMILIN1 exerts a wide range of functions mainly associated with its gC1q domain. Besides providing functional significance for adhesion and migration, the direct interaction between α4β1 integrin and EMILIN1-gC1q regulates cell proliferation, transducing net anti-proliferative effects. We have previously demonstrated that EMILIN1 degradation by neutrophil elastase (NE) is a specific mechanism leading to the loss of functions disabling its regulatory properties. In this study we further analysed the proteolytic activity of NE, MMP-3, MMP-9, and MT1-MMP on EMILIN1 and found that MMP-3 and MT1-MMP partially cleaved EMILIN1 but without affecting the functional properties associated with the gC1q domain, whereas NE was able to fully impair the interaction of gC1q with the α4β1 integrin by cleaving this domain outside of the E933 integrin binding site. By a site direct mutagenesis approach we mapped the bond between S913 and R914 residues and selected the NE-resistant R914W mutant still able to interact with the α4β1 integrin after NE treatment. Functional studies showed that NE impaired the EMILIN1-α4β1 integrin interaction by cleaving the gC1q domain in a region crucial for its proper structural conformation, paving the way to better understand NE effects on EMILIN1-cell interaction in pathological context. PMID:28074935

  20. Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin

    PubMed Central

    Figueira, Cláudio Pereira; Carvalhal, Djalma Gomes Ferrão; Almeida, Rafaela Andrade; Hermida, Micely d’ El-Rei; Touchard, Dominique; Robert, Phillipe; Pierres, Anne; Bongrand, Pierre; dos-Santos, Washington LC

    2015-01-01

    Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55–89] μm2 for uninfected and 41 [34–51] μm2 for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm2 s−1 ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm2 s−1 ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis. PMID:26249106

  1. Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin.

    PubMed

    Figueira, Cláudio Pereira; Carvalhal, Djalma Gomes Ferrão; Almeida, Rafaela Andrade; Hermida, Micely d' El-Rei; Touchard, Dominique; Robert, Phillipe; Pierres, Anne; Bongrand, Pierre; dos-Santos, Washington L C

    2015-08-07

    Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55-89] μm(2) for uninfected and 41 [34-51] μm(2) for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm(2) s(-1) ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm(2) s(-1) ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis.

  2. Nitric oxide/cGMP pathway signaling actively down-regulates α4β1-integrin affinity: an unexpected mechanism for inducing cell de-adhesion

    PubMed Central

    2011-01-01

    Background Integrin activation in response to inside-out signaling serves as the basis for rapid leukocyte arrest on endothelium, migration, and mobilization of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule, which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α4β1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic progenitors, stem cells, hematopoietic cancer cells, and others. VLA-4 conformation is rapidly up-regulated by inside-out signaling through Gαi-coupled GPCRs and down-regulated by Gαs-coupled GPCRs. However, other signaling pathways, which include nitric oxide-dependent signaling, have been implicated in the regulation of cell adhesion. The goal of the current report was to study the effect of nitric oxide/cGMP signaling pathway on VLA-4 conformational regulation. Results Using fluorescent ligand binding to evaluate the integrin activation state on live cells in real-time, we show that several small molecules, which specifically modulate nitric oxide/cGMP signaling pathway, as well as a cell permeable cGMP analog, can rapidly down-modulate binding of a VLA-4 specific ligand on cells pre-activated through three Gαi-coupled receptors: wild type CXCR4, CXCR2 (IL-8RB), and a non-desensitizing mutant of formyl peptide receptor (FPR ΔST). Upon signaling, we detected rapid changes in the ligand dissociation rate. The dissociation rate after inside-out integrin de-activation was similar to the rate for resting cells. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by nitric oxide had a statistically significant effect on real-time cell aggregation. Conclusions We conclude that nitric oxide/cGMP signaling pathway can rapidly down-modulate the affinity state of the VLA-4 binding pocket, especially under the condition of sustained Gαi-coupled GPCR signaling

  3. Prostaglandin E2 upregulates β1 integrin expression via the E prostanoid 1 receptor/nuclear factor κ-light-chain-enhancer of activated B cells pathway in non-small-cell lung cancer cells.

    PubMed

    Bai, Xiaoming; Yang, Qinyi; Shu, Wei; Wang, Jie; Zhang, Li; Ma, Juan; Xia, Shukai; Zhang, Min; Cheng, Shanyu; Wang, Yipin; Leng, Jing

    2014-05-01

    The prostaglandin E2 (PGE2) E prostanoid (EP)1 receptor shown to be associated with lung cancer cell invasion. However, the mechanism of EP1 receptor-mediated cell migration remains to be elucidated. β1 integrin is an essential regulator of the tumorigenic properties of non-small-cell lung carcinoma (NSCLC) cells. To date, little is known regarding the association between the EP1 receptor and β1 integrin expression. The present study investigated the effect of EP1 receptor activation on β1 integrin expression and cell migration in NSCLC cells. A total of 34 patients with clinical diagnosis of NSCLC and 10 patients with benign disease were recruited for the present study. The expression levels of the EP1 receptor and β1 integrin expression were studied in resected lung tissue using immunohistochemistry. A statistical analysis was performed using Stata se12.0 software. The effects of PGE2, EP1 agonist 17-phenyl trinor-PGE2 (17-PT-PGE2) and the nuclear factor κ-B (NF-κB) inhibitor on β1 integrin expression were investigated on A549 cells. The expression of β1 integrin and the phosphorylation of NF-κB‑p65 Ser536 was investigated by western blot analysis. Cell migration was assessed by a transwell assay. The results demonstrated that β1 integrin and EP1 receptor expression exhibited a positive correlation of evident significance in the 44 samples. The in vitro migration assay revealed that cell migration was increased by 30% when the cells were treated with 5 µM 17-PT-PGE2 and that the pre-treatment of β1 integrin monoclonal antibody inhibited 17-PT-PGE2‑mediated cell migration completely. PGE2 and 17-PT-PGE2 treatment increased β1 integrin expression. RNA interference against the EP1 receptor blocked the PGE2-mediated β1 integrin expression in A549 cells. Treatment with 17-PT-PGE2 induced NF-κB activation, and the selective NF-κB inhibitor pyrrolidinedithiocarbamate inhibited 17-PT-PGE2-mediated β1 integrin expression. In conclusion, the present

  4. Evidence that activation of ASIC1a by acidosis increases osteoclast migration and adhesion by modulating integrin/Pyk2/Src signaling pathway.

    PubMed

    Li, X; Ye, J-X; Xu, M-H; Zhao, M-D; Yuan, F-L

    2017-07-01

    Activated acid-sensing ion channel 1a (ASIC1a) is involved in acid-induced osteoclastogenesis by regulating activation of the transcription factor NFATc1. These results indicated that ASIC1a activation by extracellular acid may cause osteoclast migration and adhesion through Ca(2+)-dependent integrin/Pyk2/Src signaling pathway. Osteoclast adhesion and migration are responsible for osteoporotic bone loss. Acidic conditions promote osteoclastogenesis. ASIC1a in osteoclasts is associated with acid-induced osteoclastogenesis through modulating transcription factor NFATc1 activation. However, the influence and the detailed mechanism of ASIC1a in regulating osteoclast adhesion and migration, in response to extracellular acid, are not well characterized. In this study, knockdown of ASIC1a was achieved in bone marrow macrophage cells using small interfering RNA (siRNA). The adhesion and migration abilities of osteoclast precursors and osteoclasts were determined by adhesion and migration assays, in vitro. Bone resorption was performed to measure osteoclast function. Cytoskeletal changes were assessed by F-actin ring formation. αvβ3 integrin expression in osteoclasts was measured by flow cytometry. Western blotting and co-immunoprecipitation were performed to measure alterations in integrin/Pyk2/Src signaling pathway. Our results showed that blockade of ASIC1a using ASIC1a-siRNA inhibited acid-induced osteoclast precursor migration and adhesion, as well as osteoclast adhesion and bone resorption; we also demonstrated that inhibition of ASIC1a decreased the cell surface αvβ3 integrin and β3 protein expression. Moreover, blocking of ASIC1a inhibited acidosis-induced actin ring formation and reduced Pyk2 and Src phosphorylation in osteoclasts and also inhibited the acid-induced association of the αvβ3 integrin/Src/Pyk2. Together, these results highlight a key functional role of ASIC1a/αvβ3 integrin/Pyk2/Src signaling pathway in migration and adhesion of osteoclasts.

  5. Molecular magnetic resonance imaging of activated hepatic stellate cells with ultrasmall superparamagnetic iron oxide targeting integrin αvβ3 for staging liver fibrosis in rat model

    PubMed Central

    Zhang, Caiyuan; Liu, Huanhuan; Cui, Yanfen; Li, Xiaoming; Zhang, Zhongyang; Zhang, Yong; Wang, Dengbin

    2016-01-01

    Purpose To evaluate the expression level of integrin αvβ3 on activated hepatic stellate cells (HSCs) at different stages of liver fibrosis induced by carbon tetrachloride (CCl4) in rat model and the feasibility to stage liver fibrosis by using molecular magnetic resonance imaging (MRI) with arginine-glycine-aspartic acid (RGD) peptide modified ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) specifically targeting integrin αvβ3. Materials and methods All experiments received approval from our Institutional Animal Care and Use Committee. Thirty-six rats were randomly divided into three groups of 12 subjects each, and intraperitoneally injected with CCl4 for either 3, 6, or 9 weeks. Controls (n=10) received pure olive oil. The change in T2* relaxation rate (ΔR2*) pre- and postintravenous administration of RGD-USPIO or naked USPIO was measured by 3.0T clinical MRI and compared by one-way analysis of variance or the Student’s t-test. The relationship between expression level of integrin αvβ3 and liver fibrotic degree was evaluated by Spearman’s ranked correlation. Results Activated HSCs were confirmed to be the main cell types expressing integrin αvβ3 during liver fibrogenesis. The protein level of integrin αv and β3 subunit expressed on activated HSCs was upregulated and correlated well with the progression of liver fibrosis (r=0.954, P<0.001; r=0.931, P<0.001, respectively). After injection of RGD-USPIO, there is significant difference in ΔR2* among rats treated with 0, 3, 6, and 9 weeks of CCl4 (P<0.001). The accumulation of iron particles in fibrotic liver specimen is significantly greater for RGD-USPIO than naked USPIO after being injected with equal dose of iron. Conclusion Molecular MRI of integrin αvβ3 expressed on activated HSCs by using RGD-USPIO may distinguish different liver fibrotic stages in CCl4 rat model and shows promising to noninvasively monitor the progression of the liver fibrosis and therapeutic response to

  6. Nck-Interacting Ste20 Kinase Couples Eph Receptors to c-Jun N-Terminal Kinase and Integrin Activation

    PubMed Central

    Becker, Elena; Huynh-Do, Uyen; Holland, Sacha; Pawson, Tony; Daniel, Tom O.; Skolnik, Edward Y.

    2000-01-01

    The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62dok, RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62dok most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs. PMID:10669731

  7. Integrins and Cell Metabolism: An Intimate Relationship Impacting Cancer

    PubMed Central

    Ata, Rehman; Antonescu, Costin N.

    2017-01-01

    Integrins are important regulators of cell survival, proliferation, adhesion and migration. Once activated, integrins establish a regulated link between the extracellular matrix and the cytoskeleton. Integrins have well-established functions in cancer, such as in controlling cell survival by engagement of many specific intracellular signaling pathways and in facilitating metastasis. Integrins and associated proteins are regulated by control of transcription, membrane traffic, and degradation, as well as by a number of post-translational modifications including glycosylation, allowing integrin function to be modulated to conform to various cellular needs and environmental conditions. In this review, we examine the control of integrin function by cell metabolism, and the impact of this regulation in cancer. Within this context, nutrient sufficiency or deprivation is sensed by a number of metabolic signaling pathways such as AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and hypoxia-inducible factor (HIF) 1, which collectively control integrin function by a number of mechanisms. Moreover, metabolic flux through specific pathways also controls integrins, such as by control of integrin glycosylation, thus impacting integrin-dependent cell adhesion and migration. Integrins also control various metabolic signals and pathways, establishing the reciprocity of this regulation. As cancer cells exhibit substantial changes in metabolism, such as a shift to aerobic glycolysis, enhanced glucose utilization and a heightened dependence on specific amino acids, the reciprocal regulation of integrins and metabolism may provide important clues for more effective treatment of various cancers. PMID:28106780

  8. Presynaptic calcium channels and α3-integrins are complexed with synaptic cleft laminins, cytoskeletal elements and active zone components.

    PubMed

    Carlson, Steven S; Valdez, Gregorio; Sanes, Joshua R

    2010-11-01

    At chemical synapses, synaptic cleft components interact with elements of the nerve terminal membrane to promote differentiation and regulate function. Laminins containing the β2 subunit are key cleft components, and they act in part by binding the pore-forming subunit of a pre-synaptic voltage-gated calcium channel (Ca(v)α) (Nishimune et al. 2004). In this study, we identify Ca(v)α-associated intracellular proteins that may couple channel-anchoring to assembly or stabilization of neurotransmitter release sites called active zones. Using Ca(v)α-antibodies, we isolated a protein complex from Torpedo electric organ synapses, which resemble neuromuscular junctions but are easier to isolate in bulk. We identified 10 components of the complex: six cytoskeletal proteins (α2/β2 spectrins, plectin 1, AHNAK/desmoyokin, dystrophin, and myosin 1), two active zone components (bassoon and piccolo), synaptic laminin, and a calcium channel β subunit. Immunocytochemistry confirmed these proteins in electric organ synapses, and PCR analysis revealed their expression by developing mammalian motor neurons. Finally, we show that synaptic laminins also interact with pre-synaptic integrins containing the α3 subunit. Together with our previous finding that a distinct synaptic laminin interacts with SV2 on nerve terminals (Son et al. 2000), our results identify three paths by which synaptic cleft laminins can send developmentally important signals to nerve terminals.

  9. Structural basis of activation-dependent binding of ligand-mimetic antibody AL-57 to integrin LFA-1

    SciTech Connect

    Zhang, Hongmin; Liu, Jin-huan; Yang, Wei; Springer, Timothy; Shimaoka, Motomu; Wang, Jia-huai

    2010-09-21

    The activity of integrin LFA-1 ({alpha}{sub L}{beta}{sub 2}) to its ligand ICAM-1 is regulated through the conformational changes of its ligand-binding domain, the I domain of {alpha}{sub L} chain, from an inactive, low-affinity closed form (LA), to an intermediate-affinity form (IA), and then finally, to a high-affinity open form (HA). A ligand-mimetic human monoclonal antibody AL-57 (activated LFA-1 clone 57) was identified by phage display to specifically recognize the affinity-upregulated I domain. Here, we describe the crystal structures of the Fab fragment of AL-57 in complex with IA, as well as in its unligated form. We discuss the structural features conferring AL-57's strong selectivity for the high affinity, open conformation of the I domain. The AL-57-binding site overlaps the ICAM-1 binding site on the I domain. Furthermore, an antibody Asp mimics an ICAM Glu by forming a coordination to the metal-ion dependent adhesion site (MIDAS). The structure also reveals better shape complementarity and a more hydrophobic interacting interface in AL-57 binding than in ICAM-1 binding. The results explain AL-57's antagonistic mimicry of LFA-1's natural ligands, the ICAM molecules.

  10. STAT3 activation in pressure-overloaded feline myocardium: role for integrins and the tyrosine kinase BMX.

    PubMed

    Willey, Christopher D; Palanisamy, Arun P; Johnston, Rebecca K; Mani, Santhosh K; Shiraishi, Hirokazu; Tuxworth, William J; Zile, Michael R; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

    2008-06-27

    Growth, survival and cytoskeletal rearrangement of cardiomyocytes are critical for cardiac hypertrophy. Signal transducer and activator of transcription-3 (STAT3) activation is an important cardioprotective factor associated with cardiac hypertrophy. Although STAT3 activation has been reported via signaling through Janus Kinase 2 (JAK2) in several cardiac models of hypertrophy, the importance of other nonreceptor tyrosine kinases (NTKs) has not been explored. Utilizing an in vivo feline right ventricular pressure-overload (RVPO) model of hypertrophy, we demonstrate that in 48 h pressure-overload (PO) myocardium, STAT3 becomes phosphorylated and redistributed to detergent-insoluble fractions with no accompanying JAK2 activation. PO also caused increased levels of phosphorylated STAT3 in both cytoplasmic and nuclear fractions. To investigate the role of other NTKs, we used our established in vitro cell culture model of hypertrophy where adult feline cardiomyocytes are embedded three-dimensionally (3D) in type-I collagen and stimulated with an integrin binding peptide containing an Arg-Gly-Asp (RGD) motif that we have previously shown to recapitulate the focal adhesion complex (FAC) formation of 48 h RVPO. RGD stimulation of adult cardiomyocytes in vitro caused both STAT3 redistribution and activation that were accompanied by the activation and redistribution of c-Src and the TEC family kinase, BMX, but not JAK2. However, infection with dominant negative c-Src adenovirus was unable to block RGD-stimulated changes on either STAT3 or BMX. Further analysis in vivo in 48 h PO myocardium showed the presence of both STAT3 and BMX in the detergent-insoluble fraction with their complex formation and phosphorylation. Therefore, these studies indicate a novel mechanism of BMX-mediated STAT3 activation within a PO model of cardiac hypertrophy that might contribute to cardiomyocyte growth and survival.

  11. 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

  12. DDR1 promotes E-cadherin stability via inhibition of integrin-β1-Src activation-mediated E-cadherin endocytosis

    PubMed Central

    Chen, Hong-Ru; Yeh, Yi-Chun; Liu, Ching-Yi; Wu, Yu-Ting; Lo, Fang-Yu; Tang, Ming-Jer; Wang, Yang-Kao

    2016-01-01

    Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase of collagen, is primarily expressed in epithelial cells. Activation of DDR1 stabilises E-cadherin located on the cell membrane; however, the detailed mechanism of DDR1-stabilised E-cadherin remains unclear. We performed DDR1 knockdown (Sh-DDR1) on Mardin-Darby canine kidney cells to investigate the mechanism of DDR1-stabilised E-cadherin. Sh-DDR1 decreased junctional localisation, increased endocytosis of E-cadherin, and increased physical interactions between E-cadherin and clathrin. Treatment of the dynamin inhibitor Dyngo 4a suppressed Sh-DDR1-induced E-cadherin endocytosis. In addition, the phosphorylation level of Src tyrosine 418 was increased in Sh-DDR1 cell junctions, and inhibition of Src activity decreased Sh-DDR1-induced E-cadherin endocytosis. To characterise the molecular mechanisms, blocking integrin β1 decreased Src activity and E-cadherin junctional localisation in Sh-DDR1 cells. Photoconversion results showed that inhibition of Src activity rescued E-cadherin membrane stability and that inhibition of integrin β1-Src signalling decreased stress fibres and rescued E-cadherin membrane stability in Sh-DDR1 cells. Taken together, DDR1 stabilised membrane localisation of E-cadherin by inhibiting the integrin β1-Src-mediated clathrin-dependent endocytosis pathway. PMID:27824116

  13. Integrin αvβ8-Mediated TGF-β Activation by Effector Regulatory T Cells Is Essential for Suppression of T-Cell-Mediated Inflammation

    PubMed Central

    Worthington, John J.; Kelly, Aoife; Smedley, Catherine; Bauché, David; Campbell, Simon; Marie, Julien C.; Travis, Mark A.

    2015-01-01

    Summary Regulatory T (Treg) cells play a pivotal role in suppressing self-harmful T cell responses, but how Treg cells mediate suppression to maintain immune homeostasis and limit responses during inflammation is unclear. Here we show that effector Treg cells express high amounts of the integrin αvβ8, which enables them to activate latent transforming growth factor-β (TGF-β). Treg-cell-specific deletion of integrin αvβ8 did not result in a spontaneous inflammatory phenotype, suggesting that this pathway is not important in Treg-cell-mediated maintenance of immune homeostasis. However, Treg cells lacking expression of integrin αvβ8 were unable to suppress pathogenic T cell responses during active inflammation. Thus, our results identify a mechanism by which Treg cells suppress exuberant immune responses, highlighting a key role for effector Treg-cell-mediated activation of latent TGF-β in suppression of self-harmful T cell responses during active inflammation. PMID:25979421

  14. Induction of endothelial cell activation by a triple helical alpha2beta integrin ligand, derived from type I collagen alpha1(I)496-507.

    PubMed

    Baronas-Lowell, Diane; Lauer-Fields, Janelle L; Fields, Gregg B

    2004-01-09

    Endothelial cell activation involves the elevated expression of cell adhesion molecules, chemoattractants, chemokines, and cytokines. These expression profiles may be regulated by integrin-mediated cell signaling pathways. In the current study, an alpha2beta1 integrin triple helical peptide ligand derived from type I collagen residues alpha1(I)496-507 was examined for induction of human aortic endothelial cell (HAEC) activation. In addition, a "miniextracellular matrix" composed of a mixture of the alpha1(I)496-507 ligand and a second, alpha-helical ligand incorporating the endothelial cell proliferating region of SPARC (secreted protein acidic and rich in cysteine) was studied for induction of HAEC activation. Following HAEC adhesion to alpha1(I)496-507, mRNA expression of E-selectin-1, vascular and intercellular cell adhesion molecules-1, and monocytic chemoattractant protein-1 was stimulated, whereas that of endothelin-1 was inhibited. Enzyme-linked immunosorbent assay analysis demonstrated that E-selectin-1 and monocytic chemoattractant protein-1 expression was also stimulated, whereas endothelin-1 protein expression diminished. Engagement of the alpha2beta1 integrin initiated a HAEC response similar to that of tumor necrosis factor-alpha-induced HAECs but was not sufficient to induce an inflammatory response. Addition of the SPARC119-122 region had only a slight effect on HAEC activation. Other cell-extracellular matrix interactions appear to be required to elicit an inflammatory response. The alpha2beta1 integrin specific triple helical peptide ligand described herein represents a more general in vitro model system by which gene expression and protein production profiles induced by binding to a single cellular receptor type can be quantified.

  15. Transcription of the Transforming Growth Factor β Activating Integrin β8 Subunit Is Regulated by SP3, AP-1, and the p38 Pathway*

    PubMed Central

    Markovics, Jennifer A.; Araya, Jun; Cambier, Stephanie; Jablons, David; Hill, Arthur; Wolters, Paul J.; Nishimura, Stephen L.

    2010-01-01

    Integrin αvβ8 is a critical regulator of transforming growth factor β activation in vasculogenesis during development, immune regulation, and endothelial/epithelial-mesenchymal homeostasis. Recent studies have suggested roles for integrin β8 in the pathogenesis of chronic obstructive pulmonary disease, brain arteriovenous malformations, and select cancers (Araya, J., Cambier, S., Markovics, J. A., Wolters, P., Jablons, D., Hill, A., Finkbeiner, W., Jones, K., Broaddus, V. C., Sheppard, D., Barzcak, A., Xiao, Y., Erle, D. J., and Nishimura, S. L. (2007) J. Clin. Invest. 117, 3551–3562; Su, H., Kim, H., Pawlikowska, L., Kitamura, H., Shen, F., Cambier, S., Markovics, J., Lawton, M. T., Sidney, S., Bollen, A. W., Kwok, P. Y., Reichardt, L., Young, W. L., Yang, G. Y., and Nishimura, S. L. (2010) Am. J. Pathol. 176, 1018–1027; Culhane, A. C., and Quackenbush, J. (2009) Cancer Res. 69, 7480–7485; Cambier, S., Mu, D. Z., O'Connell, D., Boylen, K., Travis, W., Liu, W. H., Broaddus, V. C., and Nishimura, S. L. (2000) Cancer Res. 60, 7084–7093). Here we report the first identification and characterization of the promoter for ITGB8. We show that a SP binding site and a cyclic AMP response element (CRE) in the ITGB8 core promoter are required for its expression and that Sp1, Sp3, and several AP-1 transcription factors form a complex that binds to these sites in a p38-dependent manner. Furthermore, we demonstrate the requirement for Sp3, ATF-2, and p38 for the transcription and protein expression of integrin β8. Additionally, reduction of SP3 or inhibition of p38 blocks αvβ8-mediated transforming growth factor β activation. These results place integrin β8 expression and activity under the control of ubiquitous transcription factors in a stress-activated and pro-inflammatory pathway. PMID:20519498

  16. GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF) UPREGULATES β1 INTEGRIN AND INCREASES MIGRATION OF HUMAN TROPHOBLAST SWAN 71 CELLS VIA PI3K AND MAPK ACTIVATION

    PubMed Central

    Furmento, Verónica A.; Marino, Julieta; Blank, Viviana C.; Cayrol, María Florencia; Cremaschi, Graciela A.; Aguilar, Rubén C.; Roguin, Leonor P.

    2017-01-01

    Multiple cytokines and growth factors expressed at the fetal-maternal interface are involved in the regulation of trophoblast functions and placental growth, but the role of G-CSF has not been completely established. Based on our previous study showing that G-CSF increases the activity of matrix metalloproteinase-2 and the release of vascular endothelial growth factor in Swan 71 human trophoblast cells, in this work we explore the possible contribution of G-CSF to cell migration and the G-CSF-triggered signaling pathway. We found that G-CSF induced morphological changes on actin cytoskeleton consistent with a migratory cell phenotype. G-CSF also up-regulated the expression levels of β1 integrin and promoted Swan 71 cell migration. By using selective pharmacological inhibitors and dominant negative mutants we showed that PI3K, Erk 1/2 and p38 pathways are required for promoting Swan 71 cell motility. It was also demonstrated that PI3K behaved as an upstream regulator of Erk 1/2 and p38 MAPK. In addition, the increase of β1 integrin expression was dependent on PI3K activation. In conclusion, our results indicate that G-CSF stimulates β1 integrin expression and Swan 71 cell migration by activating PI3K and MAPK signaling pathways, suggesting that G-CSF should be considered as an additional regulatory factor that contributes to a successful embryo implantation and to the placenta development. PMID:26992288

  17. Granulocyte colony-stimulating factor (G-CSF) upregulates β1 integrin and increases migration of human trophoblast Swan 71 cells via PI3K and MAPK activation.

    PubMed

    Furmento, Verónica A; Marino, Julieta; Blank, Viviana C; Cayrol, María Florencia; Cremaschi, Graciela A; Aguilar, Rubén C; Roguin, Leonor P

    2016-03-15

    Multiple cytokines and growth factors expressed at the fetal-maternal interface are involved in the regulation of trophoblast functions and placental growth, but the role of G-CSF has not been completely established. Based on our previous study showing that G-CSF increases the activity of matrix metalloproteinase-2 and the release of vascular endothelial growth factor in Swan 71 human trophoblast cells, in this work we explore the possible contribution of G-CSF to cell migration and the G-CSF-triggered signaling pathway. We found that G-CSF induced morphological changes on actin cytoskeleton consistent with a migratory cell phenotype. G-CSF also up-regulated the expression levels of β1 integrin and promoted Swan 71 cell migration. By using selective pharmacological inhibitors and dominant negative mutants we showed that PI3K, Erk 1/2 and p38 pathways are required for promoting Swan 71 cell motility. It was also demonstrated that PI3K behaved as an upstream regulator of Erk 1/2 and p38 MAPK. In addition, the increase of β1 integrin expression was dependent on PI3K activation. In conclusion, our results indicate that G-CSF stimulates β1 integrin expression and Swan 71 cell migration by activating PI3K and MAPK signaling pathways, suggesting that G-CSF should be considered as an additional regulatory factor that contributes to a successful embryo implantation and to the placenta development.

  18. Clonorchis sinensis excretory-secretory products promote the migration and invasion of cholangiocarcinoma cells by activating the integrin β4-FAK/Src signaling pathway.

    PubMed

    Pak, Jhang Ho; Bashir, Qudsia; Kim, In Ki; Hong, Sung-Jong; Maeng, Sejung; Bahk, Young Yil; Kim, Tong-Soo

    2017-03-08

    Cholangiocarcinoma (CCA) is a slow-growing but highly metastatic cancer. Its metastatic potential largely explains its high mortality rate. A recognized risk factor for CCA development is infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis. We previously reported that the excretory-secretory products (ESPs) of C. sinensis promoted the three-dimensional aggregation and invasion of CCA cells. In the present study, a quantitative real-time PCR array of extracellular matrix (ECM) and adhesion molecules was used to examine the regulatory mechanism of ESP-mediated CCA cell migration and invasion. In particular, the expression levels of integrin α isoforms and β4 were upregulated in response to ESPs. Increased expression of integrin β4 was probably correlated with activation of focal adhesion kinase (FAK) and the steroid receptor coactivator (Src) family kinase and the subsequent activation of two downstream focal adhesion molecules, paxillin and vinculin. Moreover, inhibition of FAK/Src activation reduced paxillin and vinculin phosphorylation and attenuated ESP-induced CCA cell migration and invasion. These findings suggest that the integrin β4-FAK/Src signaling axis may play a crucial role in clonorchiasis-associated CCA metastasis during tumor progression.

  19. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:25563751

  1. 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.

  2. Fibronectin type III5 repeat contains a novel cell adhesion sequence, KLDAPT, which binds activated alpha4beta1 and alpha4beta7 integrins.

    PubMed

    Moyano, J V; Carnemolla, B; Domínguez-Jiménez, C; García-Gila, M; Albar, J P; Sánchez-Aparicio, P; Leprini, A; Querzé, G; Zardi, L; Garcia-Pardo, A

    1997-10-03

    The region of fibronectin encompassing type III repeats 4-6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact with cells as already shown for other heparin binding domains of fibronectin. A computer search based on homologies with known active sites in fibronectin revealed the sequence KLDAPT located in FN-III5. A synthetic peptide containing this sequence induced lymphoid cell adhesion upon treatment with the activating anti-beta1 monoclonal antibody (mAb) TS2/16 or with Mn2+, indicating that KLDAPT was binding to an integrin. A recombinant fragment containing repeat III5 (FN-III5) also mediated adhesion of TS2/16/Mn2+-treated cells while the FN-III6 fragment did not. Soluble KLDAPT peptide inhibited cell adhesion to FN-III5 as well as to a 38-kDa fibronectin fragment and VCAM-1, two previously known ligands for alpha4beta1 integrin. KLDAPT also competed with the binding of soluble alkaline phosphatase-coupled VCAM-Ig to Mn2+-treated alpha4beta1. Furthermore, mAbs anti-alpha4 and anti-alpha4beta7, but not mAbs to other integrins, inhibited cell adhesion to FN-III5 and KLDAPT. These results therefore establish a cell adhesive function for the FN-III5 repeat and show that KLDAPT is a novel fibronectin ligand for activated alpha4 integrins.

  3. Down-regulation of ARNT promotes cancer metastasis by activating the fibronectin/integrin β1/FAK axis

    PubMed Central

    Huang, Chi-Ruei; Lee, Chung-Ta; Chang, Kwang-Yu; Chang, Wen-Chang; Liu, Yao-Wen; Lee, Jenq-Chang; Chen, Ben-Kuen

    2015-01-01

    The aryl hydrocarbon receptor nuclear translocator (ARNT) is broadly involved in regulating tumorigenesis by inducing genes that are involved in tumor growth and angiogenesis. Tumorigenesis usually involves normoxic conditions. However, the role of ARNT in tumor metastasis during normoxia remains unclear. Here, we demonstrate that ARNT protein levels were decreased in late-stage human colorectal cancer using immunohistochemical analysis. Down-regulation of ARNT protein promoted cancer cell migration and invasion, which was mediated by activation of the fibronectin/integrin β1/FAK signaling axis. In addition, the enhancement of migration and invasion in ANRT knockdown cells was blocked when ARNT was restored in the cells. In xenografts in severe combined immunodeficiency mice, tumor growth was significantly inhibited in the ARNT-knockdown condition. However, the tail-vein injection animal model revealed that the depletion of ARNT-induced metastatic lung colonies was further enhanced when ARNT expression was recovered post-injection. Interestingly, chemotherapeutic drugs inhibited ARNT expression and promoted the invasion of residual tumor cells. These results suggest that ARNT may play a positive role during tumor growth (either in early-stage tumor growth or in organ metastases), but plays a negative role in tumor migration and invasion. Therefore, the efficiency of ARNT-targeted therapy during different cancer stages should be carefully evaluated. PMID:25839165

  4. Plant toxin β-ODAP activates integrin β1 and focal adhesion: A critical pathway to cause neurolathyrism

    PubMed Central

    Tan, Rui-Yue; Xing, Geng-Yan; Zhou, Guang-Ming; Li, Feng-Min; Hu, Wen-Tao; Lambein, Fernand; Xiong, Jun-Lan; Zhang, Sheng-Xiang; Kong, Hai-Yan; Zhu, Hao; Li, Zhi-Xiao; Xiong, You-Cai

    2017-01-01

    Neurolathyrism is a unique neurodegeneration disease caused by β-N-oxalyl-L-α, β- diaminopropionic (β-ODAP) present in grass pea seed (Lathyrus stativus L.) and its pathogenetic mechanism is unclear. This issue has become a critical restriction to take full advantage of drought-tolerant grass pea as an elite germplasm resource under climate change. We found that, in a human glioma cell line, β-ODAP treatment decreased mitochondrial membrane potential, leading to outside release and overfall of Ca2+ from mitochondria to cellular matrix. Increased Ca2+ in cellular matrix activated the pathway of ECM, and brought about the overexpression of β1 integrin on cytomembrane surface and the phosphorylation of focal adhesion kinase (FAK). The formation of high concentration of FA units on the cell microfilaments further induced overexpression of paxillin, and then inhibited cytoskeleton polymerization. This phenomenon turned to cause serious cell microfilaments distortion and ultimately cytoskeleton collapse. We also conducted qRT-PCR verification on RNA-sequence data using 8 randomly chosen genes of pathway enrichment, and confirmed that the data was statistically reliable. For the first time, we proposed a relatively complete signal pathway to neurolathyrism. This work would help open a new window to cure neurolathyrism, and fully utilize grass pea germplasm resource under climate change. PMID:28094806

  5. Tenascin-C enhances pancreatic cancer cell growth and motility and affects cell adhesion through activation of the integrin pathway.

    PubMed

    Paron, Igor; Berchtold, Sonja; Vörös, Julia; Shamarla, Madhavi; Erkan, Mert; Höfler, Heinz; Esposito, Irene

    2011-01-01

    Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells. Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition. Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt. TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and might therefore play a role in PDAC spreading and metastasis in vivo.

  6. CB₁ cannabinoid receptors promote maximal FAK catalytic activity by stimulating cooperative signaling between receptor tyrosine kinases and integrins in neuronal cells.

    PubMed

    Dalton, George D; Peterson, Lynda J; Howlett, Allyn C

    2013-08-01

    Tyrosine phosphorylation (Tyr-P) of focal adhesion kinase (FAK) regulates FAK activation. Phosphorylated FAK Tyr 397 binds Src family kinases (Src), which in turn directly phosphorylate FAK Tyr 576/577 to produce maximal FAK enzymatic activity. CB₁ cannabinoid receptors (CB₁) are abundantly expressed in the nervous system and influence FAK activation by presently unknown mechanisms. The current investigation determined that CB₁-stimulated maximal FAK catalytic activity is mediated by Gi/o proteins in N18TG2 neuronal cells, and that G12/13 regulation of Rac1 and RhoA occurs concomitantly. Immunoblotting analyses using antibodies against FAK phospho-Tyr 397 and phospho-Tyr 576/577 demonstrated that the time-course of CB₁-stimulated FAK 576/577 Tyr-P occurred in three phases: Phase I (0-2 min) maximal Tyr-P, Phase II (5-20 min) rapid decline in Tyr-P, and Phase III (>20 min) plateau in Tyr-P at submaximal levels. In contrast, FAK 397 Tyr-P was monophasic and significantly lower in magnitude. FAK 397 Tyr-P and Phase I FAK 576/577 Tyr-P involved protein tyrosine phosphatase (PTP1B and Shp1/Shp2)-mediated Src activation, Protein Kinase A (PKA) inhibition, and integrin activation. Phase I maximal FAK 576/577 Tyr-P also required cooperative signaling between receptor tyrosine kinases (RTKs) and integrins. The integrin antagonist RGDS peptide, Flk-1 vascular endothelial growth factor receptor (VEGFR) antagonist SU5416, and epidermal growth factor receptor (EGFR) antagonist AG 1478 blocked Phase I FAK 576/577 Tyr-P. CB₁ agonists failed to stimulate FAK Tyr-P in the absence of integrin activation upon suspension in serum-free culture media. In contrast, cells grown on the integrin ligands fibronectin and laminin displayed increased FAK 576/577 Tyr-P that was augmented by CB₁ agonists and blocked by the Src inhibitor PP2 and Flk-1 VEGFR antagonist SU5416. Taken together, these studies have identified a complex integrative pathway utilized by CB₁ to stimulate

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

    PubMed

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

    2017-01-01

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

  8. Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells

    PubMed Central

    Lee, Hye Shin; Cheerathodi, Mujeeburahiman; Chaki, Sankar P.; Reyes, Steve B.; Zheng, Yanhua; Lu, Zhimin; Paidassi, Helena; DerMardirossian, Celine; Lacy-Hulbert, Adam; Rivera, Gonzalo M.

    2015-01-01

    Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell's leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells. PMID:25666508

  9. Endothelial-monocyte activating polypeptide II alters fibronectin based endothelial cell adhesion and matrix assembly via alpha5 beta1 integrin

    SciTech Connect

    Schwarz, Margaret A. . E-mail: m.schwarz@umdnj.edu; Zheng, Hiahua; Liu, Jie; Corbett, Siobhan; Schwarz, Roderich E.

    2005-12-10

    Mature Endothelial-Monocyte Activating Polypeptide (mEMAP) II functions as a potent antiangiogenic peptide. Although the anti-tumor effect of mEMAP II has been described, little is known regarding its mechanism of action. Observations that mEMAP II induced apoptosis only in a subset of migrating and proliferating endothelial cells (EC) suggests a targeted effect on cells engaged in angiogenic activities which are known to rely upon cell adhesion and migration. Indeed, we demonstrate that mEMAP II inhibited fibronectin (FN) dependent microvascular EC (MEC) adhesion and spreading and we show that this depends upon the alpha5 beta1 integrin. Immunofluorescence analysis demonstrated that mEMAP II-dependent blockade of FN-alpha5 beta1 interactions was associated with disassembly of both actin stress fiber networks and FN matrix. These findings suggest that mEMAP II blocks MEC adhesion and spreading on fibronectin, via a direct interaction with the integrin alpha5 beta1, thus implicating that alpha5 integrin may be a mediator of mEMAP II's antiangiogenic function.

  10. Integrin α4β1 controls G9a activity that regulates epigenetic changes and nuclear properties required for lymphocyte migration

    PubMed Central

    Zhang, Xiaohong; Cook, Peter C.; Zindy, Egor; Williams, Craig J.; Jowitt, Thomas A.; Streuli, Charles H.; MacDonald, Andrew S.; Redondo-Muñoz, Javier

    2016-01-01

    The mechanical properties of the cell nucleus change to allow cells to migrate, but how chromatin modifications contribute to nuclear deformability has not been defined. Here, we demonstrate that a major factor in this process involves epigenetic changes that underpin nuclear structure. We investigated the link between cell adhesion and epigenetic changes in T-cells, and demonstrate that T-cell adhesion to VCAM1 via α4β1 integrin drives histone H3 methylation (H3K9me2/3) through the methyltransferase G9a. In this process, active G9a is recruited to the nuclear envelope and interacts with lamin B1 during T-cell adhesion through α4β1 integrin. G9a activity not only reorganises the chromatin structure in T-cells, but also affects the stiffness and viscoelastic properties of the nucleus. Moreover, we further demonstrated that these epigenetic changes were linked to lymphocyte movement, as depletion or inhibition of G9a blocks T-cell migration in both 2D and 3D environments. Thus, our results identify a novel mechanism in T-cells by which α4β1 integrin signaling drives specific chromatin modifications, which alter the physical properties of the nucleus and thereby enable T-cell migration. PMID:26657637

  11. Estradiol Facilitates Functional Integration of iPSC-Derived Dopaminergic Neurons into Striatal Neuronal Circuits via Activation of Integrin α5β1

    PubMed Central

    Nishimura, Kaneyasu; Doi, Daisuke; Samata, Bumpei; Murayama, Shigeo; Tahara, Tsuyoshi; Onoe, Hirotaka; Takahashi, Jun

    2016-01-01

    Summary For cell transplantation therapy for Parkinson's disease (PD) to be realized, the grafted neurons should be integrated into the host neuronal circuit to restore the lost neuronal function. Here, using wheat-germ agglutinin-based transsynaptic tracing, we show that integrin α5 is selectively expressed in striatal neurons that are innervated by midbrain dopaminergic (DA) neurons. In addition, we found that integrin α5β1 was activated by the administration of estradiol-2-benzoate (E2B) in striatal neurons of adult female rats. Importantly, we observed that the systemic administration of E2B into hemi-parkinsonian rat models facilitates the functional integration of grafted DA neurons derived from human induced pluripotent stem cells into the host striatal neuronal circuit via the activation of integrin α5β1. Finally, methamphetamine-induced abnormal rotation was recovered earlier in E2B-administered rats than in rats that received other regimens. Our results suggest that the simultaneous administration of E2B with stem cell-derived DA progenitors can enhance the efficacy of cell transplantation therapy for PD. PMID:26997644

  12. VEGF-integrin interplay controls tumor growth and vascularization

    NASA Astrophysics Data System (ADS)

    de, Sarmishtha; Razorenova, Olga; McCabe, Noel Patrick; O'Toole, Timothy; Qin, Jun; Byzova, Tatiana V.

    2005-05-01

    Cross-talk between the major angiogenic growth factor, VEGF, and integrin cell adhesion receptors has emerged recently as a critical factor in the regulation of angiogenesis and tumor development. However, the molecular mechanisms and consequences of this intercommunication remain unclear. Here, we define a mechanism whereby integrin v3, through activation, clustering, and signaling by means of p66 Shc (Src homology 2 domain containing), regulates the production of VEGF in tumor cells expressing this integrin. Tumors with "activatable" but not "inactive" 3 integrin secrete high levels of VEGF, which in turn promotes extensive neovascularization and augments tumor growth in vivo. This stimulation of VEGF expression depends upon the ability of v3 integrin to cluster and promote phosphorylation of p66 Shc. These observations identify a link between 3 integrins and VEGF in tumor growth and angiogenesis and, therefore, may influence anti-integrin as well as anti-VEGF therapeutic strategies. activation | angiogenesis | Src homology 2 domain containing

  13. A pH-responsive cell-penetrating peptide-modified liposomes with active recognizing of integrin αvβ3 for the treatment of melanoma.

    PubMed

    Shi, Kairong; Li, Jianping; Cao, Zhonglian; Yang, Ping; Qiu, Yue; Yang, Bo; Wang, Yang; Long, Yang; Liu, Yayuan; Zhang, Qianyu; Qian, Jun; Zhang, Zhirong; Gao, Huile; He, Qin

    2015-11-10

    The use of pH-responsive cell-penetrating peptides (CPPs) is an attractive strategy for drug delivery in vivo, however, they still could not actively target to the desired sites. Here, we designed a pH-responsive CPP (TR) with the ability of active targeting to integrin αvβ3, which was a tandem peptide consisted of active targeting ligand peptide (c(RGDfK)) and pH-responsive CPP (TH). The targeting efficiency of TR with integrin was evaluated by molecular simulation and docking studies. The affinity assays of TR peptide modified liposomes (TR-Lip) at pH7.4 and pH6.5 demonstrated adequately the pH-responsive binding efficacy of TR-Lip with integrin αvβ3. The cellular uptake of CFPE-labeled TR-Lip on integrin αvβ3-overexpressing B16F10 cells was 41.67-, 30.67-, and 11.90-fold higher than that of CFPE-labeled PEG-, RGD-, and TH-modified liposomes at pH6.5, respectively, suggesting that TR-Lip could not only actively target to αvβ3-overexpressing cells compared to TH-Lip, but also significantly increased cellular uptake compared to RGD-Lip. At the concentration of 20μg/mL paclitaxel (PTX), the killing activity of PTX-loaded TR-Lip (PTX-TR-Lip) against B16F10 cells was 1.80-, 1.45-, 1.30-, 1.15-time higher than that of PTX-loaded PEG-, RGD-, TH-modified liposomes and free PTX at pH6.5, respectively. In vivo imaging displayed the maximum accumulation of DiD-labeled TR-Lip at tumor sites compared to the other groups. Tumor inhibition rate of B16F10 tumor-bearing mice treated with PTX-TR-Lip was 85.04%, relative to that of PBS. In B16F10 tumor-bearing mice, PTX-TR-Lip showed significantly higher survival rate compared with the other groups. Collectively, all the results in vitro and in vivo suggested that TR-Lip would be a potential delivery system for PTX to treat integrin αvβ3-overexpressing tumor-bearing mice.

  14. Cross-Scale Integrin Regulation Organizes ECM and Tissue Topology.

    PubMed

    Jülich, Dörthe; Cobb, Garrett; Melo, Ana M; McMillen, Patrick; Lawton, Andrew K; Mochrie, Simon G J; Rhoades, Elizabeth; Holley, Scott A

    2015-07-06

    The diverse morphologies of animal tissues are underlain by different configurations of adherent cells and extracellular matrix (ECM). Here, we elucidate a cross-scale mechanism for tissue assembly and ECM remodeling involving Cadherin 2, the ECM protein Fibronectin, and its receptor Integrin α5. Fluorescence cross-correlation spectroscopy within the zebrafish paraxial mesoderm mesenchyme reveals a physical association between Integrin α5 on adjacent cell membranes. This Integrin-Integrin complex correlates with conformationally inactive Integrin. Cadherin 2 stabilizes both the Integrin association and inactive Integrin conformation. Thus, Integrin repression within the adherent mesenchymal interior of the tissue biases Fibronectin fibrillogenesis to the tissue surface lacking cell-cell adhesions. Along nascent somite boundaries, Cadherin 2 levels decrease, becoming anti-correlated with levels of Integrin α5. Simultaneously, Integrin α5 clusters and adopts the active conformation and then commences ECM assembly. This cross-scale regulation of Integrin activation organizes a stereotypic pattern of ECM necessary for vertebrate body elongation and segmentation.

  15. Canstatin inhibits hypoxia-induced apoptosis through activation of integrin/focal adhesion kinase/Akt signaling pathway in H9c2 cardiomyoblasts

    PubMed Central

    Yamawaki, Hideyuki

    2017-01-01

    A hypoxic stress which causes apoptosis of cardiomyocytes is the main problem in the ischemic heart disease. Canstatin, a non-collagenous fragment of type IV collagen α2 chain, is an endogenous anti-angiogenic factor. We have previously reported that canstatin has a cytoprotective effect on cardiomyoblasts. In the present study, we examined the effects of canstatin on hypoxia-induced apoptosis in H9c2 cardiomyoblasts. Cell counting assay was performed to determine a cell viability. Western blotting was performed to detect expression of cleaved casepase-3 and phosphorylation of focal adhesion kinase (FAK) and Akt. Immunocytochemical staining was performed to observe a distribution of αv integrin. Hypoxia (1% O2, 48 h) significantly decreased cell viability and increased cleaved caspase-3 expression. Canstatin (10–250 ng/ml) significantly inhibited these changes in a concentration-dependent manner. Cilengitide (1 μM), an αvβ3 and αvβ5 integrin inhibitor, significantly prevented the protective effects of canstatin on cell viability. Canstatin significantly increased phosphorylation of FAK and Akt under hypoxic condition, which were inhibited by cilengitide. LY294002, an inhibitor of phosphatidylinositol-3 kinase/Akt pathway, suppressed the canstatin-induced Akt phosphorylation and reversed the protective effects of canstatin. It was observed that hypoxia caused a localization of αv integrin to focal adhesion. In summary, we for the first time clarified that canstatin inhibits hypoxia-induced apoptosis via FAK and Akt pathways through activating integrins in H9c2 cardiomyoblasts. PMID:28235037

  16. Integrin signaling in atherosclerosis.

    PubMed

    Finney, Alexandra C; Stokes, Karen Y; Pattillo, Christopher B; Orr, A Wayne

    2017-06-01

    Atherosclerosis, a chronic lipid-driven inflammatory disease affecting large arteries, represents the primary cause of cardiovascular disease in the world. The local remodeling of the vessel intima during atherosclerosis involves the modulation of vascular cell phenotype, alteration of cell migration and proliferation, and propagation of local extracellular matrix remodeling. All of these responses represent targets of the integrin family of cell adhesion receptors. As such, alterations in integrin signaling affect multiple aspects of atherosclerosis, from the earliest induction of inflammation to the development of advanced fibrotic plaques. Integrin signaling has been shown to regulate endothelial phenotype, facilitate leukocyte homing, affect leukocyte function, and drive smooth muscle fibroproliferative remodeling. In addition, integrin signaling in platelets contributes to the thrombotic complications that typically drive the clinical manifestation of cardiovascular disease. In this review, we examine the current literature on integrin regulation of atherosclerotic plaque development and the suitability of integrins as potential therapeutic targets to limit cardiovascular disease and its complications.

  17. Galphas-coupled receptor signaling actively down-regulates α4β1-integrin affinity: A possible mechanism for cell de-adhesion

    PubMed Central

    Chigaev, Alexandre; Waller, Anna; Amit, Or; Sklar, Larry A

    2008-01-01

    Background Activation of integrins in response to inside-out signaling serves as a basis for leukocyte arrest on endothelium, and migration of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule (i.e. change in the affinity for the ligand and molecular unbending (extension)), which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α4β1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic stem cells, hematopoietic cancer cells, and others. Affinity and extension of VLA-4 are both rapidly up-regulated by inside-out signaling through several Gαi-coupled GPCRs. The goal of the current report was to study the effect of Gαs-coupled GPCRs upon integrin activation. Results Using real-time fluorescent ligand binding to assess affinity and a FRET based assay to probe α4β1-integrin unbending, we show that two Gαs-coupled GPCRs (H2-histamine receptor and β2-adrenergic receptor) as well as several cAMP agonists can rapidly down modulate the affinity of VLA-4 activated through two Gαi-coupled receptors (CXCR4 and FPR) in U937 cells and primary human peripheral blood monocytes. This down-modulation can be blocked by receptor-specific antagonists. The Gαs-induced responses were not associated with changes in the expression level of the Gαi-coupled receptors. In contrast, the molecular unbending of VLA-4 was not significantly affected by Gαs-coupled GPCR signaling. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by Gαs-coupled GPCR had a statistically significant effect upon cell aggregation. Conclusion We conclude that Gαs-coupled GPCRs can rapidly down modulate the affinity state of VLA-4 binding pocket through a cAMP dependent pathway. This plays an essential role in the regulation of cell adhesion. We discuss several possible implications of this described phenomenon. PMID:18534032

  18. Morus alba Leaf Lectin (MLL) Sensitizes MCF-7 Cells to Anoikis by Inhibiting Fibronectin Mediated Integrin-FAK Signaling through Ras and Activation of P38 MAPK

    PubMed Central

    Saranya, Jayaram; Shilpa, Ganesan; Raghu, Kozhiparambil G.; Priya, Sulochana

    2017-01-01

    Lectins are a unique class of carbohydrate binding proteins/glycoproteins, and many of them possess anticancer properties. They can induce cell cycle arrest and apoptosis, inhibit protein synthesis, telomerase activity and angiogenesis in cancer cells. In the present study, we have demonstrated the effect of Morus alba leaf lectin (MLL) on anoikis induction in MCF-7 cells. Anoikis induction in cancer cells has a significant role in preventing early stage metastasis. MLL treatment in monolayers of MCF-7 cells caused significant detachment of cells in a time and concentration dependent manner. The detached cells failed to re-adhere and grew even to culture plates coated with different matrix proteins. DNA fragmentation, membrane integrity studies, annexin V staining, caspase 9 activation and upregulation of Bax/Bad confirmed that the detached cells underwent apoptosis. Upregulation of matrix metalloproteinase 9 (MMP-9) caused a decrease in fibronectin (FN) production which facilitated the cells to detach by blocking the FN mediated downstream signaling. On treatment with MLL, we have observed downregulation of integrin expression, decreased phosphorylation of focal adhesion kinase (FAK), loss in FAK-integrin interaction and active Ras. MLL treatment downregulated the levels of phosphorylated Akt and PI3K. Also, we have studied the effect of MLL on two stress activated protein kinases p38 MAPK and JNK. p38 MAPK activation was found to be elevated, but there was no change in the level of JNK. Thus our study substantiated the possible antimetastatic effect of MLL by inducing anoikis in MCF-7 cells by activation of caspase 9 and proapoptotic Bax/Bad by blockage of FN mediated integrin/FAK signaling and partly by activation of p38 MAPK. PMID:28223935

  19. Keratins Stabilize Hemidesmosomes through Regulation of β4-Integrin Turnover.

    PubMed

    Seltmann, Kristin; Cheng, Fang; Wiche, Gerhard; Eriksson, John E; Magin, Thomas M

    2015-06-01

    Epidermal integrity and wound healing depend on remodeling of cell-matrix contacts including hemidesmosomes. Mutations in β4-integrin and plectin lead to severe epidermolysis bullosa (EB). Whether mutations in keratins K5 or K14, which cause EB simplex, also compromise cell-matrix adhesion through altering hemidesmosomal components is not well investigated. In particular, the dependence of β4-integrin endocytosis and turnover on keratins remains incompletely understood. Here, we show that the absence of keratins causes loss of plectin-β4-integrin interaction and elevated β4-integrin phosphorylation at Ser1354 and Ser1362. This triggered a caveolin-dependent endocytosis of β4-integrin but not of other integrins through Rab5 and Rab11 compartments in keratinocytes. Expressing a phospho-deficient β4-integrin mutant reduces β4-integrin endocytosis and rescues plectin localization in keratin-free cells. β4-integrin phosphorylation in the absence of keratins resulted from elevated Erk1/2 activity downstream of increased EGFR and PKCα signaling. Further, increased Erk1/2 phosphorylation and altered plectin localization occur in keratin-deficient mouse epidermis in vivo. Strikingly, expression of the K14-R125P EBS mutant also resulted in plectin mislocalization and elevated β4-integrin turnover, suggesting disease relevance. Our data underscore a major role of keratins in controlling β4-integrin endocytosis involving a plectin-Erk1/2-dependent mechanism relevant for epidermal differentiation and pathogenesis.

  20. 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.

  1. Sialic acid associated with αvβ3 integrin mediates HIV-1 Tat protein interaction and endothelial cell proangiogenic activation.

    PubMed

    Chiodelli, Paola; Urbinati, Chiara; Mitola, Stefania; Tanghetti, Elena; Rusnati, Marco

    2012-06-08

    Sialic acid (NeuAc) is a major anion on endothelial cells (ECs) that regulates different biological processes including angiogenesis. NeuAc is present in the oligosaccharidic portion of integrins, receptors that interact with extracellular matrix components and growth factors regulating cell adhesion, migration, and proliferation. Tat is a cationic polypeptide that, once released by HIV-1(+) cells, accumulates in the extracellular matrix, promoting EC adhesion and proangiogenic activation by engaging α(v)β(3). By using two complementary approaches (NeuAc removal by neuraminidase or its masking by NeuAc-binding lectin from Maackia amurensis, MAA), we investigated the presence of NeuAc on endothelial α(v)β(3) and its role in Tat interaction, EC adhesion, and proangiogenic activation. α(v)β(3) immunoprecipitation with biotinylated MAA or Western blot analysis of neuraminidase-treated ECs demonstrated that NeuAc is associated with both the α(v) and the β(3) subunits. Surface plasmon resonance analysis demonstrated that the masking of α(v)β(3)-associated NeuAc by MAA prevents Tat/α(v)β(3) interaction. MAA and neuraminidase prevent α(v)β(3)-dependent EC adhesion to Tat, the consequent FAK and ERK1/2 phosphorylation, and EC proliferation, migration, and regeneration in a wound-healing assay. Finally, MAA inhibits Tat-induced neovascularization in the ex vivo human artery ring sprouting assay. The inhibitions are specific because the NeuAc-unrelated lectin from Ulex europaeus is ineffective on Tat. Also, MAA and neuraminidase affect only weakly integrin-dependent EC adhesion and proangiogenic activation by fibronectin. In conclusion, NeuAc is associated with endothelial α(v)β(3) and mediates Tat-dependent EC adhesion and proangiogenic activation. These data point to the possibility to target integrin glycosylation for the treatment of angiogenesis/AIDS-associated pathologies.

  2. Phospholipase D Activity Regulates Integrin-mediated Cell Spreading and Migration by Inducing GTP-Rac Translocation to the Plasma Membrane

    PubMed Central

    Chae, Young Chan; Kim, Jung Hwan; Kim, Kyung Lock; Kim, Hyun Wook; Lee, Hye Young; Heo, Won Do; Meyer, Tobias; Suh, Pann-Ghill

    2008-01-01

    Small GTPase Rac is a crucial regulator of actin cytoskeletal rearrangement, and it plays an important role in cell spreading, migration, mitogenesis, phagocytosis, superoxide generation, and axonal growth. It is generally accepted that Rac activity is regulated by the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle. But, it is suggested that in addition to Rac-GTP loading, membrane localization is required for the initiation of downstream effector signaling. However, the molecular mechanisms that control the targeting of GTP-Rac to the plasma membrane remain largely unknown. Here, we have uncovered a signaling pathway linking phospholipase D (PLD) to the localized functions of Rac1. We show that PLD product phosphatidic acid (PA) acts as a membrane anchor of Rac1. The C-terminal polybasic motif of Rac1 is responsible for direct interaction with PA, and Rac1 mutated in this region is incapable of translocating to the plasma membrane and of activating downstream target p21-activated kinase upon integrin activation. Finally, we show that PA induces dissociation of Rho-guanine nucleotide dissociation inhibitor from Rac1 and that PA-mediated Rac1 localization is important for integrin-mediated lamellipodia formation, cell spreading, and migration. These results provide a novel molecular mechanism for the GTP-Rac1 localization through the elevating PLD activity, and they suggest a general mechanism for diverse cellular functions that is required localized Rac activation. PMID:18480413

  3. Impaired cross-activation of β3 integrin and VEGFR-2 on endothelial progenitor cells with aging decreases angiogenesis in response to hypoxia.

    PubMed

    Di, Qun; Cheng, Zeen; Kim, Weon; Liu, Zexuan; Song, Hui; Li, Xiang; Nan, Yongshan; Wang, Chengya; Cheng, Xianwu

    2013-10-03

    The mechanism by which vascular regeneration declines with aging is not fully understood. An interaction between integrin and vascular endothelial growth factor receptor-2 (VEGFR-2) plays a substantial role in angiogenesis. Here, we investigated whether aging impairs this interaction in endothelial progenitor cells (EPCs) under hypoxia. Aging reduced the blood flow and vessel density in ischemic muscles in mice. Levels of phosphorylated Src (p-Src), p-β3, and p-VEGFR-2 in acute ischemia were reduced in the muscles of aged mice compared to young mice. The hypoxia-inducible factor (HIF)-1α stabilizer deferoxamine improved the age-related impairment of angiogenesis, but this effect was diminished by LY290004, an inhibitor of phosphatidylinositol 3-kinase. Deferoxamine improved the reduction in chronic ischemia-induced β3-integrin and VEGFR-2 phosphorylation in the muscles of aged mice; this effect was also diminished by LY290004. In EPCs, we identified the molecular requirements for VEGF-mediated β3-integrin and VEGFR-2 cross-activation in vitronectin-induced cell adhesion under acute hypoxia. We demonstrated that c-Src controlled the adhesion- and VEGF-induced β3 tyrosine phosphorylation in hypoxia. Aging enhanced the hypoxia-induced EPC apoptosis and impaired several c-Src-related VEGF-induced receptor events, including β3 tyrosine activation, ligand binding, cell adhesion, and tubulogenesis in cultured EPCs of animals and those of humans. These data suggest that the aging-related decline in angiogenic action in response to ischemia is mediated by the impairment of cross-activation between β3 and VEGFR-2 in EPCs, which is partially associated with decreased HIF-1α stability. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  4. The serine/threonine kinase Ndr2 controls integrin trafficking and integrin-dependent neurite growth.

    PubMed

    Rehberg, Kati; Kliche, Stefanie; Madencioglu, Deniz A; Thiere, Marlen; Müller, Bettina; Meineke, Bernhard Manuel; Freund, Christian; Budinger, Eike; Stork, Oliver

    2014-04-09

    Integrins have been implicated in various processes of nervous system development, including proliferation, migration, and differentiation of neuronal cells. In this study, we show that the serine/threonine kinase Ndr2 controls integrin-dependent dendritic and axonal growth in mouse hippocampal neurons. We further demonstrate that Ndr2 is able to induce phosphorylation at the activity- and trafficking-relevant site Thr(788/789) of β1-integrin to stimulate the PKC- and CaMKII-dependent activation of β1-integrins, as well as their exocytosis. Accordingly, Ndr2 associates with integrin-positive early and recycling endosomes in primary hippocampal neurons and the surface expression of activated β1-integrins is reduced on dendrites of Ndr2-deficient neurons. The role of Ndr2 in dendritic differentiation is also evident in vivo, because Ndr2-null mutant mice show arbor-specific alterations of dendritic complexity in the hippocampus. This indicates a role of Ndr2 in the fine regulation of dendritic growth; in fact, treatment of primary neurons with Semaphorin 3A rescues Ndr2 knock-down-induced dendritic growth deficits but fails to enhance growth beyond control level. Correspondingly, Ndr2-null mutant mice show a Semaphorin 3A(-/-)-like phenotype of premature dendritic branching in the hippocampus. The results of this study show that Ndr2-mediated integrin trafficking and activation are crucial for neurite growth and guidance signals during neuronal development.

  5. TMPRSS4 regulates levels of integrin α5 in NSCLC through miR-205 activity to promote metastasis.

    PubMed

    Larzabal, L; de Aberasturi, A L; Redrado, M; Rueda, P; Rodriguez, M J; Bodegas, M E; Montuenga, L M; Calvo, A

    2014-02-04

    TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown. miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC. miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G0/G1 cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin α5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin α5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin α5 levels. We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin α5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC.

  6. TMPRSS4 regulates levels of integrin α5 in NSCLC through miR-205 activity to promote metastasis

    PubMed Central

    Larzabal, L; de Aberasturi, A L; Redrado, M; Rueda, P; Rodriguez, M J; Bodegas, M E; Montuenga, L M; Calvo, A

    2014-01-01

    Background: TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown. Methods: miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC. Results: miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G0/G1 cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin α5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin α5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin α5 levels. Conclusion: We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin α5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC. PMID:24434435

  7. The phosphatase of regenerating liver 3 (PRL-3) promotes cell migration through Arf-activity-dependent stimulation of integrin α5 recycling.

    PubMed

    Krndija, Denis; Münzberg, Christin; Maass, Ulrike; Hafner, Margit; Adler, Guido; Kestler, Hans A; Seufferlein, Thomas; Oswald, Franz; von Wichert, Götz

    2012-08-15

    The formation of metastasis is one of the most critical problems in oncology. The phosphatase of regenerating liver 3 (PRL-3) is a new target in colorectal cancer, mediating metastatic behavior through a promigratory function. However, detailed explanations for this effect have remained elusive. Here we show that PRL-3 interacts with the ADP-ribosylation factor 1 (Arf1). PRL-3 colocalizes with Arf1 in an endosomal compartment and associates with transmembrane proteins such as the transferrin receptor and α5 integrins. PRL-3 interacts with Arf1 through a distinct motif and regulates activation of Arf1. PRL-3-mediated migration depends on expression and activation of Arf1 and is sensitive to treatment with Brefeldin A. We also demonstrate that PRL-3 modulates recycling of α5 integrins and that its phosphatase activity as well as Arf activation and compartmentalization with Arf1 are required for this effect. In summary our data identify a new function for PRL-3 and show that Arf1 is a new PRL-3-dependent mediator of enhanced migration of cancer cells through enhanced recycling of matrix receptors.

  8. A novel solid lipid nanoparticle formulation for active targeting to tumor α(v) β(3) integrin receptors reveals cyclic RGD as a double-edged sword.

    PubMed

    Shuhendler, Adam J; Prasad, Preethy; Leung, Michael; Rauth, Andrew M; Dacosta, Ralph S; Wu, Xiao Yu

    2012-09-01

    The overexpression of α(v) β(3) integrin receptors on tumor cells and tumor vascular endothelium makes it a useful target for imaging, chemotherapy and anti-angiogenic therapy. However integrin-targeted delivery of therapeutics by nanoparticles have provided only marginal, if any, enhancement of therapeutic effect. This work was thus focused on the development of novel α(v) β(3) -targeted near infrared light-emitting solid lipid nanoparticles (SLN) through conjugation to the α(v) β(3) integrin-specific ligand cyclic Arg-Gly-Asp (cRGD), and the assessment of the effects of α(v) β(3) targeting on nanoparticle biodistribution. Since our previously developed non-targeted "stealth" SLN showed little hepatic accumulation, unlike most reported liposomes and micelles, they served as a reference for quantifying the effects of cRGD-conjugation on tumor uptake and whole animal biodistribution of SLN. Non-targeted SLN, actively targeted (RGD-SLN) and blocked RGD-SLN were prepared to contain near infrared quantum dots for live animal imaging. They were injected intravenously to nude mice bearing xenograft orthotopic human breast tumors or dorsal window chamber breast tumors. Tumor micropharmacokinetics of various SLN formulations were determined using intravital microscopy, and whole animal biodistribution was followed over time by optical imaging. The active tumor targeting with cRGD was found to be a "double-edged sword": while the specificity of RGD-SLN accumulation in tumor blood vessels and their tumor residence time increased, their distribution in the liver, spleen, and kidneys was significantly greater than the non-targeted SLN, leaving a smaller amount of nanoparticles in the tumor tissue. Nevertheless the enhanced specificity and retention of RGD-SLN in tumor neovasculature could make this novel formulation useful for tumor neovascular-specific therapies and imaging applications. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Structure of a Complete Integrin Ectodomain in a Physiologic Resting State and Activation and Deactivation by Applied Forces

    SciTech Connect

    Zhu, Jianghai; Luo, Bing-Hao; Xiao, Tsan; Zhang, Chengzhong; Nishida, Noritaka; Springer, Timothy A.

    2009-11-10

    The complete ectodomain of integrin {alpha}{sub IIb}{beta}{sub 3} reveals a bent, closed, low-affinity conformation, the {beta} knee, and a mechanism for linking cytoskeleton attachment to high affinity for ligand. Ca and Mg ions in the recognition site, including the synergistic metal ion binding site (SyMBS), are loaded prior to ligand binding. Electrophilicity of the ligand-binding Mg ion is increased in the open conformation. The {beta}{sub 3} knee passes between the {beta}{sub 3}-PSI and {alpha}{sub IIB}-knob to bury the lower {beta} leg in a cleft, from which it is released for extension. Different integrin molecules in crystals and EM reveal breathing that appears on pathway to extension. Tensile force applied to the extended ligand-receptor complex stabilizes the closed, low-affinity conformation. By contrast, an additional lateral force applied to the {beta} subunit to mimic attachment to moving actin filaments stabilizes the open, high-affinity conformation. This mechanism propagates allostery over long distances and couples cytoskeleton attachment of integrins to their high-affinity state.

  10. Binding of alphaherpesvirus glycoprotein H to surface α4β1-integrins activates calcium-signaling pathways and induces phosphatidylserine exposure on the plasma membrane.

    PubMed

    Azab, Walid; Gramatica, Andrea; Herrmann, Andreas; Osterrieder, Nikolaus

    2015-10-20

    Intracellular signaling connected to integrin activation is known to induce cytoplasmic Ca(2+) 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 Ca(2+) levels. EHV-1 expressing EHV-4 gH (gH4) in lieu of authentic gH1 failed to induce Ca(2+) release, while EHV-4 with gH1 triggered significant Ca(2+) 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 Ca(2+) 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 Ca(2+) 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. Herpesviruses are a large family of enveloped viruses that infect a wide range of hosts, causing a variety of diseases. These viruses have developed a number of strategies for successful entry into different cell types. We and others have shown that alphaherpesviruses, including EHV-1 and herpes simplex virus 1 (HSV-1), can route their entry pathway and do so by manipulation of cell signaling cascades to ensure viral genome delivery to nuclei. We show here that the interaction between EHV-1 gH and cellular α4β1-integrins is necessary to induce emptying of ER calcium stores, which induces phosphatidylserine exposure on the plasma membrane

  11. Homo-oligomerization of transmembrane α-domain of integrin.

    PubMed

    Shamloo, Amir; Nikbin, Ehsan; Mehboudi, Nastaran; Damirchi, Behzad

    2014-01-01

    Integrins contribute to form focal adhesions complex. Therefore, simulation of integrin interactions can be helpful in clarifying the mechanism of focal adhesion formation. Interactions of integrins can also initiate signal transduction in the focal adhesions. Since integrins contain α and β subunits that are separated in an active state, studying both subunits separately is crucial, since, in the active state of integrins, the distance between these subunits is long enough that they do not influence one another significantly. Thus, this study aims to investigate the tendency of α subunits of integrins to form homodimers. All simulations were carried out via MARTINI coarse grain (CG) molecular dynamics technique. α subunits were placed in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer at a distance of 5 nm, and they were allowed to diffuse in the lipid bilayer. All simulations showed that α subunits have a tendency to form stable dimers.

  12. Vimocin and vidapin, cyclic KTS peptides, are dual antagonists of α1β1/α2β1 integrins with antiangiogenic activity.

    PubMed

    Momic, Tatjana; Katzehendler, Jehoshua; Benny, Ofra; Lahiani, Adi; Cohen, Gadi; Noy, Efrat; Senderowitz, Hanoch; Eble, Johannes A; Marcinkiewicz, Cezary; Lazarovici, Philip

    2014-09-01

    Obtustatin and viperistatin, members of the disintegrin protein family, served as lead compounds for the synthesis of linear and cyclic peptides containing the KTS binding motif. The most active linear peptide, a viperistatin analog, indicated the importance of Cys(19) and Cys(29), as well as the presence of Arg at position 24 for their biologic activity, and was used as the basic sequence for the synthesis of cyclic peptides. Vimocin (compound 6) and vidapin (compound 10) showed a high potency (IC50 = 0.17 nM) and intermediate efficacy (20 and 40%) in inhibition of adhesion of α1/α2 integrin overexpressor cells to respective collagens. Vimocin was more active in inhibition of the wound healing (53%) and corneal micropocket (17%) vascularization, whereas vidapin was more potent in inhibition of migration in the Matrigel tube formation assay (90%). Both compounds similarly inhibited proliferation (50-90%) of endothelial cells, and angiogenesis induced by vascular endothelial growth factor (80%) and glioma (55%) in the chorioallantoic membrane assay. These peptides were not toxic to endothelial cell cultures and caused no acute toxicity upon intravenous injection in mice, and were stable for 10-30 hours in human serum. The in vitro and in vivo potency of the peptides are consistent with conformational ensembles and "bioactive" space shared by obtustatin and viperistatin. These findings suggest that vimocin and vidapin can serve as dual α1β1/α2β1 integrin antagonists in antiangiogenesis and cancer therapy.

  13. β8 integrin expression and activation of TGF-β by intestinal dendritic cells is determined by both tissue microenvironment and cell lineage

    PubMed Central

    Boucard-Jourdin, Mathilde; Kugler, David; Endale Ahanda, Marie-Laure; This, Sébastien; De Calisto, Jaime; Zhang, Ailiang; Mora, J. Rodrigo; Stuart, Lynda M.; Savill, John; Lacy-Hulbert, Adam; Paidassi, Helena

    2016-01-01

    Activation of TGF-β by dendritic cells (DCs) expressing αvβ8 integrin is essential for the generation of intestinal regulatory T cells (Tregs) that in turn promote tolerance to intestinal antigens. We have recently shown that αvβ8 integrin is preferentially expressed by CD103+ DCs, and confers their ability to activate TGF-β and generate Tregs. However, how these DCs become specialized for this vital function is unknown. Here we show that β8 expression is controlled by a combination of factors that include DC lineage, and signals derived from the tissue microenvironment and microbiota. Specifically, our data demonstrate that TGF-β itself, along with retinoic acid (RA) and Toll-like receptor (TLR) signaling, drive expression of αvβ8 in DCs. However, these signals only result in high levels of β8 expression in cells of the cDC1 lineage, CD8α+ or CD103+CD11b- DCs, and this is associated with epigenetic changes in the Itgb8 locus. Together, these data provide a key illustrative example of how microenvironmental factors and cell lineage drive the generation of regulatory αvβ8-expressing DCs specialized for activation of TGF-β to facilitate Treg generation. PMID:27481847

  14. Vicrostatin – An Anti-Invasive Multi-Integrin Targeting Chimeric Disintegrin with Tumor Anti-Angiogenic and Pro-Apoptotic Activities

    PubMed Central

    Minea, Radu O.; Helchowski, Corey M.; Zidovetzki, Samuel J.; Costa, Fritz K.; Swenson, Stephen D.; Markland, Francis S.

    2010-01-01

    Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., αvβ3, αvβ5, and α5β1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN. PMID:20532165

  15. Nanoparticle-formulated siRNA targeting integrins inhibits hepatocellular carcinoma progression in mice

    PubMed Central

    Bogorad, Roman L; Yin, Hao; Zeigerer, Anja; Nonaka, Hidenori; Ruda, Vera; Zerial, Marino; Anderson, Daniel G; Koteliansky, Victor

    2014-01-01

    Integrins play an important role during development, regulating cell differentiation, proliferation and survival. Here we show that knockdown of integrin subunits slows down the progression of hepatocellular carcinoma (HCC). Using nanoparticulate delivery of short interfering RNAs targeting β1 and αv integrin subunits we downregulate all integrin receptors in hepatocytes. Short-term integrin knockdown (two weeks) does not cause apparent structural or functional perturbations of normal liver tissue. Alterations in liver morphology accumulate upon sustained integrin downregulation (seven weeks). The integrin knockdown leads to significant retardation of HCC progression, reducing proliferation and increasing tumour cell death. This tumour retardation is accompanied by reduced activation of MET oncogene as well as expression of its mature form on the cell surface. Our data suggest that transformed proliferating cells from HCC are more sensitive to knockdown of integrins than normal quiescent hepatocytes, highlighting the potential of siRNA-mediated inhibition of integrins as an anti-cancer therapeutic approach. PMID:24844798

  16. Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion

    PubMed Central

    Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Gentilini, María Virginia; Velásquez, Lis Noelia; Fossati, Carlos Alberto; Giambartolomei, Guillermo Hernán

    2015-01-01

    Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-β, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage. PMID:26459511

  17. 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.

  18. C6-ceramide nanoliposome suppresses tumor metastasis by eliciting PI3K and PKCζ tumor-suppressive activities and regulating integrin affinity modulation.

    PubMed

    Zhang, Pu; Fu, Changliang; Hu, Yijuan; Dong, Cheng; Song, Yang; Song, Erqun

    2015-03-20

    Nanoliposomal formulation of C6-ceramide, a proapoptotic sphingolipid metabolite, presents an effective way to treat malignant tumor. Here, we provide evidence that acute treatment (30 min) of melanoma and breast cancer cells with nanoliposomal C6-ceramide (NaL-C6) may suppress cell migration without inducing cell death. By employing a novel flow migration assay, we demonstrated that NaL-C6 decreased tumor extravasation under shear conditions. Compared with ghost nanoliposome, NaL-C6 triggered phosphorylation of PI3K and PKCζ and dephosphorylation of PKCα. Concomitantly, activated PKCζ translocated into cell membrane. siRNA knockdown or pharmacological inhibition of PKCζ or PI3K rescued NaL-C6-mediated suppression of tumor migration. By inducing dephosphorylation of paxillin, PKCζ was responsible for NaL-C6-mediated stress fiber depolymerization and focal adhesion disassembly in the metastatic tumor cells. PKCζ and PI3K regulated cell shear-resistant adhesion in a way that required integrin αvβ3 affinity modulation. In conclusion, we identified a novel role of acute nanoliposomal ceramide treatment in reducing integrin affinity and inhibiting melanoma metastasis by conferring PI3K and PKCζ tumor-suppressive activities.

  19. Adhesion of ADP-activated platelets to intact endothelium under stagnation point flow in vitro is mediated by the integrin alphaIIbeta3.

    PubMed

    Reininger, A J; Korndörfer, M A; Wurzinger, L J

    1998-05-01

    As we demonstrated earlier, platelets adhere to intact endothelium provided they are activated and convectively transported against the endothelial surface. To identify the platelet receptors involved we superfused cultured endothelium with activated platelet rich plasma (PRP) by means of the Stagnation Point Flow Adhesio- Aggregometer while blocking various platelet receptors. Inhibition was performed with the tetrapeptide RGDS, the non-peptide Ro-43-8857, or a monoclonal antibody directed against integrin alphaIIbeta3. Platelet deposition was video-recorded and quantified by image analysis. Infusion of RGDS or Ro-43-8857 into ADP-stimulated PRP completely prevented adhesion as well as subsequent aggregation. Interrupting the inhibitor infusion while ADP stimulation persisted, prompted adhesion and aggregation, demonstrating the reversibility of the inhibition. Platelet adhesion was irreversibly blocked by preincubation of the PRP with the moab against alphaIIbeta3. Its specific binding was confirmed by immunoelectron microscopy. Our results suggest that platelet adhesion to intact endothelium is mediated via platelet integrin alphaIIbeta3.

  20. 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

  1. Tumour exosome integrins determine organotropic metastasis

    SciTech Connect

    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.; Sandstrom, Per; Jørgen Labori, Knut; 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

    2015-10-28

    Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. In this paper, 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. In conclusion, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

  2. Tumour exosome integrins determine organotropic metastasis

    DOE PAGES

    Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; ...

    2015-10-28

    Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. In this paper, 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,more » 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. In conclusion, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.« less

  3. 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.

  4. Altered vascular endothelium integrin expression in psoriasis.

    PubMed Central

    Creamer, D.; Allen, M.; Sousa, A.; Poston, R.; Barker, J.

    1995-01-01

    Considerable evidence indicates that microvascular changes observed in psoriasis are a result of vascular proliferation. A critical step in the sequence of events leading to neovascularization involves interactions between endothelial cells and extracellular matrix proteins mediated in part by the integrin family of adhesion molecules. A number of endothelial integrins have been shown to participate in neovascularization, including members of the beta 1, beta 3, and beta 4 subfamilies. To investigate the role of these integrins in psoriasis, specimens of lesional and nonlesional skin were taken from 10 patients with active, untreated plaque disease. Vascular endothelium was labeled with monoclonal antibodies specific for alpha 2, alpha 5, alpha 6, beta 1, av beta 3, and beta 4 integrins. The use of image analysis permitted quantification of immunoperoxidase staining and comparison of endothelial labeling in lesional and nonlesional skin. There was a significant increase in endothelial staining of av beta 3 integrin in lesional compared with nonlesional skin, both in superficial and deep vasculature. In contrast, there was a significant decrease in endothelial beta 4 staining in lesional compared with nonlesional superficial dermal vessels, alpha 2, alpha 5, alpha 6, and beta 1 staining showed no significant difference between the two groups. These results demonstrate an important role of av beta 3 and beta 4 integrins in the microvascular changes of psoriatic lesions. Images Figure 1 Figure 3 PMID:7495291

  5. Structure and mechanics of integrin-based cell adhesion

    PubMed Central

    Goodman, Simon; Xiong, Jian-Ping

    2007-01-01

    Summary of Recent Advances Integrins are α/β heterodimeric adhesion glycoprotein receptors that regulate a wide variety of dynamic cellular processes such as cell migration, phagocytosis and growth and development. X-ray crystallography of the integrin ectodomain revealed its modular architecture and defined its metal-dependent interaction with extracellular ligands. This interaction is regulated from inside the cell (inside-out activation), through the short cytoplasmic α and β integrin tails, which also mediate biochemical and mechanical signals transmitted to the cytoskeleton by the ligand-occupied integrins, which effect major changes in cell shape, behavior and fate. Recent advances in the structural elucidation of integrins and integrin binding cytoskeleton proteins are the subject of this review. PMID:17928215

  6. 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

  7. Osteopontin Undergoes Polymerization in Vivo and Gains Chemotactic Activity for Neutrophils Mediated by Integrin α9β1*

    PubMed Central

    Nishimichi, Norihisa; Hayashita-Kinoh, Hiromi; Chen, Chun; Matsuda, Haruo; Sheppard, Dean; Yokosaki, Yasuyuki

    2011-01-01

    Osteopontin (OPN) is an integrin-binding inflammatory cytokine that undergoes polymerization catalyzed by transglutaminase 2. We have previously reported that polymeric OPN (polyOPN), but not unpolymerized OPN (OPN*), attracts neutrophils in vitro by presenting an acquired binding site for integrin α9β1. Among many in vitro substrates for transglutaminase 2, only a few have evidence for in vivo polymerization and concomitant function. Although polyOPN has been identified in bone and aorta, the in vivo functional significance of polyOPN is unknown. To determine whether OPN polymerization contributes to neutrophil recruitment in vivo, we injected OPN* into the peritoneal space of mice. Polymeric OPN was detected by immunoblotting in the peritoneal wash of mice injected with OPN*, and both intraperitoneal and plasma OPN* levels were higher in mice injected with a polymerization-incompetent mutant, confirming that OPN* polymerizes in vivo. OPN* injection induced neutrophil accumulation, which was significantly less following injection of a mutant OPN that was incapable of polymerization. The importance of in vivo polymerization was further confirmed with cystamine, a transglutaminase inhibitor, which blocked the polymerization and attenuated OPN*-mediated neutrophil recruitment. The thrombin-cleaved N-terminal fragment of OPN, another ligand for α9β1, was not responsible for neutrophil accumulation because a thrombin cleavage-incompetent mutant recruited similar numbers of neutrophils as wild type OPN*. Neutrophil accumulation in response to both wild type and thrombin cleavage-incompetent OPN* was reduced in mice lacking the integrin α9 subunit in leukocytes, indicating that α9β1 is required for polymerization-induced recruitment. We have illustrated a physiological role of molecular polymerization by demonstrating acquired chemotactic properties for OPN. PMID:21321126

  8. Integrin extension enables ultrasensitive regulation by cytoskeletal force.

    PubMed

    Li, Jing; Springer, Timothy A

    2017-05-02

    Integrins undergo large-scale conformational changes upon activation. Signaling events driving integrin activation have previously been discussed conceptually, but not quantitatively. Here, recent measurements of the intrinsic ligand-binding affinity and free energy of each integrin conformational state on the cell surface, together with the length scales of conformational change, are used to quantitatively compare models of activation. We examine whether binding of cytoskeletal adaptors to integrin cytoplasmic domains is sufficient for activation or whether exertion of tensile force by the actin cytoskeleton across the integrin-ligand complex is also required. We find that only the combination of adaptor binding and cytoskeletal force provides ultrasensitive regulation. Moreover, switch-like activation by force depends on the large, >130 Å length-scale change in integrin extension, which is well tailored to match the free-energy difference between the inactive (bent-closed) and active (extended-open) conformations. The length scale and energy cost in integrin extension enable activation by force in the low pN range and appear to be the key specializations that enable cell adhesion through integrins to be coordinated with cytoskeletal dynamics.

  9. β1 integrin

    PubMed Central

    Howe, Grant A.; Addison, Christina L.

    2012-01-01

    Historically, a hallmark of tumorigenesis was the ability to grow in an anchorage-independent manner. Hence, tumors were thought to proliferate and survive independently of integrin attachment to the substratum. However, recent data suggest that integrins regulate not only tumor cell proliferation, survival and migration, but may also influence their response to anti-cancer agents. Interestingly, these influences are largely masked by growth of tumor cells in the standard, yet artificial, environment of 2D cell culture, but are readily apparent under 3D in vitro culture conditions and in tumor growth in vivo. We, and others, have recently demonstrated that the β1 integrin subunit controls the growth and invasion of prostate tumor cells in 3D culture conditions. Recently, the importance of integrins has also been demonstrated using tissue specific conditional knockout strategies in transgenic mouse tumor models, where they control primary tumor growth and dictate the site of metastatic spread. Furthermore, integrin-extracellular matrix interactions may modulate the response of tumors to standard chemotherapy agents or radiation. Taken together, these results highlight the important role of integrins in regulating tumor growth and metastasis; however, point out that the evaluation of their contribution to these processes requires appropriate contextual modeling. PMID:22568952

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

    PubMed

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

    2014-11-03

    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. © 2014 The Authors.

  11. The progenitor cell marker NG2/MPG promotes chemoresistance by activation of integrin-dependent PI3K/Akt signaling.

    PubMed

    Chekenya, M; Krakstad, C; Svendsen, A; Netland, I A; Staalesen, V; Tysnes, B B; Selheim, F; Wang, J; Sakariassen, P Ø; Sandal, T; Lønning, P E; Flatmark, T; Enger, P Ø; Bjerkvig, R; Sioud, M; Stallcup, W B

    2008-09-04

    Chemoresistance represents a major problem in the treatment of many malignancies. Overcoming this obstacle will require improved understanding of the mechanisms responsible for this phenomenon. The progenitor cell marker NG2/melanoma proteoglycan (MPG) is aberrantly expressed by various tumors, but its role in cell death signaling and its potential as a therapeutic target are largely unexplored. We have assessed cytotoxic drug-induced cell death in glioblastoma spheroids from 15 patients, as well as in five cancer cell lines that differ with respect to NG2/MPG expression. The tumors were treated with doxorubicin, etoposide, carboplatin, temodal, cisplatin and tumor necrosis factor (TNF)alpha. High NG2/MPG expression correlated with multidrug resistance mediated by increased activation of alpha3beta1 integrin/PI3K signaling and their downstream targets, promoting cell survival. NG2/MPG knockdown with shRNAs incorporated into lentiviral vectors attenuated beta1 integrin signaling revealing potent antitumor effects and further sensitized neoplastic cells to cytotoxic treatment in vitro and in vivo. Thus, as a novel regulator of the antiapoptotic response, NG2/MPG may represent an effective therapeutic target in several cancer subtypes.

  12. The progenitor cell marker NG2/MPG promotes chemoresistance by activation of integrin-dependent PI3K/Akt signalling

    PubMed Central

    Chekenya, M; Krakstad, C; Svendsen, A; Netland, IA; Staalesen, V; Tysnes, B.B; Selheim, F; Wang, J; Sakariassen, P.Ø; Sandal, T; Lønning, P.E; Flatmark, T; Enger, P.Ø; Bjerkvig, R; Sioud, M; Stallcup, WB

    2009-01-01

    Chemoresistance represents a major problem in the treatment of many malignancies. Overcoming this obstacle will require improved understanding of the mechanisms responsible for the phenomenon. The progenitor cell marker NG2/MPG is aberrantly expressed by various tumors, but its role in cell death signalling and its potential as a therapeutic target is largely unexplored. We have assessed cytotoxic drug-induced cell death in glioblastoma (GBM) spheroids from fifteen patients, as well as in five cancer cell lines that differ with respect to NG2/MPG expression. The tumors were treated with doxorubicin, etoposide, carboplatin, temodal, cisplatin and TNFα. High NG2/MPG expression correlated with multi-drug resistance mediated by increased activation of α3β1 integrin/PI3K signalling and their downstream targets, promoting cell survival. NG2/MPG knockdown with shRNAs incorporated into lentiviral vectors attenuated β1 integrin signalling revealing potent anti-tumor effects and further sensitized neoplastic cells to cytotoxic treatment in vitro and in vivo. Thus, as a novel regulator of the anti-apoptotic response, NG2/MPG may represent an effective therapeutic target in several cancer subtypes. PMID:18469852

  13. CXCR6-CXCL16 axis promotes prostate cancer by mediating cytoskeleton rearrangement via Ezrin activation and αvβ3 integrin clustering

    PubMed Central

    Singh, Rajesh; Kapur, Neeraj; Mir, Hina; Singh, Nalinaksha; Lillard, James W.; Singh, Shailesh

    2016-01-01

    Cytoskeletal rearrangement is required for migration and invasion, which are the key steps of cancer metastasis. Ezrin and integrin co-ordinate these processes by regulating cellular adhesion and cytoskeletal polymerization-depolymerization. It is also well established that chemokine-chemokine receptor axis plays a crucial role in regulating cancer cell migration and invasion. In this study, we show involvement of CXC chemokine receptor 6 (CXCR6) and its only natural ligand CXCL16 in pathobiology of prostate cancer (PCa). CXCR6 is highly expressed in PCa tissues and cell lines (LNCaP and PC3), relative to normal tissue and cells. CXCR6 expression in PCa tissues correlated with higher Gleason score. Similarly, aggressive PCa cells (PC3) show high CXCR6 compared to less aggressive LNCaP. Besides, PC3 cells show higher MMPs expression compared to LNCaP cells following CXCL16 stimulation. Intriguingly, CXCR6-CXCL16 interaction in PCa cells promotes Ezrin activation, αvβ3 integrin clustering and capping at the leading edge in FAK/PI3K/PKC dependent manner, thereby modifying cellular adhesion as well as motility. Together these results demonstrate that CXCL16 stimulation changes cytoskeletal dynamics resulting in enhanced migration, invasion and adhesion to endothelial cells, ultimately enabling PCa cells to achieve their metastatic goal. PMID:26799186

  14. Ablation of beta1 integrin in mammary epithelium reveals a key role for integrin in glandular morphogenesis and differentiation.

    PubMed

    Naylor, Matthew J; Li, Na; Cheung, Julia; Lowe, Emma T; Lambert, Elise; Marlow, Rebecca; Wang, Pengbo; Schatzmann, Franziska; Wintermantel, Timothy; Schüetz, Günther; Clarke, Alan R; Mueller, Ulrich; Hynes, Nancy E; Streuli, Charles H

    2005-11-21

    Integrin-mediated adhesion regulates the development and function of a range of tissues; however, little is known about its role in glandular epithelium. To assess the contribution of beta1 integrin, we conditionally deleted its gene in luminal epithelia during different stages of mouse mammary gland development and in cultured primary mammary epithelia. Loss of beta1 integrin in vivo resulted in impaired alveologenesis and lactation. Cultured beta1 integrin-null cells displayed abnormal focal adhesion function and signal transduction and could not form or maintain polarized acini. In vivo, epithelial cells became detached from the extracellular matrix but remained associated with each other and did not undergo overt apoptosis. beta1 integrin-null mammary epithelial cells did not differentiate in response to prolactin stimulation because of defective Stat5 activation. In mice where beta1 integrin was deleted after the initiation of differentiation, fewer defects in alveolar morphology occurred, yet major deficiencies were also observed in milk protein and milk fat production and Stat5 activation, indicating a permissive role for beta1 integrins in prolactin signaling. This study demonstrates that beta1 integrin is critical for the alveolar morphogenesis of a glandular epithelium and for maintenance of its differentiated function. Moreover, it provides genetic evidence for the cooperation between integrin and cytokine signaling pathways.

  15. Unexpected diversity of cnidarian integrins: expression during coral gastrulation

    PubMed Central

    2008-01-01

    Background Adhesion mediated through the integrin family of cell surface receptors is central to early development throughout the Metazoa, playing key roles in cell-extra cellular matrix adhesion and modulation of cadherin activity during the convergence and extension movements of gastrulation. It has been suggested that Caenorhabditis elegans, which has a single β and two α integrins, might reflect the ancestral integrin complement. Investigation of the integrin repertoire of anthozoan cnidarians such as the coral Acropora millepora is required to test this hypothesis and may provide insights into the original roles of these molecules. Results Two novel integrins were identified in Acropora. AmItgα1 shows features characteristic of α integrins lacking an I-domain, but phylogenetic analysis gives no clear indication of its likely binding specificity. AmItgβ2 lacks consensus cysteine residues at positions 8 and 9, but is otherwise a typical β integrin. In situ hybridization revealed that AmItgα1, AmItgβ1, and AmItgβ2 are expressed in the presumptive endoderm during gastrulation. A second anthozoan, the sea anemone Nematostella vectensis, has at least four β integrins, two resembling AmItgβ1 and two like AmItgβ2, and at least three α integrins, based on its genomic sequence. Conclusion In two respects, the cnidarian data do not fit expectations. First, the cnidarian integrin repertoire is more complex than predicted: at least two βs in Acropora, and at least three αs and four βs in Nematostella. Second, whereas the bilaterian αs resolve into well-supported groups corresponding to those specific for RGD-containing or laminin-type ligands, the known cnidarian αs are distinct from these. During early development in Acropora, the expression patterns of the three known integrins parallel those of amphibian and echinoderm integrins. PMID:18466626

  16. Combination therapy targeting integrins reduces glioblastoma tumor growth through antiangiogenic and direct antitumor activity and leads to activation of the pro-proliferative prolactin pathway

    PubMed Central

    2013-01-01

    Background Tumors may develop resistance to specific angiogenic inhibitors via activation of alternative pathways. Therefore, multiple angiogenic pathways should be targeted to achieve significant angiogenic blockade. In this study we investigated the effects of a combined application of the angiogenic inhibitors endostatin and tumstatin in a model of human glioblastoma multiforme. Results Inhibitors released by stably transfected porcine aortic endothelial cells (PAE) showed anti-angiogenic activity in proliferation and wound-healing assays with endothelial cells (EC). Interestingly, combination of endostatin and tumstatin (ES + Tum) also reduced proliferation of glioma cells and additionally induced morphological changes and apoptosis in vitro. Microencapsulated PAE-cells producing these inhibitors were applied for local therapy in a subcutaneous glioblastoma model. When endostatin or tumstatin were applied separately, in vivo tumor growth was inhibited by 58% and 50%, respectively. Combined application of ES + Tum, in comparison, resulted in a significantly more pronounced inhibition of tumor growth (83%). cDNA microarrays of tumors treated with ES + Tum revealed an up-regulation of prolactin receptor (PRLR). ES + Tum-induced up-regulation of PRLR in glioma cells was also found in in vitro. Moreover, exogenous PRLR overexpression in vitro led to up-regulation of its ligand prolactin and increased proliferation suggesting a functional autocrine growth loop in these cells. Conclusion Our data indicate that integrin-targeting factors endostatin and tumstatin act additively by inhibiting glioblastoma growth via reduction of vessel density but also directly by affecting proliferation and viability of tumor cells. Treatment with the ES + Tum-combination activates the PRLR pro-proliferative pathway in glioblastoma. Future work will show whether the prolactin signaling pathway represents an additional target to improve therapeutic strategies in this

  17. 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.

  18. The interaction between caveolin-1 and Rho-GTPases promotes metastasis by controlling the expression of alpha5-integrin and the activation of Src, Ras and Erk.

    PubMed

    Arpaia, E; Blaser, H; Quintela-Fandino, M; Duncan, G; Leong, H S; Ablack, A; Nambiar, S C; Lind, E F; Silvester, J; Fleming, C K; Rufini, A; Tusche, M W; Brüstle, A; Ohashi, P S; Lewis, J D; Mak, T W

    2012-02-16

    Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130(Cas), which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1-Rho-GTPase interaction in metastasis, we disrupted Cav1-Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130(Cas) phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130(Cas)/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades.

  19. The Talin Head Domain Reinforces Integrin-Mediated Adhesion by Promoting Adhesion Complex Stability and Clustering

    PubMed Central

    Ellis, Stephanie J.; Lostchuck, Emily; Goult, Benjamin T.; Bouaouina, Mohamed; Fairchild, Michael J.; López-Ceballos, Pablo; Calderwood, David A.; Tanentzapf, Guy

    2014-01-01

    Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development. PMID:25393120

  20. 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

  1. Integrin signaling is critical for pathological angiogenesis

    PubMed Central

    Mahabeleshwar, Ganapati H.; Feng, Weiyi; Phillips, David R.; Byzova, Tatiana V.

    2006-01-01

    The process of postnatal angiogenesis plays a crucial role in pathogenesis of numerous diseases, including but not limited to tumor growth/metastasis, diabetic retinopathy, and in tissue remodeling upon injury. However, the molecular events underlying this complex process are not well understood and numerous issues remain controversial, including the regulatory function of integrin receptors. To analyze the role of integrin phosphorylation and signaling in angiogenesis, we generated knock-in mice that express a mutant β3 integrin unable to undergo tyrosine phosphorylation. Two distinct models of pathological angiogenesis revealed that neovascularization is impaired in mutant β3 knock-in mice. In an ex vivo angiogenesis assay, mutant β3 knock-in endothelial cells did not form complete capillaries in response to vascular endothelial growth factor (VEGF) stimulation. At the cellular level, defective tyrosine phosphorylation in mutant β3 knock-in cells resulted in impaired adhesion, spreading, and migration of endothelial cells. At the molecular level, VEGF stimulated complex formation between VEGF receptor-2 and β3 integrin in wild-type but not in mutant β3 knock-in endothelial cells. Moreover, phosphorylation of VEGF receptor-2 was significantly reduced in cells expressing mutant β3 compared to wild type, leading to impaired integrin activation in these cells. These findings provide novel mechanistic insights into the role of integrin–VEGF axis in pathological angiogenesis. PMID:17030947

  2. 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.

  3. Integrin inhibition promotes atypical anoikis in glioma cells

    PubMed Central

    Silginer, M; Weller, M; Ziegler, U; Roth, P

    2014-01-01

    Integrins regulate cellular adhesion and transmit signals important for cell survival, proliferation and motility. They are expressed by glioma cells and may contribute to their malignant phenotype. Integrin inhibition may therefore represent a promising therapeutic strategy. GL-261 and SMA-560 glioma cells grown under standard conditions uniformly detached and formed large cell clusters after integrin gene silencing or pharmacological inhibition using EMD-121974, a synthetic Arg-Gly-Asp-motif peptide, or GLPG0187, a nonpeptidic integrin inhibitor. After 120 h, the clusters induced by integrin inhibition decayed and cells died. In contrast, when cells were cultured under stem cell (sphere) conditions, no disaggregation became apparent upon integrin inhibition, and cell death was not observed. As poly-HEMA-mediated detachment had similar effects on cell viability as integrin inhibition, we postulated that cell death may result from detachment alone, which was confirmed using various permissive and nonpermissive substrates. No surrogate markers of apoptosis were detected and electron microscopy confirmed that necrosis represents the dominant morphology of detachment-induced cell death. In addition, integrin inhibition resulted in the induction of autophagy that represents a survival signal. When integrins were inhibited in nonsphere glioma cells, the TGF-β pathway was strongly impaired, whereas no such effect was observed in glioma cells cultured under sphere conditions. Cell death induced by integrin inhibition was rescued by the addition of recombinant transforming growth factor-β (TGF-β) and accelerated by exposure to the TGF-β receptor inhibitor, SD-208. In summary, cell death following integrin inhibition is detachment mediated, represents an atypical form of anoikis involving necrosis as well as autophagy, and is modulated by TGF-β pathway activity. PMID:24457956

  4. Cytoplasmic salt bridge formation in integrin αvß3 stabilizes its inactive state affecting integrin-mediated cell biological effects.

    PubMed

    Müller, Martina A; Brunie, Leonora; Bächer, Anne-Sophie; Kessler, Horst; Gottschalk, Kay-Eberhard; Reuning, Ute

    2014-11-01

    Heterodimeric integrin receptors are mediators of cell adhesion, motility, invasion, proliferation, and survival. By this, they are crucially involved in (tumor) cell biological behavior. Integrins trigger signals bidirectionally across cell membranes: by outside-in, following binding of protein ligands of the extracellular matrix, and by inside-out, where proteins are recruited to ß-integrin cytoplasmic tails resulting in conformational changes leading to increased integrin binding affinity and integrin activation. Computational modeling and experimental/mutational approaches imply that associations of integrin transmembrane domains stabilize the low-affinity integrin state. Moreover, a cytoplasmic interchain salt bridge is discussed to contribute to a tight clasp of the α/ß-membrane-proximal regions; however, its existence and physiological relevance for integrin activation are still a controversial issue. In order to further elucidate the functional role of salt bridge formation, we designed mutants of the tumor biologically relevant integrin αvß3 by mutually exchanging the salt bridge forming amino acid residues on each chain (αvR995D and ß3D723R). Following transfection of human ovarian cancer cells with different combinations of wild type and mutated integrin chains, we showed that loss of salt bridge formation strengthened αvß3-mediated adhesion to vitronectin, provoked recruitment of cytoskeletal proteins, such as talin, and induced integrin signaling, ultimately resulting in enhanced cell migration, proliferation, and activation of integrin-related signaling molecules. These data support the notion of a functional relevance of integrin cytoplasmic salt bridge disruption during integrin activation. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Monocyte migration to arthritis in the rat utilizes both CD11/CD18 and very late activation antigen 4 integrin mechanisms

    PubMed Central

    1995-01-01

    In human and experimental models of arthritis, blood monocytes migrate into the inflamed synovium and joint space. The mechanisms required for monocyte migration across the vascular endothelium in joints is poorly defined. Radiolabeled rat blood monocytes were used to measure monocyte migration to the inflamed joints of rats with adjuvant arthritis, and the role of monocyte adhesion molecules was analyzed. Monocyte accumulation in the inflamed joints was maximal 14-21 d after immunization with adjuvant, when arthritis had fully developed. Blocking mAbs to lymphocyte function-associated antigen 1 (LFA-1), Mac- 1, and very late activation antigen 4 (VLA-4) were used to evaluate the role of these integrins in the migration. Migration to the joints was not inhibited by treatment of the animals with mAb to LFA-1, Mac-1, or VLA-4 alone, and was partially (50%) inhibited in only the most arthritic joint, the talar joint, by the combination of mAb to LFA-1 plus Mac-1. In contrast, this combination inhibited migration to dermal inflammation induced by C5ades Arg, endotoxin, tumor necrosis factor alpha, and polyinosine-cytosine by 60-70%. When mAbs to LFA-1 and VLA-4 were combined, migration to all the inflamed joints was strongly inhibited (80-98%, depending on the joint). Treatment with the combination of the three mAbs to LFA-1, Mac-1, and VLA-4 completely eliminated monocyte migration to all joints and dermal inflammation. The results show that 51Cr blood monocytes can be used to quantify monocyte migration to arthritic joints in the rat. LFA-1 alone or VLA-4 alone is sufficient to mediate most of this migration, and either LFA-1 or VLA-4 is required for monocyte migration to joint inflammation. These results indicate that both the VLA-4 and LFA-1 integrins should be therapeutic targets for suppression of monocyte infiltration of joints in arthritis. PMID:7532681

  6. Integrins as Therapeutic Targets: Successes and Cancers

    PubMed Central

    Raab-Westphal, Sabine; Marshall, John F.; Goodman, Simon L.

    2017-01-01

    Integrins are transmembrane receptors that are central to the biology of many human pathologies. Classically mediating cell-extracellular matrix and cell-cell interaction, and with an emerging role as local activators of TGFβ, they influence cancer, fibrosis, thrombosis and inflammation. Their ligand binding and some regulatory sites are extracellular and sensitive to pharmacological intervention, as proven by the clinical success of seven drugs targeting them. The six drugs on the market in 2016 generated revenues of some US$3.5 billion, mainly from inhibitors of α4-series integrins. In this review we examine the current developments in integrin therapeutics, especially in cancer, and comment on the health economic implications of these developments. PMID:28832494

  7. Real-time analysis of integrin-dependent transendothelial migration and integrin-independent interstitial motility of leukocytes.

    PubMed

    Shulman, Ziv; Alon, Ronen

    2012-01-01

    The role of integrins in leukocyte migration across endothelial barriers is widely accepted. In contrast, the contribution of integrins to interstitial motility of leukocytes is still elusive. Chemokine binding to G-protein-coupled receptors expressed on the surface of leukocytes plays key roles in both of these processes by directly activating integrin conformations favorable for ligand binding and integrin microclustering. Chemokines can also serve as weak adhesive ligands and potent inducers of actin cytoskeleton remodeling. Real-time assays utilizing live imaging microscopy have been implemented to dissect these versatile roles of chemokines in different leukocyte migration processes. Here, we review several in vitro assays useful for exploring the contribution of chemokine signals and shear forces to integrin activation and function during various stages of leukocyte transendothelial migration. In addition, we describe a new assay that assesses the contribution of chemokines to integrin-independent interstitial leukocyte motility. These assays can also follow the outcome of specific genetic or biochemical manipulations of either the leukocyte or the endothelial barrier on distinct migratory steps. Following fixation, subcellular changes in the distribution of integrin subsets and of specific integrin-associated adaptors can be further dissected by immunofluorescence tools and by ultrastructural electron microscopic analysis.

  8. Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function.

    PubMed

    Serini, Guido; Valdembri, Donatella; Zanivan, Sara; Morterra, Giulia; Burkhardt, Constanze; Caccavari, Francesca; Zammataro, Luca; Primo, Luca; Tamagnone, Luca; Logan, Malcolm; Tessier-Lavigne, Marc; Taniguchi, Masahiko; Püschel, Andreas W; Bussolino, Federico

    2003-07-24

    The motility and morphogenesis of endothelial cells is controlled by spatio-temporally regulated activation of integrin adhesion receptors, and integrin activation is stimulated by major determinants of vascular remodelling. In order for endothelial cells to be responsive to changes in activator gradients, the adhesiveness of these cells to the extracellular matrix must be dynamic, and negative regulators of integrins could be required. Here we show that during vascular development and experimental angiogenesis, endothelial cells generate autocrine chemorepulsive signals of class 3 semaphorins (SEMA3 proteins) that localize at nascent adhesive sites in spreading endothelial cells. Disrupting endogenous SEMA3 function in endothelial cells stimulates integrin-mediated adhesion and migration to extracellular matrices, whereas exogenous SEMA3 proteins antagonize integrin activation. Misexpression of dominant negative SEMA3 receptors in chick embryo endothelial cells locks integrins in an active conformation, and severely impairs vascular remodelling. Sema3a null mice show vascular defects as well. Thus during angiogenesis endothelial SEMA3 proteins endow the vascular system with the plasticity required for its reshaping by controlling integrin function.

  9. Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion.

    PubMed

    Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Gentilini, María Virginia; Velásquez, Lis Noelia; Fossati, Carlos Alberto; Giambartolomei, Guillermo Hernán; Delpino, María Victoria

    2015-10-12

    Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-β, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. 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

  11. 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

  12. The NPIY motif in the integrin beta1 tail dictates the requirement for talin-1 in outside-in signaling.

    PubMed

    Nieves, Bethsaida; Jones, Christopher W; Ward, Rachel; Ohta, Yasutaka; Reverte, Carlos G; LaFlamme, Susan E

    2010-04-15

    Protein interactions with the integrin beta-subunit cytoplasmic domain (beta-tail) are essential for adhesion-dependent processes, including cell spreading and the connection of integrins with actin filaments at adhesion sites. Talin-1 binds to the conserved membrane-proximal NPxY motif of beta-tails (NPIY in beta1 integrin) promoting the inside-out activation of integrins and providing a linkage between integrins and the actin cytoskeleton. Here, we characterize the role of interactions between talin-1 and beta-tail downstream of integrin activation, in the context of recombinant integrins containing either the wild type (WT) or the (YA) mutant beta1A tail, with a tyrosine to alanine substitution in the NPIY motif. In addition to inhibiting integrin activation, the YA mutation suppresses cell spreading, integrin signaling, focal adhesion and stress-fiber formation, as well as microtubule assembly. Constitutive activation of the mutant integrin restores these integrin-dependent processes, bringing into question the importance of the NPIY motif downstream of integrin activation. Depletion of talin-1 using TLN1 siRNA demonstrated that talin-1 is required for cell spreading, focal adhesion and stress-fiber formation, as well as microtubule assembly, even when cells are adhered by constitutively activated WT integrins. Depletion of talin-1 does not inhibit these processes when cells are adhered by constitutively activated mutant integrins, suggesting that the binding of an inhibitory protein to the NPIY motif negatively regulates integrin function when talin-1 is depleted. We identified filamin A (FLNa) as this inhibitory protein; it binds to the beta1A tail in an NPIY-dependent manner and inhibition of FLNa expression in talin-1-depleted cells restores integrin function when cells are adhered by constitutively activated WT integrins. FLNa binds FilGAP, which is a negative regulator of Rac activation. Expression of the dominant inhibitory mutant, Fil

  13. Aberrant mucosal mast cell protease expression in the enteric epithelium of nematode-infected mice lacking the integrin alphavbeta6, a transforming growth factor-beta1 activator.

    PubMed

    Knight, Pamela A; Brown, Jeremy K; Wright, Steven H; Thornton, Elisabeth M; Pate, Judith A; Miller, Hugh R P

    2007-10-01

    Infection of mice with the nematode Trichinella spiralis triggers recruitment and differentiation of intraepithelial intestinal mucosal mast cells expressing mouse mast cell protease 1 (Mcpt-1), which contributes to expulsion of the parasite. Expression of Mcpt-1 is transforming growth factor (TGF)-beta1-dependent in vitro. TGF-beta1, which is secreted within tissues as a biologically inactive complex with latency-associated peptide, requires extracellular modification to become functionally active. The integrin-alpha(nu)beta(6) mediates local activation of TGF-beta(1) in association with epithelia. Using T. spiralis-infected beta(6)(-/-) mice, we show accumulation of mucosal mast cells in the lamina propria of the small intestine with minimal recruitment into the epithelial compartment. This was accompanied by a coordinate reduction in expression of both Mcpt-1 and -2 in the jejunum and increased tryptase expression, whereas Mcpt-9 became completely undetectable. In contrast, the cytokine stem cell factor, a regulator of mast cell differentiation and survival, was significantly up-regulated in T. spiralis-infected beta(6)(-/-) mice compared with infected beta(6)(+/+) controls. Despite these changes, beta(6)(-/-) mice still appeared to expel the worms normally. We postulate that compromised TGF-beta(1) activation within the gastrointestinal epithelial compartment is a major, but not the only, contributing factor to the observed changes in mucosal mast cell protease and epithelial cytokine expression in beta(6)(-/-) mice.

  14. 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.

  15. Integrin Dynamics Produce a Delayed Stage of Long-Term Potentiation and Memory Consolidation

    PubMed Central

    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.

    2012-01-01

    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

  16. Modulation of Wnt3a-mediated nuclear beta-catenin accumulation and activation by integrin-linked kinase in mammalian cells.

    PubMed

    Oloumi, A; Syam, S; Dedhar, S

    2006-12-14

    The Wnt gene family encodes secreted signaling molecules that play important roles in tumorgenesis and embryogenesis. The canonical Wnt signaling pathway regulates target gene expression via the stabilization and nuclear translocation of the cytoplasmic pool of beta-catenin. The activation of integrin-linked kinase (ILK) is also known to regulate the stabilization and subsequent nuclear translocation of beta-catenin in several epithelial cell models. We now report that molecular and pharmacological inhibition of ILK activity in mammalian cells directly modulates Wnt signaling by suppressing the stabilization and nuclear translocation of beta-catenin, as well as beta-catenin/Lef-mediated transcription. Inhibition of ILK activity, but not phosphatidylinositol-3 kinase (PI3K) or MEK activities suppresses nuclear beta-catenin stabilization in cells stably expressing Wnt3a as well as in cells exposed to either Wnt3a conditioned media or purified Wnt3a. Furthermore, ILK inhibition reverses the Wnt3a-induced suppression of beta-catenin phosphorylation that accompanies beta-catenin stabilization. In addition, we show that ILK can be identified in a complex with Wnt pathway components such as adenomatous polyposis coli and GSK-3. Upon treatment of L cells with Wnt3a-CM, glycogen synthase kinase-3 (GSK-3beta) becomes highly phosphorylated on Ser 9, which is completely abolished upon inhibition of ILK activity. However, acute exposure of L cells to purified Wnt3a does not result in the stimulation of GSK-3beta Ser 9 phosphorylation, despite beta-catenin stabilization. Together our data demonstrate that ILK activity can modulate acute Wnt3a mediated beta-catenin phosphorylation, stabilization and nuclear activation in a PI3K-independent manner, as well as the more prolonged PI3K-dependent secondary effects of Wnt signaling on GSK-3 phosphorylation. Finally, we suggest that a novel small molecule inhibitor of ILK, QLT-0267, may be a useful tool in the regulation of pathological

  17. Silencing of VAMP3 inhibits cell migration and integrin-mediated adhesion

    SciTech Connect

    Luftman, Kevin; Hasan, Nazarul; Day, Paul; Hardee, Deborah; Hu Chuan

    2009-02-27

    Integrins are transmembrane receptors for cell adhesion to the extracellular matrix. In cell migration, integrins are endocytosed from the plasma membrane or the cell surface, transported in vesicles and exocytosed actively at the cell front. In the present study, we examined the roles of VAMP3, a SNARE protein that mediates exocytosis, in cell migration and integrin trafficking. Small interfering RNA (siRNA)-induced silencing of VAMP3 inhibited chemotactic cell migration by more than 60% without affecting cell proliferation. VAMP3 silencing reduced the levels of {beta}1 integrin at the cell surface but had no effect on total cellular {beta}1 integrin, indicating that VAMP3 is required for trafficking of {beta}1 integrin to the plasma membrane. Furthermore, VAMP3 silencing diminished cell adhesion to laminin but not to fibronectin or collagen. Taken together, these data suggest that VAMP3-dependent integrin trafficking is crucial in cell migration and cell adhesion to laminin.

  18. Integrin alpha 2 beta 1-independent activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for alpha 2 beta 1-independent platelet reactivity.

    PubMed Central

    Morton, L F; Hargreaves, P G; Farndale, R W; Young, R D; Barnes, M J

    1995-01-01

    The platelet reactivities of two simple collagen-like synthetic peptides, Gly-Lys-Hyp-(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly and Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly, were investigated. Both peptides adopted a stable triple-helical conformation in solution. Following cross-linking, both peptides proved to be highly platelet-aggregatory, more active than collagen fibres, inducing aggregation at concentrations as low as 20 ng/ml. These peptides formed microaggregates in solution, and cross-linking was thought to stabilize these structures, allowing expression of their platelet reactivity at 37 degrees C. Like collagen fibres, the peptides caused platelet secretion and release of arachidonate from platelet membrane lipids as well as activation of integrin alpha IIb beta 3 culminating in aggregation. Monoclonal antibodies directed against the integrin alpha 2 beta 1 failed to prevent aggregation release of arachidonate or platelet adhesion to the peptides. Our results indicate that collagen can activate platelets by a mechanism that is independent of integrin alpha 2 beta 1 and for which collagen tertiary and quaternary structures are sufficient alone for activity without the involvement of highly specific cell-recognition sequences. Images Figure 5 PMID:7534064

  19. Lysophosphatidic Acid Increases Proximal Tubule Cell Secretion of Profibrotic Cytokines PDGF-B and CTGF through LPA2- and Gαq-Mediated Rho and αvβ6 Integrin-Dependent Activation of TGF-β

    PubMed Central

    Geng, Hui; Lan, Rongpei; Singha, Prajjal K.; Gilchrist, Annette; Weinreb, Paul H.; Violette, Shelia M.; Weinberg, Joel M.; Saikumar, Pothana; Venkatachalam, Manjeri A.

    2013-01-01

    After ischemia-reperfusion injury (IRI), kidney tubules show activated transforming growth factor β (TGF-β) signaling and increased expression of profibrotic peptides, platelet-derived growth factor-B (PDGF-B) and connective tissue growth factor (CTGF). If tubule repair after IRI is incomplete, sustained paracrine activity of these peptides can activate interstitial fibroblast progenitors and cause fibrosis. We show that lysophosphatidic acid (LPA), a ubiquitous phospholipid that is increased at sites of injury and inflammation, signals through LPA2 receptors and Gαq proteins of cultured proximal tubule cells to transactivate latent TGF-β in a Rho/Rho-kinase and αvβ6 integrin-dependent manner. Active TGF-β peptide then initiates signaling to increase the production and secretion of PDGF-B and CTGF. In a rat model of IRI, increased TGF-β signaling that was initiated early during reperfusion did not subside during recovery, but progressively increased, causing tubulointerstitial fibrosis. This was accompanied by correspondingly increased LPA2 and β6 integrin proteins and elevated tubule expression of TGF-β1, together with PDGF-B and CTGF. Treatment with a pharmacological TGF-β type I receptor antagonist suppressed TGF-β signaling, decreased the expression of β6 integrin, PDGF-B, and CTGF, and ameliorated fibrosis. We suggest that LPA-initiated autocrine signaling is a potentially important mechanism that gives rise to paracrine profibrotic signaling in injured kidney tubule cells. PMID:22885106

  20. Mechanical stimulation of human tendon stem/progenitor cells results in upregulation of matrix proteins, integrins and MMPs, and activation of p38 and ERK1/2 kinases.

    PubMed

    Popov, Cvetan; Burggraf, Martina; Kreja, Ludwika; Ignatius, Anita; Schieker, Matthias; Docheva, Denitsa

    2015-03-13

    Tendons are dense connective tissues subjected periodically to mechanical stress upon which complex responsive mechanisms are activated. These mechanisms affect not only the development of these tissues but also their healing. Despite of the acknowledged importance of the mechanical stress for tendon function and repair, the mechanotransduction mechanisms in tendon cells are still unclear and the elucidation of these mechanisms is a key goal in tendon research. Tendon stem/progenitor cells (TSPC) possess common adult stem cell characteristics, and are suggested to actively participate in tendon development, tissue homeostasis as well as repair. This makes them an important cell population for tendon repair, and also an interesting research target for various open questions in tendon cell biology. Therefore, in our study we focused on TSPC, subjected them to five different mechanical protocols, and investigated the gene expression changes by using semi-quantitative, quantitative PCR and western blotting technologies. Among the 25 different genes analyzed, we can convincingly report that the tendon-related genes - fibromodulin, lumican and versican, the collagen I-binding integrins - α1, α2 and α11, the matrix metalloproteinases - MMP9, 13 and 14 were strongly upregulated in TSPC after 3 days of mechanical stimulation with 8% amplitude. Molecular signaling analyses of five key integrin downstream kinases suggested that mechanical stimuli are mediated through ERK1/2 and p38, which were significantly activated in 8% biaxial-loaded TSPC. Our results demonstrate the positive effect of 8% mechanical loading on the gene expression of matrix proteins, integrins and matrix metalloproteinases, and activation of integrin downstream kinases p38 and ERK1/2 in TSPC. Taken together, our study contributes to better understanding of mechanotransduction mechanisms in TPSC, which in long term, after further translational research between tendon cell biology and orthopedics, can be

  1. Differential Role of β1C and β1A Integrin Cytoplasmic Variants in Modulating Focal Adhesion Kinase, Protein Kinase B/AKT, and Ras/Mitogen-activated Protein Kinase Pathways

    PubMed Central

    Fornaro, Mara; Steger, Craig A.; Bennett, Anton M.; Wu, J. Julie; Languino, Lucia R.

    2000-01-01

    The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The β1 integrin subunits, β1C and β1A, that contain variant cytoplasmic domains differentially affect cell proliferation; β1C inhibits proliferation, whereas β1A promotes it. We investigated the ability of β1C and β1A to modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human β1C or human β1A. The different cytodomains of either β1C or β1A did not affect either association with the endogenous α2, αV, and α5 subunits or cell adhesion to fibronectin or TS2/16, a mAb to human β1. Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing β1C showed significantly inhibited extracellular signal–regulated kinase (ERK) 2 activation compared with β1A stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed β1C by TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, β1A engagement induced activation of both proteins. We show that Ras activation was strongly reduced in β1C stable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued β1C-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in β1C stable cell lines was attributable to an inhibitory effect of β1C on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued β1C antiproliferative effect. These findings show that the β1C variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings

  2. βig-h3 Promotes Human Osteosarcoma Cells Metastasis by Interacting with Integrin α2β1 and Activating PI3K Signaling Pathway

    PubMed Central

    Sun, Zhen; Gao, Bo; He, Shu; Han, Yue-Hu; Fan, Jing; Yang, Liu; Tang, Juan; Luo, Zhuo-Jing

    2014-01-01

    Osteosarcoma, the most common primary bone tumor in children and young adolescents, is characterized by local invasion and distant metastasis. But the detailed mechanisms of osteosarcoma metastasis are not well known. In the present study, we found that βig-h3 promotes metastatic potential of human osteosarcoma cells in vitro and in vivo. Furthermore, βig-h3 co-localized with integrin α2β1 in osteosarcoma cells. But βig-h3 did not change integrin α2β1 expression in Saos-2 cells. Interaction of βig-h3 with integrin α2β1 mediates metastasis of human osteosarcoma cells. The second FAS1 domain of βig-h3 but not the first FAS1 domain, the third FAS1 domain or the fourth FAS1 domain mediates human osteosarcoma cells metastasis, which is the α2β1 integrin-interacting domain. We further demonstrated that PI3K/AKT signaling pathway is involved in βig-h3-induced human osteosarcoma cells metastasis process. Together, these results reveal βig-h3 enhances the metastasis potentials of human osteosarcoma cells via integrin α2β1-mediated PI3K/AKT signal pathways. The discovery of βig-h3-mediated pathway helps us to understand the mechanism of human osteosarcoma metastasis and provides evidence for the possibility that βig-h3 can be a potential therapeutic target for osteosarcoma treatment. PMID:24595049

  3. βig-h3 promotes human osteosarcoma cells metastasis by interacting with integrin α2β1 and activating PI3K signaling pathway.

    PubMed

    Guo, Yun-Shan; Zhao, Rui; Ma, Jie; Cui, Wei; Sun, Zhen; Gao, Bo; He, Shu; Han, Yue-Hu; Fan, Jing; Yang, Liu; Tang, Juan; Luo, Zhuo-Jing

    2014-01-01

    Osteosarcoma, the most common primary bone tumor in children and young adolescents, is characterized by local invasion and distant metastasis. But the detailed mechanisms of osteosarcoma metastasis are not well known. In the present study, we found that βig-h3 promotes metastatic potential of human osteosarcoma cells in vitro and in vivo. Furthermore, βig-h3 co-localized with integrin α2β1 in osteosarcoma cells. But βig-h3 did not change integrin α2β1 expression in Saos-2 cells. Interaction of βig-h3 with integrin α2β1 mediates metastasis of human osteosarcoma cells. The second FAS1 domain of βig-h3 but not the first FAS1 domain, the third FAS1 domain or the fourth FAS1 domain mediates human osteosarcoma cells metastasis, which is the α2β1 integrin-interacting domain. We further demonstrated that PI3K/AKT signaling pathway is involved in βig-h3-induced human osteosarcoma cells metastasis process. Together, these results reveal βig-h3 enhances the metastasis potentials of human osteosarcoma cells via integrin α2β1-mediated PI3K/AKT signal pathways. The discovery of βig-h3-mediated pathway helps us to understand the mechanism of human osteosarcoma metastasis and provides evidence for the possibility that βig-h3 can be a potential therapeutic target for osteosarcoma treatment.

  4. 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

  5. Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry.

    PubMed

    Gianni, Tatiana; Massaro, Raffaele; Campadelli-Fiume, Gabriella

    2015-07-21

    Herpes simplex virus (HSV) is an important human pathogen. It enters cells through an orchestrated process that requires four essential glycoproteins, gD, gH/gL, and gB, activated in cascade fashion by receptor-binding and signaling. gH/gL heterodimer is conserved across the Herpesviridae family. HSV entry is enabled by gH/gL interaction with αvβ6- or αvβ8-integrin receptors. We report that the interaction of virion gH/gL with integrins resulted in gL dissociation and its release in the medium. gL dissociation occurred if all components of the entry apparatus-receptor-bound gD and gB-were present and was prevented if entry was blocked by a neutralizing monoclonal antibody to gH or by a mutation in gH. We propose that (i) gL dissociation from gH/gL is part of the activation of HSV glycoproteins, critical for HSV entry; and (ii) gL is a functional inhibitor of gH and maintains gH in an inhibited form until receptor-bound gD and integrins signal to gH/gL.

  6. Rap1 and integrin inside-out signaling.

    PubMed

    Katagiri, Koko; Kinashi, Tatsuo

    2012-01-01

    In leukocytes, integrins play important roles in adhesive interactions with endothelium, antigen-presenting cells, and effector functions such as cytotoxicity. This chapter describes methods to study Ras proximity 1 (Rap1), a signaling molecule that has been increasingly recognized as an important regulator of integrin-mediated cell adhesion in the immune system as well as hemostasis. Rap1 is activated by a wide variety of external stimuli including chemokines and antigens. Signaling via Rap1 transmits an inside-out signal to the integrins, thereby increasing adhesiveness to ligands such as immunoglobulin superfamily proteins as well as extracellular matrix proteins and plasma proteins. This process induces leukocyte cell adhesion to the endothelium and antigen-presenting cells. In addition to integrin regulation, activated Rap1 induces cell polarity of lymphocytes, which is coordinated with LFA-1 redistribution to the leading edge.

  7. Integrin-mediated adhesion complex

    PubMed Central

    Sebé-Pedrós, Arnau

    2010-01-01

    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. PMID:21057645

  8. Effects of riboflavin and ultraviolet light treatment on platelet thrombus formation on collagen via integrin αIIbβ3 activation.

    PubMed

    Terada, Chikahiro; Mori, Junpei; Okazaki, Hitoshi; Satake, Masahiro; Tadokoro, Kenji

    2014-07-01

    The adoption of pathogen reduction technology (PRT) is considered for the implementation of safer platelet (PLT) transfusion. However, the effects of PRT treatment on PLT thrombus formation under blood flow have not yet been fully clarified. Leukoreduced PLT concentrates (PCs) obtained by plateletpheresis were treated with riboflavin and ultraviolet light (Mirasol PRT). PC samples were passed through a column filled with collagen-coated beads at a fixed shear rate after 1, 3, and 5 days of storage. The thrombus formation ability was evaluated by measuring collagen column retention rate. The change in the activation state of integrin αIIbβ3 on PLTs during storage was examined by flow cytometry. The retention rate of the PRT-treated PLTs was significantly higher than that of the control PLTs on the day of treatment and decreased with storage but remained higher than those of the control during storage. This modification did not correlate with the total αIIbβ3 or fibrinogen binding on the PLTs but correlated significantly with PAC-1 binding. Mn(2+) -induced αIIbβ3 activation also fully restored the retention rate in the Day 5 PRT-treated PLTs along with the increase in PAC-1 binding. Riboflavin-based PRT treatment of PCs leads to the enhancement of thrombus formation on collagen, which is related to the activation status of αIIbβ3, which does not bind to fibrinogen but binds to PAC-1. The impact of this finding on the hemostatic or even thrombogenic potential in vivo must await clinical evaluation. © 2014 AABB.

  9. 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

  10. beta1-integrin cytoplasmic subdomains involved in dominant negative function.

    PubMed

    Retta, S F; Balzac, F; Ferraris, P; Belkin, A M; Fässler, R; Humphries, M J; De Leo, G; Silengo, L; Tarone, G

    1998-04-01

    The beta1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms ("common" region) and a distal subdomain specific for each isoform ("variable" region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used beta1A and beta1B isoforms as well as four mutants lacking the entire cytoplasmic domain (beta1TR), the variable region (beta1COM), or the common region (beta1 deltaCOM-B and beta1 deltaCOM-A). By expressing these constructs in Chinese hamster ovary and beta1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227-238, 1996), we show that beta1B, beta1COM, beta1 deltaCOM-B, and beta1 deltaCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, beta1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that beta1B interferes in a dominant negative manner with beta1A and beta3/beta5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the beta1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the beta1B isoform.

  11. The MIG-2/integrin interaction strengthens cell-matrix adhesion and modulates cell motility.

    PubMed

    Shi, Xiaohua; Ma, Yan-Qing; Tu, Yizeng; Chen, Ka; Wu, Shan; Fukuda, Koichi; Qin, Jun; Plow, Edward F; Wu, Chuanyue

    2007-07-13

    Integrin-mediated cell-matrix adhesion plays an important role in control of cell behavior. We report here that MIG-2, a widely expressed focal adhesion protein, interacts with beta1 and beta3 integrin cytoplasmic domains. Integrin binding is mediated by a single site within the MIG-2 FERM domain. Functionally, the MIG-2/integrin interaction recruits MIG-2 to focal adhesions. Furthermore, using alphaIIbbeta3 integrin-expressing Chinese hamster ovary cells, a well described model system for integrin activation, we show that MIG-2 promotes integrin activation and enhances cell-extracellular matrix adhesion. Although MIG-2 is expressed in many cell types, it is deficient in certain colon cancer cells. Expression of MIG-2, but not of an integrin binding-defective MIG-2 mutant, in MIG-2-null colon cancer cells strengthened cell-matrix adhesion, promoted focal adhesion formation, and reduced cell motility. These results suggest that the MIG-2/integrin interaction is an important element in the cellular control of integrin-mediated cell-matrix adhesion and that loss of this interaction likely contributes to high motility of colon cancer cells.

  12. β3 integrin expression is required for invadopodia-mediated ECM degradation in lung carcinoma cells

    PubMed Central

    Morales, Xabier; Salvo, Elizabeth; Garasa, Saray; Ortiz de Solórzano, Carlos; Martínez, Alfredo; Larrayoz, Ignacio M.; Rouzaut, Ana

    2017-01-01

    Cancer related deaths are primarily due to tumor metastasis. To facilitate their dissemination to distant sites, cancer cells develop invadopodia, actin-rich protrusions capable of degrading the surrounding extracellular matrix (ECM). We aimed to determine whether β3 integrin participates in invadopodia formed by lung carcinoma cells, based on our previous findings of specific TGF-β induction of β3 integrin dependent metastasis in animal models of lung carcinoma. In this study, we demonstrate that lung carcinoma cells form invadopodia in response to TGF-β exposure. Invadopodia formation and degradation activity is dependent on β3 integrin expression since β3 integrin deficient cells are not able to degrade gelatin-coated surfaces. Even more, transient over-expression of SRC did not restore invadopodia formation in β3 integrin deficient cells. Finally, we observed that blockade of PLC-dependent signaling leads to more intense labeling for β3 integrin in invadopodia. Our results suggest that β3 integrin function, and location, in lung cancer cells are essential for invadopodia formation, and this integrin regulates the activation of different signal pathways necessary for the invasive structure. β3 integrin has been associated with poor prognosis and increased metastasis in several carcinoma types, including lung cancer. Our findings provide new evidence to support the use of targeted therapies against this integrin to combat the onset of metastases. PMID:28767724

  13. Pro-metastatic NEDD9 regulates individual cell migration via caveolin-1-dependent trafficking of integrins

    PubMed Central

    Kozyulina, Polina Y.; Loskutov, Yuriy V.; Kozyreva, Varvara K.; Rajulapati, Anuradha; Ice, Ryan J.; Jones, Brandon. C.; Pugacheva, Elena N.

    2014-01-01

    The dissemination of tumor cells relies on efficient cell adhesion and migration, which in turn depends upon endocytic trafficking of integrins. In the current work, it was found that depletion of pro-metastatic protein, NEDD9, in breast cancer (BC) cells results in a significant decrease in individual cell migration due to impaired trafficking of ligand-bound integrins. NEDD9 deficiency does not affect the expression or internalization of integrins but heightens caveolae-dependent trafficking of ligand-bound integrins to early endosomes. Increase in mobility of ligand-bound integrins is concomitant with an increase in tyrosine phosphorylation of caveolin-1 (CAV1) and volume of CAV1-vesicles. NEDD9 directly binds to CAV1 and co-localizes within CAV1 vesicles. In the absence of NEDD9, the trafficking of ligand-bound integrins from early to late endosomes is impaired, resulting in a significant decrease in degradation of ligand/integrin complexes and an increase in recycling of ligand-bound integrins from early endosomes back to the plasma membrane without ligand disengagement, thus leading to low adhesion and migration. Re-expression of NEDD9 or decrease in the amount of active, tyrosine 14 phosphorylated (Tyr14) CAV1 in NEDD9 depleted cells rescues the integrin trafficking deficiency and restores cellular adhesion and migration capacity. Collectively, these findings indicate that NEDD9 orchestrates trafficking of ligand-bound integrins through the attenuation of CAV1 activity. PMID:25319010

  14. Low-Intensity Pulsed Ultrasound Treatment at an Early Osteoarthritis Stage Protects Rabbit Cartilage From Damage via the Integrin/Focal Adhesion Kinase/Mitogen-Activated Protein Kinase Signaling Pathway.

    PubMed

    Xia, Peng; Shen, Shihao; Lin, Qiang; Cheng, Kai; Ren, Shasha; Gao, Mingxia; Li, Xueping

    2015-11-01

    To investigate whether low-intensity pulsed ultrasound (US) has different protective effects on early and late rabbit osteoarthritis cartilage via the integrin/focal adhesion kinase (FAK)/mitogen-activated protein kinase (MAPK) signaling pathway. Thirty-six New Zealand White rabbits were divided into early control, early osteoarthritis, early treatment, late control, late osteoarthritis, and late treatment groups. The early and late osteoarthritis and treatment groups underwent anterior cruciate ligament transection. The remaining groups underwent sham operations with knee joint exposure. The early and late treatment groups were exposed to low-intensity pulsed US 4 and 8 weeks after surgery. After 6 weeks of US exposure, pathologic changes on the articular surface of the femoral condyle were assessed by modified Mankin scores. Expression of type II collagen, matrix metalloproteinase, integrin β1, phosphorylated FAK, and MAPKs (including extracellular signal-regulated kinase 1/2, MAPK 38, and c-Jun N-terminal kinase) was assessed by Western blot analysis. Cartilage damage was less severe in the early treatment group than the early osteoarthritis group. The Mankin score was significantly lower in the early treatment group than the early osteoarthritis group (P < .05). There was no significant difference in cartilage damage or Mankin score between the late treatment and late osteoarthritis groups. There was a significant increase in type II collagen expression but a significant decrease in matrix metalloproteinase 13 expression in the early treatment group compared to the early osteoarthritis group, whereas no significant difference was found between the late treatment and late osteoarthritis groups. Integrin β1 and phosphorylated FAK expression was significantly higher, and phosphorylated extracellular signal-regulated kinase 1/2 and phosphorylated MAPK 38 expression was significantly lower in the early treatment group than the early osteoarthritis group. Our

  15. Periodontal Pathogens Invade Gingiva and Aortic Adventitia and Elicit Inflammasome Activation in αvβ6 Integrin-Deficient Mice

    PubMed Central

    Velsko, Irina M.; Chukkapalli, Sasanka S.; Rivera-Kweh, Mercedes F.; Zheng, Donghang; Aukhil, Ikramuddin; Lucas, Alexandra R.; Larjava, Hannu

    2015-01-01

    The American Heart Association supports an association between periodontal diseases and atherosclerosis but not a causal association. This study explores the use of the integrin β6−/− mouse model to study the causality. We investigated the ability of a polymicrobial consortium of Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum to colonize the periodontium and induce local and systemic inflammatory responses. Polymicrobially infected Itgβ6−/− mice demonstrate greater susceptibility to gingival colonization/infection, with severe gingival inflammation, apical migration of the junctional epithelium, periodontal pocket formation, alveolar bone resorption, osteoclast activation, bacterial invasion of the gingiva, a greater propensity for the bacteria to disseminate hematogenously, and a strong splenic T cell cytokine response. Levels of atherosclerosis risk factors, including serum nitric oxide, oxidized low-density lipoprotein, serum amyloid A, and lipid peroxidation, were significantly altered by polybacterial infection, demonstrating an enhanced potential for atherosclerotic plaque progression. Aortic gene expression revealed significant alterations in specific Toll-like receptor (TLR) and nucleotide-binding domain- and leucine-rich-repeat-containing receptor (NLR) pathway genes in response to periodontal bacterial infection. Histomorphometry of the aorta demonstrated larger atherosclerotic plaques in Itgβ6−/− mice than in wild-type (WT) mice but no significant difference in atherosclerotic plaque size between mice with polybacterial infection and mice with sham infection. Fluorescence in situ hybridization demonstrated active invasion of the aortic adventitial layer by P. gingivalis. Our observations suggest that polybacterial infection elicits distinct aortic TLR and inflammasome signaling and significantly increases local aortic oxidative stress. These results are the first to demonstrate the

  16. Periodontal pathogens invade gingiva and aortic adventitia and elicit inflammasome activation in αvβ6 integrin-deficient mice.

    PubMed

    Velsko, Irina M; Chukkapalli, Sasanka S; Rivera-Kweh, Mercedes F; Zheng, Donghang; Aukhil, Ikramuddin; Lucas, Alexandra R; Larjava, Hannu; Kesavalu, Lakshmyya

    2015-12-01

    The American Heart Association supports an association between periodontal diseases and atherosclerosis but not a causal association. This study explores the use of the integrin β6(-/-) mouse model to study the causality. We investigated the ability of a polymicrobial consortium of Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum to colonize the periodontium and induce local and systemic inflammatory responses. Polymicrobially infected Itgβ6(-/-) mice demonstrate greater susceptibility to gingival colonization/infection, with severe gingival inflammation, apical migration of the junctional epithelium, periodontal pocket formation, alveolar bone resorption, osteoclast activation, bacterial invasion of the gingiva, a greater propensity for the bacteria to disseminate hematogenously, and a strong splenic T cell cytokine response. Levels of atherosclerosis risk factors, including serum nitric oxide, oxidized low-density lipoprotein, serum amyloid A, and lipid peroxidation, were significantly altered by polybacterial infection, demonstrating an enhanced potential for atherosclerotic plaque progression. Aortic gene expression revealed significant alterations in specific Toll-like receptor (TLR) and nucleotide-binding domain- and leucine-rich-repeat-containing receptor (NLR) pathway genes in response to periodontal bacterial infection. Histomorphometry of the aorta demonstrated larger atherosclerotic plaques in Itgβ6(-/-) mice than in wild-type (WT) mice but no significant difference in atherosclerotic plaque size between mice with polybacterial infection and mice with sham infection. Fluorescence in situ hybridization demonstrated active invasion of the aortic adventitial layer by P. gingivalis. Our observations suggest that polybacterial infection elicits distinct aortic TLR and inflammasome signaling and significantly increases local aortic oxidative stress. These results are the first to demonstrate the mechanism of

  17. Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins.

    PubMed

    Kim, Minsoo; Carman, Christopher V; Springer, Timothy A

    2003-09-19

    Although critical for development, immunity, wound healing, and metastasis, integrins represent one of the few classes of plasma membrane receptors for which the basic signaling mechanism remains a mystery. We investigated cytoplasmic conformational changes in the integrin LFA-1 (alphaLbeta2) in living cells by measuring fluorescence resonance energy transfer between cyan fluorescent protein-fused and yellow fluorescent protein-fused alphaL and beta2 cytoplasmic domains. In the resting state these domains were close to each other, but underwent significant spatial separation upon either intracellular activation of integrin adhesiveness (inside-out signaling) or ligand binding (outside-in signaling). Thus, bidirectional integrin signaling is accomplished by coupling extracellular conformational changes to an unclasping and separation of the alpha and beta cytoplasmic domains, a distinctive mechanism for transmitting information across the plasma membrane.

  18. The FN13 peptide inhibits human tumor cells invasion through the modulation of alpha v beta 3 integrins organization and the inactivation of ILK pathway.

    PubMed

    Zoppi, Nicoletta; Ritelli, Marco; Salvi, Alessandro; Colombi, Marina; Barlati, Sergio

    2007-06-01

    We report the effect of the stable expression of a 13 amino acid human fibronectin (FN) peptide (FN13) on the organization of the FN extracellular matrix (ECM) and of FN integrin receptors (FNRs), in relationship with the inhibition of cellular invasion, in three FN-ECM defective human tumor-derived cell lines: SK-Hep1C3, hepatoma, ACN, neuroblastoma, and SK-OV-3, ovary carcinoma. All these cell lines stably expressing the FN13 peptide, organized an FN-ECM, disorganized alpha v beta 1 integrins and inactivated the ILK pathway, with the loss of secretion of MMP-9. This was associated with the inhibition of cell invasion in Matrigel matrix only in SK-Hep1C3 and ACN, but not in SK-OV-3 cells. Analysis of the integrin receptors organization showed that the FN13 expressing cells SK-Hep1C3 and ACN organized alpha v beta 3 integrins, whereas SK-OV-3 organized alpha v beta 5 dimers. The functional block of alpha v beta 5 integrins, with an inactivating anti-alpha v beta 5 antibody, led to the induction of alpha v beta 3 integrins also in SK-OV-3 cells, and to the inhibition of cell invasion. These data show that in the human tumor cells studied FN13 inhibits the in vitro invasion through the dissociation of alpha v beta 1 dimers, leading to ILK pathway inactivation, only when the organization of alpha v beta 3 integrins is induced in the plasma membrane.

  19. Integrin alpha(3)-subunit expression modulates alveolar epithelial cell monolayer formation.

    PubMed

    Lubman, R L; Zhang, X L; Zheng, J; Ocampo, L; Lopez, M Z; Veeraraghavan, S; Zabski, S M; Danto, S I; Borok, Z

    2000-07-01

    We investigated expression of the alpha(3)-integrin subunit by rat alveolar epithelial cells (AECs) grown in primary culture as well as the effects of monoclonal antibodies with blocking activity against the alpha(3)-integrin subunit on AEC monolayer formation. alpha(3)-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. alpha(3)- and beta(1)-integrin subunits coprecipitated in immunoprecipitation experiments with alpha(3)- and beta(1)-subunit-specific antibodies, consistent with their association as the alpha(3)beta(1)-integrin receptor at the cell membrane. Treatment with blocking anti-alpha(3) monoclonal antibody from day 0 delayed development of transepithelial resistance, reduced transepithelial resistance through day 5 compared with that in untreated AECs, and resulted in large subconfluent patches in monolayers viewed by scanning electron microscopy on day 3. These data indicate that alpha(3)- and beta(1)-integrin subunits are expressed in AEC monolayers where they form the heterodimeric alpha(3)beta(1)-integrin receptor at the cell membrane. Blockade of the alpha(3)-integrin subunit inhibits formation of confluent AEC monolayers. We conclude that the alpha(3)-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the alpha(3)beta(1)-integrin receptor in AEC adhesion.

  20. The molecular basis of talin2’s high affinity toward β1-integrin

    PubMed Central

    Yuan, Yaxia; Li, Liqing; Zhu, Yanyan; Qi, Lei; Azizi, Latifeh; Hytönen, Vesa P.; Zhan, Chang-Guo; Huang, Cai

    2017-01-01

    Talin interacts with β-integrin tails and actin to control integrin activation, thus regulating focal adhesion dynamics and cell migration. There are two talin genes, Tln1 and Tln2, which encode talin1 and talin2, and it is generally believed that talin2 functions redundantly with talin1. However, we show here that talin2 has a higher affinity to β1-integrin tails than talin1. Mutation of talin2 S339 to leucine, which can cause Fifth Finger Camptodactyly, a human genetic disease, completely disrupted its binding to β–integrin tails. Also, substitution of talin1 C336 with Ser enhanced the affinity of talin1, whereas substitution of talin2 S339 with Cys diminished that of talin2. Further computational modeling analysis shows that talin2 S339 formed a hydrogen bond with E353, which is critical for inducing key hydrogen bonds between talin2 N326 and β1-integrin R760, and between talin2 K327 and β1-integrin D759. Mutation at any of these residues significantly diminished the interaction of talin2 with β1- integrin tails. These hydrogen bonds were not observed in talin1/β1-integrin, but did exist in talin1C336S/β1-integrin complex. These results suggest that talin2 S339 forms a hydrogen bond with E353 to mediate its high affinity to β1-integrin. PMID:28155884

  1. Major Host Factors Involved in Epithelial Cell Invasion of Campylobacter jejuni: Role of Fibronectin, Integrin Beta1, FAK, Tiam-1, and DOCK180 in Activating Rho GTPase Rac1

    PubMed Central

    Boehm, Manja; Krause-Gruszczynska, Malgorzata; Rohde, Manfred; Tegtmeyer, Nicole; Takahashi, Seiichiro; Oyarzabal, Omar A.; Backert, Steffen

    2011-01-01

    Host cell entry by the food-borne pathogen Campylobacter jejuni has been reported as one of the primary reasons of tissue damage in infected humans, however, molecular invasion mechanisms and cellular factors involved in this process are widely unclear. Here we used knockout cell lines derived from fibronectin−/−, integrin beta1−/−, and focal adhesion kinase (FAK)−/− deficient mice and corresponding wild-type (WT) controls, to study C. jejuni-induced signaling cascades involved in the bacterial invasion process. Using high resolution scanning electron microscopy, GTPase pull-downs, G-LISA, and gentamicin protection assays we found that each of these host cell factors is indeed required for activation of the small Rho GTPase member Rac1 and maximal host cell invasion of this pathogen. Interestingly, membrane ruffling, tight engulfment of bacteria and invasion were only seen during infection of WT control cells, but not in fibronectin−/−, integrin beta1−/−, and FAK−/− knockout cell lines. We also demonstrate that C. jejuni activates FAK autophosphorylation activity at Y-397 and phosphorylation of Y-925, which is required for stimulating two downstream guanine exchange factors, DOCK180 and Tiam-1, which are upstream of Rac1. Small interfering (si) RNA studies further show that DOCK180 and Tiam-1 act cooperatively to trigger Rac1 activation and C. jejuni invasion. Moreover, mutagenesis data indicate that the bacterial fibronectin-binding protein CadF and the intact flagellum are involved in Rho GTPase activation and host cell invasion. Collectively, our results suggest that C. jejuni infection of host epithelial target cells hijacks a major fibronectin → integrin beta1 → FAK → DOCK180/Tiam-1 signaling cascade, which has a crucial role for Rac1 GTPase activity and bacterial entry into host target cells. PMID:22919583

  2. A novel integrin function in innate immunity from Chinese mitten crab (Eriocheir sinensis).

    PubMed

    Huang, Ying; Zhao, Ling-Ling; Feng, Jin-Ling; Zhu, Huan-Xi; Huang, Xin; Ren, Qian; Wang, Wen

    2015-10-01

    Integrins belong to a superfamily of conserved α β heterodimeric cell surface receptors that have critical function in cell migration, differentiation, and survival. In this study, an integrin called EsIntegrin was identified from Chinese mitten crab Eriocheir sinensis. EsIntegrin cDNA is 4415 bp long with a 2457 bp open reading frame that encodes an 818 amino acid protein. EsIntegrin contains a signal peptide, an integrin beta subunit (N-terminal portion of extracellular region) INB domain, an epidermal growth factor (hEGF) domain, an integrin B tail domain, a transmembrane region, and an integrin b cyt domain. EsIntegrin was mainly expressed in hemocytes and the heart, with a relatively lower expression level in gills, nerves, intestine, hepatopancreas, muscles, and eyestalk. When healthy crabs were challenged with LPS, PGN, Staphyloccocus aureus, or Vibrio parahaemolyticus, EsIntegrin expression level was upregulated significantly. Recombinant EsIntegrin has agglutination activity to Gram-positive (e.g., S. aureus and Bacillus subtilis) and Gram-negative bacteria (e.g., V. parahaemolyticus and Aeromonas hydrophila) in the presence of calcium. Furthermore, rEsIntegrin could not only bind to various bacteria such as S. aureus, Micrococcus luteus, B. subtilis, Bacillus megaterium, Bacillus thuringiensis, V. parahaemolyticus, Vibrio anguillarum, A. hydrophila, Vibrio natriegens, and Escherichia coli, but this compound also helped crabs in clearing virulent Gram-negative bacterium, V. parahaemolyticus, in vivo. These data suggested that EsIntegrin might function as cellular receptor that is involved in anti-bacterial immunity from E. sinensis.

  3. Integrin-β1 regulates chondrocyte proliferation and apoptosis through the upregulation of GIT1 expression.

    PubMed

    Zhang, Long-Qiang; Zhao, Guang-Zong; Xu, Xiao-Yan; Fang, Jun; Chen, Jing-Ming; Li, Ji-Wen; Gao, Xue-Jian; Hao, Li-Juan; Chen, Yun-Zhen

    2015-04-01

    Chondrocytes play a critical role in the repair process of osteoarthritis, which is also known as degenerative arthritis. Integrins, as the key family of cell surface receptors, are responsible for the regulation of chondrocyte proliferation, differentiation, survival and apoptosis through the recruitment and activation of downstream adaptor proteins. Moreover, G-protein-coupled receptor kinase interacting protein-1 (GIT1) exerts its effects on cell proliferation and migration through interaction with various cytokines. It has been previously suggested that GIT1 acts as a vital protein downstream of the integrin-mediated pathway. In the present study, we investigated the effects of integrin-β1 on cell proliferation and apoptosis, as well as the underlying mechanisms in chondrocytes in vitro. Following transfection with a vector expressing integrin-β1, our results revealed that the overexpression of integrin-β1 enhanced GIT1 expression, whereas the knockdown of integrin-β1 by siRNA suppressed GIT1 expression. However, no significant effect was observed on integrin-β1 expression following the enforced overexpression of GIT1, which suggests that GIT1 is localized downstream of integrin-β1. In other words, integrin-β1 regulates the expression of GIT1. Furthermore, this study demonstrated that integrin-β1 and GIT1 increased the expression levels of aggrecan and type II collagen, thus promoting chondrocyte proliferation; however, they inhibited chondrocyte apoptosis. Taken together, our data demonstrate that integrin-β1 plays a vital role in chondrocyte proliferation, differentiation and apoptosis. GIT1 exerts effects similar to those of integrin-β1 and is a downstream target of integrin-β1.

  4. LFA-1 and Mac-1 integrins bind to the serine/threonine-rich domain of thrombomodulin

    SciTech Connect

    Kawamoto, Eiji; Okamoto, Takayuki; Takagi, Yoshimi; Honda, Goichi; Suzuki, Koji; Imai, Hiroshi; Shimaoka, Motomu

    2016-05-13

    LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding of PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells. - Highlights: • LFA-1 and Mac-1 integrins bind to the anti-coagulant molecule thrombomodulin. • The serine/threonine-rich domain of thrombomodulin is essential to interact with the LFA-1 and Mac-1 integrins on PBMCs. • Integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells.

  5. Effect of D to E mutation of the RGD motif in rhodostomin on its activity, structure, and dynamics: importance of the interactions between the D residue and integrin.

    PubMed

    Chen, Chiu-Yueh; Shiu, Jia-Hau; Hsieh, Yao-Husn; Liu, Yu-Chen; Chen, Yen-Chin; Chen, Yi-Chun; Jeng, Wen-Yih; Tang, Ming-Jer; Lo, Szecheng J; Chuang, Woei-Jer

    2009-09-01

    Rhodostomin (Rho) is a snake venom protein containing an RGD motif that specifically inhibits the integrin-binding function. Rho produced in Pichia pastoris inhibits platelet aggregation with a K(I) of 78 nM as potent as native Rho. In contrast, its D51E mutant inhibits platelet aggregation with a K(I) of 49 muM. Structural analysis of Rho and its D51E mutant showed that they have the same tertiary fold with three two-stranded antiparallel beta-sheets. There are no structural backbone differences between the RG[D/E] loop which extends outward from the protein core and the RG[D/E] sequence at its apex in a four-residue RG[D/E]M type I turn. Two minor differences between Rho and its D51E mutant were only found from their backbone dynamics and 3D structures. The R(2) value of E51 is 13% higher than that of the D51 residue. A difference in the charge separation of 1.76 A was found between the sidechains of positive (R49) and negative residues (D51 or E51).The docking of Rho into integrin alphavbeta3 showed that the backbone amide and carbonyl groups of the D51 residue of Rho were formed hydrogen bonds with the integrin residues R216 and R214, respectively. In contrast, these hydrogen bonds were absent in the D51E mutant-integrin complex. Our findings suggest that the interactions between both the sidechain and backbone of the D residue of RGD-containing ligands and integrin are important for their binding. Copyright 2009 Wiley-Liss, Inc.

  6. Human platelet activation by Escherichia coli: roles for FcγRIIA and integrin αIIbβ3

    PubMed Central

    Watson, Callum N.; Kerrigan, Steven W.; Cox, Dermot; Henderson, Ian R.; Watson, Steve P.; Arman, Mònica

    2016-01-01

    Abstract Gram-negative Escherichia coli cause diseases such as sepsis and hemolytic uremic syndrome in which thrombotic disorders can be found. Direct platelet–bacterium interactions might contribute to some of these conditions; however, mechanisms of human platelet activation by E. coli leading to thrombus formation are poorly understood. While the IgG receptor FcγRIIA has a key role in platelet response to various Gram-positive species, its role in activation to Gram-negative bacteria is poorly defined. This study aimed to investigate the molecular mechanisms of human platelet activation by E. coli, including the potential role of FcγRIIA. Using light-transmission aggregometry, measurements of ATP release and tyrosine-phosphorylation, we investigated the ability of two E. coli clinical isolates to activate platelets in plasma, in the presence or absence of specific receptors and signaling inhibitors. Aggregation assays with washed platelets supplemented with IgGs were performed to evaluate the requirement of this plasma component in activation. We found a critical role for the immune receptor FcγRIIA, αIIbβ3, and Src and Syk tyrosine kinases in platelet activation in response to E. coli. IgG and αIIbβ3 engagement was required for FcγRIIA activation. Moreover, feedback mediators adenosine 5’-diphosphate (ADP) and thromboxane A2 (TxA2) were essential for platelet aggregation. These findings suggest that human platelet responses to E. coli isolates are similar to those induced by Gram-positive organisms. Our observations support the existence of a central FcγRIIA-mediated pathway by which human platelets respond to both Gram-negative and Gram-positive bacteria. PMID:27025455

  7. Muscle beta1D integrin reinforces the cytoskeleton-matrix link: modulation of integrin adhesive function by alternative splicing.

    PubMed

    Belkin, A M; Retta, S F; Pletjushkina, O Y; Balzac, F; Silengo, L; Fassler, R; Koteliansky, V E; Burridge, K; Tarone, G

    1997-12-15

    Expression of muscle-specific beta1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, beta1 integrin-minus cells leads to their phenotypic conversion. beta1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their beta1A-expressing counterparts. The transfected beta1D is targeted to focal adhesions and efficiently displaces the endogenous beta1A and alphavbeta3 integrins from the sites of cell-matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in beta1D transfectants. Whereas a significant part of cellular beta1A integrin is extractable in digitonin, the majority of the transfected beta1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of beta1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, beta1A interacts more strongly with alpha-actinin, than beta1D. Inside-out driven activation of the beta1D ectodomain increases ligand binding and fibronectin matrix assembly by beta1D transfectants. Phenotypic effects of beta1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of beta1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton- matrix link in muscle cells. This reflects the major role for beta1D integrin in muscle, where extremely stable association is required for contraction.

  8. Muscle β1D Integrin Reinforces the Cytoskeleton–Matrix Link: Modulation of Integrin Adhesive Function by Alternative Splicing

    PubMed Central

    Belkin, Alexey M.; Retta, S. Francesco; Pletjushkina, Olga Y.; Balzac, Fiorella; Silengo, Lorenzo; Fassler, Reinhard; Koteliansky, Victor E.; Burridge, Keith; Tarone, Guido

    1997-01-01

    Expression of muscle-specific β1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, β1 integrin-minus cells leads to their phenotypic conversion. β1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their β1A-expressing counterparts. The transfected β1D is targeted to focal adhesions and efficiently displaces the endogenous β1A and αvβ3 integrins from the sites of cell–matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in β1D transfectants. Whereas a significant part of cellular β1A integrin is extractable in digitonin, the majority of the transfected β1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of β1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, β1A interacts more strongly with α-actinin, than β1D. Inside-out driven activation of the β1D ectodomain increases ligand binding and fibronectin matrix assembly by β1D transfectants. Phenotypic effects of β1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of β1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton– matrix link in muscle cells. This reflects the major role for β1D integrin in muscle, where extremely stable association is required for contraction. PMID:9396762

  9. Activation of the alpha 4 beta 1 integrin through the beta 1 subunit induces recognition of the RGDS sequence in fibronectin

    PubMed Central

    1994-01-01

    Lymphocyte attachment to fibronectin is mainly mediated by the interaction of alpha 5 beta 1 and alpha 4 beta 1 integrins with the RGD and CS-1/Hep II sites, respectively. We have recently shown that the anti-beta 1 mAb TS2/16 can convert the partly active alpha 4 beta 1 present on certain hemopoietic cells that recognizes CS-1 but not Hep II, to a high avidity form that binds both ligands. In this report we have studied whether mAb TS2/16 also affects alpha 4 beta 1 ligand specificity. Incubation of the B cell lines Ramos and Daudi (which lack alpha 5 beta 1) with mAb TS2/16 induced specific attachment to an 80-kD fragment which lacks CS-1 and Hep II and contains the RGD sequence. mAbs anti-alpha 4 and the synthetic peptides CS-1 and IDAPS inhibited adhesion to the 80-kD fragment thus implying alpha 4 beta 1 as the receptor for this fragment. Interestingly, the synthetic peptide GRGDSPC and a 15-kD peptic fibronectin fragment containing the RGD sequence also inhibited B cell adhesion to the 80-kD fragment. Because we have previously shown that RGD peptides do not affect the constitutive function of alpha 4 beta 1, we tested whether TS2/16- activated alpha 4 beta 1 acquired the capacity to recognize RGD. Indeed RGD peptides inhibited TS2/16-treated B cell adhesion to a 38-kD fragment containing CS-1 and Hep II but did not affect binding of untreated cells to this fragment. An anti-fibronectin mAb reactive with an epitope on or near the RGD sequence also efficiently inhibited cell adhesion to the 80-kD fragment, indicating that the RGD sequence is a novel adhesive ligand for activated alpha 4 beta 1. These results emphasize the role of alpha 4 beta 1 as a receptor with different ligand specificities according to the activation state, a fact that may be important for lymphocyte migration, localization, and function. PMID:7517944

  10. 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.

  11. Kindlin-1 regulates integrin dynamics and adhesion turnover.

    PubMed

    Margadant, Coert; Kreft, Maaike; Zambruno, Giovanna; Sonnenberg, Arnoud

    2013-01-01

    Loss-of-function mutations in the gene encoding the integrin co-activator kindlin-1 cause Kindler syndrome. We report a novel kindlin-1-deficient keratinocyte cell line derived from a Kindler syndrome patient. Despite the expression of kindlin-2, the patient's cells display several hallmarks related to reduced function of β1 integrins, including abnormal cell morphology, cell adhesion, cell spreading, focal adhesion assembly, and cell migration. Defective cell adhesion was aggravated by kindlin-2 depletion, indicating that kindlin-2 can compensate to a certain extent for the loss of kindlin-1. Intriguingly, β1 at the cell-surface was aberrantly glycosylated in the patient's cells, and its expression was considerably reduced, both in cells in vitro and in the patient's epidermis. Reconstitution with wild-type kindlin-1 but not with a β1-binding defective mutant restored the aberrant β1 expression and glycosylation, and normalized cell morphology, adhesion, spreading, and migration. Furthermore, the expression of wild-type kindlin-1, but not of the integrin-binding-defective mutant, increased the stability of integrin-mediated cell-matrix adhesions and enhanced the redistribution of internalized integrins to the cell surface. Thus, these data uncover a role for kindlin-1 in the regulation of integrin trafficking and adhesion turnover.

  12. Kindlin-1 Regulates Integrin Dynamics and Adhesion Turnover

    PubMed Central

    Margadant, Coert; Kreft, Maaike; Zambruno, Giovanna; Sonnenberg, Arnoud

    2013-01-01

    Loss-of-function mutations in the gene encoding the integrin co-activator kindlin-1 cause Kindler syndrome. We report a novel kindlin-1-deficient keratinocyte cell line derived from a Kindler syndrome patient. Despite the expression of kindlin-2, the patient’s cells display several hallmarks related to reduced function of β1 integrins, including abnormal cell morphology, cell adhesion, cell spreading, focal adhesion assembly, and cell migration. Defective cell adhesion was aggravated by kindlin-2 depletion, indicating that kindlin-2 can compensate to a certain extent for the loss of kindlin-1. Intriguingly, β1 at the cell-surface was aberrantly glycosylated in the patient’s cells, and its expression was considerably reduced, both in cells in vitro and in the patient’s epidermis. Reconstitution with wild-type kindlin-1 but not with a β1-binding defective mutant restored the aberrant β1 expression and glycosylation, and normalized cell morphology, adhesion, spreading, and migration. Furthermore, the expression of wild-type kindlin-1, but not of the integrin-binding-defective mutant, increased the stability of integrin-mediated cell-matrix adhesions and enhanced the redistribution of internalized integrins to the cell surface. Thus, these data uncover a role for kindlin-1 in the regulation of integrin trafficking and adhesion turnover. PMID:23776470

  13. Integrin-Associated Complexes Form Hierarchically with Variable Stoichiometry during Nascent Adhesion Formation

    PubMed Central

    Bachir, Alexia I.; Zareno, Jessica; Moissoglu, Konstadinos; Plow, Edward; Gratton, Enrico; Horwitz, Alan R.

    2014-01-01

    Summary Background A complex network of putative molecular interactions underlies the architecture and function of cell-matrix adhesions. Most of these interactions are implicated from co-immunoprecipitation studies using expressed components; but few have been demonstrated or characterized functionally in living cells. Results We introduce fluorescence fluctuation methods to determine, at high spatial and temporal resolution, ‘when’ and ‘where’ molecular complexes form and their stoichiometry in nascent adhesions (NAs). We focus on integrin-associated molecules implicated in integrin-activation and in the integrin-actin linkage in NAs and show that these molecules form integrin containing complexes hierarchically within the adhesion itself. Integrin and kindlin reside in a molecular complex as soon as adhesions are visible; talin, while also present early, associates with the integrin-kindlin complex only after NAs have formed and in response to myosin II activity. Furthermore, talin and vinculin association precedes the formation of the integrin-talin complex. Finally, α-actinin enters NAs periodically and in clusters that transiently associate with integrins. The absolute number and stoichiometry of these molecules varies among the molecules studied and changes as adhesions mature. Conclusions These observations suggest a working model for NA assembly, whereby transient α-actinin- integrin complexes help nucleate NAs within the lamellipodium. Subsequently integrin complexes containing kindlin, but not talin, emerge. Once NAs have formed, myosin II activity promotes talin association with the integrin-kindlin complex in a stoichiometry consistent with each talin molecule linking two integrin-kindlin complexes. PMID:25088556

  14. Integrins promote cytokinesis through the RSK signaling axis

    PubMed Central

    Mathew, Shomita S.; Nieves, Bethsaida; Sequeira, Sharon; Sambandamoorthy, Savitha; Pumiglia, Kevin; Larsen, Melinda; LaFlamme, Susan E.

    2014-01-01

    ABSTRACT Cytokinesis is the final stage in cell division. Although integrins can regulate cytokinesis, the mechanisms involved are not fully understood. In this study, we demonstrate that integrin-regulated ERK (extracellular signal-related kinase) and RSK (p90 ribosomal S6 kinase) signaling promotes successful cytokinesis. Inhibiting the activation of ERK and RSK in CHO cells by a mutation in the integrin β1 cytoplasmic tail or with pharmacological inhibitors results in the accumulation of cells with midbodies and the formation of binucleated cells. Activation of ERK and RSK signaling by the expression of constitutively active RAF1 suppresses the mutant phenotype in a RSK-dependent manner. Constitutively active RSK2 also restores cytokinesis inhibited by the mutant integrin. Importantly, the regulatory role of the RSK pathway is not specific to CHO cells. MCF-10A human mammary epithelial cells and HPNE human pancreatic ductal epithelial cells exhibit a similar dependence on RSK for successful cytokinesis. In addition, depriving mitotic MCF10A cells of integrin-mediated adhesion by incubating them in suspension suppressed ERK and RSK activation and resulted in a failure of cytokinesis. Furthermore, inhibition of RSK or integrins within the 3D context of a developing salivary gland organ explant also leads to an accumulation of epithelial cells with midbodies, suggesting a similar defect in cytokinesis. Interestingly, neither ERK nor RSK regulates cytokinesis in human fibroblasts, suggesting cell-type specificity. Taken together, our results identify the integrin–RSK signaling axis as an important regulator of cytokinesis in epithelial cells. We propose that the proper interaction of cells with their microenvironment through integrins contributes to the maintenance of genomic stability by promoting the successful completion of cytokinesis. PMID:24284076

  15. Integrin binding: Sticking around vessels

    NASA Astrophysics Data System (ADS)

    Blatchley, Michael R.; Gerecht, Sharon

    2017-09-01

    A study demonstrates that controlled integrin binding on a biomaterial was capable of promoting vascular cell sprouting and formation of a non-leaky blood vessel network in a healthy and diseased state.

  16. Corneal integrins and their functions.

    PubMed

    Stepp, Mary Ann

    2006-07-01

    Integrins were first described just over 20 years ago and have been studied in the cornea by many groups interested in how the cornea functions in health and disease. There are a minimum of 12 different integrin heterodimers reported to be expressed by the major resident cells of the cornea: the corneal and limbal epithelial cells, keratocytes/fibroblasts, and corneal endothelial cells. These different integrin heterodimers play important and varied roles in maintaining the cornea and organizing how its cells interact with their surrounding extracellular matrix to maintain corneal clarity. In this review, an overview of the discovery and functions of integrins is provided along with a description of the current state of our knowledge of this large family of important proteins. While we have learned a lot about corneal integrins over the past 20 years, there is still much to learn. Areas where gaps in our knowledge of integrin functions in the cornea are slowing our progress in understanding corneal diseases and dystrophies at a molecular level are highlighted.

  17. Induction of Cell Scattering by Expression of β1 Integrins in β1-Deficient Epithelial Cells Requires Activation of Members of the Rho Family of Gtpases and Downregulation of Cadherin and Catenin Function

    PubMed Central

    Gimond, Clotilde; van der Flier, Arjan; van Delft, Sanne; Brakebusch, Cord; Kuikman, Ingrid; Collard, John G.; Fässler, Reinhard; Sonnenberg, Arnoud

    1999-01-01

    Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and β1 integrins influence each other using two different β1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of β1A or the cytoplasmic splice variant β1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of β1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of β1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-β1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM–cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length β1A. This indicates that the disruption of cell–cell adhesion is not simply the consequence of the stimulated cell migration. Expression of β1 integrins in GE11 cells resulted in a decrease in cadherin and α-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of α-catenin protein levels by β1 integrins is likely to play a role in the morphological transition, since overexpression of α-catenin in GE11 cells before β1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of β1A

  18. Integrin β1, Osmosensing, and Chemoresistance in Mouse Ehrlich Carcinoma Cells.

    PubMed

    Sørensen, Belinda Halling; Rasmussen, Line Jee Hartmann; Broberg, Bjørn Sindballe; Klausen, Thomas Kjær; Sauter, Daniel Peter Rafael; Lambert, Ian Henry; Aspberg, Anders; Hoffmann, Else Kay

    2015-01-01

    Altered expression of the integrin family of cell adhesion receptors has been associated with initiation, progression, and metastasis of solid tumors as well as in the development of chemoresistance. Here, we investigated the role of integrins, in particular integrin β1, in cell volume regulation and drug-induced apoptosis in adherent and non-adherent Ehrlich ascites cell lines. Adhesion phenotypes were verified by colorimetric cell-adhesion-assay. Quantitative real-time PCR and western blot were used to compare expression levels of integrin subunits. Small interfering RNA was used to silence integrin β1 expression. Regulatory volume decrease (RVD) after cell swelling was studied with calcein-fluorescence-self-quenching and Coulter counter analysis. Taurine efflux was estimated with tracer technique. Caspase assay was used to determine apoptosis. We show that adherent cells have stronger fibronectin binding and a significantly increased expression of integrin α5, αv, and β1 at mRNA and protein level, compared to non-adherent cells. Knockdown of integrin β1 reduced RVD of the adherent but not of the non-adherent cells. Efflux of taurine was unaffected. In contrast to non-adherent, adherent cells exhibited chemoresistance to chemotherapeutic drugs (cisplatin and gemcitabine). However, knockdown of integrin β1 promoted cisplatin-induced caspase activity in adherent cells. Our data identifies integrin β1 as a part of the osmosensing machinery and regulator of cisplatin resistance in adherent Ehrlich cells.

  19. Down-regulation of β3-integrin inhibits bone metastasis of small cell lung cancer.

    PubMed

    Li, Na; Zhang, Jian-ping; Guo, Shan; Min, Jie; Liu, Li-li; Su, Hai-chuan; Feng, Ying-ming; Zhang, He-long

    2012-03-01

    Bone is one of the most frequent targets of small cell lung cancer (SCLC) metastasis, but the molecular mechanism remains unclear. β3-integrin plays an important role in invasion of various kinds of tumors. Yet, its role in bone-metastasis of SCLC is still unknown. In this study, we first examined the expression of β3-integrin in SBC-5 and SBC-3 cells by real-time PCR, western blot and immunofluorescence. We found that, compared to none bone-metastatic SBC-3 cells, β3-integrin was highly expressed in SBC-5 cells, a specific bone-metastatic SCLC cells line characterized in our previous study. We next constructed β3-integrin siRNA and transfected SBC-5 cell line, and found that β3-integrin siRNA significantly down-regulated the β3-integrin mRNA level and protein expression in SBC-5 cell line. We further found that inhibition of β3-integrin significantly reduced tumor cell proliferation and induced apoptosis. In addition, the β3-integrin down-regulated cells presented significant decrease in cell adhesion, migration and invasion activity. Our results suggest the β3-integrin has an essential effect on tumor cell proliferation and progression, and may be a potential therapeutic target for the prevention of skeletal metastases of lung cancer.

  20. 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.

  1. Autocrine transforming growth factor-{beta}1 activation mediated by integrin {alpha}V{beta}3 regulates transcriptional expression of laminin-332 in Madin-Darby canine kidney epithelial cells.

    PubMed

    Moyano, Jose V; Greciano, Patricia G; Buschmann, Mary M; Koch, Manuel; Matlin, Karl S

    2010-11-01

    Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed. Addition of active transforming growth factor (TGF)-β1 to confluent MDCK monolayers is sufficient to induce transcription of the LM α3 gene and LM-332 protein expression via the TGF-β type I receptor (TβR-I) and the Smad2-Smad4 complex. Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-β1 secretion and activation mediated by integrin αVβ3 because neutralizing antibodies block LM-332 production in subconfluent cells. In confluent cells, latent TGF-β1 is secreted apically, whereas TβR-I and integrin αVβ3 are localized basolaterally. Disruption of the epithelial barrier by mechanical injury activates TGF-β1, leading to LM-332 expression. Together, our data suggest a novel mechanism for triggering the production of LM-332 after epithelial injury.

  2. Autocrine Transforming Growth Factor-β1 Activation Mediated by Integrin αVβ3 Regulates Transcriptional Expression of Laminin-332 in Madin-Darby Canine Kidney Epithelial Cells

    PubMed Central

    Greciano, Patricia G.; Buschmann, Mary M.; Koch, Manuel; Matlin, Karl S.

    2010-01-01

    Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed. Addition of active transforming growth factor (TGF)-β1 to confluent MDCK monolayers is sufficient to induce transcription of the LM α3 gene and LM-332 protein expression via the TGF-β type I receptor (TβR-I) and the Smad2–Smad4 complex. Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-β1 secretion and activation mediated by integrin αVβ3 because neutralizing antibodies block LM-332 production in subconfluent cells. In confluent cells, latent TGF-β1 is secreted apically, whereas TβR-I and integrin αVβ3 are localized basolaterally. Disruption of the epithelial barrier by mechanical injury activates TGF-β1, leading to LM-332 expression. Together, our data suggest a novel mechanism for triggering the production of LM-332 after epithelial injury. PMID:20844080

  3. Urokinase-type plasminogen activator receptor interaction with β1 integrin is required for platelet-derived growth factor-AB-induced human mesenchymal stem/stromal cell migration.

    PubMed

    Chabot, Valérie; Dromard, Cécile; Rico, Angélique; Langonné, Alain; Gaillard, Julien; Guilloton, Fabien; Casteilla, Louis; Sensebé, Luc

    2015-09-29

    Mesenchymal stem cells (MSC) are well described for their role in tissue regeneration following injury. Migratory properties of endogenous or administrated MSC are critical for tissue repair processes. Platelet-derived growth factor (PDGF) is a chemotactic growth factor that elicits mesenchymal cell migration. However, it is yet to be elucidated if signaling pathways other than direct activation of PDGF receptor (PDGF-R) are involved in PDGF-induced cell migration. Knocking down and co-immunoprecipitation approaches were used to evaluate urokinase-type plasminogen activator receptor (uPAR) requirement and its interactions with proteins involved in migration mechanisms, in human MSC induced to migrate under PDGF-AB effect. We demonstrated that uPAR activation and its association with β1-integrin are required for PDGF-AB-induced migration. This phenomenon takes place in MSC derived from bone marrow and from adipose tissue. We showed that PDGF-AB downstream signaling requires other effector molecules in MSC such as the uPA/uPAR system and β1 integrin signaling pathway known for their role in migration. These findings provide new insights in molecular mechanisms of PDGF-AB-induced migration of human MSC that may be relevant to control MSC function and tissue remodeling after injury.

  4. 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.

  5. 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.

  6. Computational Study of Bond Dissociation Enthalpies for Lignin Model Compounds: $\\beta$-5 Arylcoumaran

    SciTech Connect

    Beste, Ariana; Buchanan III, A C; Younker, Jarod M

    2012-01-01

    The biopolymer lignin is a potential source of valuable chemicals. The $\\beta$-5 linkage comprises $\\sim$10\\% of the linkages in lignin. Density Functional Theory (DFT) was used to calculate the $\\alpha$C-O and $\\alpha$C-$\\beta$C bond dissociation enthalpies (BDEs) for $\\beta$-5 models with varied substituents, which are important for understanding initial lignin decomposition. The $\\alpha$C-O ($\\alpha$C-$\\beta$C) BDEs were in the range of 40-44 (57-62) kcal/mol. The products resulting from either homolysis are bi-radicals with multi-determinant character in the singlet electronic state. Multiconfiguration self-consistent field (MCSCF) theory results were used to verify that unrestricted DFT and broken-symmetry DFT were sufficient to study these reactions.

  7. Platelet collagen receptor integrin alpha2beta1 activation involves differential participation of ADP-receptor subtypes P2Y1 and P2Y12 but not intracellular calcium change.

    PubMed

    Jung, S M; Moroi, M

    2001-06-01

    In agonist-induced platelet activation, the collagen platelet receptor integrin alpha2beta1 is activated to high-affinity states through ADP involvement [Jung, S.M. & Moroi, M. (2000) J. Biol. Chem. 275, 8016-8026]. Here we determined the ADP-receptor subtypes involved and their relative contributions to alpha2beta1 activation (assessed by soluble-collagen binding) using the P2Y12 antagonist AR-C69931MX and P2Y1 antagonists adenosine 3',5'-diphosphate (Ado(3,5)PP) and adenosine 3'-phosphate 5'-phosphosulfate (AdoPPS). All three inhibited alpha2beta1 activation induced by low or high ADP, low thrombin, or low collagen-related peptide (CRP) concentrations; however, AR-C69931MX was markedly more inhibitory than the P2Y1 antagonists, suggesting the greater contribution of P2Y12. Inhibition patterns by various combinations of AR-C69931MX, AdoPPS, and wortmannin suggested that P2Y1 and P2Y12 mediate alpha2beta1 activation through different pathways, with possible involvement of phosphoinositide 3-kinase in both. Low concentrations of the acetoxy-methyl derivative of 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (calcium chelator) markedly decreased alpha2beta1 activation by low thrombin or CRP, but did not affect that by low or high ADP. Measurements of intracellular Ca2+ level (fluorimetric method) and alpha2beta1 activation (soluble-collagen binding) in the same platelet preparation indicated that alpha2beta1 activation via ADP receptors was independent of intracellular Ca2+ release. Our data indicate that integrin alpha2beta1 activation by ADP occurs through an inside-out signaling mechanism involving differential contributions by P2Y1 and P2Y12 wherein each contributes to some portion of the activation, with the stronger contribution of P2Y12. Furthermore, intracellular Ca2+ increase is not directly related to integrin alpha2beta1 activation, meaning that it is separate from the calcium mobilization pathways that these two ADP receptors are involved in.

  8. Loss of miR-200b promotes invasion via activating the Kindlin-2/integrin β1/AKT pathway in esophageal squamous cell carcinoma: An E-cadherin-independent mechanism

    PubMed Central

    Zhang, Hai-Feng; Alshareef, Abdulraheem; Wu, Chengsheng; Li, Shang; Jiao, Ji-Wei; Cao, Hui-Hui; Lai, Raymond; Xu, Li-Yan; Li, En-Min

    2015-01-01

    Our previous studies have shown that loss of miR-200b enhances the invasiveness of esophageal squamous cell carcinoma (ESCC) cells. However, whether the miR-200-ZEB1/2-E-cadherin regulatory cascade, a master regulator of epithelial-to-mesenchymal transition (EMT), is involved in the regulation of ESCC invasion remains elusive. Here, we show that miR-200b represses ESCC cell invasion in vivo without altering the expression of E-cadherin and vimentin, two surrogate markers of EMT. However, an inverse correlation was observed between the expression levels of miR-200b and ZEB1/2 in both ESCC cell lines (n = 7, P < 0.05) and ESCC tumor samples (n = 88, P < 0.05). Methylation of E-cadherin gene was found to block the regulation of E-cadherin by the miR-200b-ZEB1/2 axis, indicating that an E-cadherin-independent mechanism can mediate the biological function of miR-200b in ESCC. We revealed that miR-200b suppresses the integrin β1-AKT pathway via targeting Kindlin-2 to mitigate ESCC cell invasiveness. In two independent cohorts of ESCC samples (n = 20 and n = 53, respectively), Kindlin-2 expression positively correlated with the activation status of both the integrin signaling pathway and the PI3K-AKT signaling pathway (both P < 0.01). These data highlight that suppression of the Kindlin-2-integrin β1-AKT regulatory axis is an alternative mechanism underlying the tumor suppressor function of miR-200b in ESCC. PMID:26334393

  9. Loss of miR-200b promotes invasion via activating the Kindlin-2/integrin β1/AKT pathway in esophageal squamous cell carcinoma: An E-cadherin-independent mechanism.

    PubMed

    Zhang, Hai-Feng; Alshareef, Abdulraheem; Wu, Chengsheng; Li, Shang; Jiao, Ji-Wei; Cao, Hui-Hui; Lai, Raymond; Xu, Li-Yan; Li, En-Min

    2015-10-06

    Our previous studies have shown that loss of miR-200b enhances the invasiveness of esophageal squamous cell carcinoma (ESCC) cells. However, whether the miR-200-ZEB1/2-E-cadherin regulatory cascade, a master regulator of epithelial-to-mesenchymal transition (EMT), is involved in the regulation of ESCC invasion remains elusive. Here, we show that miR-200b represses ESCC cell invasion in vivo without altering the expression of E-cadherin and vimentin, two surrogate markers of EMT. However, an inverse correlation was observed between the expression levels of miR-200b and ZEB1/2 in both ESCC cell lines (n = 7, P < 0.05) and ESCC tumor samples (n = 88, P < 0.05). Methylation of E-cadherin gene was found to block the regulation of E-cadherin by the miR-200b-ZEB1/2 axis, indicating that an E-cadherin-independent mechanism can mediate the biological function of miR-200b in ESCC. We revealed that miR-200b suppresses the integrin β1-AKT pathway via targeting Kindlin-2 to mitigate ESCC cell invasiveness. In two independent cohorts of ESCC samples (n = 20 and n = 53, respectively), Kindlin-2 expression positively correlated with the activation status of both the integrin signaling pathway and the PI3K-AKT signaling pathway (both P < 0.01). These data highlight that suppression of the Kindlin-2-integrin β1-AKT regulatory axis is an alternative mechanism underlying the tumor suppressor function of miR-200b in ESCC.

  10. Integrin priming dynamics: mechanisms of integrin antagonist-promoted alphaIIbbeta3:PAC-1 molecular recognition.

    PubMed

    Hantgan, Roy R; Stahle, Mary C

    2009-09-08

    This investigation addressed the paradox that disintegrins and small RGD-ligands readily bind to the resting alphaIIbbeta3 integrin, while macromolecules with similar integrin recognition motifs require an activated, or primed, receptor. Three structurally similar pharmaceutical integrin antagonists (eptifibatide, tirofiban, and roxifiban) were each incubated with resting alphaIIbbeta3; after drug wash-out, the receptor's ability to recognize PAC-1, an activation-dependent IgM with an RYD integrin-targeting site was measured. Their promotion of PAC-1:alphaIIbbeta3 binding (solid phase assay), eptifibatide > tirofiban > roxifiban, correlated with their ability to shift the receptor to an open conformer, as measured by analytical ultracentrifugation. Surface plasmon resonance (SPR) demonstrated that PAC-1 bound rapidly (k(on) approximately 5 x 10(5) l/mol-s, 25 degrees C) and tightly (Kd approximately 1 nM) to eptifibatide-primed integrins, captured on a biosensor using an IgG specific for alphaIIb's cytoplasmic domain. Varying the interval between integrin capture and antagonist dissociation indicated that transiently primed alphaIIbbeta3 retains the ability to rapidly bind PAC-1 from 2-90 min, although the dissociation rate increased at later times, indicative of a weakening of the complex. Fluorescence anisotropy (fluorophore-tagged analogue exchange assay) demonstrated that eptifibatide dissociates rapidly from alphaIIbbeta3 (half-time <2 min), consistent with the priming window determined by SPR. van't Hoff analysis of alphaIIbbeta3:PAC-1's temperature-dependent Kd indicated entropy/enthalpy compensation, similar to (resting) integrin binding to the disintegrin echistatin. Eyring analysis of k(on) yielded DeltaG degrees approximately 10 kcal/mol for PAC-1 binding to primed alphaIIbbeta3, 3 kcal/mol lower than that of echistatin. These observations suggest that priming lowers the transition-state energy barrier, enabling rapid macromolecular ligand binding to

  11. 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.

  12. 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.

  13. Integrins and epithelial cell polarity.

    PubMed

    Lee, Jessica L; Streuli, Charles H

    2014-08-01

    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.

  14. Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S [beta]5-subunit

    SciTech Connect

    Blackburn, Christopher; Gigstad, Kenneth M.; Hales, Paul; Garcia, Khristofer; Jones, Matthew; Bruzzese, Frank J.; Barrett, Cynthia; Liu, Jane X.; Soucy, Teresa A.; Sappal, Darshan S.; Bump, Nancy; Olhava, Edward J.; Fleming, Paul; Dick, Lawrence R.; Tsu, Christopher; Sintchak, Michael D.; Blank, Jonathan L.

    2012-04-30

    The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome used on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the {beta}5 (chymotrypsin-like) site over the {beta}1 (caspase-like) and {beta}2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC{sub 50} values for the human 20S {beta}5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin-luciferase reporter, activation of NF{Kappa}B (nuclear factor {Kappa}B) in response to TNF-{alpha} (tumor necrosis factor-{alpha}) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the {beta}5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the {beta}5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.

  15. The opposing roles of laminin-binding integrins in cancer.

    PubMed

    Ramovs, Veronika; Te Molder, Lisa; Sonnenberg, Arnoud

    2017-01-01

    Integrins play an important role in cell adhesion by linking the cytoskeleton of cells to components in the extracellular matrix. In this capacity, integrins cooperate with different cell surface receptors, including growth factor receptors and G-protein coupled receptors, to regulate intracellular signaling pathways that control cell polarization, spreading, migration, survival, and gene expression. A distinct subfamily of molecules in the integrin family of adhesion receptors is formed by receptors that mediate cell adhesion to laminins, major components of the basement membrane that lie under clusters of cells or surround them, separating them from other cells and/or adjacent connective tissue. During the past decades, many studies have provided evidence for a role of laminin-binding integrins in tumorigenesis, and both tumor-promoting and suppressive activities have been identified. In this review we discuss the dual role of the laminin-binding integrins α3β1 and α6β4 in tumor development and progression, and examine the factors and mechanisms involved in these opposing effects.

  16. Integrin β4 in EMT: an implication of renal diseases.

    PubMed

    Wang, Qi; Wang, Yan; Huang, Xiaoyan; Liang, Wei; Xiong, Zibo; Xiong, Zuying

    2015-01-01

    Renal fibrosis is a main cause of chronic renal failure. Epithelial-to-mesenchymal transition (EMT) markers play a role in renal fibrosis. Transforming growth factor-β1 (TGF-β1) has been shown to initiate and complete the whole EMT process. It is now well accepted that loss of E-cadherin, EMT marker α-SMA, and connective tissue growth factor (CTGF) expression are key events in the EMT process. We found that by stimulating human renal proximal tubular epithelial (HK-2) cells with TGF-β1, the expression of E-cadherin was down regulated and the expression of α-SMA and CTGF were up regulated in a dose dependent manner. In our present study we also found that integrin β4 and peroxisome proliferators-activated receptor-γ (PPAR-γ) play roles in EMT process, with TGF-β1 stimulation increasing integrin β4 expression in HK2 cells. Integrin β4 and PPARγ were detected in tubulointerstitial tissues, immunohistochemistry analysis showed enhanced expression of integrin β4 in early stage, with over-expression at later stage. In contrast, the expression of PPARγ showed little increased in early stage, but was dramatically decreased at later stage. This is consistent with TGF-β1 inducing EMT. Our immune-precipitation studies show that integrin β4 disassociation with PPARγ is present in E-cadherin signaling. It suggests that PPARγ has a role in EMT inhibition.

  17. Sequence 274-368 in the beta 3-subunit of the integrin alpha IIb beta 3 provides a ligand recognition and binding domain for the gamma-chain of fibrinogen that is independent of platelet activation.

    PubMed

    Alemany, M; Concord, E; Garin, J; Vinçon, M; Giles, A; Marguerie, G; Gulino, D

    1996-01-15

    Several bacterial-expressed recombinant fragments encompassing the extracellular part of the beta 3 subunit of the integrin alpha IIb beta 3 were shown to recognize and bind soluble and immobilized forms of fibrinogen. Two of them, designated as rIII-11 (beta 3 274-368) and rIII-13 (beta 3 274-403), did not contain the established RGD-ligand binding sequence. In fact, they interacted, in a Ca(2+)-independent manner, with the C-terminal part of the fibrinogen gamma chain. Both beta 3 fragments blocked the participation of fibrinogen in the induction of platelet aggregation induced by adenosine diphosphate. Fragment rIII-13 was recognized by the anti-beta 3 monoclonal antibody B2A. This antibody, which possesses an epitope exposed on both resting and activated platelets, inhibited fibrinogen binding as well as platelet adhesion and aggregation. In conclusion, the results demonstrated that the 274-368 sequence of the beta 3 subunit of integrin alpha IIb beta 3 constitutes a fibrinogen ligand binding domain, distinct from the RGD-binding site, that is required for both platelet adhesion and aggregation.

  18. Proteolytic degradation of the RGD-binding and non-RGD-binding conformers of human platelet integrin glycoprotein IIb/IIIa: clues for identification of regions involved in the receptor's activation.

    PubMed Central

    Calvete, J J; Mann, K; Schäfer, W; Fernandez-Lafuente, R; Guisán, J M

    1994-01-01

    The human integrin glycoprotein (GP)IIb/IIIa plays a central role in haemostasis as an inducible receptor for fibrinogen and other RGD-containing adhesive proteins at the platelet plasma membrane. Expression of the fibrinogen receptor on platelet activation involves conformational changes in the quaternary structure of GPIIb/IIIa. Little is known, however, about the nature of this conformational transition. Given that isolated GPIIb/IIIa contains a mixture of RGD-binding and non-RGD-binding heterodimers, we used limited proteolysis as a tool for investigating the structural differences between the two conformers. Comparison of their fragmentation patterns shows that, whereas in the non-RGD-binding form of GPIIb/IIIa the N-terminal half of the heavy chain of GPIIb (GPIIbH) and the central region of GPIIIa are cleaved by endoproteinase Arg-C, these domains associate tightly with one another in the RGD-binding GPIIb/IIIa and are thus protected from proteolysis. In addition, the C-terminal half of GPIIb becomes more susceptible to degradation in the non-RGD-binding GPIIb/IIIa conformer. Our interpretation, in the context of available structural and functional data, is that a major relative reorientation of the GPIIbH and GPIIIa extracellular domains takes place along the subunit interface during the conformational transition of the platelet integrin. Images Figure 1 PMID:8129707

  19. Fibronectin Fragment Activation of Proline-rich Tyrosine Kinase PYK2 Mediates Integrin Signals Regulating Collagenase-3 Expression by Human Chondrocytes through a Protein Kinase C-dependent Pathway*

    PubMed Central

    Loeser, Richard F.; Forsyth, Christopher B.; Samarel, Allen M.; Im, Hee-Jeong

    2010-01-01

    Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the α5β1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the α5β1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKCδ inhibitor rottlerin. Furthermore, PKCδ translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after co-transfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves

  20. Rac recruits high-affinity integrin alphavbeta3 to lamellipodia in endothelial cell migration.

    PubMed

    Kiosses, W B; Shattil, S J; Pampori, N; Schwartz, M A

    2001-03-01

    Integrin alphavbeta3 has an important role in the proliferation, survival, invasion and migration of vascular endothelial cells. Like other integrins, alphavbeta3 can exist in different functional states with respect to ligand binding. These changes involve both affinity modulation, by which conformational changes in the integrin heterodimer govern affinity for individual extracellular matrix proteins, and avidity modulation, by which changes in lateral mobility and integrin clustering affect the binding of cells to multivalent matrices. Here we have used an engineered monoclonal antibody Fab (antigen-binding fragment) named WOW-1, which binds to activated integrins alphavbeta3 and alphavbeta5 from several species, to investigate the role of alphavbeta3 activation in endothelial cell behaviour. Because WOW-1 is monovalent, it is insensitive to changes in integrin clustering and therefore reports only changes in affinity. WOW-1 contains an RGD tract in its variable region and binds only to unoccupied, high-affinity integrins. By using WOW-1, we have identified the selective recruitment of high-affinity integrins as a mechanism by which lamellipodia promote formation of new adhesions at the leading edge in cell migration.

  1. Targeting of Alpha-V Integrins Reduces Malignancy of Bladder Carcinoma

    PubMed Central

    van der Horst, Geertje; Bos, Lieke; van der Mark, Maaike; Cheung, Henry; Heckmann, Bertrand; Clément-Lacroix, Philippe; Lorenzon, Giocondo; Pelger, Rob C. M.; Bevers, Rob F. M.; van der Pluijm, Gabri

    2014-01-01

    Low survival rates of metastatic cancers emphasize the need for a drug that can prevent and/or treat metastatic cancer. αv integrins are involved in essential processes for tumor growth and metastasis and targeting of αv integrins has been shown to decrease angiogenesis, tumor growth and metastasis. In this study, the role of αv integrin and its potential as a drug target in bladder cancer was investigated. Treatment with an αv integrin antagonist as well as knockdown of αv integrin in the bladder carcinoma cell lines, resulted in reduced malignancy invitro, as illustrated by decreased proliferative, migratory and clonogenic capacity. The CDH1/CDH2 ratio increased, indicating a shift towards a more epithelial phenotype. This shift appeared to be associated with downregulation of EMT-inducing transcription factors including SNAI2. The expression levels of the self-renewal genes NANOG and BMI1 decreased as well as the number of cells with high Aldehyde Dehydrogenase activity. In addition, self-renewal ability decreased as measured with the urosphere assay. In line with these observations, knockdown or treatment of αv integrins resulted in decreased metastatic growth in preclinical invivo models as assessed by bioluminescence imaging. In conclusion, we show that αv integrins are involved in migration, EMT and maintenance of Aldehyde Dehydrogenase activity in bladder cancer cells. Targeting of αv integrins might be a promising approach for treatment and/or prevention of metastatic bladder cancer. PMID:25247809

  2. Integrins synergise to induce expression of the MRTF-A-SRF target gene ISG15 for promoting cancer cell invasion.

    PubMed

    Hermann, Michaela-Rosemarie; Jakobson, Madis; Colo, Georgina P; Rognoni, Emanuel; Jakobson, Maili; Kupatt, Christian; Posern, Guido; Fässler, Reinhard

    2016-04-01

    Integrin-mediated activation of small GTPases induces the polymerisation of G-actin into various actin structures and the release of the transcriptional co-activator MRTF from G-actin. Here we report that pan-integrin-null fibroblasts seeded on fibronectin and expressing β1- and/or αV-class integrin contained different G-actin pools, nuclear MRTF-A (also known as MKL1 or MAL) levels and MRTF-A-SRF activities. The nuclear MRTF-A levels and activities were highest in cells expressing both integrin classes, lower in cells expressing β1 integrins and lowest in cells expressing the αV integrins. Quantitative proteomics and transcriptomics analyses linked the differential MRTF-A activities to the expression of the ubiquitin-like modifier interferon-stimulated gene 15 (ISG15), which is known to modify focal adhesion and cytoskeletal proteins. The malignant breast cancer cell line MDA-MB-231 expressed high levels of β1 integrins, ISG15 and ISGylated proteins, which promoted invasive properties, whereas non-invasive MDA-MB-468 and MCF-7 cell lines expressed low levels of β1 integrins, ISG15 and ISGylated proteins. Our findings suggest that integrin-adhesion-induced MRTF-A-SRF activation and ISG15 expression constitute a newly discovered signalling circuit that promotes cell migration and invasion. © 2016. Published by The Company of Biologists Ltd.

  3. Binding of Autotaxin to Integrins Localizes Lysophosphatidic Acid Production to Platelets and Mammalian Cells*

    PubMed Central

    Fulkerson, Zachary; Wu, Tao; Sunkara, Manjula; Kooi, Craig Vander; Morris, Andrew J.; Smyth, Susan S.

    2011-01-01

    Autotaxin (ATX) is a secreted lysophospholipase D that generates the bioactive lipid mediator lysophosphatidic acid (LPA). We and others have reported that ATX binds to integrins, but the function of ATX-integrin interactions is unknown. The recently reported crystal structure of ATX suggests a role for the solvent-exposed surface of the N-terminal tandem somatomedin B-like domains in binding to platelet integrin αIIbβ3. The opposite face of the somatomedin B-like domain interacts with the catalytic phosphodiesterase (PDE) domain to form a hydrophobic channel through which lysophospholipid substrates enter and leave the active site. Based on this structure, we hypothesize that integrin-bound ATX can access cell surface substrates and deliver LPA to cell surface receptors. To test this hypothesis, we investigated the integrin selectivity and signaling pathways that promote ATX binding to platelets. We report that both platelet β1 and β3 integrins interact in an activation-dependent manner with ATX via the SMB2 domain. ATX increases thrombin-stimulated LPA production by washed platelets ∼10-fold. When incubated under conditions to promote integrin activation, ATX generates LPA from CHO cells primed with bee venom phospholipase A2, and ATX-mediated LPA production is enhanced more than 2-fold by CHO cell overexpression of integrin β3. The effects of ATX on platelet and cell-associated LPA production, but not hydrolysis of small molecule or detergent-solubilized substrates, are attenuated by point mutations in the SMB2 that impair integrin binding. Integrin binding therefore localizes ATX activity to the cell surface, providing a mechanism to generate LPA in the vicinity of its receptors. PMID:21832043

  4. Role of Integrin in Mechanical Loading of Osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, Ruth; Demsky, Caroline

    2000-01-01

    Mechanical forces generated by gravity, weightbearing, and muscle contraction play a key role in the genesis and maintenance of skeletal structure. The molecular mechanisms that mediate changes in osteoblast activity in response to altered patterns of skeletal loading are not known, and a better understanding of these processes may be essential for developing effective treatment strategies to prevent disuse osteoporosis. We have elucidated specific integrin/ECM (extracellular matrix) interactions that are required for osteoblast differentiation and survival and have developed a useful loading system to further explore the molecular basis of mechano-sensitivity of osteoblasts. The long term goal of our collaborative research is to understand how the ECM and cell adhesion proteins and integrins interaction to mediate the response of osteoblasts and their progenitors to mechanical loading. We suggest that integrin/ECM interactions are crucial for basic cellular processes, including differentiation and survival, as well as to participate in detecting and mediating cellular responses to mechanical stimuli.

  5. Semaphorin 7A promotes axon outgrowth through integrins and MAPKs.

    PubMed

    Pasterkamp, R Jeroen; Peschon, Jacques J; Spriggs, Melanie K; Kolodkin, Alex L

    2003-07-24

    Striking parallels exist between immune and nervous system cellular signalling mechanisms. Molecules originally shown to be critical for immune responses also serve neuronal functions, and similarly neural guidance cues can modulate immune function. We show here that semaphorin 7A (Sema7A), a membrane-anchored member of the semaphorin family of guidance proteins previously known for its immunomodulatory effects, can also mediate neuronal functions. Unlike many other semaphorins, which act as repulsive guidance cues, Sema7A enhances central and peripheral axon growth and is required for proper axon tract formation during embryonic development. Unexpectedly, Sema7A enhancement of axon outgrowth requires integrin receptors and activation of MAPK signalling pathways. These findings define a previously unknown biological function for semaphorins, identify an unexpected role for integrins and integrin-dependent intracellular signalling in mediating semaphorin responses, and provide a framework for understanding and interfering with Sema7A function in both immune and nervous systems.

  6. A Small Molecule, Which Competes with MAdCAM-1, Activates Integrin α4β7 and Fails to Prevent Mucosal Transmission of SHIV-SF162P3

    PubMed Central

    Arrode-Brusés, Géraldine; Goode, Diana; Kleinbeck, Kyle; Wilk, Jolanta; Frank, Ines; Byrareddy, Siddappa; Arthos, James; Grasperge, Brooke; Blanchard, James; Zydowsky, Thomas; Gettie, Agegnehu; Martinelli, Elena

    2016-01-01

    Mucosal HIV-1 transmission is inefficient. However, certain viral and host characteristics may play a role in facilitating HIV acquisition and systemic expansion. Cells expressing high levels of integrin α4β7 have been implicated in favoring the transmission process and the infusion of an anti-α4β7 mAb (RM-Act-1) prior to, and during a repeated low-dose vaginal challenge (RLDC) regimen with SIVmac251 reduced SIV acquisition and protected the gut-associated lymphoid tissues (GALT) in the macaques that acquired SIV. α4β7 expression is required for lymphocyte trafficking to the gut lamina propria and gut inductive sites. Several therapeutic strategies that target α4β7 have been shown to be effective in treating inflammatory conditions of the intestine, such as inflammatory bowel disease (IBD). To determine if blocking α4β7 with ELN, an orally available anti-α4 small molecule, would inhibit SHIV-SF162P3 acquisition, we tested its ability to block MAdCAM-1 (α4β7 natural ligand) and HIV-gp120 binding in vitro. We studied the pharmacokinetic profile of ELN after oral and vaginal delivery in macaques. Twenty-six macaques were divided into 3 groups: 9 animals were treated with ELN orally, 9 orally and vaginally and 8 were used as controls. All animals were challenged intra-vaginally with SHIV-SF162P3 using the RLDC regimen. We found that ELN did not protect macaques from SHIV acquisition although it reduced the SHIV-induced inflammatory status during the acute phase of infection. Notably, integrins can exist in different activation states and, comparing the effect of ELN and the anti-α4β7 mAb RM-Act-1 that reduced susceptibility to SIV infection, we determined that ELN induces the active conformation of α4β7, while RM-Act-1 inhibits its activation through an allosteric mechanism. These results suggest that inhibition of α4β7 activation may be necessary to reduce susceptibility to SIV/SHIV infection and highlight the complexity of anti-integrins

  7. Conditional deletion of beta1-integrin from the developing lens leads to loss of the lens epithelial phenotype.

    PubMed

    Simirskii, Vladimir N; Wang, Yan; Duncan, Melinda K

    2007-06-15

    Beta1-integrins are cell surface receptors that participate in sensing the cell's external environment. We used the Cre-lox system to delete beta1-integrin in all lens cells as the lens vesicle transitions into the lens. Adult mice lacking beta1-integrin in the lens are microphthalmic due to apoptosis of the lens epithelium and neonatal disintegration of the lens fibers. The first morphological alterations in beta1-integrin null lenses are seen at 16.5 dpc when the epithelium becomes disorganized and begins to upregulate the fiber cell markers beta- and gamma-crystallins, the transcription factors cMaf and Prox1 and downregulate Pax6 levels demonstrating that beta1-integrin is essential to maintain the lens epithelial phenotype. Furthermore, beta1-integrin null lens epithelial cells upregulate the expression of alpha-smooth muscle actin and nuclear Smad4 and downregulate Smad6 suggesting that beta1-integrin may brake TGFbeta family signaling leading to epithelial-mesenchymal transitions in the lens. In contrast, beta1-integrin null lens epithelial cells show increased E-cadherin immunoreactivity which supports the proposed role of beta1-integrins in mediating complete EMT in response to TGFbeta family members. Thus, beta1-integrin is required to maintain the lens epithelial phenotype and block inappropriate activation of some aspects of the lens fiber cell differentiation program.

  8. Integrin-beta3 clusters recruit clathrin-mediated endocytic machinery in the absence of traction force.

    PubMed

    Yu, Cheng-han; Rafiq, Nisha Bte Mohd; Cao, Fakun; Zhou, Yuhuan; Krishnasamy, Anitha; Biswas, Kabir Hassan; Ravasio, Andrea; Chen, Zhongwen; Wang, Yu-Hsiu; Kawauchi, Keiko; Jones, Gareth E; Sheetz, Michael P

    2015-10-28

    The turnover of integrin receptors is critical for cell migration and adhesion dynamics. Here we find that force development at integrins regulates adaptor protein recruitment and endocytosis. Using mobile RGD (Arg-Gly-Asp) ligands on supported lipid membranes (RGD membranes) and rigid RGD ligands on glass (RGD-glass), we find that matrix force-dependent integrin signals block endocytosis. Dab2, an adaptor protein of clathrin-mediated endocytosis, is not recruited to activated integrin-beta3 clusters on RGD-glass; however, it is recruited to integrin-mediated adhesions on RGD membranes. Further, when force generation is inhibited on RGD-glass, Dab2 binds to integrin-beta3 clusters. Dab2 binding to integrin-beta3 excludes other adhesion-related adaptor proteins, such as talin. The clathrin-mediated endocytic machinery combines with Dab2 to facilitate the endocytosis of RGD-integrin-beta3 clusters. From these observations, we propose that loss of traction force on ligand-bound integrin-beta3 causes recruitment of Dab2/clathrin, resulting in endocytosis of integrins.

  9. Integrin-beta3 clusters recruit clathrin-mediated endocytic machinery in the absence of traction force

    PubMed Central

    Yu, Cheng-han; Rafiq, Nisha Bte Mohd; Cao, Fakun; Zhou, Yuhuan; Krishnasamy, Anitha; Biswas, Kabir Hassan; Ravasio, Andrea; Chen, Zhongwen; Wang, Yu-Hsiu; Kawauchi, Keiko; Jones, Gareth E.; Sheetz, Michael P.

    2015-01-01

    The turnover of integrin receptors is critical for cell migration and adhesion dynamics. Here we find that force development at integrins regulates adaptor protein recruitment and endocytosis. Using mobile RGD (Arg-Gly-Asp) ligands on supported lipid membranes (RGD membranes) and rigid RGD ligands on glass (RGD-glass), we find that matrix force-dependent integrin signals block endocytosis. Dab2, an adaptor protein of clathrin-mediated endocytosis, is not recruited to activated integrin-beta3 clusters on RGD-glass; however, it is recruited to integrin-mediated adhesions on RGD membranes. Further, when force generation is inhibited on RGD-glass, Dab2 binds to integrin-beta3 clusters. Dab2 binding to integrin-beta3 excludes other adhesion-related adaptor proteins, such as talin. The clathrin-mediated endocytic machinery combines with Dab2 to facilitate the endocytosis of RGD-integrin-beta3 clusters. From these observations, we propose that loss of traction force on ligand-bound integrin-beta3 causes recruitment of Dab2/clathrin, resulting in endocytosis of integrins. PMID:26507506

  10. 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.

  11. Detection of an Integrin-Binding Mechanoswitch within Fibronectin during Tissue Formation and Fibrosis.

    PubMed

    Cao, Lizhi; Nicosia, John; Larouche, Jacqueline; Zhang, Yuanyuan; Bachman, Haylee; Brown, Ashley C; Holmgren, Lars; Barker, Thomas H

    2017-07-25

    Fibronectin (Fn) is an extracellular matrix protein that orchestrates complex cell adhesion and signaling through cell surface integrin receptors during tissue development, remodeling, and disease, such as fibrosis. Fn is sensitive to mechanical forces in its tandem type III repeats, resulting in extensive molecular enlongation. As such, it has long been hypothesized that cell- and tissue-derived forces may activate an "integrin switch" within the critical integrin-binding ninth and 10th type III repeats-conferring differential integrin-binding specificity, leading to differential cell responses. Yet, no direct evidence exists to prove the hypothesis nor demonstrate the physiological existence of the switch. We report direct experimental evidence for the Fn integrin switch both in vitro and ex vivo using a scFv engineered to detect the transient, force-induced conformational change, representing an opportunity for detection and targeting of early molecular signatures of cell contractile forces in tissue repair and disease.

  12. Biology and structure of leukocyte β 2 integrins and their role in inflammation

    PubMed Central

    Arnaout, M. Amin

    2016-01-01

    Integrins comprise a large family of αβ heterodimeric cell adhesion receptors that are expressed on all cells except red blood cells and that play essential roles in the regulation of cell growth and function. The leukocyte integrins, which include members of the β 1, β 2, β 3, and β 7 integrin family, are critical for innate and adaptive immune responses but also can contribute to many inflammatory and autoimmune diseases when dysregulated. This review focuses on the β 2 integrins, the principal integrins expressed on leukocytes. We review their discovery and role in host defense, the structural basis for their ligand recognition and activation, and their potential as therapeutic targets. PMID:27781085

  13. Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis

    PubMed Central

    Fan, Zhichao; McArdle, Sara; Marki, Alex; Mikulski, Zbigniew; Gutierrez, Edgar; Engelhardt, Britta; Deutsch, Urban; Ginsberg, Mark; Groisman, Alex; Ley, Klaus

    2016-01-01

    Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high-affinity conformation with an ‘open' headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E−H+ conformation. E−H+ β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation. PMID:27578049

  14. Galectin-1 sensitizes carcinoma cells to anoikis via the fibronectin receptor α5β1-integrin

    PubMed Central

    Sanchez-Ruderisch, H; Detjen, K M; Welzel, M; André, S; Fischer, C; Gabius, H-J; Rosewicz, S

    2011-01-01

    Anoikis resistance is a hallmark of transformed epithelial cells. Here, we show that treatment of anoikis-resistant carcinoma cell lines with the endogenous lectin galectin-1 (Gal-1) promoted apoptosis via interaction with the unligated fibronectin receptor α5β1-integrin. Gal-1 efficiency correlated with expression of α5β1-integrin, and transfection of the α5-subunit into deficient cell lines conferred Gal-1 binding and anoikis stimulation. Furthermore, Gal-1 and the α5- and β1-integrin subunits co-precipitated in Gal-1-stimulated cells undergoing anoikis. Other members of the galectin family failed to be active. The functional interaction between Gal-1 and α5β1-integrin was glycan dependent with α2,6-sialylation representing a switch-off signal. Desialylation of cell surface glycans resulted in increased electrophoretic mobility of α5β1-integrin and facilitated Gal-1 binding and anoikis stimulation. On the level of signaling, Gal-1-stimulated anoikis was prevented by filipin, which impaired the internalization of α5β1-integrin via cholesterol-enriched microdomains, and by pretreatment with a caspase-8 inhibitor. We propose that Gal-1/α5β1-integrin interaction participates in the control of epithelial integrity and integrin sialylation may enable carcinoma cells to evade this Gal-1-dependent control mechanism. PMID:21113146

  15. 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.

  16. 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.

  17. Cell adhesion and integrin expression are modulated by oxidative stress in EA.hy 926 cells.

    PubMed

    Lamari, Foudil; Braut-Boucher, Francoise; Pongnimitprasert, Nushjira; Bernard, Maguy; Foglietti, Marie-Jose; Derappe, Christian; Aubery, Michele

    2007-07-01

    The effects of oxidative stress on integrin-mediated cell adhesion to the extracellular matrix (ECM) and related apoptosis were investigated using the EA.hy926 endothelial cells treated (or not) with two oxidants: the hypoxanthine/xanthine oxidase system (HX/XO) or the tert-butyl hydroperoxide (t-BHP) which both increased cell apoptosis. Cell adhesion onto vitronectin (Vn) and fibronectin (Fn) was increased at low concentrations of HX/XO (up to 5 mU/ml) or t-BHP (up to 125 microM) and prevented ROS-induced apoptosis. Flow cytometry analysis of integrin expression showed that the expression of integrin alphav and alpha5 subunits was, respectively, increased and decreased. Cell adhesion inhibition experiments using function-blocking monoclonal antibodies against integrin subunits indicated that alphavbeta1 and alphavbeta3 integrins were involved in adhesion of cells to Vn, and alphavbeta3 integrin played a major role in oxidant-treated cells. For adhesion to Fn, alpha5beta1 and alphavbeta1 integrins were required for oxidant-treated cells. Taken together, the results suggest that reactive oxygen species (ROS) produced either by HX/XO or t-BHP could affect expression and/or activation of specific integrins in the interaction of EA.hy926 cells with ECM.

  18. Tumor Targeting via Integrin Ligands

    PubMed Central

    Marelli, Udaya Kiran; Rechenmacher, Florian; Sobahi, Tariq Rashad Ali; Mas-Moruno, Carlos; Kessler, Horst

    2013-01-01

    Selective and targeted delivery of drugs to tumors is a major challenge for an effective cancer therapy and also to overcome the side-effects associated with current treatments. Overexpression of various receptors on tumor cells is a characteristic structural and biochemical aspect of tumors and distinguishes them from physiologically normal cells. This abnormal feature is therefore suitable for selectively directing anticancer molecules to tumors by using ligands that can preferentially recognize such receptors. Several subtypes of integrin receptors that are crucial for cell adhesion, cell signaling, cell viability, and motility have been shown to have an upregulated expression on cancer cells. Thus, ligands that recognize specific integrin subtypes represent excellent candidates to be conjugated to drugs or drug carrier systems and be targeted to tumors. In this regard, integrins recognizing the RGD cell adhesive sequence have been extensively targeted for tumor-specific drug delivery. Here we review key recent examples on the presentation of RGD-based integrin ligands by means of distinct drug-delivery systems, and discuss the prospects of such therapies to specifically target tumor cells. PMID:24010121

  19. Alphavbeta integrins play an essential role in BMP-2 induction of osteoblast differentiation.

    PubMed

    Lai, Chung-Fang; Cheng, Su-Li

    2005-02-01

    Both integrins and BMP-2 exert similar effects on osteoblasts. We examined the relationship between the alphav-containing integrins (alphavbeta) and BMP-2 in osteoblast function. BMP-2 stimulates alphavbeta expression. BMP-2 receptors co-localize/overlap with alphavbeta integrins, and the intact function of alphavbeta is essential in BMP-2 activity. Bone morphogenetic protein (BMP)-2 not only induces osteoblast differentiation and bone matrix mineralization, but also stimulates osteoblast migration on and adhesion to bone matrix proteins. The alphavbeta- and beta1- (alphabeta1) containing integrins mediate osteoblast interaction with many bone matrix proteins and play important roles in osteoblast adhesion, migration, and differentiation. Because alphavbeta integrins and BMP-2 share common effects on osteoblasts, we analyzed their relationship in osteoblast function. The effects of BMP-2 on integrin expression were determined by surface labeling/immunoprecipitation and cell adhesion to matrix proteins. Confocal analysis of the immunostained cells and co-immunoprecipitation of cell extracts were used to study the spatial relationship between integrins and BMP-2 receptors. A function-blocking anti-alphavbeta integrin antibody (L230) was employed to investigate the roles of alphavbeta integrins in BMP-2 function. Human osteoblasts (HOBs) express alphabeta1, alphavbeta3, alphavbeta5, alphavbeta6, and alphavbeta8 integrins at focal adhesion sites. BMP-2 increases the levels of these integrins on osteoblast surface and enhances HOB adhesion to osteopontin and vitronectin. Immunoprecipitation and immunostaining analyses show that BMP-2 receptors co-localize or overlap with alphavbeta and alphabeta1 integrins. Incubation of HOBs with L230 abolishes the antiproliferative effect of BMP-2 and reduces the capacity of BMP-2 to stimulate alkaline phosphatase activity and the expression of osteocalcin, osteopontin, and bone sialoprotein. Furthermore, L230 prevents BMP-2 induction

  20. Integrin β1, myosin light chain kinase and myosin IIA are required for activation of PI3K-AKT signaling following MEK inhibition in metastatic triple negative breast cancer

    PubMed Central

    Choi, Cheolwon; Kwon, Junyeob; Lim, Sunyoung; Helfman, David M.

    2016-01-01

    The effectiveness of targeted therapies against the Ras-ERK signaling pathway are limited due to adaptive resistance of tumor cells. Inhibition of the Ras-ERK pathway can result in activation of the PI3K-AKT pathway, thereby diminishing the therapeutic effects of targeting ERK signaling. Here we investigated the crosstalk between the Ras-ERK and PI3K-AKT pathways in MDA-MB-231 breast cancer cell lines that have a preference to metastasize to lung (LM2), brain (BrM2) or bone (BoM2). Inhibition of the Ras-ERK pathway reduced motility in both parental and BoM2 cells. In contrast, inhibition of the Ras-ERK pathway in BrM2 and LM2 cells resulted in activation of PI3K-AKT signaling that was responsible for continued cell motility. Analysis of the cross talk between Ras-ERK and PI3K-AKT signaling pathways revealed integrin β1, myosin light chain kinase (MLCK) and myosin IIA are required for the activation of PI3K-AKT following inhibition of the Ras-ERK pathway. Furthermore, feedback activation of the PI3K-AKT pathway following MEK suppression was independent of the epidermal growth factor receptor. Thus, integrin β1, MLCK, and myosin IIA are factors in the development of resistance to MEK inhibitors. These proteins could provide an opportunity to develop markers and therapeutic targets in a subgroup of triple negative breast cancer (TNBC) that exhibit resistance against MEK inhibition. PMID:27563827

  1. Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

    PubMed Central

    1994-01-01

    Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin. PMID:7507494

  2. Functional Effect of the Mutations Similar to the Cleavage during Platelet Activation at Integrin β3 Cytoplasmic Tail when Expressed in Mouse Platelets

    PubMed Central

    Huang, Jiansong; Long, Zhangbiao; Zhou, Yulan; Liu, Ping; Tao, Lanlan; Ruan, Zheng; Xiao, Bing; Zhang, Wei; Li, Dongya; Dai, Kesheng; Mao, Jianhua; Xi, Xiaodong

    2016-01-01

    Previous studies in Chinese hamster ovary cells showed that truncational mutations of β3 at sites of F754 and Y759 mimicking calpain cleavage regulate integrin signaling. The roles of the sequence from F754 to C-terminus and the conservative N756ITY759 motif in platelet function have yet to be elaborated. Mice expressing β3 with F754 and Y759 truncations, or NITY deletion (β3-ΔTNITYRGT, β3-ΔRGT, or β3-ΔNITY) were established through transplanting the homozygous β3-deficient mouse bone marrow cells infected by the GFP tagged MSCV MigR1 retroviral vector encoding different β3 mutants into lethally radiated wild-type mice. The platelets were harvested for soluble fibrinogen binding and platelet spreading on immobilized fibrinogen. Platelet adhesion on fibrinogen- and collagen-coated surface under flow was also tested to assess the ability of the platelets to resist hydrodynamic drag forces. Data showed a drastic inhibition of the β3-ΔTNITYRGT platelets to bind soluble fibrinogen and spread on immobilized fibrinogen in contrast to a partially impaired fibrinogen binding and an almost unaffected spreading exhibited in the β3-ΔNITY platelets. Behaviors of the β3-ΔRGT platelets were consistent with the previous observations in the β3-ΔRGT knock-in platelets. The adhesion impairment of platelets with the β3 mutants under flow was in different orders of magnitude shown as: β3-ΔTNITYRGT>β3-ΔRGT>β3-ΔNITY to fibrinogen-coated surface, and β3-ΔTNITYRGT>β3-ΔNITY>β3-ΔRGT to collagen-coated surface. To evaluate the interaction of the β3 mutants with signaling molecules, GST pull-down and immunofluorescent assays were performed. Results showed that β3-ΔRGT interacted with kindlin but not c-Src, β3-ΔNITY interacted with c-Src but not kindlin, while β3-ΔTNITYRGT did not interact with both proteins. This study provided evidence in platelets at both static and flow conditions that the calpain cleavage-related sequences of integrin β3, i.e. T755

  3. Modeled microgravity disrupts collagen I/integrin signaling during osteoblastic differentiation of human mesenchymal stem cells.

    PubMed

    Meyers, Valerie E; Zayzafoon, Maid; Gonda, Steve R; Gathings, William E; McDonald, Jay M

    2004-11-01

    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 7 days culture in modeled microgravity (MMG). One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen (Col I)-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix (ECM) proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of MMG on integrin expression and function in hMSC. We demonstrate that 7 days of culture in MMG leads to reduced expression of the ECM protein, Col I. Conversely, MMG consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin subunit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt protein kinase (Akt) is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-mitogen activated protein kinase (MAPK) pathway is evidenced by a reduction in Ras and extracellular signal-related protein kinase (ERK) activation. Taken together, our findings indicate that MMG decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.

  4. Early storage lesions in apheresis platelets are induced by the activation of the integrin αIIbβ₃ and focal adhesion signaling pathways.

    PubMed

    Thiele, Thomas; Iuga, Cristina; Janetzky, Susann; Schwertz, Hansjorg; Gesell Salazar, Manuela; Fürll, Birgit; Völker, Uwe; Greinacher, Andreas; Steil, Leif

    2012-12-05

    Production and storage of platelet concentrates (PC) induce protein changes in platelets leading to impaired platelet function. This study aimed to identify signaling pathways involved in the development of early platelet storage lesions in apheresis-PCs stored in plasma or additive solution (PAS). Apheresis-PCs from four donors were stored in plasma or in PAS at 22°C (n=4 each). Platelets were analyzed at day 0 (production day) and after 1, 6 and 9 days of storage. Platelet response to agonists (TRAP, collagen, ADP) and to hypotonic shock decreased, CD62P expression increased in both storage media over time. Using DIGE 1550 protein spots were monitored and compared to baseline values at day 0. Platelets in plasma displayed changes in 352 spots (166/day 1, 263/day 6 and 201/day 9); in PAS 325 spots changed (202/day 1, 221/day 6, 200/day 9). LC-ESI-MS/MS analysis of 405 platelet proteins revealed 32 proteins changed during storage in plasma (9/day 1, 15/day 6 and 26/day 9) and 28 in PAS (5/day 1, 20/day 6, 26/day 9). Ingenuity pathway analysis found integrin-αII(b)β(3) and focal adhesion signaling pathways involved in early alterations, being confirmed by Western blotting. Corresponding mRNAs in platelets were identified by next generation sequencing for 84 changed proteins. Integrin-αII(b)β(3) and focal adhesion signaling cause irreversible early storage lesions in apheresis platelets. This article is part of a Special Issue entitled: Integrated omics.

  5. The Phosphorylation and Distribution of Cortactin Downstream of Integrin α9β1 Affects Cancer Cell Behaviour

    PubMed Central

    Høye, Anette M.; Couchman, John R.; Wewer, Ulla M.; Yoneda, Atsuko

    2016-01-01

    Integrins, a family of heterodimeric adhesion receptors are implicated in cell migration, development and cancer progression. They can adopt conformations that reflect their activation states and thereby impact adhesion strength and migration. Integrins in an intermediate activation state may be optimal for migration and we have shown previously that fully activated integrin α9β1 corresponds with less migratory behaviour in melanoma cells. Here, we aimed to identify components associated with the activation status of α9β1. Using cancer cell lines with naturally occuring high levels of this integrin, activation by α9β1-specific ligands led to upregulation of fibronectin matrix assembly and tyrosine phosphorylation of cortactin on tyrosine 470 (Y470). Specifically, cortactin phosphorylated on Y470, but not Y421, redistributed together with α9β1 to focal adhesions where active β1 integrin also localises, upon integrin activation. This was commensurate with reduced migration. The localisation and phosphorylation of cortactin Y470 was regulated by Yes kinase and PTEN phosphatase. Cortactin levels influenced fibronectin matrix assembly and active β1 integrin on the cell surface, being inversely correlated with migratory behaviour. This study underlines the complex interplay between cortactin and α9β1 integrin that regulates cell-extracellular matrix interactions. PMID:27339664

  6. 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.

  7. The role of integrin-β/FAK in cyclic mechanical stimulation in MG-63 cells.

    PubMed

    Yang, Min; Xiao, Li-Wei; Liao, Er-Yuan; Wang, Qiao-Jing; Wang, Bei-Bei; Lei, Jia-Xuan

    2014-01-01

    This study aims to explore the function of Integrin-β/FAK in the mechanical signal transduction and the connection with downstream ERK signal pathways. Human osteosarcoma MG63 cell lines were used in this study. The effects of mechanical strain on the Integrin-β₁ expression, FAK and ERK signal pathway in Human osteosarcoma MG63 cells were detected using RT-PCR and Western-blotting methods. The localization of FAK in Human osteosarcoma MG63 cells were determined using immunofluorescent method. The interaction between Integrin-β₁ and FAK were detected by using co-immunoprecipitation method. The expression of Integrin-β₁ shows a notable bimodel distribution, mechanical strain stimulation can promote Integrin-β₁ expression and the phosphorylation of FAK and ERK, mechanical strain activated FAK and ERK mediated by Integrin-β₁. Integrin-β₁ may play an important role in osteoblast proliferation differentiation process, it might feel external strain stimulation through ECM composition and makes FAK phosphated through the interaction with FAK, thus causing a series of activation of signal molecules. Finally it reduces MAPK (ERK) activation and cellular responses to finish mechanical signal transduction.

  8. Integrin αvβ3 and thyroid hormones promote expansion of progenitors in embryonic neocortex.

    PubMed

    Stenzel, Denise; Wilsch-Bräuninger, Michaela; Wong, Fong Kuan; Heuer, Heike; Huttner, Wieland B

    2014-02-01

    Neocortex expansion during evolution is associated with the enlargement of the embryonic subventricular zone, which reflects an increased self-renewal and proliferation of basal progenitors. In contrast to human, the vast majority of mouse basal progenitors lack self-renewal capacity, possibly due to lack of a basal process contacting the basal lamina and downregulation of cell-autonomous production of extracellular matrix (ECM) constituents. Here we show that targeted activation of the ECM receptor integrin αvβ3 on basal progenitors in embryonic mouse neocortex promotes their expansion. Specifically, integrin αvβ3 activation causes an increased cell cycle re-entry of Pax6-negative, Tbr2-positive intermediate progenitors, rather than basal radial glia, and a decrease in the proportion of intermediate progenitors committed to neurogenic division. Interestingly, integrin αvβ3 is the only known cell surface receptor for thyroid hormones. Remarkably, tetrac, a thyroid hormone analog that inhibits the binding of thyroid hormones to integrin αvβ3, completely abolishes the intermediate progenitor expansion observed upon targeted integrin αvβ3 activation, indicating that this expansion requires the binding of thyroid hormones to integrin αvβ3. Convergence of ECM and thyroid hormones on integrin αvβ3 thus appears to be crucial for cortical progenitor proliferation and self-renewal, and hence for normal brain development and the evolutionary expansion of the neocortex.

  9. Antiplatelet activity of Phellinus baummii methanol extract is mediated by cyclic AMP elevation and inhibition of collagen-activated integrin-α(IIb) β₃ and MAP kinase.

    PubMed

    Kamruzzaman, S M; Endale, Mehari; Oh, Won-Jun; Park, Seung-Chun; Kim, Tae-Hwan; Lee, In-Kyoung; Cho, Jae Youl; Park, Hwa-Jin; Kim, Sang Keun; Yun, Bong-Sik; Rhee, Man Hee

    2011-11-01

    Phellinus baumii is a mushroom that has been used as folk medicine against various diseases and is reported to have antidiabetic, anticancer, antioxidant, antiinflammatory and antihypertensive activities. However, information on the effects of P. baumii extract in platelet function is limited. Therefore, the aim of this study was to examine the impact of a P. baumii methanol extract (PBME) on platelet activation and to investigate the mechanism behind its antiplatelet activity. PBME effects on agonist-induced platelet aggregation, granule secretion, [Ca²⁺](i) mobilization, α(IIb) β₃ activation, cyclic AMP release and mitogen-activated protein kinase (MAPK) phosphorylations were studied using rat platelets. PBME dose-dependently inhibited collagen, thrombin and ADP-induced platelet aggregation with an IC₅₀ of 51.0 ± 2.4, 54.0 ± 2.1 and 53.0 ± 4.3 μg/mL, respectively. Likewise, thrombin-induced [Ca²⁺](i) and collagen-activated ATP secretions were suppressed in PBME treated platelets. Aggregation and ATP secretion were also markedly attenuated by PBME alone or in combination with PP2 (Src inhibitor) and U-73122 (PLC inhibitor) in collagen-stimulated platelets. Besides, PBME treatment elevated basal cyclic AMP levels and inhibited collagen-induced integrin-α(IIb) β₃ activation. Moreover, PBME attenuated extracellular-signal-regulated protein kinase 2 (ERK2) and c-Jun N-terminal kinase 1 (JNK1) phosphorylations. Further PD98059 (ERK inhibitor) and SP60025 (JNK inhibitor) reduced collagen-induced platelet aggregation and ATP secretion. In conclusion, the observed PBME antiplatelet activity may be mediated by activation of cyclic AMP and inhibition of ERK2 and JNK1 phosphorylations. Finally, these data suggest that PBME may have therapeutic potential for the treatment of cardiovascular diseases that involve aberrant platelet function. Copyright © 2011 John Wiley & Sons, Ltd.

  10. Transforming Growth Factor-β1 Triggers Hepatocellular Carcinoma Invasiveness via α3β1 Integrin

    PubMed Central

    Giannelli, Gianluigi; Fransvea, Emilia; Marinosci, Felice; Bergamini, Carlo; Colucci, Silvia; Schiraldi, Oronzo; Antonaci, Salvatore

    2002-01-01

    Metastasis occurrence in the course of hepatocellular carcinoma (HCC) severely affects prognosis and survival. We have shown that HCC invasive cells express α3β1-integrin whereas noninvasive cells do not. Here we show that transforming growth factor (TGF)-β1 stimulates α3-integrin expression at a transcriptional level in noninvasive HCC cells, causing transformation into a motile and invasive phenotype. Such activities are inhibited by neutralizing anti-α3- but not anti-α6-integrin monoclonal antibodies. HCC invasive cells secrete abundant levels of active TGF-β1 in comparison with noninvasive cells, but in the latter, addition of active matrix metalloproteinases-2 increases the concentration of active TGF-β1. In this way, the cells express α3-integrin at a transcriptional level and acquire motility on Ln-5. By contrast, an anti-TGF-β1-neutralizing antibody reduces α3-integrin expression and the invasive ability of HCC invading cells. In HCC patients, TGF-β1 serum concentrations and α3-integrin expression are strongly correlated. The integrin, absent in normal and peritumoral liver parenchyma, is abundantly expressed in HCC primary and metastatic tissue. In particular, patients with metastasis show higher levels of TGF-β1 serum concentrations and stronger expression of TGF-β1 and α3-integrin in HCC tissues. In conclusion, TGF-β1 may play an important role in HCC invasiveness by stimulating α3-integrin expression, and could therefore be an important target for new therapies. PMID:12107103

  11. Primary afferent second messenger cascades interact with specific integrin subunits in producing inflammatory hyperalgesia.

    PubMed

    Dina, Olayinka A; Hucho, Tim; Yeh, Jenny; Malik-Hall, Misbah; Reichling, David B; Levine, Jon D

    2005-05-01

    We recently reported that hyperalgesia induced by the inflammatory mediator prostaglandin E(2) (PGE(2)) requires intact alpha1, alpha3 and beta1 integrin subunit function, whereas epinephrine-induced hyperalgesia depends on alpha5 and beta1. PGE(2)-induced hyperalgesia is mediated by protein kinase A (PKA), while epinephrine-induced hyperalgesia is mediated by a combination of PKA, protein kinase Cepsilon (PKCepsilon) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK). We hypothesized that inflammatory mediator-induced hyperalgesia involves specific interactions between different subsets of integrin subunits and particular second messenger species. In the present study, function-blocking anti-integrin antibodies and antisense oligodeoxynucleotides were used to elucidate these interactions in rat. Hyperalgesia produced by an activator of adenylate cyclase (forskolin) depended on alpha1, alpha3 and beta1 integrins. However, hyperalgesia induced by activation of the cascade at a point farther downstream (by cAMP analog or PKA catalytic subunit) was independent of any integrins tested. In contrast, hyperalgesia induced by a specific PKCepsilon agonist depended only on alpha5 and beta1 integrins. Hyperalgesia induced by agonism of MAPK/ERK depended on all four integrin subunits tested (alpha1, alpha3, alpha5 and beta1). Finally, disruption of lipid rafts antagonized hyperalgesia induced by PGE(2) and by forskolin, but not that induced by epinephrine. Furthermore, alpha1 integrin, but not alpha5, was present in detergent-resistant membrane fractions (which retain lipid raft components). These observations suggest that integrins play a critical role in inflammatory pain by interacting with components of second messenger cascades that mediate inflammatory hyperalgesia, and that such interaction with the PGE(2)-activated pathway may be organized by lipid rafts.

  12. Specific crosstalk between EGFR and integrin αvβ5 promotes carcinoma cell invasion and metastasis

    PubMed Central

    Ricono, Jill M.; Huang, Miller; Barnes, Leo A.; Lau, Steven K.; Weis, Sara M.; Schlaepfer, David D.; Hanks, Steven K.; Cheresh, David A.

    2008-01-01

    Tyrosine kinase receptors and integrins play essential roles in tumor cell invasion and metastasis. Previously, we demonstrated that EGF stimulation of pancreatic carcinoma cells leads to invasion and metastasis that was blocked by antagonists of integrin αvβ5. Here we show EGF stimulates metastasis of carcinoma cells via a Src dependent phosphorylation of p130 CAS leading to activation of Rap1, a small GTPase involved in integrin activation. Specifically, EGFR induced Src activity leads to phosphorylation of a region within the CAS substrate domain which is essential for Rap1 and αvβ5 activation. This pathway induces αvβ5-mediated invasion and metastasis in vivo yet does not influence primary tumor growth or activation of other integrins on these cells. These findings demonstrate crosstalk between a tyrosine kinase receptor and an integrin involved in carcinoma cell invasion and metastasis and may explain in part how inhibitors of EGFR impact malignant disease. PMID:19208836

  13. Molecular Basis of the Ligand Binding Specificity of αvβ8 Integrin*

    PubMed Central

    Ozawa, Akio; Sato, Yuya; Imabayashi, Tsukasa; Uemura, Toshihiko; Takagi, Junichi; Sekiguchi, Kiyotoshi

    2016-01-01

    αvβ8 is an integrin that recognizes an Arg-Gly-Asp (RGD) motif and interacts with fibronectin, vitronectin, and latent TGF-β1. We comprehensively determined the binding activity of the αvβ8 integrin toward 25 secreted proteins having an RGD motif. The αvβ8 integrin strongly bound to latent TGF-β1 but showed marginal activity for other RGD-containing proteins, including fibronectin and vitronectin. Site-directed mutagenesis of latent TGF-β1 demonstrated that the high affinity binding of αvβ8 integrin to latent TGF-β1 was defined by Leu-218 immediately following the RGD motif within the latency-associated peptide of TGF-β1. Consistent with the critical role of Leu-218 in latent TGF-β1 recognition by αvβ8 integrin, a 9-mer synthetic peptide containing an RGDL sequence strongly inhibited interactions of latent TGF-β1 with αvβ8 integrin, whereas a 9-mer peptide with an RGDA sequence was ∼60-fold less inhibitory. Because αvβ3 integrin did not exhibit strong binding to latent TGF-β1 or distinguish between RGDL- and RGDA-containing peptides, we explored the mechanism by which the integrin β8 subunit defines the high affinity binding of latent TGF-β1 by αvβ8 integrin. Production of a series of swap mutants of integrin β8 and β3 subunits indicated that the high affinity binding of αvβ8 integrin with latent TGF-β1 was ensured by interactions between the Leu-218 residue and the β8 I-like domain, with the former serving as an auxiliary recognition residue defining the restricted ligand specificity of αvβ8 integrin toward latent TGF-β1. In support of this conclusion, high affinity binding toward the αvβ8 integrin was conferred on fibronectin by substitution of its RGDS motif with an RGDL sequence. PMID:27033701

  14. Mixed Extracellular Matrix Ligands Synergistically Modulate Integrin Adhesion and Signaling

    PubMed Central

    Reyes, Catherine D.; Petrie, Timothy A.; García, Andrés J

    2008-01-01

    Cell adhesion to extracellular matrix (ECM) components through cell-surface integrin receptors is essential to the formation, maintenance and repair of numerous tissues, and therefore represents a central theme in the design of bioactive materials that successfully interface with the body. While the adhesive responses associated with a single ligand have been extensively analyzed, the effects of multiple integrin subtypes binding to multivalent ECM signals remain poorly understood. In the present study, we generated a high throughput platform of non-adhesive surfaces presenting well-defined, independent densities of two integrin-specific engineered ligands for the type I collagen (COL-I) receptor α2β1 and the fibronectin (FN) receptor α5β1 to evaluate the effects of integrin cross-talk on adhesive responses. Engineered surfaces displayed ligand density-dependent adhesive effects, and mixed ligand surfaces significantly enhanced cell adhesion strength and focal adhesion assembly compared to single FN and COL-I ligand surfaces. Moreover, surfaces presenting mixed COL-I/FN ligands synergistically enhanced FAK activation compared to the single ligand substrates. The enhanced adhesive activities of the mixed ligand surfaces also promoted elevated proliferation rates. Our results demonstrate interplay between multivalent ECM ligands in adhesive responses and downstream cellular signaling. PMID:18613064

  15. Integrins control motile strategy through a Rho–cofilin pathway

    PubMed Central

    Danen, Erik H.J.; van Rheenen, Jacco; Franken, Willeke; Huveneers, Stephan; Sonneveld, Petra; Jalink, Kees; Sonnenberg, Arnoud

    2005-01-01

    During wound healing, angiogenesis, and tumor invasion, cells often change their expression profiles of fibronectin-binding integrins. Here, we show that β1 integrins promote random migration, whereas β3 integrins promote persistent migration in the same epithelial cell background. Adhesion to fibronectin by αvβ3 supports extensive actin cytoskeletal reorganization through the actin-severing protein cofilin, resulting in a single broad lamellipod with static cell–matrix adhesions at the leading edge. Adhesion by α5β1 instead leads to the phosphorylation/inactivation of cofilin, and these cells fail to polarize their cytoskeleton but extend thin protrusions containing highly dynamic cell–matrix adhesions in multiple directions. The activity of the small GTPase RhoA is particularly high in cells adhering by α5β1, and inhibition of Rho signaling causes a switch from a β1- to a β3-associated mode of migration, whereas increased Rho activity has the opposite effect. Thus, alterations in integrin expression profiles allow cells to modulate several critical aspects of the motile machinery through Rho GTPases. PMID:15866889

  16. Soluble CD40 ligand stimulates CD40-dependent activation of the β2 integrin Mac-1 and protein kinase C zeda (PKCζ) in neutrophils: implications for neutrophil-platelet interactions and neutrophil oxidative burst.

    PubMed

    Jin, Rong; Yu, Shiyong; Song, Zifang; Zhu, Xiaolei; Wang, Cuiping; Yan, Jinchuan; Wu, Fusheng; Nanda, Anil; Granger, D Neil; Li, Guohong

    2013-01-01

    Recent work has revealed an essential involvement of soluble CD40L (sCD40L) in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40), i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ) is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b) and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better understanding of the

  17. 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.

  18. Integrins as architects of cell behavior

    PubMed Central

    Streuli, Charles H.

    2016-01-01

    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

  19. 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.

  20. Akt1 mediates prostate cancer cell microinvasion and chemotaxis to metastatic stimuli via integrin β3 affinity modulation

    PubMed Central

    Goc, A; Liu, J; Byzova, T V; Somanath, P R

    2012-01-01

    Background: Activation of Akt and increased expression of integrin β3 are the two most important changes that have been linked to the attainment of metastatic potential by prostate cancer cells. However, a direct link between Akt activity and inside-out activation of integrin β3 in mediating prostate cancer cell metastatic properties is not established. Methods: Using functional and biochemical approaches, we examined the role of Akt1 in the affinity modulation of integrin β3 in prostate cancer cells. Results: Although expression of murine TRAMP and human PC3 cells with constitutively active Akt1 (CA-Akt1) enhanced their affinity for integrin αvβ3 specific ligands and motility on various extracellular matrix proteins, the reverse was observed with the expression of dominant-negative Akt1 (DN-Akt1). Although enhanced motility and transendothelial migration of CA-Akt1-expressing cells were blunted by co-expression with DN-integrin β3 or upon pre-treatment with integrin β3-blocking antibodies (LM 609), impaired motility and transendothelial migration of DN-Akt1-expressing cells were rescued by pre-treatment of prostate cancer cells with integrin β3-activating antibodies, AP7.4. Conclusion: Our data is the first to demonstrate a link between Akt1 activity and affinity modulation of integrin β3 in the regulation of prostate cancer cell motility, transendothelial migration and chemotaxis to metastatic stimuli. PMID:22767145

  1. 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

  2. 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.

  3. The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases

    PubMed Central

    1995-01-01

    Interaction of cells with extracellular matrix via integrin adhesion receptors plays an important role in a wide range of cellular: functions, for example cell growth, movement, and differentiation. Upon interaction with substrate, integrins cluster and associate with a variety of cytoplasmic proteins to form focal complexes and with the actin cytoskeleton. Although the intracellular signals induced by integrins are at present undefined, it is thought that they are mediated by proteins recruited to the focal complexes. It has been suggested, for example, that after recruitment to focal adhesions p125FAK can activate the ERK1/2 MAP kinase cascade. We have previously reported that members of the rho family of small GTPases can trigger the assembly of focal complexes when activated in cells. Using microinjection techniques, we have now examined the role of the extracellular matrix and of the two GTP-binding proteins, rac and rho, in the assembly of integrin complexes in both mouse and human fibroblasts. We find that the interaction of integrins with extracellular matrix alone is not sufficient to induce integrin clustering and focal complex formation. Similarly, activation of rho or rac by extracellular growth factors does not lead to focal complex formation in the absence of matrix. Focal complexes are only assembled in the presence of both matrix and functionally active members of the rho family. In agreement with this, the interaction of integrins with matrix in the absence of rho/rac activity is unable to activate the ERK1/2 kinases in Swiss 3T3 cells. In fact, ERK1/2 can be activated fully by growth factors in the absence of matrix and it seems unlikely, therefore, that the adhesion dependence of fibroblast growth is mediated through the ras/MAP kinase pathway. We conclude that extracellular matrix is not sufficient to trigger focal complex assembly and subsequent integrin-dependent signal transduction in the absence of functionally active members of the rho

  4. Macrophage integrins modulate response to ultra-high molecular weight polyethylene particles and direct particle-induced osteolysis.

    PubMed

    Zaveri, Toral D; Dolgova, Natalia V; Lewis, Jamal S; Hamaker, Kiri; Clare-Salzler, Michael J; Keselowsky, Benjamin G

    2017-01-01

    Aseptic loosening due to peri-prosthetic osteolysis is one of the primary causes for failure of artificial joint replacements. Implant-derived wear particles, often ultra-high molecular weight polyethylene (UHMWPE) microparticles, initiate an inflammatory cascade upon phagocytosis by macrophages, which leads to osteoclast recruitment and activation, ultimately resulting in osteolysis. Investigation into integrin receptors, involved in cellular interactions with biomaterial-adsorbed adhesive proteins, is of interest to understand and modulate inflammatory processes. In this work, we investigate the role of macrophage integrins Mac-1 and RGD-binding integrins in response to UHMWPE wear particles. Using integrin knockout mice as well as integrin blocking techniques, reduction in macrophage phagocytosis and inflammatory cytokine secretion is demonstrated when these receptors are either absent or blocked. Along this line, various opsonizing proteins are shown to differentially modulate microparticle uptake and macrophage secretion of inflammatory cytokines. Furthermore, using a calvarial osteolysis model it is demonstrated that both Mac-1 integrin and RGD-binding integrins modulate the particle induced osteolysis response to UHMWPE microparticles, with a 40% decrease in the area of osteolysis by the absence or blocking of these integrins, in vivo. Altogether, these findings indicate Mac-1 and RGD-binding integrins are involved in macrophage-directed inflammatory responses to UHMWPE and may serve as therapeutic targets to mitigate wear particle induced peri-prosthetic osteolysis for improved performance of implanted joints.

  5. 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.

  6. 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.

  7. CW-EPR studies revealed different motional properties and oligomeric states of the integrin β1a transmembrane domain in detergent micelles or liposomes

    PubMed Central

    Yu, Lu; Wang, Wei; Ling, Shenglong; Liu, Sanling; Xiao, Liang; Xin, Yanlong; Lai, Chaohua; Xiong, Ying; Zhang, Longhua; Tian, Changlin

    2015-01-01

    Integrins are heterodimeric membrane proteins that regulate essential processes: cell migration, cell growth, extracellular matrix assembly and tumor metastasis. Each integrin α or β subunit contains a large extracellular domain, a single transmembrane (TM) domain, and a short cytoplasmic tail. The integrin TM domains are important for heterodimeric association and dissociation during the conversion from inactive to active states. Moreover, integrin clustering occurs by homo-oligomeric interactions between the TM helices. Here, the transmembrane and cytoplasmic (TMC) domains of integrin β1a were overexpressed, and the protein was purified in detergent micelles and/or reconstituted in liposomes. To investigate the TM domain conformational properties of integrin β1a, 26 consecutive single cysteine mutants were generated for site-directed spin labeling and continuous-wave electron paramagnetic resonance (CW-EPR) mobility and accessibility analyses. The mobility analysis identified two integrin β1a-TM regions with different motional properties in micelles and a non-continuous integrin β1a-TM helix with high immobility in liposomes. The accessibility analysis verified the TM range (Val737-Lys752) of the integrin β1a-TMC in micelles. Further mobility and accessibility comparisons of the integrin β1a-TMC domains in micelles or liposomes identified distinctively different oligomeric states of integrin β1a-TM, namely a monomer embedded in detergent micelles and leucine-zipper-like homo-oligomeric clusters in liposomes. PMID:25597475

  8. CCM proteins control endothelial β1 integrin dependent response to shear stress

    PubMed Central

    Macek Jilkova, Zuzana; Lisowska, Justyna; Manet, Sandra; Verdier, Claude; Deplano, Valerie; Geindreau, Christian; Faurobert, Eva; Albigès-Rizo, Corinne; Duperray, Alain

    2014-01-01

    ABSTRACT Hemodynamic shear stress from blood flow on the endothelium critically regulates vascular function in many physiological and pathological situations. Endothelial cells adapt to shear stress by remodeling their cytoskeletal components and subsequently by changing their shape and orientation. We demonstrate that β1 integrin activation is critically controlled during the mechanoresponse of endothelial cells to shear stress. Indeed, we show that overexpression of the CCM complex, an inhibitor of β1 integrin activation, blocks endothelial actin rearrangement and cell reorientation in response to shear stress similarly to β1 integrin silencing. Conversely, depletion of CCM2 protein leads to an elongated “shear-stress-like” phenotype even in the absence of flow. Taken together, our findings reveal the existence of a balance between positive extracellular and negative intracellular signals, i.e. shear stress and CCM complex, for the control of β1 integrin activation and subsequent adaptation of vascular endothelial cells to mechanostimulation by fluid shear stress. PMID:25432514

  9. Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

    PubMed Central

    Morgan, Mark R.; Hamidi, Hellyeh; Bass, Mark D.; Warwood, Stacey; Ballestrem, Christoph; Humphries, Martin J.

    2013-01-01

    Summary Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration. PMID:23453597

  10. Syndecan-4 phosphorylation is a control point for integrin recycling.

    PubMed

    Morgan, Mark R; Hamidi, Hellyeh; Bass, Mark D; Warwood, Stacey; Ballestrem, Christoph; Humphries, Martin J

    2013-03-11

    Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration.

  11. β2-Integrins in demyelinating disease: not adhering to the paradigm

    PubMed Central

    Hu, Xianzhen; Wohler, Jillian E.; Dugger, Kari J.; Barnum, Scott R.

    2010-01-01

    The β2-integrins are a subfamily of integrins expressed on leukocytes that play an essential role in leukocyte trafficking, activation, and many other functions. Studies in EAE, the animal model for multiple sclerosis, show differential requirements for β2-integrins in this disease model, ranging from critical in the case of LFA-1 (CD11a/CD18) to unimportant in the case of CD11d/CD18. Importantly, expression of β2-integrins on T cell subsets provides some clues as to the function(s) these adhesion molecules play in disease development. For example, transferred EAE studies have shown that Mac-1 (CD11b/CD18) expression on αβ T cells is critical for disease development, and the absence of LFA-1 on Tregs in recipient mice results in exacerbated disease. In this review, we summarize recent findings regarding the role of β2-integrins in demyelinating disease and new information about the role of β2-integrins with respect to alterations in Treg numbers and function. In addition, we discuss the potential for targeting β2-integrins in human demyelinating disease in light of the recent animal model studies. PMID:20007244

  12. beta2-integrins in demyelinating disease: not adhering to the paradigm.

    PubMed

    Hu, Xianzhen; Wohler, Jillian E; Dugger, Kari J; Barnum, Scott R

    2010-03-01

    The beta(2)-integrins are a subfamily of integrins expressed on leukocytes that play an essential role in leukocyte trafficking, activation, and many other functions. Studies in EAE, the animal model for multiple sclerosis, show differential requirements for beta(2)-integrins in this disease model, ranging from critical in the case of LFA-1 (CD11a/CD18) to unimportant in the case of CD11d/CD18. Importantly, expression of beta(2)-integrins on T cell subsets provides some clues as to the function(s) these adhesion molecules play in disease development. For example, transferred EAE studies have shown that Mac-1 (CD11b/CD18) expression on alphabeta T cells is critical for disease development, and the absence of LFA-1 on Tregs in recipient mice results in exacerbated disease. In this review, we summarize recent findings regarding the role of beta(2)-integrins in demyelinating disease and new information about the role of beta(2)-integrins with respect to alterations in Treg numbers and function. In addition, we discuss the potential for targeting beta(2)-integrins in human demyelinating disease in light of the recent animal model studies.

  13. Compensatory redistribution of neuroligins and N-cadherin following deletion of synaptic β1-integrin

    PubMed Central

    Mortillo, Steven; Elste, Alice; Ge, Yongchao; Patil, Shekhar B.; Hsiao, Kuangfu; Huntley, George W.; Davis, Ronald L.; Benson, Deanna L.

    2012-01-01

    β1-containing integrins are required for persistent synaptic potentiation in hippocampus and regulate hippocampal-dependent learning. Based largely on indirect evidence, there is a prevailing assumption that β1-integrins are localized at synapses, where they contribute to synapse adhesion and signaling, but this has not been examined directly. Here, we investigate the fine localization of β1-integrin in adult mouse hippocampus using high-resolution immunogold labeling, with a particular emphasis on synaptic labeling patterns. We find that β1-integrins localize to synapses in CA1 and are concentrated postsynaptically. At the postsynaptic membrane, β1-integrins are found more commonly clustered near active zone centers rather than at the peripheral edges. In mice harboring a conditional deletion of β1-integrins, labeling for N-cadherin and Neuroligins increases. Western blots show increased levels of N-cadherin in total lysates and Neuroligins increase selectively in synaptosomes. These data suggest there is a dynamic, compensatory adjustment of synaptic adhesion. Such adjustment is specific only for certain cell adhesion molecules (CAMs), because labeling for SynCAM is unchanged. Together our findings demonstrate unequivocally that β1-integrin is an integral synaptic adhesion protein, and suggest that adhesive function at the synapse reflects a cooperative and dynamic network of multiple CAM families. PMID:22488504

  14. αvβ3 integrins negatively regulate cellular forces by phosphorylation of its distal NPXY site.

    PubMed

    Milloud, Rachel; Destaing, Olivier; de Mets, Richard; Bourrin-Reynard, Ingrid; Oddou, Christiane; Delon, Antoine; Wang, Irène; Albigès-Rizo, Corinne; Balland, Martial

    2017-03-01

    Integrins are key receptors that allow cells to sense and respond to their mechanical environment. Although they bind the same ligand, β1 and β3 integrins have distinct and cooperative roles in mechanotransduction. Using traction force microscopy on unconstrained cells, we show that deleting β3 causes traction forces to increase, whereas the deletion of β1 integrin results in a strong decrease of contractile forces. Consistently, loss of β3 integrin also induces an increase in β1 integrin activation. Using a genetic approach, we identified the phosphorylation of the distal NPXY domain as an essential process for β3 integrin to be able to modulate traction forces. Loss of β3 integrins also impacted cell shape and the spatial distribution of traction forces, by causing forces to be generated closer to the cell edge, and the cell shape. Our results emphasize the role of β3 integrin in spatial distribution of cellular forces. We speculate that, by modulating its affinity with kindlin, β3 integrins may be able to locate near the cell edge where it can control β1 integrin activation and clustering. Tensional homeostasis at the single cell level is performed by the ability of β3 adhesions to negatively regulate the activation degree and spatial localization of β1 integrins. By combining genetic approaches and new tools to analyze traction distribution and cell morphology on a population of cells we were able to identify the molecular partners involved in cellular forces regulation. © 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  15. Integrin binding angiopoietin-1 monomers reduce cardiac hypertrophy

    PubMed Central

    Dallabrida, Susan M.; Ismail, Nesreen S.; Pravda, Elke A.; Parodi, Emily M.; Dickie, Renee; Durand, Ellen M.; Lai, Jean; Cassiola, Flavia; Rogers, Rick A.; Rupnick, Maria A.

    2008-01-01

    Angiopoietins were thought to be endothelial cell-specific via the tie2 receptor. We showed that angiopoietin-1 (ang1) also interacts with integrins on cardiac myocytes (CMs) to increase survival. Because ang1 monomers bind and activate integrins (not tie2), we determined their function in vivo. We examined monomer and multimer expressions during physiological and pathological cardiac remodeling and overexpressed ang1 monomers in phenylephrine-induced cardiac hypertrophy. Cardiac ang1 levels (mRNA, protein) increased during postnatal development and decreased with phenylephrine-induced cardiac hypertrophy, whereas tie2 phosphorylations were unchanged. We found that most or all of the changes during cardiac remodeling were in monomers, offering an explanation for unchanged tie2 activity. Heart tissue contains abundant ang1 monomers and few multimers (Western blotting). We generated plasmids that produce ang1 monomers (ang1–256), injected them into mice, and confirmed cardiac expression (immunohistochemistry, RT-PCR). Ang1 monomers localize to CMs, smooth muscle cells, and endothelial cells. In phenylephrine-induced cardiac hypertrophy, ang1–256 reduced left ventricle (LV)/tibia ratios, fetal gene expressions (atrial and brain natriuretic peptides, skeletal actin, β-myosin heavy chain), and fibrosis (collagen III), and increased LV prosurvival signaling (akt, MAPKp42/44), and AMPKT172. However, tie2 phosphorylations were unchanged. Ang1–256 increased integrin-linked kinase, a key regulator of integrin signaling and cardiac health. Collectively, these results suggest a role for ang1 monomers in cardiac remodeling.—Dallabrida, S. M., Ismail, N. S., Pravda, E. A., Parodi, E. M., Dickie, R., Durand, E. M., Lai, J., Cassiola, F., Rogers, R. A., Rupnick, M. A. Integrin binding angiopoietin-1 monomers reduce cardiac hypertrophy. PMID:18502941

  16. The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin gamma chains in integrin binding by laminins.

    PubMed

    Ido, Hiroyuki; Nakamura, Aya; Kobayashi, Reiko; Ito, Shunsuke; Li, Shaoliang; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2007-04-13

    Laminins are the major cell-adhesive proteins in the basement membrane, consisting of three subunits termed alpha, beta, and gamma. The putative binding site for integrins has been mapped to the G domain of the alpha chain, although trimerization with beta and gamma chains is necessary for the G domain to exert its integrin binding activity. The mechanism underlying the requirement of beta and gamma chains in integrin binding by laminins remains poorly understood. Here, we show that the C-terminal region of the gamma chain is involved in modulation of the integrin binding activity of laminins. We found that deletion of the C-terminal three but not two amino acids within the gamma1 chain completely abrogated the integrin binding activity of laminin-511. Furthermore, substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the gamma1 chain, also abolished the integrin binding activity, underscoring the role of Glu-1607 in integrin binding by the laminin. We also found that the conserved Glu residue of the gamma2 chain is necessary for integrin binding by laminin-332, suggesting that the same mechanism operates in the modulation of the integrin binding activity of laminins containing either gamma1 or gamma2 chains. However, the peptide segment modeled after the C-terminal region of gamma1 chain was incapable of either binding to integrin or inhibiting integrin binding by laminin-511, making it unlikely that the Glu residue is directly recognized by integrin. These results, together, indicate a novel mechanism operating in ligand recognition by laminin binding integrins.

  17. Involvement of proline-rich tyrosine kinase 2 in platelet activation: tyrosine phosphorylation mostly dependent on alphaIIbbeta3 integrin and protein kinase C, translocation to the cytoskeleton and association with Shc through Grb2.

    PubMed Central

    Ohmori, T; Yatomi, Y; Asazuma, N; Satoh, K; Ozaki, Y

    2000-01-01

    Proline-rich tyrosine kinase 2 (Pyk2) (also known as RAFTK, CAKbeta or CADTK) has been identified as a member of the focal adhesion kinase (FAK) family of protein-tyrosine kinases and it has been suggested that the mode of Pyk2 activation is distinct from that of FAK. In the present study we investigated the mode of Pyk2 activation in human platelets. When platelets were stimulated with thrombin, Pyk2, as well as FAK, was markedly tyrosine-phosphorylated, in a manner mostly dependent on alphaIIbbeta3 integrin-mediated aggregation. The residual Pyk2 tyrosine phosphorylation observed in the absence of platelet aggregation was completely abolished by pretreatment with BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester]. The Pyk2 phosphorylation was inhibited by protein kinase C (PKC) inhibitors at concentrations that inhibited platelet aggregation. In contrast, direct activation of PKC with the active phorbol ester PMA induced the tyrosine phosphorylation of Pyk2 and FAK but only when platelets were fully aggregated with the exogenous addition of fibrinogen (the ligand for alphaIIbbeta3 integrin). Furthermore, PMA-induced Pyk2 (and FAK) tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. The activation of the von Willebrand factor (vWF)--glycoprotein Ib pathway with botrocetin together with vWF failed to induce Pyk2 (and FAK) tyrosine phosphorylation. Most Pyk2 and FAK was present in the cytosol and membrane skeleton fractions in unstimulated platelets. When platelets were stimulated with thrombin, both Pyk2 and FAK were translocated to the cytoskeleton in an aggregation-dependent manner. In immunoprecipitation studies, Pyk2, as well as FAK, seemed to associate with Shc through Grb2. With the use of glutathione S-transferase fusion proteins containing Shc-SH2, Grb2-SH2, and Grb2 N-terminal and C-terminal SH3 domains, it was implied that the proline-rich region of Pyk2 (and FAK) binds to the N

  18. An agent based model of integrin clustering: Exploring the role of ligand clustering, integrin homo-oligomerization, integrin-ligand affinity, membrane crowdedness and ligand mobility

    NASA Astrophysics Data System (ADS)

    Jamali, Yousef; Jamali, Tahereh; Mofrad, Mohammad R. K.

    2013-07-01

    Integrins are cell-surface protein heterodimers that coordinate cellular responses to mechanochemical cues from the extracellular matrix (ECM) and stimulate the assembly of small adhesion complexes, which are the initial sites of cell-ECM adhesion. Clustering of integrins is known to mediate signaling through a variety of signal transduction pathways. Yet, the molecular mechanisms of integrin clustering are poorly understood. In this paper, we develop computational models, using agent based modeling (ABM) techniques, to explore two key underlying mechanisms of integrin clustering, namely ligand organization and integrin homo-oligomerization. Our models help to shed light on the potential roles ligand clustering and integrin homo-oligomerization may play in controlling integrin clustering. A potential mechanism for the clustering of integrin is discussed and the effects of other parameters such as integrin-ligand affinity, membrane crowdedness and ligand mobility on integrin clustering are examined.

  19. A dual role for integrin-linked kinase in platelets: regulating integrin function and α-granule secretion

    PubMed Central

    Sage, Tanya; Stevens, Joanne M.; Jordan, Peter A.; Jones, Sarah; Barrett, Natasha E.; St-Arnaud, Rene; Frampton, Jonathan; Dedhar, Shoukat; Gibbins, Jonathan M.

    2008-01-01

    Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with β1- and β3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLCγ2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in α-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for α-granule secretion and therefore may play a central role in the regulation of platelet function. PMID:18772455

  20. Laminin isoforms and their integrin receptors in glioma cell migration and invasiveness: Evidence for a role of alpha5-laminin(s) and alpha3beta1 integrin.

    PubMed

    Kawataki, Tomoyuki; Yamane, Tetsu; Naganuma, Hirofumi; Rousselle, Patricia; Andurén, Ingegerd; Tryggvason, Karl; Patarroyo, Manuel

    2007-11-01

    Glioma cell infiltration of brain tissue often occurs along the basement membrane (BM) of blood vessels. In the present study we have investigated the role of laminins, major structural components of BMs and strong promoters of cell migration. Immunohistochemical studies of glioma tumor tissue demonstrated expression of alpha2-, alpha3-, alpha4- and alpha5-, but not alpha1-, laminins by the tumor vasculature. In functional assays, alpha3 (Lm-332/laminin-5)- and alpha5 (Lm-511/laminin-10)-laminins strongly promoted migration of all glioma cell lines tested. alpha1-Laminin (Lm-111/laminin-1) displayed lower activity, whereas alpha2 (Lm-211/laminin-2)- and alpha4 (Lm-411/laminin-8)-laminins were practically inactive. Global integrin phenotyping identified alpha3beta1 as the most abundant integrin in all the glioma cell lines, and this laminin-binding integrin exclusively or largely mediate the cell migration. Moreover, pretreatment of U251 glioma cells with blocking antibodies to alpha3beta1 integrin followed by intracerebral injection into nude mice inhibited invasion of the tumor cells into the brain tissue. The cell lines secreted Lm-211, Lm-411 and Lm-511, at different ratios. The results indicate that glioma cells secrete alpha2-, alpha4- and alpha5-laminins and that alpha3- and alpha5-laminins, found in brain vasculature, selectively promote glioma cell migration. They identify alpha3beta1 as the predominant integrin and laminin receptor in glioma cells, and as a brain invasion-mediating integrin.

  1. Role of β7 integrins in intestinal lymphocyte homing and retention

    PubMed Central

    Gorfu, Gezahegn; Rivera-Nieves, Jesús; Ley, Klaus

    2009-01-01

    Lymphocytes involved in intestinal immune response are found in organized immune inductive sites of the gut-associated lymphoid tissues (GALT) such as Peyer’s patches (PP), mesenteric lymph nodes (MLN) and diffuse effector sites of gut epithelium and lamina propria (LP). β7 integrins are responsible for efficient trafficking and retention of lymphocytes in these sites. Naïve and effector lymphocytes use α4β7 integrin to extravasate from blood to gut mucosal tissues of GALT, MLN and LP via interactions with Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1). The αEβ7 integrin facilitates retention of effector and memory lymphocytes in the gut epithelial layer via interactions with E-cadherin. Mucosal dendritic cells (DCs) regulate the expression of the gut homing receptors α4β7 integrin and the chemokine receptor CCR9 on activated effector and regulatory lymphocytes in a retinoic acid-dependent manner. CD103 (αE integrin) identifies a subset of mucosal DCs in MLN and small intestine LP that have an enhanced ability to induce gut-tropic receptors on responding lymphocytes. The interactions between β7 integrin and their ligands are also implicated in the pathogenesis and progression of inflammatory bowel diseases (IBDs), intestinal parasitic infections and graft-versus-host diseases. During intestinal inflammation, β7 integrin-dependent and -independent pathways contribute to lymphocytes recruitment to the intestinal tissues and disease pathogenesis. Recent works have explored the potential of therapeutic targeting of α4 and β7 integrins in IBDs. Here, we review the current understanding of the role of β7 integrins in intestinal lymphocyte trafficking and retention in health and disease. PMID:19860663

  2. Spatiotemporal segregation of endothelial cell integrin and nonintegrin extracellular matrix-binding proteins during adhesion events

    PubMed Central

    1990-01-01

    Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine- aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important

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

    PubMed

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

    2014-05-02

    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.

  4. Fundamentally different roles for LFA-1, Mac-1 and alpha4-integrin in neutrophil chemotaxis.

    PubMed

    Heit, Bryan; Colarusso, Pina; Kubes, Paul

    2005-11-15

    Although the LFA-1, Mac-1 and alpha(4) integrins are required for chemotaxis, it is unknown how they are regulated or what specific role they play. Previously we demonstrated that fMLP and IL-8 induce chemotaxis via the p38 MAPK and phosphoinositide 3-kinase (PI3K) pathways, respectively. Here we show that these chemoattractants also activate and use Mac-1 and LFA-1 in a differential manner during chemotaxis. Using integrin-specific substrata, we demonstrate that cell movement in response to IL-8 is mediated by Mac-1, whereas LFA-1 is required for directional migration. By contrast, chemotaxis to fMLP requires Mac-1 for cell movement, whereas LFA-1 and alpha(4)-integrin are required for directional migration. On serum protein, which contains ligands for LFA-1, Mac-1 and alpha(4)-integrin, chemotaxis to fMLP is dependent on Mac-1, whereas chemotaxis to IL-8 is dependent on LFA-1. These results suggest that Mac-1 is the dominant integrin involved in chemotaxis to fMLP, and LFA-1 is the dominant integrin involved in chemotaxis to IL-8. Consistent with these observations, higher quantities of high-affinity Mac-1 are found on cells chemotaxing to fMLP then on cells chemotaxing to IL-8. Moreover, a much larger quantity of clustered LFA-1 was found on cells migrating to IL-8 compared to cells moving towards fMLP. When cells are presented with competing gradients of fMLP and IL-8, they preferentially migrate towards fMLP and activate/utilize integrins in a manner identical to fMLP alone. Under the same conditions, p38 MAPK inhibition abolishes the preferential migration to fMLP; instead, the cells migrate preferentially towards IL-8. The activation and utilization of integrins under these conditions are consistent with patterns observed with IL-8 alone. Together, these data suggest that fMLP and IL-8 differentially activate integrins for use during chemotaxis, that p38 MAPK is a major mediator in the activation and utilization of integrins, and selective integrin

  5. Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase

    PubMed Central

    1995-01-01

    Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti- phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas

  6. Probing the acidic residue within the integrin binding site of laminin-511 that interacts with the metal ion-dependent adhesion site of α6β1 integrin.

    PubMed

    Taniguchi, Yukimasa; Li, Shaoliang; Takizawa, Mamoru; Oonishi, Eriko; Toga, Junko; Yagi, Emiko; Sekiguchi, Kiyotoshi

    2017-06-03

    Laminins are major cell-adhesive proteins of basement membranes that interact with integrins in a divalent cation-dependent manner. Laminin-511 consists of α5, β1, and γ1 chains, of which three laminin globular domains of the α5 chain (α5/LG1-3) and a Glu residue in the C-terminal tail of chain γ1 (γ1-Glu1607) are required for binding to integrins. However, it remains unsettled whether the Glu residue in the γ1 tail is involved in integrin binding by coordinating the metal ion in the metal ion-dependent adhesion site of β1 integrin (β1-MIDAS), or by stabilizing the conformation of α5/LG1-3. To address this issue, we examined whether α5/LG1-3 contain an acidic residue required for integrin binding that is as critical as the Glu residue in the γ1 tail; to achieve this, we undertook exhaustive alanine substitutions of the 54 acidic residues present in α5/LG1-3 of the E8 fragment of laminin-511 (LM511E8). Most of the alanine mutants possessed α6β1 integrin binding activities comparable with wild-type LM511E8. Alanine substitution for α5-Asp3198 and Asp3219 caused mild reduction in integrin binding activity, and that for α5-Asp3218 caused severe reduction, possibly resulting from conformational perturbation of α5/LG1-3. When α5-Asp3218 was substituted with asparagine, the resulting mutant possessed significant binding activity to α6β1 integrin, indicating that α5-Asp3218 is not directly involved in integrin binding through coordination with the metal ion in β1-MIDAS. Given that substitution of γ1-Glu1607 with glutamine nullified the binding activity to α6β1 integrin, these results, taken together, support the possibility that the critical acidic residue coordinating the metal ion in β1-MIDAS is Glu1607 in the γ1 tail, but no such residue is present in α5/LG1-3. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Multiple non-catalytic ADAMs are novel integrin α4 ligands.

    PubMed

    Wang, Lei; Hoggard, Jason A; Korleski, Erica D; Long, Gideon V; Ree, Brandy C; Hensley, Kenneth; Bond, Stephen R; Wolfsberg, Tyra G; Chen, JianMing; Zeczycki, Tonya N; Bridges, Lance C

    2017-09-14

    The ADAM (a disintegrin and metalloprotease) protein family uniquely exhibits both catalytic and adhesive properties. In the well-defined process of ectodomain shedding, ADAMs transform latent, cell-bound substrates into soluble, biologically active derivatives to regulate a spectrum of normal and pathological processes. In contrast, the integrin ligand properties of ADAMs are not fully understood. Emerging models posit that ADAM-integrin interactions regulate shedding activity by localizing or sequestering the ADAM sheddase. Interestingly, 8 of the 21 human ADAMs are predicted to be catalytically inactive. Unlike their catalytically active counterparts, integrin recognition of these "dead" enzymes has not been largely reported. The present study delineates the integrin ligand properties of a group of non-catalytic ADAMs. Here we report that human ADAM11, ADAM23, and ADAM29 selectively support integrin α4-dependent cell adhesion. This is the first demonstration that the disintegrin-like domains of multiple catalytically inactive ADAMs are ligands for a select subset of integrin receptors that also recognize catalytically active ADAMs.

  8. Integrins in the Spotlight of Cancer

    PubMed Central

    Bianconi, Daniela; Unseld, Matthias; Prager, Gerald W.

    2016-01-01

    Integrins are heterodimeric cell surface receptors that bind to different extracellular ligands depending on their composition and regulate all processes which enable multicellular life. In cancer, integrins trigger and play key roles in all the features that were once described as the Hallmarks of Cancer. In this review, we will discuss the contribution of integrins to these hallmarks, including uncontrolled and limitless proliferation, invasion of tumor cells, promotion of tumor angiogenesis and evasion of apoptosis and resistance to growth suppressors, by highlighting the latest findings. Further on, given the paramount role of integrins in cancer, we will present novel strategies for integrin inhibition that are starting to emerge, promising a hopeful future regarding cancer treatment. PMID:27929432

  9. 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

  10. Modulation of the expression of integrins on glial cells during experimental autoimmune encephalomyelitis. A central role for TNF-alpha.

    PubMed Central

    Previtali, S. C.; Archelos, J. J.; Hartung, H. P.

    1997-01-01

    Integrins comprise a group of adhesion receptors involved in cell-cell and cell-extracellular matrix interactions. Evidence is accumulating that integrins expressed on mononuclear cells play a central role in the induction of autoimmune diseases of the central nervous system. The effects of integrins on glial cell behavior, myelination, and angiogenesis suggest that they may also have a role in resolving inflammation in the nervous system and in promoting tissue repair. We investigated the temporospatial expression of integrins in the rat central nervous system during the course of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. A higher expression of alpha v- and beta 4-integrin subunits in astrocytes and alpha 2 integrin in oligodendrocytes was observed in active lesions of experimental autoimmune encephalomyelitis, in comparison with controls. Proinflammatory cytokines, primarily TNF-alpha, also enhanced alpha v, beta 4, and alpha 2 expression in purified glial cells ex vivo. Furthermore, we observed that the expression of some integrin subunits was modulated in the cerebral vasculature during inflammation. Our results suggest an active role for glial and vascular integrins in the regulation of autoimmune diseases of the central nervous system, opening an avenue for new potential immunotherapies. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 9 PMID:9358769

  11. MAGP2 controls Notch via interactions with RGD binding integrins: Identification of a novel ECM-integrin-Notch signaling axis.

    PubMed

    Deford, Peter; Brown, Kasey; Richards, Rae Lee; King, Aric; Newburn, Kristin; Westover, Katherine; Albig, Allan R

    2016-02-01

    Canonical Notch signaling involves Notch receptor activation via interaction with cell surface bound Notch ligand. Recent findings also indicate that Notch signaling may be modulated by cross-talk with other signaling mechanisms. The ECM protein MAGP2 was previously shown to regulate Notch in a cell type dependent manner, although the molecular details of this interaction have not been dissected. Here, we report that MAGP2 cell type specific control of Notch is independent of individual Notch receptor-ligand combinations but dependent on interaction with RGD binding integrins. Overexpressed MAGP2 was found to suppress transcriptional activity from the Notch responsive Hes1 promoter activity in endothelial cells, while overexpression of a RGD→RGE MAGP2 mutant increased Notch signaling in the same cell type. This effect was not unique to MAGP2 since the RGD domain of the ECM protein EGFL7 was also found to be an important modulator of Hes1 promoter activity. Independently of MAGP2 or EGFL7, inhibition of RGD-binding integrins with soluble RGD peptides also increased accumulation of active N1ICD fragments and Notch responsive promoter activity independently of changes in Notch1, Jag1, or Dll4 expression. Finally, β1 or β3 integrin blocking antibodies also enhanced Notch signaling. Collectively, these results answer the question of how MAGP2 controls cell type dependent Notch signaling, but more importantly uncover a new mechanism to understand how extracellular matrices and cellular environments impact Notch signaling.

  12. Targeting β1-Integrin Signaling Enhances Regeneration in Aged and Dystrophic Muscle in Mice

    PubMed Central

    Rozo, Michelle; Li, Liangji; Fan, Chen-Ming

    2016-01-01

    Interactions between stem cells and their microenvironment, or niche, are essential for stem cell maintenance and function. Our knowledge of the niche for the skeletal muscle stem cell, i.e. the satellite cell (SC), is incomplete. Here we show that β1-integrin is an essential niche molecule that maintains SC homeostasis, and sustains the expansion and self-renewal of this stem cell pool during regeneration. We further show that β1-integrin cooperates with FGF-2, a potent growth factor for SCs, to synergistically activate their common downstream effectors Erk and Akt. Importantly, SCs in aged mice display altered β1-integrin activity and insensitivity to FGF-2. Augmenting β1-integrin activity with a monoclonal antibody restores FGF-2 sensitivity and improves regeneration after experimentally-induced muscle injury. The same treatment also enhances regeneration and function of dystrophic muscles in mdx mice. Therefore, β1-integrin senses the SC niche to maintain responsiveness to FGF-2, and this integrin represents a potential therapeutic target for pathological conditions of the muscle in which the stem cell niche is compromised. PMID:27376575

  13. The interrelationship of alpha4 integrin and matrix metalloproteinase-2 in the pathogenesis of experimental autoimmune encephalomyelitis.

    PubMed

    Graesser, D; Mahooti, S; Haas, T; Davis, S; Clark, R B; Madri, J A

    1998-11-01

    Previous studies have suggested that surface expression of alpha4 integrin by autoreactive T-cell clones is necessary for the clones to induce experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis. To provide direct evidence for this phenomenon, we have transfected alpha4 integrin into C19alpha4-LO, a myelin basic protein-reactive T-cell clone that does not express alpha4 integrin and does not induce EAE when adoptively transferred into a susceptible mouse strain. Transfection of alpha4 integrin converted this clone to an alpha4 integrin-expressing clone that induced EAE. We then examined potential mechanisms by which alpha4 integrin may facilitate the disease process. C19 T-cell clones adhered equally to a monolayer of microvascular endothelial cells, regardless of level of alpha4 integrin expression. However, in contrast to T-cell clones that do not express alpha4 integrin, T-cell clones that express alpha4 integrin (endogenously or by transfection) transmigrated through an endothelial cell layer and subendothelial matrix at an enhanced rate and adhered to recombinant vascular cell adhesion molecule-1 (rVCAM-1) and the CS1 fragment of fibronectin, and after adhesion to these ligands, a matrix-degrading metalloproteinase (MMP-2) was induced and activated. The clones were also shown to constitutively express the membrane-type matrix metalloproteinase (MT1-MMP), an enzyme that activates MMP-2. GM6001 and UK-221,316, inhibitors of metalloproteinases, reduced alpha4 integrin-mediated transmigration and EAE induction by C19 T-cell clones. In addition, we studied a second EAE-inducing T-cell clone, MM4, which constitutively expresses alpha4 integrin and MMP-2. Engagement of alpha4 integrin on the MM4 clone up-regulated the expression and activation of MMP-2, without changing the expression of MT1-MMP. MMP inhibitors also reduced transmigration of and EAE induction by the MM4 T-cell clone. These studies demonstrate directly that

  14. Dominant Suppression of β1 Integrin by Ectopic CD98-ICD Inhibits Hepatocellular Carcinoma Progression.

    PubMed

    Wu, Bo; Zhou, Yang; Wang, Yu; Yang, Xiang-Min; Liu, Zhen-Yu; Li, Jiang-Hua; Feng, Fei; Chen, Zhi-Nan; Jiang, Jian-Li

    2016-11-10

    Hepatocellular carcinoma (HCC) is currently the third most common cause of cancer-related death in the Asia-Pacific region. Our previous work showed that knockdown of CD98 significantly inhibits malignant HCC cell phenotypes in vitro and in vivo. The level of CD98 in the membrane is tightly regulated to mediate complex processes associated with cell-cell communication and intracellular signaling. In addition, the intracellular domain of CD98 (CD98-ICD) seems to be of vital importance for recycling CD98 to the membrane after it is endocytosed. The intracellular and transmembrane domains of CD98 associate with β-integrins (primarily β1 but also β3), and this association is essential for CD98 mediation of integrin-like signaling and complements dominant suppression of β1-integrin. We speculated that isolated CD98-ICD would similarly suppress β1-integrin activation and inhibit the malignant behaviors of cancer cells. In particular, the exact role of CD98-ICD has not been studied independently in HCC. In this study, we found that ectopic expression of CD98-ICD inhibited the malignant phenotypes of HCC cells, and the mechanism possibly i