Sample records for disintegrins integrin selective

  1. ADAM disintegrin-like domain recognition by the lymphocyte integrins α4β1 and α4β7

    PubMed Central

    Bridges, Lance C.; Sheppard, Dean; Bowditch, Ron D.

    2004-01-01

    The ADAM (a disintegrin and metalloprotease) family of proteins possess both proteolytic and adhesive domains. We have established previously that the disintegrin domain of ADAM28, an ADAM expressed by human lymphocytes, is recognized by the integrin α4β1. The present study characterizes the integrin binding properties of the disintegrin-like domains of human ADAM7, ADAM28 and ADAM33 with the integrins α4β1, α4β7 and α9β1. Cell-adhesion assays demonstrated that, similar to ADAM28, the ADAM7 disintegrin domain supported α4β1-dependent Jurkat cell adhesion, whereas the ADAM33 disintegrin domain did not. The lymphocyte integrin α4β7 was also found to recognize both disintegrin domains of ADAM7 and ADAM28, but not of ADAM33. This is the first demonstration that mammalian disintegrins are capable of interacting with α4β7. All three disintegrin domains supported α9β1-dependent cell adhesion. Recognition by both α4β1 and α4β7 of ADAM7 and ADAM28 was activation-dependent, requiring either the presence of Mn2+ or an activating monoclonal antibody for cell attachment. Charge-to-alanine mutagenesis experiments revealed that the same residues within an individual ADAM disintegrin domain function in recognizing multiple integrins. However, the residues within a specific region of each ADAM disintegrin-like domain required for integrin binding were distinct. These results establish that ADAM7 and ADAM28 are recognized by the leucocyte integrins α4β1, α4β7 and α9β1. ADAM33 exclusively supported only α9β1-dependent adhesion. PMID:15504110

  2. Selective Modulation of Integrin-mediated Cell Migration by Distinct ADAM Family MembersV⃞

    PubMed Central

    Huang, Jing; Bridges, Lance C.; White, Judith M.

    2005-01-01

    A disintegrin and a metalloprotease (ADAM) family members have been implicated in many biological processes. Although it is recognized that recombinant ADAM disintegrin domains can interact with integrins, little is known about ADAM-integrin interactions in cellular context. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (α4β1, α5β1, or both), and cell migration on full-length fibronectin or on its α4β1 or α5β1 binding fragments was studied. We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAM12 inhibited cell migration mediated by the α4β1 but not the α5β1 integrin. ADAM17 had the reciprocal effect; it inhibited α5β1- but not α4β1-mediated cell migration. ADAM19 and ADAM33 inhibited migration mediated by both α4β1 and α5β1 integrins. A point mutation in the ADAM12 disintegrin loop partially reduced the inhibitory effect of ADAM12 on cell migration on the α4β1 binding fragment of fibronectin, whereas mutations that block metalloprotease activity had no effect. Our results indicate that distinct ADAMs can modulate cell migration mediated by specific integrins in a pattern dictated, at least in part, by their disintegrin domains. PMID:16079176

  3. Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

    PubMed Central

    Takahashi, Yuji; Bigler, Dora; Ito, Yasuhiko; White, Judith M.

    2001-01-01

    ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions. PMID:11294888

  4. Sequence-specific interaction between the disintegrin domain of mouse ADAM 3 and murine eggs: role of beta1 integrin-associated proteins CD9, CD81, and CD98.

    PubMed

    Takahashi, Y; Bigler, D; Ito, Y; White, J M

    2001-04-01

    ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-alpha6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-alpha6 mAb, or by mAbs against either the alphav or beta3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other beta1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg beta1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface "tetraspan web" facilitates fertilization and that it may do so by fostering ADAM-integrin interactions.

  5. A family of cellular proteins related to snake venom disintegrins.

    PubMed

    Weskamp, G; Blobel, C P

    1994-03-29

    Disintegrins are short soluble integrin ligands that were initially identified in snake venom. A previously recognized cellular protein with a disintegrin domain was the guinea pig sperm protein PH-30, a protein implicated in sperm-egg membrane binding and fusion. Here we present peptide sequences that are characteristic for several cellular disintegrin-domain proteins. These peptide sequences were deduced from cDNA sequence tags that were generated by polymerase chain reaction from various mouse tissue and a mouse muscle cell line. Northern blot analysis with four sequence tags revealed distinct mRNA expression patterns. Evidently, cellular proteins containing a disintegrin domain define a superfamily of potential integrin ligands that are likely to function in important cell-cell and cell-matrix interactions.

  6. Crystal structure of the disintegrin heterodimer from saw-scaled viper (Echis carinatus) at 1.9 A resolution.

    PubMed

    Bilgrami, Sameeta; Yadav, Savita; Kaur, Punit; Sharma, Sujata; Perbandt, Markus; Betzel, Christian; Singh, Tej P

    2005-08-23

    Disintegrins constitute a family of potent polypeptide inhibitors of integrins. Integrins are transmembrane heterodimeric molecules involved in cell-cell and cell-extracellular matrix interactions. They are involved in many diseases such as cancer and thrombosis. Thus, disintegrins have a great potential as anticancer and antithrombotic agents. A novel heterodimeric disintegrin was isolated from the venom of saw-scaled viper (Echis carinatus) and was crystallized. The crystals diffracted to 1.9 A resolution and belonged to space group P4(3)2(1)2. The data indicated the presence of a pseudosymmetry. The structure was solved by applying origin shifts to the disintegrin homodimer schistatin solved in space group I4(1)22 with similar cell dimensions. The structure refined to the final R(cryst)/R(free) factors of 0.213/0.253. The notable differences are observed between the loops, (Gln39-Asp48) containing the important Arg42-Gly43-Asp44, of the present heterodimer and schistatin. These differences are presumably due to the presence of two glycines at positions 43 and 46 that allow the molecule to adopt variable conformations. A comparative analysis of the surface-charge distributions of various disintegrins showed that the charge distribution on monomeric disintegrins occurred uniformly over the whole surface of the molecule, while in the dimeric disintegrins, the charge is distributed only on one face. Such a feature may be important in the binding of two integrins to a single dimeric disintegrin. The phylogenetic analysis developed on the basis of amino acid sequence and three-dimensional structures indicates that the protein diversification and evolution presumably took place from the medium disintegrins and both the dimeric and short disintegrins evolved from them.

  7. Disintegrins from Hematophagous Sources

    PubMed Central

    Assumpcao, Teresa C. F.; Ribeiro, José M. C.; Francischetti, Ivo M. B.

    2012-01-01

    Bloodsucking arthropods are a rich source of salivary molecules (sialogenins) which inhibit platelet aggregation, neutrophil function and angiogenesis. Here we review the literature on salivary disintegrins and their targets. Disintegrins were first discovered in snake venoms, and were instrumental in our understanding of integrin function and also for the development of anti-thrombotic drugs. In hematophagous animals, most disintegrins described so far have been discovered in the salivary gland of ticks and leeches. A limited number have also been found in hookworms and horseflies, and none identified in mosquitoes or sand flies. The vast majority of salivary disintegrins reported display a RGD motif and were described as platelet aggregation inhibitors, and few others as negative modulator of neutrophil or endothelial cell functions. This notably low number of reported disintegrins is certainly an underestimation of the actual complexity of this family of proteins in hematophagous secretions. Therefore an algorithm was created in order to identify the tripeptide motifs RGD, KGD, VGD, MLD, KTS, RTS, WGD, or RED (flanked by cysteines) in sialogenins deposited in GenBank database. The search included sequences from various blood-sucking animals such as ticks (e.g., Ixodes sp., Argas sp., Rhipicephalus sp., Amblyommasp.), tabanids (e.g., Tabanus sp.), bugs (e.g., Triatoma sp., Rhodnius prolixus), mosquitoes (e.g., Anopheles sp., Aedes sp., Culex sp.), sand flies (e.g., Lutzomyia sp., Phlebotomus sp.), leeches (e.g., Macrobdella sp., Placobdella sp.) and worms (e.g., Ancylostoma sp.). This approach allowed the identification of a remarkably high number of novel putative sialogenins with tripeptide motifs typical of disintegrins (>450 sequences) whose biological activity remains to be verified. This database is accessible online as a hyperlinked worksheet and displays biochemical, taxonomic, and gene ontology aspects for each putative disintegrin. It is also freely

  8. Disintegrins from hematophagous sources.

    PubMed

    Assumpcao, Teresa C F; Ribeiro, José M C; Francischetti, Ivo M B

    2012-05-01

    Bloodsucking arthropods are a rich source of salivary molecules (sialogenins) which inhibit platelet aggregation, neutrophil function and angiogenesis. Here we review the literature on salivary disintegrins and their targets. Disintegrins were first discovered in snake venoms, and were instrumental in our understanding of integrin function and also for the development of anti-thrombotic drugs. In hematophagous animals, most disintegrins described so far have been discovered in the salivary gland of ticks and leeches. A limited number have also been found in hookworms and horseflies, and none identified in mosquitoes or sand flies. The vast majority of salivary disintegrins reported display a RGD motif and were described as platelet aggregation inhibitors, and few others as negative modulator of neutrophil or endothelial cell functions. This notably low number of reported disintegrins is certainly an underestimation of the actual complexity of this family of proteins in hematophagous secretions. Therefore an algorithm was created in order to identify the tripeptide motifs RGD, KGD, VGD, MLD, KTS, RTS, WGD, or RED (flanked by cysteines) in sialogenins deposited in GenBank database. The search included sequences from various blood-sucking animals such as ticks (e.g., Ixodes sp., Argas sp., Rhipicephalus sp., Amblyommasp.), tabanids (e.g., Tabanus sp.), bugs (e.g., Triatoma sp., Rhodnius prolixus), mosquitoes (e.g., Anopheles sp., Aedes sp., Culex sp.), sand flies (e.g., Lutzomyia sp., Phlebotomus sp.), leeches (e.g., Macrobdella sp., Placobdella sp.) and worms (e.g., Ancylostoma sp.). This approach allowed the identification of a remarkably high number of novel putative sialogenins with tripeptide motifs typical of disintegrins (>450 sequences) whose biological activity remains to be verified. This database is accessible online as a hyperlinked worksheet and displays biochemical, taxonomic, and gene ontology aspects for each putative disintegrin. It is also freely

  9. Importance of interaction between nerve growth factor and α9β1 integrin in glial tumor angiogenesis

    PubMed Central

    Walsh, Erin M.; Kim, Richard; Del Valle, Luis; Weaver, Michael; Sheffield, Joel; Lazarovici, Philip; Marcinkiewicz, Cezary

    2012-01-01

    NGF is a growth factor for which the role in the promotion of angiogenesis is still not completely understood. We found that NGF promotes the pathological neovascularization process in glioma through a direct interaction with α9β1 integrin, which is up-regulated on microvascular endothelial cells in cancer tissue. We propagated gHMVEC primary cells using a new method of immune-selection, and these cells demonstrated α9β1 integrin-dependent binding of NGF in a cell adhesion assay. Moreover, NGF induced gHMVEC proliferation and chemotaxis inhibited by specific blockers of α9β1 integrin, such as MLD-disintegrins and monoclonal antibody Y9A2. A Matrigel tube formation assay revealed that NGF significantly increased capillary-like growth from gHMVEC to a level comparable to treatment with VEGF. The snake venom disintegrin, VLO5, inhibited the agonistic effect of both growth factors, whereas the effect of Y9A2 was not statistically significant. Angiogenesis exogenously induced by NGF  was also α9β1-integrin dependent in an embryonic quail CAM system. However, angiogenesis pathologically induced by developing glioma in this system was only sensitive for inhibition with MLD-disintegrin, suggesting a more complex effect of cancer cells on the neovascularization process. The anti-angiogenic effect of MLD-disintegrins is probably related to their pro-apoptotic ability induced in activated tumoral endothelial cells. Therefore, the molecular basis of these disintegrins may be useful for developing new angiostatic pharmaceuticals for application in cancer therapy. PMID:22611032

  10. Development of a chimeric recombinant disintegrin as a cost-effective anticancer agent with promising translational potential

    PubMed Central

    Minea, Radu; Helchowski, Corey; Rubino, Barbara; Brodmann, Kyle; Swenson, Stephen; Markland, Francis

    2011-01-01

    Vicrostatin (VCN) is a chimeric recombinant disintegrin generated in Origami B (DE3) E. coli as a genetic fusion between the C-terminal tail of a viperid disintegrin echistatin and crotalid disintegrin contortrostatin (CN). The therapeutic modulation of multiple integrin pathways via soluble disintegrins was previously shown by us and others to elicit potent anti-angiogenic and anti-metastatic effects in several animal cancer models. Despite these favorable attributes, these polypeptides are notoriously difficult to produce recombinantly in significant quantity due to their structure which requires the correct pairing of multiple disulfide bonds for biological activity. In this report, we show that VCN can be reliably produced in large amounts (yields in excess of 200mg of active purified disintegrin per liter of bacterial culture) in Origami B (DE3), an E. coli expression strain engineered to support the folding of disulfide-rich heterologous proteins directly in its oxidative cytoplasmic compartment. VCN retains the integrin binding specificity of both parental molecules it was derived from, but with a different binding affinity profile. While competing for the same integrin receptors that are preferentially upregulated in the tumor microenvironment, VCN exerts a potent inhibitory effect on endothelial cell (EC) migration and tube formation in a dose-dependent manner, by forcing these cells to undergo significant actin cytoskeleton reorganization when exposed to this agent in vitro. Moreover, VCN has a direct effect on breast cancer cells inhibiting their in vitro motility. In an effort to address our main goal of developing a clinically relevant delivery method for recombinant disintegrins, VCN was efficiently packaged in liposomes (LVCN) and evaluated in vivo in an animal breast cancer model. Our data demonstrate that LVCN is well tolerated, its intravenous administration inducing a significant delay in tumor growth and an increase in animal survival, results

  11. MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

    PubMed Central

    1996-01-01

    Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein. PMID:8647900

  12. CLONING, EXPRESSION, AND HEMOSTATIC ACTIVITIES OF A DISINTEGRIN, r-MOJASTIN 1, FROM THE MOHAVE RATTLESNAKE (Crotalus scutulatus scutulatus)

    PubMed Central

    Sánchez, Elda E.; Lucena, Sara E.; Reyes, Steven; Soto, Julio G.; Cantu, Esteban; Lopez-Johnston, Juan Carlos; Guerrero, Belsy; Salazar, Ana Maria; Rodríguez-Acosta, Alexis; Galán, Jacob A.; Tao, W. Andy; Pérez, John C.

    2012-01-01

    Interactions with exposed subendothelial extracellular proteins and cellular integrins (endothelial cells, platelets and lymphocytes) can cause alterations in the hemostatic system associated with atherothrombotic processes. Many molecules found in snake venoms induce pathophysiological changes in humans, cause edema, hemorrhage, and necrosis. Disintegrins are low molecular weight, non-enzymatic proteins found in snake venom that mediate changes by binding to integrins of platelets or other cells and prevent binding of the natural ligands such as fibrinogen, fibronectin or vitronectin. Disintegrins are of great biomedical importance due to their binding affinities resulting in the inhibition of platelet aggregation, adhesion of cancer cells, and induction of signal transduction pathways. RT-PCR was used to obtain a 216 bp disintegrin cDNA from a C. s. scutulatus snake venom gland. The cloned recombinant disintegrin called r-mojastin 1 codes for 71 amino acids, including 12 cysteines, and an RGD binding motif. r-Mojastin 1 inhibited platelet adhesion to fibronectin with an IC50 of 58.3 nM and ADP-induced platelet aggregation in whole blood with an IC50 of 46 nM. r-Mojastin 1 was also tested for its ability to inhibit platelet ATP release using PRP resulting with an IC50 of 95.6 nM. MALDI-TOF mass spectrum analysis showed that r-mojastin has a mass of 7.9509 kDa. PMID:20598348

  13. Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2

    PubMed Central

    De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

    2016-01-01

    Integrins are heterodimeric cell-surface adhesion molecules comprising one of possible 18 α-chains and one of possible 8 β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalised by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalisation by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with AP2 C-µ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions. PMID:26779610

  14. Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2.

    PubMed

    De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

    2016-02-01

    Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-μ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions.

  15. Identification of metalloprotease/disintegrins in Xenopus laevis testis with a potential role in fertilization.

    PubMed

    Shilling, F M; Krätzschmar, J; Cai, H; Weskamp, G; Gayko, U; Leibow, J; Myles, D G; Nuccitelli, R; Blobel, C P

    1997-06-15

    Proteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell-cell interactions. We have identified PCR sequence tags encoding the disintegrin domain of five distinct MDC proteins from Xenopus laevis testis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. Northern blot analysis revealed that the mRNA for xMDC16 was only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products were expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence, as predicted from the full-length cDNA, contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibit X. laevis fertilization. Cyclic and linear xMDC16 peptides inhibited fertilization in a concentration-dependent manner, whereas xMDC16 peptides that were scrambled or had certain amino acid replacements in the predicted integrin-binding domain did not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibited fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 did not. These results are discussed in the context of a model in which multiple MDC protein-receptor interactions are necessary for fertilization to occur.

  16. β1 Integrin is an Adhesion Protein for Sperm Binding to Eggs

    PubMed Central

    Baessler, Keith A.; Lee, Younjoo; Sampson, Nicole S.

    2009-01-01

    We investigated the role of β1 integrin in mammalian fertilization and the mode of inhibition of fertilinβ-derived polymers. We determined that polymers displaying the Glu-Cys-Asp peptide from the fertilinβ disintegrin domain mediate inhibition of mammalian fertilization through a β1 integrin receptor on the egg surface. Inhibition of fertilization is a consequence of competition with sperm binding to the cell surface, not activation of an egg-signaling pathway. The presence of the β1 integrin on the egg surface increases the rate of sperm attachment, but does not alter the total number of sperm that can attach or fuse to the egg. We conclude that the presence of β1 integrin enhances the initial adhesion of sperm to the egg plasma membrane and that subsequent attachment and fusion are mediated by additional egg and sperm proteins present in the β1 integrin complex. Therefore, the mechanisms by which sperm fertilize wild-type and β1 knockout eggs are different. PMID:19338281

  17. Inhibition of melanoma cell motility by the snake venom disintegrin eristostatin

    PubMed Central

    Tian, Jing; Paquette-Straub, Carrie; Sage, E. Helene; Funk, Sarah E.; Patel, Vivek; Galileo, Deni; McLane, Mary Ann

    2007-01-01

    Eristostatin, an RGD-containing disintegrin isolated from the venom of Eristicophis macmahoni, inhibits lung or liver colonization of melanoma cells in a mouse model. In this study, transwell migration and in vitro wound closure assays were used to determine the effect of eristostatin on the migration of melanoma cells. Eristostatin significantly impaired the migration of 5 human melanoma cell lines. Furthermore, it specifically inhibited cell migration on fibronectin in a concentration-dependent manner, but not that on collagen IV or laminin. In contrast, eristostatin was found to have no effect on cell proliferation or angiogenesis. These results indicate that the interaction between eristostatin and melanoma cells may involve fibronectin-binding integrins that mediate cell migration. Mutations to alanine of seven residues within the RGD loop of eristostatin and four residues outside the RGD loop of eristostatin resulted in significantly less potency in both platelet aggregation and wound closure assays. For six of the mutations, however, decreased activity was found only in the latter assay. We conclude that a different mechanism and/or integrin is involved in these two cell activities. PMID:17316731

  18. Expression of Selected Integrins and Selectins in Bullous Pemphigoid

    PubMed Central

    Żebrowska, Agnieszka; Sysa-Jędrzejowska, Anna; Wągrowska-Danilewicz, Małgorzata; Joss-Wichman, Ewa; Erkiert-Polguj, Anna; Waszczykowska, Elżbieta

    2007-01-01

    Blister development in bullous pemphigoid (BP) results from destruction of hemidesmosomes and basement membrane components within the dermoepidermal junction by autoantibodies. Adhesion molecules can take part in pathogenesis of this disease. The aim of the study was to determine the localization and expression of L- and E-selectins and β1, β3, and β4 integrins by immunohistochemistry in skin lesions of 21 patients with BP, compared with 10 healthy subjects. Expression of L and E selectins and β1, β3 integrins was detected mainly in basal keratinocytes and in inflammatory infiltrates in the dermis, expression of β4 integrin was irregular and was detected mainly in dermal part of the blister, while in the control group only weak and single expression of the examined molecules was detected in basal keratinocytes and endothelium cells. The obtained results reveal the important role of selected selectins and integrins in development of skin lesions in BP. PMID:17515951

  19. Evidence that Distinct States of the Integrin α6β1 Interact with Laminin and an ADAM

    PubMed Central

    Chen, M.S.; Almeida, E.A.C.; Huovila, A.-P.J.; Takahashi, Y.; Shaw, L.M.; Mercurio, A.M.; White, J.M.

    1999-01-01

    Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin α6β1, on mouse eggs and on α6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin β). In the present study we tested the hypothesis that different states of α6β1 interact with fertilin and laminin, an extracellular matrix ligand for α6β1. Using α6-transfected cells we found that treatments (e.g., with phorbol myristate acetate or MnCl2) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding. Next, we compared the ability of fluorescent beads coated with either fertilin β or with the laminin E8 fragment to bind to eggs. In Ca2+-containing media, fertilin β beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin β and by the α6 integrin subunit. In Ca2+-containing media, laminin E8 beads did not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding. Treatment of eggs with Mn2+ dramatically increased laminin E8 bead binding, and inhibited fertilin bead binding. Our results provide the first evidence that different states of an integrin (α6β1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2). PMID:9971748

  20. Isolation and characterization of an anti-leishmanial disintegrin from Cerastes cerastes venom.

    PubMed

    Allane, Dihia; Oussedik-Oumehdi, Habiba; Harrat, Zoubir; Seve, Michel; Laraba-Djebari, Fatima

    2018-02-01

    Investigating new antimicrobial and antiparasitic components from Viperidae venoms represents an alternative therapeutic strategy. In this study, we report the characterization of a disintegrin isolated from Cerastes cerastes venom, exhibiting antiparasitic activity on Leishmania infantum promastigotes. Indeed, isolated disintegrin, referred to Disintegrin_Cc, induced 84.75% of parasiticidal activity and deep morphological alterations on the parasites. SDS-PAGE analysis indicated that this disintegrin was homogenous. This dimeric disintegrin of 14,193.97 Da contains an RGD domain and four intramolecular disulfide bridges. It presents a high percentage of identity with other related snake disintegrins. Predicted 3D structure indicated that this peptide shares partial homology with well-known active antimicrobial peptides. Disintegrin_Cc inhibited 80% of arachidonic acid-induced platelet aggregation. The obtained results suggest that the isolated molecule plays a dual role as a disintegrin and as an anti-leishmanial compound. This component could be useful as a drug in the treatment of leishmaniasis. © 2017 Wiley Periodicals, Inc.

  1. Characterisation of a novel, high affinity and selective αvβ6 integrin RGD-mimetic radioligand.

    PubMed

    Hall, Eleanor R; Bibby, Lloyd I; Slack, Robert J

    2016-10-01

    The alpha-v beta-6 (αvβ6) integrin has been identified as playing a key role in the activation of transforming growth factor-β (TGFβ) that is hypothesised to be pivotal in the development of cancer and fibrotic diseases. Therefore, the αvβ6 integrin is an attractive therapeutic target for these debilitating diseases and a drug discovery programme to identify small molecule αvβ6 selective arginyl-glycinyl-aspartic acid (RGD)-mimetics was initiated within GlaxoSmithKline. The primary aim of this study was to pharmacologically characterise the binding to αvβ6 of a novel clinical candidate, compound 1, using a radiolabelled form. Radioligand binding studies were completed with [(3)H]compound 1 against the human and mouse soluble protein forms of αvβ6 to determine accurate affinity estimates and binding kinetics. The selectivity of compound 1 for the RGD integrin family was also determined using saturation binding studies (αvβ1, αvβ3, αvβ5, αvβ8, α5β1 and α8β1 integrins) and fibrinogen-induced platelet aggregation (αIIbβ3 integrin). In addition, the relationship between divalent metal cation type and concentration and αvβ6 RGD site binding was also investigated. Compound 1 has been demonstrated to bind with extremely high affinity and selectivity for the αvβ6 integrin and has the potential as a clinical tool and therapeutic for investigating the role of αvβ6 in a range of disease states both pre-clinically and clinically. In addition, this is the first study that has successfully applied radioligand binding to the RGD integrin field to accurately determine the affinity and selectivity profile of a small molecule RGD-mimetic. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. 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.

  3. A ligand-specific blockade of the integrin Mac-1 selectively targets pathologic inflammation while maintaining protective host-defense.

    PubMed

    Wolf, Dennis; Anto-Michel, Nathaly; Blankenbach, Hermann; Wiedemann, Ansgar; Buscher, Konrad; Hohmann, Jan David; Lim, Bock; Bäuml, Marina; Marki, Alex; Mauler, Maximilian; Duerschmied, Daniel; Fan, Zhichao; Winkels, Holger; Sidler, Daniel; Diehl, Philipp; Zajonc, Dirk M; Hilgendorf, Ingo; Stachon, Peter; Marchini, Timoteo; Willecke, Florian; Schell, Maximilian; Sommer, Björn; von Zur Muhlen, Constantin; Reinöhl, Jochen; Gerhardt, Teresa; Plow, Edward F; Yakubenko, Valentin; Libby, Peter; Bode, Christoph; Ley, Klaus; Peter, Karlheinz; Zirlik, Andreas

    2018-02-06

    Integrin-based therapeutics have garnered considerable interest in the medical treatment of inflammation. Integrins mediate the fast recruitment of monocytes and neutrophils to the site of inflammation, but are also required for host defense, limiting their therapeutic use. Here, we report a novel monoclonal antibody, anti-M7, that specifically blocks the interaction of the integrin Mac-1 with its pro-inflammatory ligand CD40L, while not interfering with alternative ligands. Anti-M7 selectively reduces leukocyte recruitment in vitro and in vivo. In contrast, conventional anti-Mac-1 therapy is not specific and blocks a broad repertoire of integrin functionality, inhibits phagocytosis, promotes apoptosis, and fuels a cytokine storm in vivo. Whereas conventional anti-integrin therapy potentiates bacterial sepsis, bacteremia, and mortality, a ligand-specific intervention with anti-M7 is protective. These findings deepen our understanding of ligand-specific integrin functions and open a path for a new field of ligand-targeted anti-integrin therapy to prevent inflammatory conditions.

  4. Elucidating the role of select cytoplasmic proteins in altering diffusion of integrin receptors.

    PubMed

    Sander, Suzanne; Arora, Neha; Smith, Emily A

    2012-06-01

    Cytoplasmic proteins that affect integrin diffusion in the cell membrane are identified using a combination of fluorescence recovery after photobleaching (FRAP) and RNA interference. Integrin receptors are essential for many cellular events, and alterations in lateral diffusion are one mechanism for modulating their function. In cells expressing native cytoplasmic protein concentrations and spread on a slide containing integrin extracellular ligand, 45 ± 2% of the integrin is mobile with a time-dependent 5.2 ± 0.9 × 10(-9) cm(2)/s diffusion coefficient at 1 s. The time exponent is 0.90 ± 0.07, indicating integrin diffusion moderately slows at longer times. The role of a specific cytoplasmic protein in altering integrin diffusion is revealed through changes in the FRAP curve after reducing the cytoplasmic protein's expression. Decreased expression of cytoplasmic proteins rhea, focal adhesion kinase (FAK), or steamer duck decreases the integrin mobile fraction. For rhea and FAK, there is a concomitant shift to Brownian (i.e., time-independent) diffusion at reduced concentrations of these proteins. In contrast, when the expression of actin 42A, dreadlocks, paxillin, integrin-linked kinase (ILK), or vinculin is reduced, integrin diffusion generally becomes more constrained with an increase in the integrin mobile fraction. This same change in integrin diffusion is measured in the absence of integrin extracellular ligand. The results indicate breaking the extracellular ligand-integrin-cytoskeletal linkage alters integrin diffusion properties, and, in most cases, there is no correlation between integrin and lipid diffusion properties.

  5. Integrin Targeted MR Imaging

    PubMed Central

    Tan, Mingqian; Lu, Zheng-Rong

    2011-01-01

    Magnetic resonance imaging (MRI) is a powerful medical diagnostic imaging modality for integrin targeted imaging, which uses the magnetic resonance of tissue water protons to display tissue anatomic structures with high spatial resolution. Contrast agents are often used in MRI to highlight specific regions of the body and make them easier to visualize. There are four main classes of MRI contrast agents based on their different contrast mechanisms, including T1, T2, chemical exchange saturation transfer (CEST) agents, and heteronuclear contrast agents. Integrins are an important family of heterodimeric transmembrane glycoproteins that function as mediators of cell-cell and cell-extracellular matrix interactions. The overexpressed integrins can be used as the molecular targets for designing suitable integrin targeted contrast agents for MR molecular imaging. Integrin targeted contrast agent includes a targeting agent specific to a target integrin, a paramagnetic agent and a linker connecting the targeting agent with the paramagnetic agent. Proper selection of targeting agents is critical for targeted MRI contrast agents to effectively bind to integrins for in vivo imaging. An ideal integrin targeted MR contrast agent should be non-toxic, provide strong contrast enhancement at the target sites and can be completely excreted from the body after MR imaging. An overview of integrin targeted MR contrast agents based on small molecular and macromolecular Gd(III) complexes, lipid nanoparticles and superparamagnetic nanoparticles is provided for MR molecular imaging. By using proper delivery systems for loading sufficient Gd(III) chelates or superparamagnetic nanoparticles, effective molecular imaging of integrins with MRI has been demonstrated in animal models. PMID:21547154

  6. Immunological characterization of eristostatin and echistatin binding sites on alpha IIb beta 3 and alpha V beta 3 integrins.

    PubMed Central

    Marcinkiewicz, C; Rosenthal, L A; Mosser, D M; Kunicki, T J; Niewiarowski, S

    1996-01-01

    Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with alpha IIb beta 3 genes (A5 cells) and to CHO cells transfected with alpha v beta 3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the beta 3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (alpha IIb beta 3 complex dependent) and 7E3 (alpha IIb beta 3 and alpha v beta 3 complex dependent) to A5 cells, to resting and to activated platelets and to purified alpha IIb beta 3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on alpha IIb beta 3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on alpha v beta 3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified alpha v beta 3 suggesting that alpha v beta 3 and alpha IIb beta 3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on alpha v beta 3. PMID:8760368

  7. Convulxin, a C-type lectin-like protein, inhibits HCASMCs functions via WAD-motif/integrin-αv interaction and NF-κB-independent gene suppression of GRO and IL-8.

    PubMed

    Shih, Chun-Ho; Chiang, Tin-Bin; Wang, Wen-Jeng

    2017-03-15

    Convulxin (CVX), a C-type lectin-like protein (CLPs), is a potent platelet aggregation inducer. To evaluate its potential applications in angiogenic diseases, the multimeric CVX were further explored on its mode of actions toward human coronary artery smooth muscle cells (HCASMCs). The N-terminus of β-chain of CVX (CVX-β) contains a putative disintegrin-like domain with a conserved motif upon the sequence comparison with other CLPs. Importantly, native CVX had no cytotoxic activity as examined by electrophoretic pattern. A Trp-Ala-Asp (WAD)-containing octapeptide, MTWADAEK, was thereafter synthesized and analyzed in functional assays. In the case of specific integrin antagonists as positive controls, the anti-angiogenic effects of CVX on HCASMCs were investigated by series of functional analyses. CVX showed to exhibit multiple inhibitory activities toward HCASMCs proliferation, adhesion and invasion with a dose- and integrin αvβ3-dependent fashion. However, the WAD-octapeptide exerting a minor potency could also work as an active peptidomimetic. In addition, flow cytometric analysis demonstrated both the intact CVX and synthetic peptide can specifically interact with integrin-αv on HCASMCs and CVX was shown to have a down-regulatory effect on the gene expression of CXC-chemokines, such as growth-related oncogene and interleukin-8. According to nuclear factor-κB (NF-κB) p65 translocation assay and Western blotting analysis, the NF-κB activation was not involved in the signaling events of CVX-induced gene expression. In conclusion, CVX may act as a disintegrin-like protein via the interactions of WAD-motif in CVX-β with integrin-αv on HCASMCs and it also is a gene suppressor with the ability to diminish the expression of two CXC-chemokines in a NF-κB-independent manner. Indeed, more extensive investigations are needed and might create a new avenue for the development of a novel angiostatic agent. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. The binding effectiveness of anti-r-disintegrin polyclonal antibodies against disintegrins and PII and PIII metalloproteases: An immunological survey of type A, B and A + B venoms from Mohave rattlesnakes

    PubMed Central

    Cantú, Esteban; Mallela, Sahiti; Nyguen, Matthew; Báez, Raúl; Parra, Victoria; Johnson, Rachel; Wilson, Kyle; Suntravat, Montamas; Lucena, Sara; Rodríguez-Acosta, Alexis; Sánchez, Elda E.

    2016-01-01

    Snake venoms are known to have different venom compositions and toxicity, but differences can also be found within populations of the same species contributing to the complexity of treatment of envenomated victims. One of the first well-documented intraspecies venom variations comes from the Mohave rattlesnake (Crotalus scutulatus scutulatus). Initially, three types of venoms were described; type A venom is the most toxic as a result of ~45% Mojave toxin in the venom composition, type B lacks the Mojave toxin but contains over 50% of snake venom metalloproteases (SVMPs). Also, type A + B venom contains a combination of Mojave toxin and SVMP. The use of an anti-disintegrin antibody in a simple Enzyme-Linked Immunosorbent Assay (ELISA) can be used to identify the difference between the venoms of the type A, B, and A+B Mohave rattlesnakes. This study implements the use of an anti-recombinant disintegrin polyclonal antibody (ARDPA) for the detection of disintegrins and ADAMs (a disintegrin and metalloproteases) in individual crude snake venoms of Mohave rattlesnakes (Crotalus scutulatus scutulatus) of varying geographical locations. After correlation with Western blots, coagulation activity and LD50 data, it was determined that the antibody allows for a quick and cost-efficient identification of venom types. PMID:27989783

  9. 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

  10. Identification of stepped changes of binding affinity during interactions between the disintegrin rhodostomin and integrin αIIbβ3 in living cells using optical tweezers

    NASA Astrophysics Data System (ADS)

    Hsieh, Chia-Fen; Chang, Bo-Jui; Pai, Chyi-Huey; Chen, Hsuan-Yi; Chi, Sien; Hsu, Long; Tsai, Jin-Wu; Lin, Chi-Hung

    2004-10-01

    Integrin receptors serve as both mechanical links and signal transduction mediators between the cell and its environment. Experimental evidence demonstrates that conformational changes and lateral clustering of the integrin proteins may affect their binding to ligands and regulate downstream cellular responses; however, experimental links between the structural and functional correlations of the ligand-receptor interactions are not yet elucidated. In the present report, we utilized optical tweezers to measure the dynamic binding between the snake venom rhodostomin, coated on a microparticle and functioned as a ligand, and the membrane receptor integrin alpha(IIb)beta(3) expressed on a Chinese Hamster Ovary (CHO) cell. A progressive increase of total binding affinity was found between the bead and CHO cell in the first 300 sec following optical tweezers-guided contact. Further analysis of the cumulative data revealed the presence of "unit binding force" presumably exerted by a single rhodostomin-integrin pair. Interestingly, two such units were found. Among the measurements of less total binding forces, presumably taken at the early stage of ligand-receptor interactions, a unit of 4.15 pN per molecule pair was derived. This unit force dropped to 2.54 pN per molecule pair toward the later stage of interactions when the total binding forces were relatively large. This stepped change of single molecule pair binding affinity was not found when mutant rhodostomin proteins were used as ligands (a single unit of 1.81 pN per pair was found). These results were interpreted along with the current knowledge about the conformational changes of integrins during the "molecule activation" process.

  11. Small-molecule inhibitors of FGFR, integrins and FAK selectively decrease L1CAM-stimulated glioblastoma cell motility and proliferation.

    PubMed

    Anderson, Hannah J; Galileo, Deni S

    2016-06-01

    The cell adhesion/recognition protein L1CAM (L1; CD171) has previously been shown to act through integrin, focal adhesion kinase (FAK) and fibroblast growth factor receptor (FGFR) signaling pathways to increase the motility and proliferation of glioblastoma cells in an autocrine/paracrine manner. Here, we investigated the effects of clinically relevant small-molecule inhibitors of the integrin, FAK and FGFR signaling pathways on glioblastoma-derived cells to determine their effectiveness and selectivity for diminishing L1-mediated stimulation. The effects of the FGFR inhibitor PD173074, the FAK inhibitors PF431396 and Y15 and the αvβ3/αvβ5 integrin inhibitor cilengitide were assessed in L1-positive and L1-negative variants of the human glioblastoma-derived cell lines T98G and U-118 MG. Their motility and proliferation were quantified using time-lapse microscopy and DNA content/cell cycle analyses, respectively. The application of all four inhibitors resulted in reductions in L1-mediated motility and proliferation rates of L1-positive glioblastoma-derived cells, down to the level of L1-negative cells when used at nanomolar concentrations, whereas no or much smaller reductions in these rates were obtained in L1-negative cells. In addition, we found that single inhibitor treatment resulted in maximum effects (i.e., combinations of FAK or integrin inhibitors with the FGFR inhibitor were rarely more effective). These results suggest that FAK may act as a point of convergence between the integrin and FGFR signaling pathways stimulated by L1 in these cells. We here show for the first time that small-molecule inhibitors of FGFR, integrins and FAK effectively and selectively abolish L1-stimulated migration and proliferation of glioblastoma-derived cells. Our results suggest that these inhibitors have the potential to reduce the aggressiveness of high-grade gliomas expressing L1.

  12. The disintegrin, trimucrin, suppresses LPS-induced activation of phagocytes primarily through blockade of NF-κB and MAPK activation.

    PubMed

    Hung, Yu-Chun; Hsu, Chun-Chieh; Chung, Ching-Hu; Huang, Tur-Fu

    2016-07-01

    In addition to antiplatelet activity, disintegrin, a small-mass RGD-containing polypeptide, has been shown to exert anti-inflammatory effects but the mechanism involved remains unclear. In this study, we report that trimucrin, a disintegrin from the venom of Trimeresurus mucrosquamatus, inhibits lipopolysaccharide (LPS)-induced stimulation of THP-1 and RAW 264.7 cells. We also investigate the underlying mechanism. Trimucrin decreased the release of proinflammatory cytokines including tumor necrosis factor α (TNFα), interleukin-6 (IL-6), nitric oxide, and reactive oxygen species (ROS), and inhibited the adhesion and migration of LPS-activated phagocytes. Trimucrin significantly blocked the expression of nuclear factor kappaB (NF-κB)-related downstream inducible enzymes such as inducible nitric oxide synthase (iNOS) and COX-2. In addition, its anti-inflammatory effect was associated with the decreased mitogen-activated protein kinase (MAPK) phosphorylation. Furthermore, trimucrin concentration dependently inhibited LPS-induced phosphorylation of focal adhesion kinase (FAK), PI3K, and Akt. Trimucrin also reversed the DNA-binding activity of NF-κB by suppressing the LPS-induced nuclear translocation of p65 and the cytosolic IκB release. Flow cytometric analyses showed that trimucrin bound to cells in a concentration-dependent manner. The anti-αVβ3 mAb also specifically decreased the binding of fluorescein isothiocyanate (FITC)-conjugated trimucrin. Binding assays demonstrated that integrin αVβ3 was the binding site for trimucrin on THP-1 and RAW 264.7 cells. In conclusion, we showed that trimucrin decreases the inflammatory reaction through the attenuation of iNOS expression and nitric oxide (NO) production by blocking MAP kinase and the NF-κB activation in LPS-stimulated THP-1 and RAW 264.7 cells.

  13. Isolation and characterization of two disintegrins inhibiting ADP-induced human platelet aggregation from the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sanchez, Elda E.; Galan, Jacob A.; Russell, William K.

    2006-04-01

    Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are nonenzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction, and may have potential in the treatment of strokes, heart attacks, cancers, and osteoporosis. Prior to 1983, the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake) was known to be only neurotoxic; however, now there is evidence that these snakes can contain venom with: (1) neurotoxins; (2) hemorrhagins; and (3) both neurotoxins and hemorrhagins. In this study, two disintegrins, mojastin 1 and mojastin 2, from the venom ofmore » a Mohave rattlesnake collected in central Arizona (Pinal County), were isolated and characterized. The disintegrins in these venoms were identified by mass-analyzed laser desorption ionization/time-of-flight/time-of-flight (MALDI/TOF/TOF) mass spectrometry as having masses of 7.436 and 7.636 kDa. Their amino acid sequences are similar to crotratroxin, a disintegrin isolated from the venom of the western diamondback rattlesnake (C. atrox). The amino acid sequence of mojastin 1 was identical to the amino acid sequence of a disintegrin isolated from the venom of the Timber rattlesnake (C. horridus). The disintegrins from the Mohave rattlesnake venom were able to inhibit ADP-induced platelet aggregation in whole human blood both having IC{sub 5}s of 13.8 nM, but were not effective in inhibiting the binding of human urinary bladder carcinoma cells (T24) to fibronectin.« less

  14. The influence of surface integrin binding patterns on specific biomaterial-cell interactions

    NASA Astrophysics Data System (ADS)

    Beranek, Maggi Marie

    As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second

  15. Selective binding and lateral clustering of α5β1 and αvβ3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

    PubMed Central

    Schaufler, Viktoria; Czichos-Medda, Helmi; Hirschfeld-Warnecken, Vera; Neubauer, Stefanie; Rechenmacher, Florian; Medda, Rebecca; Kessler, Horst; Geiger, Benjamin; Spatz, Joachim P.; Cavalcanti-Adam, E. Ada

    2016-01-01

    ABSTRACT Coordination of the specific functions of α5β1 and αvβ3 integrins is crucial for the precise regulation of cell adhesion, spreading and migration, yet the contribution of differential integrin-specific crosstalk to these processes remains unclear. To determine the specific functions of αvβ3 and α5β1 integrins, we used nanoarrays of gold particles presenting immobilized, integrin-selective peptidomimetic ligands. Integrin binding to the peptidomimetics is highly selective, and cells can spread on both ligands. However, spreading is faster and the projected cell area is greater on α5β1 ligand; both depend on ligand spacing. Quantitative analysis of adhesion plaques shows that focal adhesion size is increased in cells adhering to αvβ3 ligand at 30 and 60 nm spacings. Analysis of αvβ3 and α5β1 integrin clusters indicates that fibrillar adhesions are more prominent in cells adhering to α5β1 ligand, while clusters are mostly localized at the cell margins in cells adhering to αvβ3 ligand. αvβ3 integrin clusters are more pronounced on αvβ3 ligand, though they can also be detected in cells adhering to α5β1 ligand. Furthermore, α5β1 integrin clusters are present in cells adhering to α5β1 ligand, and often colocalize with αvβ3 clusters. Taken together, these findings indicate that the activation of αvβ3 integrin by ligand binding is dispensable for initial adhesion and spreading, but essential to formation of stable focal adhesions. PMID:27003228

  16. 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

  17. Integrin traffic - the update.

    PubMed

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

    2015-03-01

    Integrins are a family of transmembrane cell surface molecules that constitute the principal adhesion receptors for the extracellular matrix (ECM) and are indispensable for the existence of multicellular organisms. In vertebrates, 24 different integrin heterodimers exist with differing substrate specificity and tissue expression. Integrin-extracellular-ligand interaction provides a physical anchor for the cell and triggers a vast array of intracellular signalling events that determine cell fate. Dynamic remodelling of adhesions, through rapid endocytic and exocytic trafficking of integrin receptors, is an important mechanism employed by cells to regulate integrin-ECM interactions, and thus cellular signalling, during processes such as cell migration, invasion and cytokinesis. The initial concept of integrin traffic as a means to translocate adhesion receptors within the cell has now been expanded with the growing appreciation that traffic is intimately linked to the cell signalling apparatus. Furthermore, endosomal pathways are emerging as crucial regulators of integrin stability and expression in cells. Thus, integrin traffic is relevant in a number of pathological conditions, especially in cancer. Nearly a decade ago we wrote a Commentary in Journal of Cell Science entitled 'Integrin traffic'. With the advances in the field, we felt it would be appropriate to provide the growing number of researchers interested in integrin traffic with an update. © 2015. Published by The Company of Biologists Ltd.

  18. Exercise promotes alpha7 integrin gene transcription and protection of skeletal muscle.

    PubMed

    Boppart, Marni D; Volker, Sonja E; Alexander, Nicole; Burkin, Dean J; Kaufman, Stephen J

    2008-11-01

    The alpha7beta1 integrin is increased in skeletal muscle in response to injury-producing exercise, and transgenic overexpression of this integrin in mice protects against exercise-induced muscle damage. The present study investigates whether the increase in the alpha7beta1 integrin observed in wild-type mice in response to exercise is due to transcriptional regulation and examines whether mobilization of the integrin at the myotendinous junction (MTJ) is a key determinant in its protection against damage. A single bout of downhill running exercise selectively increased transcription of the alpha7 integrin gene in 5-wk-old wild-type mice 3 h postexercise, and an increased alpha7 chain was detected in muscle sarcolemma adjacent to tendinous tissue immediately following exercise. The alpha7B, but not alpha7A isoform, was found concentrated and colocalized with tenascin-C in muscle fibers lining the MTJ. To further validate the importance of the integrin in the protection against muscle damage following exercise, muscle injury was quantified in alpha7(-/-) mice. Muscle damage was extensive in alpha7(-/-) mice in response to both a single and repeated bouts of exercise and was largely restricted to areas of high MTJ concentration and high mechanical force near the Achilles tendon. These results suggest that exercise-induced muscle injury selectively increases transcription of the alpha7 integrin gene and promotes a rapid change in the alpha7beta integrin at the MTJ. These combined molecular and cellular alterations are likely responsible for integrin-mediated attenuation of exercise-induced muscle damage.

  19. Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation

    PubMed Central

    Wei, Xiaofan; Wang, Xiang; Zhan, Jun; Chen, Yuhan; Fang, Weigang; Zhang, Lingqiang

    2017-01-01

    Integrin activation is an indispensable step for various integrin-mediated biological functions. Kindlin-2 is known to coactivate integrins with Talin; however, molecules that restrict integrin activation are elusive. Here, we demonstrate that the E3 ubiquitin ligase Smurf1 controls the amount of Kindlin-2 protein in cells and hinders integrin activation. Smurf1 interacts with and promotes Kindlin-2 ubiquitination and degradation. Smurf1 selectively mediates degradation of Kindlin-2 but not Talin, leading to inhibition of αIIbβ3 integrin activation in Chinese hamster ovary cells and β1 integrin activation in fibroblasts. Enhanced activation of β1 integrin was found in Smurf1-knockout mouse embryonic fibroblasts, which correlates with an increase in Kindlin-2 protein levels. Similarly, a reciprocal relationship between Smurf1 and Kindlin-2 protein levels is found in tissues from colon cancer patients, suggesting that Smurf1 mediates Kindlin-2 degradation in vivo. Collectively, we demonstrate that Smurf1 acts as a brake for integrin activation by controlling Kindlin-2 protein levels, a new mechanism that permits precise modulation of integrin-mediated cellular functions. PMID:28408404

  20. 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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Whelan, Jarrett T.; Wang, Lei; Chen, Jianming

    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 Xmore » 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.« less

  2. Pharmacological Aspects of Vipera xantina palestinae Venom

    PubMed Central

    Momic, Tatjana; Arlinghaus, Franziska T.; Arien-Zakay, Hadar; Katzhendler, Jeoshua; Eble, Johannes A.; Marcinkiewicz, Cezary; Lazarovici, Philip

    2011-01-01

    In Israel, Vipera xantina palestinae (V.x.p.) is the most common venomous snake, accounting for several hundred cases of envenomation in humans and domestic animals every year, with a mortality rate of 0.5 to 2%. In this review we will briefly address the research developments relevant to our present understanding of the structure and function of V.x.p. venom with emphasis on venom disintegrins. Venom proteomics indicated the presence of four families of pharmacologically active compounds: (i) neurotoxins; (ii) hemorrhagins; (iii) angioneurin growth factors; and (iv) different types of integrin inhibitors. Viperistatin, a α1β1selective KTS disintegrin and VP12, a α2β1 selective C-type lectin were discovered. These snake venom proteins represent promising tools for research and development of novel collagen receptor selective drugs. These discoveries are also relevant for future improvement of antivenom therapy towards V.x.p. envenomation. PMID:22174978

  3. Morulustatin, A Disintegrin that Inhibits ADP-Induced Platelet Aggregation, Isolated from the Mexican Tamaulipan Rock Rattlesnake (Crotalus lepidus morulus)

    PubMed Central

    Borja, Miguel; Galan, Jacob Anthony; Cantu, Esteban; Zugasti-Cruz, Alejandro; Rodríguez-Acosta, Alexis; Lazcano, David; Lucena, Sara; Suntravat, Montamas; Sánchez, y Elda Eliza

    2016-01-01

    The Tamaulipan rock rattlesnake (Crotalus lepidus morulus) is a montane snake that occurs in the humid pine-oak forest and the upper cloud forest of the Sierra Madre Oriental in southwestern Tamaulipas, central Nuevo Leon, and southeastern Coahuila in Mexico. Venom from this rattlesnake was fractionated by High-Performance Liquid Chromatography for the purpose of discovering disintegrin molecules. Disintegrins are non-enzymatic, small molecular weight peptides that interfere with cell-cell and cell-matrix interactions by binding to various cell receptors. Eleven fractions were collected by anion exchange chromatography and pooled into six groups (I, II, III, IV, V, and VI). Proteins of the six groups were analyzed by SDS-PAGE and western blot using antibodies raised against a disintegrin. The antibodies recognized different protein bands in five (II, III, IV, V, and VI) of six groups in a molecular mass range of 7 to 105 kDa. Western blot analysis revealed fewer protein bands in the higher molecular mass range and two bands in the disintegrin weight range in group II compared with the other four groups. Proteins in group II were further separated into nine fractions using reverse phase C18 chromatography. Fraction 4 inhibited platelet aggregation and was named morulustatin, which exhibited a single band with a molecular mass of approximately 7 kDa. Mass spectrometry analysis of fraction 4 revealed the identification of disintegrin peptides LRPGAQCADGLCCDQCR (MH+ 2035.84) and AGEECDCGSPANCCDAATCK (MH+ 2328.82). Morulustatin inhibited ADP-induced platelet aggregation in human whole blood and was concentration-dependent with an IC50 of 89.5 nM ± 12. PMID:28713196

  4. 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.

  5. A screen to identify Drosophila genes required for integrin-mediated adhesion.

    PubMed Central

    Walsh, E P; Brown, N H

    1998-01-01

    Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. We have used FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F1 generation and screened for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, 3 are PS integrin genes, 2 are blistered and bloated, and the remaining 9 appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin alpha subunit genes. Mutations in 8 loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the strongest integrin mutations, cause a "tail-up" embryonic lethal phenotype, and mutant alleles of 1 of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function. PMID:9755209

  6. Dramatic and concerted conformational changes enable rhodocetin to block α2β1 integrin selectively

    PubMed Central

    Orriss, George L.; Niland, Stephan; Johanningmeier, Benjamin; Pohlentz, Gottfried; Meier, Markus; Karrasch, Simone; Estevão-Costa, Maria Inacia; Martins Lima, Augusto; Stetefeld, Jörg

    2017-01-01

    The collagen binding integrin α2β1 plays a crucial role in hemostasis, fibrosis, and cancer progression amongst others. It is specifically inhibited by rhodocetin (RC), a C-type lectin-related protein (CLRP) found in Malayan pit viper (Calloselasma rhodostoma) venom. The structure of RC alone reveals a heterotetramer arranged as an αβ and γδ subunit in a cruciform shape. RC specifically binds to the collagen binding A-domain of the integrin α2 subunit, thereby blocking collagen-induced platelet aggregation. However, until now, the molecular basis for this interaction has remained unclear. Here, we present the molecular structure of the RCγδ-α2A complex solved to 3.0 Å resolution. Our findings show that RC undergoes a dramatic structural reorganization upon binding to α2β1 integrin. Besides the release of the nonbinding RCαβ tandem, the RCγ subunit interacts with loop 2 of the α2A domain as result of a dramatic conformational change. The RCδ subunit contacts the integrin α2A domain in the “closed” conformation through its helix C. Combined with epitope-mapped antibodies, conformationally locked α2A domain mutants, point mutations within the α2A loop 2, and chemical modifications of the purified toxin protein, this molecular structure of RCγδ-α2A complex explains the inhibitory mechanism and specificity of RC for α2β1 integrin. PMID:28704364

  7. Integrins in T Cell Physiology

    PubMed Central

    Alabiso, Oscar; Galetto, Alessandra Silvia; Baldanzi, Gianluca

    2018-01-01

    From the thymus to the peripheral lymph nodes, integrin-mediated interactions with neighbor cells and the extracellular matrix tune T cell behavior by organizing cytoskeletal remodeling and modulating receptor signaling. LFA-1 (αLβ2 integrin) and VLA-4 (α4β1 integrin) play a key role throughout the T cell lifecycle from thymocyte differentiation to lymphocyte extravasation and finally play a fundamental role in organizing immune synapse, providing an essential costimulatory signal for the T cell receptor. Apart from tuning T cell signaling, integrins also contribute to homing to specific target organs as exemplified by the importance of α4β7 in maintaining the gut immune system. However, apart from those well-characterized examples, the physiological significance of the other integrin dimers expressed by T cells is far less understood. Thus, integrin-mediated cell-to-cell and cell-to-matrix interactions during the T cell lifespan still represent an open field of research. PMID:29415483

  8. αV-class integrins exert dual roles on α5β1 integrins to strengthen adhesion to fibronectin

    PubMed Central

    Bharadwaj, Mitasha; Strohmeyer, Nico; Colo, Georgina P.; Helenius, Jonne; Beerenwinkel, Niko; Schiller, Herbert B.; Fässler, Reinhard; Müller, Daniel J.

    2017-01-01

    Upon binding to the extracellular matrix protein, fibronectin, αV-class and α5β1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion. Both integrin classes have been functionally specified, however their specific roles in immediate phases of cell attachment remain uncharacterized. Here, we quantify the adhesion of αV-class and/or α5β1 integrins expressing fibroblasts initiating attachment to fibronectin (≤120 s) by single-cell force spectroscopy. Our data reveals that αV-class integrins outcompete α5β1 integrins. Once engaged, αV-class integrins signal to α5β1 integrins to establish additional adhesion sites to fibronectin, away from those formed by αV-class integrins. This crosstalk, which strengthens cell adhesion, induces α5β1 integrin clustering by RhoA/ROCK/myosin-II and Arp2/3-mediated signalling, whereas overall cell adhesion depends on formins. The dual role of both fibronectin-binding integrin classes commencing with an initial competition followed by a cooperative crosstalk appears to be a basic cellular mechanism in assembling focal adhesions to the extracellular matrix. PMID:28128308

  9. From a Helix to a Small Cycle: Metadynamics-Inspired αvβ6 Integrin Selective Ligands.

    PubMed

    Di Leva, Francesco Saverio; Tomassi, Stefano; Di Maro, Salvatore; Reichart, Florian; Notni, Johannes; Dangi, Abha; Marelli, Udaya Kiran; Brancaccio, Diego; Merlino, Francesco; Wester, Hans-Jürgen; Novellino, Ettore; Kessler, Horst; Marinelli, Luciana

    2018-04-16

    The RGD-recognizing αvβ6 integrin has only recently emerged as a major target for cancer diagnosis and therapy. Thus, the development of selective, low-molecular-weight ligands of this receptor is still in great demand. Here, a metadynamics-driven design strategy allowed us to successfully convert a helical nonapeptide into a cyclic pentapeptide (6) showing remarkable potency and αvβ6 specificity. NMR and docking studies elucidated the reasons for the high affinity and selectivity of this compound, setting the ground for the rational design of new αvβ6-specific small peptides or even peptidomimetics. In vivo PET imaging studies demonstrated the potential use of 6 for medical applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Guiding plant virus particles to integrin-displaying cells

    NASA Astrophysics Data System (ADS)

    Hovlid, Marisa L.; Steinmetz, Nicole F.; Laufer, Burkhardt; Lau, Jolene L.; Kuzelka, Jane; Wang, Qian; Hyypiä, Timo; Nemerow, Glen R.; Kessler, Horst; Manchester, Marianne; Finn, M. G.

    2012-05-01

    Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity

  11. Tensin stabilizes integrin adhesive contacts in Drosophila.

    PubMed

    Torgler, Catherine N; Narasimha, Maithreyi; Knox, Andrea L; Zervas, Christos G; Vernon, Matthew C; Brown, Nicholas H

    2004-03-01

    We report the functional characterization of the Drosophila ortholog of tensin, a protein implicated in linking integrins to the cytoskeleton and signaling pathways. A tensin null was generated and is viable with wing blisters, a phenotype characteristic of loss of integrin adhesion. In tensin mutants, mechanical abrasion is required during wing expansion to cause wing blisters, suggesting that tensin strengthens integrin adhesion. The localization of tensin requires integrins, talin, and integrin-linked kinase. The N-terminal domain and C-terminal PTB domain of tensin provide essential recruitment signals. The intervening SH2 domain is not localized on its own. We suggest a model where tensin is recruited to sites of integrin adhesion via its PTB and N-terminal domains, localizing the SH2 domain so that it can interact with phosphotyrosine-containing proteins, which stabilize the integrin link to the cytoskeleton.

  12. Trimucrin, an Arg-Gly-Asp containing disintegrin, attenuates myocardial ischemia-reperfusion injury in murine by inhibiting platelet function.

    PubMed

    Hung, Yu-Chun; Kuo, Yu-Ju; Huang, Shiang-Suo; Huang, Tur-Fu

    2017-10-15

    Trimucrin, a novel small-mass Arg-Gly-Asp (RGD)-containing disintegrin, has been demonstrated to possess anti-platelet and anti-inflammatory effect through blockade of platelet αIIbβ3 and phagocyte αvβ3 integrin. In this study, we found that the platelet-rich plasma prepared from trimucrin-treated rats platelet aggregation was diminished in response to adenosine diphosphate (ADP). We tried to determine whether trimucrin is cardioprotective in rats subjected to myocardial ischemia-reperfusion (I-R) injury. The left anterior descending coronary artery of anesthetized rats was subjected to 1h occlusion and 3h reperfusion. The animals received intravenous trimucrin or saline, and the severities of I-R-induced arrhythmia and infarction were compared. Trimucrin significantly reduced I-R-induced arrhythmias and reduced mortality, as well as infarct volume, troponin-I levels, creatine kinase, and lactate dehydrogenase activity in carotid blood compared with vehicle-treated animals during the same period. Trimucrin also improved cardiac function and survival rates after I-R injury. In addition, trimucrin concentration-dependently inhibited platelet adhesion on collagen- and fibrinogen-coated surfaces without affecting platelet counts. Trimucrin also significantly reduced neutrophil infiltration into heart tissues after I-R compared with controls. Furthermore, trimucrin treatment caused significant downregulation of Bax, Caspase-3 apoptotic proteins and upregulation of anti-apoptotic Bcl-2 protein. These results demonstrate that trimucrin exerts cardioprotective property against myocardial I-R injury mediated through antiplatele, anti-inflammatory, anti-apoptotic mechanism, as well as improvements in cardiac function. Copyright © 2017. Published by Elsevier B.V.

  13. Definition of Two Angiogenic Pathways by Distinct α_v Integrins

    NASA Astrophysics Data System (ADS)

    Friedlander, Martin; Brooks, Peter C.; Shaffer, Robert W.; Kincaid, Christine M.; Varner, Judith A.; Cheresh, David A.

    1995-12-01

    Angiogenesis depends on cytokines and vascular cell adhesion events. Two cytokine-dependent pathways of angiogenesis were shown to exist and were defined by their dependency on distinct vascular cell integrins. In vivo angiogenesis in corneal or chorioallantoic membrane models induced by basic fibroblast growth factor or by tumor necrosis factor-α depended on α_vβ_3, whereas angiogenesis initiated by vascular endothelial growth factor, transforming growth factor-α, or phorbol ester depended on α_vβ_5. Antibody to each integrin selectively blocked one of these pathways, and a cyclic peptide antagonist of both integrins blocked angiogenesis stimulated by each cytokine tested. These pathways are further distinguished by their sensitivity to calphostin C, an inhibitor of protein kinase C that blocked angiogenesis potentiated by α_vβ_5 but not by α_vβ_3.

  14. Integrins protect cardiomyocytes from ischemia/reperfusion injury

    PubMed Central

    Okada, Hideshi; Lai, N. Chin; Kawaraguchi, Yoshitaka; Liao, Peter; Copps, Jeffrey; Sugano, Yasuo; Okada-Maeda, Sunaho; Banerjee, Indroneal; Schilling, Jan M.; Gingras, Alexandre R.; Asfaw, Elizabeth K.; Suarez, Jorge; Kang, Seok-Min; Perkins, Guy A.; Au, Carol G.; Israeli-Rosenberg, Sharon; Manso, Ana Maria; Liu, Zheng; Milner, Derek J.; Kaufman, Stephen J.; Patel, Hemal H.; Roth, David M.; Hammond, H. Kirk; Taylor, Susan S.; Dillmann, Wolfgang H.; Goldhaber, Joshua I.; Ross, Robert S.

    2013-01-01

    Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7β1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of β1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7β1D exposed to I/R had a substantial reduction in infarct size compared with that of α5β1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7β1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that β1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165–175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7β1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury. PMID:24091324

  15. Fine Tuning Cell Migration by a Disintegrin and Metalloproteinases

    PubMed Central

    Theodorou, K.

    2017-01-01

    Cell migration is an instrumental process involved in organ development, tissue homeostasis, and various physiological processes and also in numerous pathologies. Both basic cell migration and migration towards chemotactic stimulus consist of changes in cell polarity and cytoskeletal rearrangement, cell detachment from, invasion through, and reattachment to their neighboring cells, and numerous interactions with the extracellular matrix. The different steps of immune cell, tissue cell, or cancer cell migration are tightly coordinated in time and place by growth factors, cytokines/chemokines, adhesion molecules, and receptors for these ligands. This review describes how a disintegrin and metalloproteinases interfere with several steps of cell migration, either by proteolytic cleavage of such molecules or by functions independent of proteolytic activity. PMID:28260841

  16. Integrin expression and integrin-mediated adhesion in vitro of human multipotent stromal cells (MSCs) to endothelial cells from various blood vessels.

    PubMed

    Semon, Julie A; Nagy, Lauren H; Llamas, Claire B; Tucker, H Alan; Lee, Ryang Hwa; Prockop, Darwin J

    2010-07-01

    Multipotent mesenchymal stromal cells (MSCs) home to damaged tissue by processes partly regulated by integrins. Integrin subunits expressed by MSCs were identified by flow cytometry (FC), immunocytochemistry (IC), and a panel of integrin-binding antibodies. In subconfluent cultures, over 80% of MSCs expressed integrin subunits beta1, beta2, and alpha3, 20%-55% expressed alpha1, alpha2, alpha4, alpha5, alpha6, and alphaV, and about 10% expressed beta3 when assayed by FC. None of the cells expressed significant levels of 13 other integrins as assayed by FC, but seven of the 13 integrins were detected by IC: beta5, alpha7, alpha8, alpha9, alpha11, alphaX, and alphaD. Expression of some integrins changed with MSC confluency: integrins beta3, alpha1, alpha3, alpha5, and alphaV increased, and alpha6 decreased. Furthermore, alpha4 was the only integrin to vary among preparations of MSCs from different donors. The results resolved some discrepancies in the literature concerning integrin expression by MSCs. We also investigated the role of specific integrins in MSC adhesion to endothelial cells (ECs) from the pulmonary artery (HPAEC), cardiac-derived microvasculature (HMVEC-C), and umbilical veins (HUVEC). In experiments with blocking antibodies to beta integrins, anti-beta5 reduced MSC adhesion to all types of ECs, anti-beta1 to both HUVEC and HPAEC, anti-beta3 to HUVEC, and anti-beta2 to HMVEC-C. With blocking antibodies to alpha integrins, anti-alphaX reduced adhesion to HPAEC and HMVEC-C, anti-alphaV to HPAEC, and both anti-alpha7 and anti-alphaD to HMVEC-C. Thus, MSCs use diverse integrins to adhere to EC from various blood vessels in vitro.

  17. 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.

  18. Blocking neutrophil integrin activation prevents ischemia-reperfusion injury.

    PubMed

    Yago, Tadayuki; Petrich, Brian G; Zhang, Nan; Liu, Zhenghui; Shao, Bojing; Ginsberg, Mark H; McEver, Rodger P

    2015-07-27

    Neutrophil recruitment, mediated by β2 integrins, combats pyogenic infections but also plays a key role in ischemia-reperfusion injury and other inflammatory disorders. Talin induces allosteric rearrangements in integrins that increase affinity for ligands (activation). Talin also links integrins to actin and other proteins that enable formation of adhesions. Structural studies have identified a talin1 mutant (L325R) that perturbs activation without impairing talin's capacity to link integrins to actin and other proteins. Here, we found that mice engineered to express only talin1(L325R) in myeloid cells were protected from renal ischemia-reperfusion injury. Dissection of neutrophil function in vitro and in vivo revealed that talin1(L325R) neutrophils had markedly impaired chemokine-induced, β2 integrin-mediated arrest, spreading, and migration. Surprisingly, talin1(L325R) neutrophils exhibited normal selectin-induced, β2 integrin-mediated slow rolling, in sharp contrast to the defective slow rolling of neutrophils lacking talin1 or expressing a talin1 mutant (W359A) that blocks talin interaction with integrins. These studies reveal the importance of talin-mediated activation of integrins for renal ischemia-reperfusion injury. They further show that neutrophil arrest requires talin recruitment to and activation of integrins. However, although neutrophil slow rolling requires talin recruitment to integrins, talin-mediated integrin activation is dispensable. © 2015 Yago et al.

  19. The regulation of integrin function by divalent cations

    PubMed Central

    Zhang, Kun; Chen, JianFeng

    2012-01-01

    Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca2+-binding motifs that have essential roles in integrin biogenesis. The function of another Ca2+-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions. PMID:22647937

  20. A Disintegrin and Metalloprotease 17 in the Cardiovascular and Central Nervous Systems.

    PubMed

    Xu, Jiaxi; Mukerjee, Snigdha; Silva-Alves, Cristiane R A; Carvalho-Galvão, Alynne; Cruz, Josiane C; Balarini, Camille M; Braga, Valdir A; Lazartigues, Eric; França-Silva, Maria S

    2016-01-01

    ADAM17 is a metalloprotease and disintegrin that lodges in the plasmatic membrane of several cell types and is able to cleave a wide variety of cell surface proteins. It is somatically expressed in mammalian organisms and its proteolytic action influences several physiological and pathological processes. This review focuses on the structure of ADAM17, its signaling in the cardiovascular system and its participation in certain disorders involving the heart, blood vessels, and neural regulation of autonomic and cardiovascular modulation.

  1. 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

  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 α 6β 4 and α 6β 1 weremore » associated with lung metastasis, while exosomal integrin α vβ 5 was linked to liver metastasis. Targeting the integrins α 6β 4 and α vβ 5 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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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 α 6β 4 and α 6β 1 weremore » associated with lung metastasis, while exosomal integrin α vβ 5 was linked to liver metastasis. Targeting the integrins α 6β 4 and α vβ 5 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

  4. An Integrin-Targeting RGDK-Tagged Nanocarrier: Anticancer Efficacy of Loaded Curcumin.

    PubMed

    Das, Krishnendu; Nimushakavi, Sahithi; Chaudhuri, Arabinda; Das, Prasanta Kumar

    2017-05-22

    Herein we report the design and development of α 5 β 1 integrin-specific noncovalent RGDK-lipopeptide-functionalized single-walled carbon nanotubes (SWNTs) that selectively deliver the anticancer drug curcumin to tumor cells. RGDK tetrapeptide-tagged amphiphiles were synthesized that efficiently disperse SWNTs with a suspension stability index of >80 % in cell culture media. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)- and lactate dehydrogenase (LDH)-based cell viability assays in tumor (B16F10 melanoma) and noncancerous (NIH3T3 mouse fibroblast) cells revealed the non-cytotoxic nature of these RGDK-lipopeptide-SWNT conjugates. Cellular uptake experiments with monoclonal antibodies against α v β 3 , α v β 5 , and α 5 β 1 integrins showed that these SWNT nanovectors deliver their cargo (Cy3-labeled oligonucleotides, Cy3-oligo) to B16F10 cells selectively via α 5 β 1 integrin. Notably, the nanovectors failed to deliver the Cy3-oligo to NIH3T3 cells. The RGDK-SWNT is capable of delivering the anticancer drug curcumin to B16F10 cells more efficiently than NIH3T3 cells, leading to selective killing of B16F10 cells. Results of Annexin V binding based flow cytometry experiments are consistent with selective killing of tumor cells through the late apoptotic pathway. Biodistribution studies in melanoma (B16F10)-bearing C57BL/6J mice showed tumor-selective accumulation of curcumin intravenously administered via RGDK-lipopeptide-SWNT nanovectors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Integrin-directed modulation of macrophage responses to biomaterials.

    PubMed

    Zaveri, Toral D; Lewis, Jamal S; Dolgova, Natalia V; Clare-Salzler, Michael J; Keselowsky, Benjamin G

    2014-04-01

    Macrophages are the primary mediator of chronic inflammatory responses to implanted biomaterials, in cases when the material is either in particulate or bulk form. Chronic inflammation limits the performance and functional life of numerous implanted medical devices, and modulating macrophage interactions with biomaterials to mitigate this response would be beneficial. The integrin family of cell surface receptors mediates cell adhesion through binding to adhesive proteins nonspecifically adsorbed onto biomaterial surfaces. In this work, the roles of integrin Mac-1 (αMβ2) and RGD-binding integrins were investigated using model systems for both particulate and bulk biomaterials. Specifically, the macrophage functions of phagocytosis and inflammatory cytokine secretion in response to a model particulate material, polystyrene microparticles were investigated. Opsonizing proteins modulated microparticle uptake, and integrin Mac-1 and RGD-binding integrins were found to control microparticle uptake in an opsonin-dependent manner. The presence of adsorbed endotoxin did not affect microparticle uptake levels, but was required for the production of inflammatory cytokines in response to microparticles. Furthermore, it was demonstrated that integrin Mac-1 and RGD-binding integrins influence the in vivo foreign body response to a bulk biomaterial, subcutaneously implanted polyethylene terephthalate. A thinner foreign body capsule was formed when integrin Mac-1 was absent (~30% thinner) or when RGD-binding integrins were blocked by controlled release of a blocking peptide (~45% thinner). These findings indicate integrin Mac-1 and RGD-binding integrins are involved and may serve as therapeutic targets to mitigate macrophage inflammatory responses to both particulate and bulk biomaterials. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Cigarette smoke extract induces select matrix metalloproteinases and integrin expression in periodontal ligament fibroblasts.

    PubMed

    Bulmanski, Zachary; Brady, Matthew; Stoute, Diana; Lallier, Thomas E

    2012-06-01

    The periodontal ligament (PDL) is the connective tissue that anchors the cementum of the teeth to the alveolar bone. PDL fibroblasts are responsible for the production of collagen and remodeling of the PDL. Periodontal disease is increased among smokers in both incidence and severity. This study examines the direct effect of smoking on PDL fibroblasts and their production of various matrix components and remodeling enzymes. PDL cells were plated for 1 day and then treated with various concentrations of cigarette smoke extract (CSE). Survival of PDL cells was quantified after exposure to CSE, and their ability to contract three-dimensional collagen gels was examined. Changes in transcript expression after CSE treatment was compared using reverse transcription-polymerase chain reaction analysis for matrix metalloproteinases (MMPs), collagens, and integrins. Treatment with CSE-induced cell death at concentrations of ≥5%. PDL-cell-induced collagen gel contraction was reduced at concentrations of 1.5% CSE. Treatment with CSE selectively increased the expression of collagen Vα3 and decreased collagen XIα1. CSE increased the expression of MMP1 and MMP3 and, to a lesser extent, MMP2 and MMP8. CSE also increased the expression of integrins α1, α2, and α10 (collagen receptors) and α9 (a tenascin receptor). This study shows that cigarette smoking has local effects on the cells of the PDL. CSE reduced survival of PDL cells and their ability to contract collagen matrices. CSE also altered the expression of molecules known to provide the structural integrity of the ligament by altering collagen synthesis and remodeling as well as cell adhesion.

  7. Integrin Expression Regulates Neuroblastoma Attachment and Migration1

    PubMed Central

    Meyer, Amy; van Golen, Cynthia M.; Kim, Bhumsoo; van Golen, Kenneth L.; Feldman, Eva L.

    2004-01-01

    Abstract Neuroblastoma (NBL) is the most common malignant disease of infancy, and children with bone metastasis have a mortality rate greater than 90%. Two major classes of proteins, integrins and growth factors, regulate the metastatic process. We have previously shown that tumorigenic NBL cells express higher levels of the type I insulin-like growth factor receptor (IGF-IR) and that β1 integrin expression is inversely proportional to tumorigenic potential in NBL. In the current study, we analyze the effect of β1 integrin and IGF-IR on NBL cell attachment and migration. Nontumorigenic S-cells express high levels of β1 integrin, whereas tumorigenic N-cells express little β1 integrin. Alterations in β1 integrin are due to regulation at the protein level, as translation is decreased in N-type cells. Moreover, inhibition of protein synthesis shows that β1 integrin is degraded more slowly in S-type cells (SHEP) than in N-type cells (SH-SY5Y and IMR32). Inhibition of α5β1 integrin prevents SHEP (but not SH-SY5Y or IMR32) cell attachment to fibronectin and increases SHEP cell migration. Increases in IGF-IR decrease β1 integrin expression, and enhance SHEP cell migration, potentially through increased expression of αvβ3. These data suggest that specific classes of integrins in concert with IGF-IR regulate NBL attachment and migration. PMID:15256055

  8. Aggregatibacter actinomycetemcomitans regulates the expression of integrins and reduces cell adhesion via integrin α5 in human gingival epithelial cells.

    PubMed

    Kochi, Shinsuke; Yamashiro, Keisuke; Hongo, Shoichi; Yamamoto, Tadashi; Ugawa, Yuki; Shimoe, Masayuki; Kawamura, Mari; Hirata-Yoshihara, Chiaki; Ideguchi, Hidetaka; Maeda, Hiroshi; Takashiba, Shogo

    2017-12-01

    Gingival epithelial cells form a physiological barrier against bacterial invasion. Excessive bacterial invasion destroys the attachment between the tooth surface and the epithelium, resulting in periodontitis. Integrins play a significant role in cell attachment; therefore, we hypothesized that bacterial infection might decrease the expressions of these integrins in gingival epithelial cells, resulting in reduced cell adhesion. Immortalized human gingival epithelial cells were co-cultured with Aggregatibacter actinomycetemcomitans Y4 (Aa Y4), and the gene expression levels of IL-8, proliferating cell nuclear antigen (PCNA), and integrins (α2, α3, α5, β4, and β6) were measured using quantitative reverse transcription polymerase chain reaction. Expression of PCNA and integrins, except integrin α5, was significantly downregulated, while expression of IL-8 and integrin α5 was significantly upregulated in the cells co-cultured with Aa Y4. The number of adherent cells significantly decreased when co-cultured with Aa Y4, as determined using cell adhesion assays. In the cells co-cultured with Aa Y4 and an integrin α5 neutralizing antibody, there was no effect on the expression of IL-8 and PCNA, while the expressions of integrins α2, α3, β4, and β6, and the number of adherent cells did not decrease. The number of invading bacteria in the cells was reduced in the presence of the antibody and increased in the presence of TLR2/4 inhibitor. Therefore, integrin α5 might be involved in Aa Y4 invasion into gingival epithelial cells, and the resulting signal transduction cascade reduces cell adhesion by decreasing the expression of integrins, while the TLR2/4 signaling cascade regulates IL-8 expression.

  9. Blocking neutrophil integrin activation prevents ischemia–reperfusion injury

    PubMed Central

    Yago, Tadayuki; Petrich, Brian G.; Zhang, Nan; Liu, Zhenghui; Shao, Bojing; Ginsberg, Mark H.

    2015-01-01

    Neutrophil recruitment, mediated by β2 integrins, combats pyogenic infections but also plays a key role in ischemia–reperfusion injury and other inflammatory disorders. Talin induces allosteric rearrangements in integrins that increase affinity for ligands (activation). Talin also links integrins to actin and other proteins that enable formation of adhesions. Structural studies have identified a talin1 mutant (L325R) that perturbs activation without impairing talin’s capacity to link integrins to actin and other proteins. Here, we found that mice engineered to express only talin1(L325R) in myeloid cells were protected from renal ischemia–reperfusion injury. Dissection of neutrophil function in vitro and in vivo revealed that talin1(L325R) neutrophils had markedly impaired chemokine-induced, β2 integrin–mediated arrest, spreading, and migration. Surprisingly, talin1(L325R) neutrophils exhibited normal selectin-induced, β2 integrin–mediated slow rolling, in sharp contrast to the defective slow rolling of neutrophils lacking talin1 or expressing a talin1 mutant (W359A) that blocks talin interaction with integrins. These studies reveal the importance of talin-mediated activation of integrins for renal ischemia–reperfusion injury. They further show that neutrophil arrest requires talin recruitment to and activation of integrins. However, although neutrophil slow rolling requires talin recruitment to integrins, talin-mediated integrin activation is dispensable. PMID:26169939

  10. Stable coordination of the inhibitory Ca2+ ion at MIDAS in integrin CD11b/CD18 by an antibody-derived ligand aspartate: Implications for integrin regulation and structure-based drug design

    PubMed Central

    Mahalingam, Bhuvaneshwari; Ajroud, Kaouther; Alonso, Jose Luis; Anand, Saurabh; Adair, Brian; Horenstein, Alberto L; Malavasi, Fabio; Xiong, Jian-Ping; Arnaout, M. Amin

    2011-01-01

    A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal-ion-dependent-adhesion site (MIDAS) in the integrin A-domain. This interaction stabilizes the A-domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A-domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking monoclonal antibody (mAb) 107 binds MIDAS of integrin CD11b/CD18 A-domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over Mg2+ at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of Ca2+ at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+. Binding of Fab 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that denticity of the ligand Asp/Glu can modify divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists. PMID:22095715

  11. Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells.

    PubMed

    Das, Lipsa; Anderson, Todd A; Gard, Jaime M C; Sroka, Isis C; Strautman, Stephanie R; Nagle, Raymond B; Morrissey, Colm; Knudsen, Beatrice S; Cress, Anne E

    2017-05-01

    Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (k actual ) of 3.25 min -1 , threefold faster than α3 integrin (1.0 min -1 ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min -1 ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min -1 ). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in k actual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8-fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the k actual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells†

    PubMed Central

    Das, Lipsa; Anderson, Todd A.; Gard, Jaime M.C.; Sroka, Isis C.; Strautman, Stephanie R.; Nagle, Raymond B.; Morrissey, Colm; Knudsen, Beatrice S.; Cress, Anne E.

    2017-01-01

    Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modelling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual) of 3.25min−1, 3-fold faster than α3 integrin (1.0 min−1), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min−1), and significantly slower than the unrelated transferrin receptor (CD71) (15 min−1). Silencing of α3 integrin protein expression in DU145, PC3 and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8 fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. This article is protected by copyright. All rights reserved PMID:27509031

  13. Selectively Targeting T- and B-Cell Lymphomas: A Benzothiazole Antagonist of α4β1 Integrin

    PubMed Central

    Carpenter, Richard D.; Andrei, Mirela; Aina, Olulanu H.; Lau, Edmond Y.; Lightstone, Felice C.; Liu, Ruiwu; Lam, Kit S.; Kurth, Mark J.

    2011-01-01

    Current cancer chemotherapeutic agents clinically deployed today are designed to be indiscriminately cytotoxic, however achieving selective targeting of cancer malignancies would allow for improved diagnostic and chemotherapeutic tools. Integrin α4β1, a heterodimeric cell surface receptor, is believed to have a relaxed conformation in normal cells and an active conformation in cancerous cells, specifically T- and B-cell lymphomas. This highly attractive yet poorly understood receptor has been selectively targeted with the bisaryl urea peptidomimetic antagonist 1. However, concerns regarding its preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl urea to a 2-arylaminobenzothiazole moiety, resulting in an analog with improved physicochemical properties, solubility and kidney:tumor ratio while maintaining potency (6; IC50 = 53 pM). The results presented herein utilized heterocyclic and solid-phase chemistry, cell adhesion assay, and in vivo optical imaging using the cyanine dye Cy5.5 conjugate. PMID:19072684

  14. A Disintegrin and Metalloproteases (ADAMs) in Cardiovascular, Metabolic and Inflammatory Diseases: Aspects for Theranostic Approaches.

    PubMed

    van der Vorst, Emiel P C; Weber, Christian; Donners, Marjo M P C

    2018-06-06

    A disintegrin and metalloproteases (ADAMs) are membrane-bound enzymes responsible for the shedding or cleavage of various cell surface molecules, such as adhesion molecules, cytokines/chemokines and growth factors. This shedding can result in the release of soluble proteins that can exert agonistic or antagonistic functions. Additionally, ADAM-mediated cleavage can render these membrane proteins inactive. This review will describe the role and association of ADAMs in various pathologies with a main focus on ADAM10 and ADAM17 in atherosclerosis, including a brief overview of atherosclerosis-related ADAM substrates. Furthermore, ADAMs involvement in other metabolic and inflammatory diseases like diabetes, sepsis, Alzheimer's disease and rheumatoid arthritis will be highlighted. Subsequently, we will briefly discuss an interesting emerging field of research, i.e. the potential implications of ADAM expression in extracellular vesicles. Finally, several ADAM-based therapeutic and diagnostic (theranostic) opportunities will be discussed, while focusing on key questions about the required specificity and selectivity. Schattauer GmbH Stuttgart.

  15. Peptides derived from central turn motifs within integrin αIIb and αV cytoplasmic tails inhibit integrin activation.

    PubMed

    Li, Xinlei; Liu, Yongqing; Haas, Thomas A

    2014-12-01

    We previously found that peptides derived from the full length of integrin αIIb and αV cytoplasmic tails inhibited their parent integrin activation, respectively. Here we showed that the cell-permeable peptides corresponding to the conserved central turn motif within αIIb and αV cytoplasmic tails, myr-KRNRPPLEED (αIIb peptide) and myr-KRVRPPQEEQ (αV peptide), similarly inhibited both αIIb and αV integrin activation. Pre-treatment with αIIb or αV peptides inhibited Mn(2+)-activated αIIbβ3 binding to soluble fibrinogen as well as the binding of αIIbβ3-expressing Chinese Hamster Ovary cells to immobilized fibrinogen. Our turn peptides also inhibited adhesion of two breast cancer cell lines (MDA-MB-435 and MCF7) to αV ligand vitronectin. These results suggest that αIIb and αV peptides share a same mechanism in regulating integrin function. Using αIIb peptide as a model, we found that replacement of RPP with AAA significantly attenuated the inhibitory activity of αIIb peptide. Furthermore, we found that αIIb peptide specifically bound to β-tubulin in cells. Our work suggests that the central motif of α tails is an anchoring point for cytoskeletons during integrin activation and integrin-mediated cell adhesion, and its function depends on the turn structure at RPP. However, post-treatment of peptides derived from the full-length tail or from the turn motif did not reverse αIIb and αV integrin activation. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. The Changing Integrin Expression and a Role for Integrin β8 in the Chondrogenic Differentiation of Mesenchymal Stem Cells

    PubMed Central

    LaPointe, Vanessa L. S.; Verpoorte, Amanda; Stevens, Molly M.

    2013-01-01

    Many cartilage tissue engineering approaches aim to differentiate human mesenchymal stem cells (hMSCs) into chondrocytes and develop cartilage in vitro by targeting cell-matrix interactions. We sought to better inform the design of cartilage tissue engineering scaffolds by understanding how integrin expression changes during chondrogenic differentiation. In three models of in vitro chondrogenesis, we studied the temporal change of cartilage phenotype markers and integrin subunits during the differentiation of hMSCs. We found that transcript expression of most subunits was conserved across the chondrogenesis models, but was significantly affected by the time-course of differentiation. In particular, ITGB8 was up-regulated and its importance in chondrogenesis was further established by a knockdown of integrin β8, which resulted in a non-hyaline cartilage phenotype, with no COL2A1 expression detected. In conclusion, we performed a systematic study of the temporal changes of integrin expression during chondrogenic differentiation in multiple chondrogenesis models, and revealed a role for integrin β8 in chondrogenesis. This work enhances our understanding of the changing adhesion requirements of hMSCs during chondrogenic differentiation and underlines the importance of integrins in establishing a cartilage phenotype. PMID:24312400

  17. Current Anti-Integrin Therapy for Ocular Disease.

    PubMed

    Gonzalez-Salinas, Roberto; Hernández-Zimbrón, Luis F; Gulias-Cañizo, Rosario; Sánchez-Vela, Mario Alberto; Ochoa-De La Paz, Lenin; Zamora, Ruben; Quiroz-Mercado, Hugo

    2017-10-31

    The integrin family of cell adhesion molecules mediates homeostasis, signal transduction, and various other interactions between the cell and the extracellular matrix. Integrins are type-1 transmembrane glycoproteins located on the cell surface, widely expressed in leukocytes, which play an important role in the inflammatory pathway. The purpose of this review is to summarize the current state of anti-integrin therapy and to assess ongoing clinical trials in ocular disease. We performed a search on PubMed, CINAHL, and Embase for the published literature available using the MeSH terms: "integrin therapy" and "αLβ2," "α4β1" and "α4β7," "αvβ3," "αvβ5," and "αvβ1" and/or "ophthalmology," and "clinical trials." We used no language restrictions. We generated searches to account for synonyms of these keywords and MESH headings as follows: (1) "integrin," "therapy," or "treatment"; (2) "clinical trials," "ophthalmology," or "ocular." In addition, the analysis included phase 2 and phase 3 clinical trials with a minimal follow-up of six months. Integrin antagonists have shown their capacity to improve signs and symptoms of patients with dry eye disease, age-related macular degeneration, diabetic macular edema, and vitreomacular traction.

  18. A novel cancer-targeting transporter with integrin αvβ3 monoclonal antibody functionalized single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Ou, Zhongmin; Wu, Baoyan; Xing, Da

    2009-08-01

    The pursuit of efficient and highly targeting-selective transporters is an active topic in cancer-targeting therapy. In this study, a novel cancer-targeting transporter with integrin αvβ3 monoclonal antibody functionalized single-walled carbon nanotubes (SWCNTs) was developed to investigate cancer cell targeting in vitro. SWCNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). PL-PEG functionalized SWCNTs were then conjugated with fluorescein isothiocyanate (FITC) labeled integrin αvβ3 monoclonal antibody to construct SWCNT-integrin αvβ3 monoclonal antibody system (denoted as SWCNT-PEG-mAb). In vitro study revealed that the system had a high efficiency in cancer cell targeting in integrin αvβ3 positive U87MG cells. Moreover, the SWCNT-PEG-mAb is stable in physiological media, and can be readily transported into U87MG cells via integrin αvβ3-mediated endocytosis in cell. In summary, the integrin αvβ3 monoclonal antibody labeled SWCNT is a potential carrier-candidate for cancer-imaging and drug-delivering in cancer-targeting therapy.

  19. Cardiac integrins the ties that bind.

    PubMed

    Simpson, D G; Reaves, T A; Shih, D T; Burgess, W; Borg, T K; Terracio, L

    1998-01-01

    An elaborate series of morphogenetic events must be precisely coordinated during development to promote the formation of the elaborate three-dimensional structure of the normal heart. In this study we focus on discussing how interconnections between the cardiac myocyte and its surrounding environment regulate cardiac form and function. In vitro experiments from our laboratories provide direct evidence that cardiac cell shape is regulated by a dynamic interaction between constituents of the extracellular matrix (ECM) and by specific members of the integrin family of matrix receptors. Our data indicates that phenotypic information is stored in the tertiary structure and chemical identity of the ECM. This information appears to be actively communicated and transduced by the α1β1 integrin molecule into an intracellular signal that regulates cardiac cell shape and myofibrillar organization. In this study we have assessed the phenotypic consequences of suppressing the expression and accumulation of the α1 integrin molecule in aligned cultures of cardiac myocytes. In related experiments we have examined how the overexpression of α2 and α5 integrin, integrins normally not present or present at very low copy number on the cell surface of neonatal cardiac myocytes, affect cardiac protein metabolism. We also consider how biochemical signals and the mechanical signals mediated by the integrins may converge on common intracellular signaling pathways in the heart. Experiments with the whole embryo culture system indicate that angiotensin II, a peptide that carries information concerning cardiac load, plays a role in controling cardiac looping and the proliferation of myofibrils during development.

  20. Integrins are Mechanosensors That Modulate Human Eosinophil Activation

    PubMed Central

    Ahmadzai, Mustafa; Small, Mike; Sehmi, Roma; Gauvreau, Gail; Janssen, Luke J.

    2015-01-01

    Eosinophil migration to the lung is primarily regulated by the eosinophil-selective family of eotaxin chemokines, which mobilize intracellular calcium (Ca2+) and orchestrate myriad changes in cell structure and function. Eosinophil function is also known to be flow-dependent, although the molecular cognate of this mechanical response has yet to be adequately characterized. Using confocal fluorescence microscopy, we determined the effects of fluid shear stress on intracellular calcium concentration ([Ca2+]i) in human peripheral blood eosinophils by perfusing cells in a parallel-plate flow chamber. Our results indicate that fluid perfusion evokes a calcium response that leads to cell flattening, increase in cell area, shape change, and non-directional migration. None of these changes are seen in the absence of a flow stimulus, and all are blocked by chelation of intracellular Ca2+ using BAPTA. These changes are enhanced by stimulating the cells with eotaxin-1. The perfusion-induced calcium response (PICR) could be blocked by pre-treating cells with selective (CDP-323) and non-selective (RGD tripeptides) integrin receptor antagonists, suggesting that α4β7/α4β1 integrins mediate this response. Overall, our study provides the first pharmacological description of a molecular mechanosensor that may collaborate with the eotaxin-1 signaling program in order to control human eosinophil activation. PMID:26539194

  1. Disintegrin and metalloproteinases (ADAMs) expression in gastroesophageal reflux disease and in esophageal adenocarcinoma.

    PubMed

    Kauttu, T; Mustonen, H; Vainionpää, S; Krogerus, L; Ilonen, I; Räsänen, J; Salo, J; Puolakkainen, P

    2017-01-01

    Clinically useful marker molecules for the progression of gastroesophageal reflux disease and Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) are lacking. Many adenocarcinomas and inflammatory conditions exhibit increased expression of ADAMs, 'a disintegrin and metalloproteinases'. We assessed the expression of five ADAMs (9, 10, 12, 17, 19) in three esophageal cell lines (Het-1A, OE19, OE33) by RT-PCR and Western blotting, and in human samples of normal esophagus, esophagitis, BE, Barrett's dysplasia, and EAC by RT-PCR, and in selected samples by immunohistochemistry. EAC patients showed increased mRNA expression of ADAMs 9, 12, 17 and 19, as compared to controls. At immunohistochemistry, ADAM9 and ADAM10 proteins were increased in EAC. Patient samples also showed increased mRNA expression of ADAM12 in esophagitis, of ADAM9 in BE, and of ADAMs 9, 12 and 19 in Barrett's dysplasia, as compared to controls. Two EAC cell lines showed increased ADAM9 mRNA. ADAM9 expression is increased in EAC. Its predecessors show increased ADAM9 mRNA expression. The importance of the alterations in ADAM expression for the development of EAC, and their use as marker molecules, warrant further studies.

  2. 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

  3. Beyond the Matrix: The Many Non-ECM Ligands for Integrins

    PubMed Central

    LaFoya, Bryce; Munroe, Jordan A.; Miyamoto, Alison; Detweiler, Michael A.; Crow, Jacob J.; Gazdik, Tana

    2018-01-01

    The traditional view of integrins portrays these highly conserved cell surface receptors as mediators of cellular attachment to the extracellular matrix (ECM), and to a lesser degree, as coordinators of leukocyte adhesion to the endothelium. These canonical activities are indispensable; however, there is also a wide variety of integrin functions mediated by non-ECM ligands that transcend the traditional roles of integrins. Some of these unorthodox roles involve cell-cell interactions and are engaged to support immune functions such as leukocyte transmigration, recognition of opsonization factors, and stimulation of neutrophil extracellular traps. Other cell-cell interactions mediated by integrins include hematopoietic stem cell and tumor cell homing to target tissues. Integrins also serve as cell-surface receptors for various growth factors, hormones, and small molecules. Interestingly, integrins have also been exploited by a wide variety of organisms including viruses and bacteria to support infectious activities such as cellular adhesion and/or cellular internalization. Additionally, the disruption of integrin function through the use of soluble integrin ligands is a common strategy adopted by several parasites in order to inhibit blood clotting during hematophagy, or by venomous snakes to kill prey. In this review, we strive to go beyond the matrix and summarize non-ECM ligands that interact with integrins in order to highlight these non-traditional functions of integrins. PMID:29393909

  4. High-Throughput Screening based Identification of Small Molecule Antagonists of Integrin CD11b/CD18 Ligand Binding

    PubMed Central

    Faridi, Mohd Hafeez; Maiguel, Dony; Brown, Brock T.; Suyama, Eigo; Barth, Constantinos J.; Hedrick, Michael; Vasile, Stefan; Sergienko, Eduard; Schürer, Stephan; Gupta, Vineet

    2010-01-01

    Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique. PMID:20188705

  5. Integrins: masters and slaves of endocytic transport.

    PubMed

    Caswell, Patrick T; Vadrevu, Suryakiran; Norman, Jim C

    2009-12-01

    Since it has become clear that adhesion receptors are trafficked through the endosomal pathway and that this can influence their function, much effort has been invested in obtaining detailed descriptions of the molecular machinery responsible for internalizing and recycling integrins. New findings indicate that integrin trafficking dictates the nature of Rho GTPase signalling during cytokinesis and cell migration. Furthermore, integrins can exert control over the trafficking of other receptors in a way that drives cancer cell invasion and tumour angiogenesis.

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

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  7. Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

    PubMed

    Walther, Christa G; Whitfield, Robert; James, David C

    2016-04-01

    The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.

  8. The Integrin Receptor in Biologically Relevant Bilayers: Insights from Molecular Dynamics Simulations.

    PubMed

    Kalli, Antreas C; Rog, Tomasz; Vattulainen, Ilpo; Campbell, Iain D; Sansom, Mark S P

    2017-08-01

    Integrins are heterodimeric (αβ) cell surface receptors that are potential therapeutic targets for a number of diseases. Despite the existence of structural data for all parts of integrins, the structure of the complete integrin receptor is still not available. We have used available structural data to construct a model of the complete integrin receptor in complex with talin F2-F3 domain. It has been shown that the interactions of integrins with their lipid environment are crucial for their function but details of the integrin/lipid interactions remain elusive. In this study an integrin/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin/talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2-F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction across cell membranes.

  9. Integrin trafficking regulated by Rab21 is necessary for cytokinesis.

    PubMed

    Pellinen, Teijo; Tuomi, Saara; Arjonen, Antti; Wolf, Maija; Edgren, Henrik; Meyer, Hannelore; Grosse, Robert; Kitzing, Thomas; Rantala, Juha K; Kallioniemi, Olli; Fässler, Reinhard; Kallio, Marko; Ivaska, Johanna

    2008-09-01

    Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.

  10. The synthesis and biological evaluation of integrin receptor targeting molecules as potential radiopharmaceuticals

    NASA Astrophysics Data System (ADS)

    Pellegrini, Paul

    This thesis reports on the synthesis, characterisation and biological evaluation of a number of metal complexes designed to interact with the alphavbeta3 integrin receptor, an important biological target that is heavily involved in angiogenesis, and thus cancer related processes. Two approaches were used to synthesise the integrin-avid targets. The first was to attach a variety of bifunctional chelators (BFC's) for the incorporation of different metal centres to a known integrin antagonist, L-748,415, developed by Merck. The BFC's used were the hydrazinonicotinamide (HYNIC) and monoamine monoamide dithiol (MAMA) systems for coordination to Tc-99m and rhenium of which was used as a characterization surrogate for the unstable Tc core. The 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA) BFC was attached for the inclusion of copper and lutetium. This 'conjugate' approach was designed to yield information on how the BFC and the linker length would affect the affinity for the integrin receptor. The second approach was an 'integrated' method where the chelation moiety was integral to the biologically relevant part of the molecule, which in the case of the alphavbeta3 integrin receptor, is the arginine-glycine-aspartic acid (RGD) mimicking sequence. Two complexes were created with a modified MAMA derivative placed between a benzimidazole moiety (arginine mimick) and the aspartic acid mimicking terminal carboxylic acid to see how it would affect binding while keeping the molecular weight relatively low. The molecules were tested in vitro against purified human alphavbeta3 integrin receptor protein in a solid phase receptor binding assay to evaluate their inhibition constants against a molecule of known high affinity and selectivity in [I125]L-775,219, the I125 labelled alphavbeta3 integrin antagonist. The radiolabelled analogues were also tested in vivo against the A375 human melanoma cell line transplanted into balb/c nude mice as well as Fischer rats implanted

  11. The effect of ligand affinity on integrins' lateral diffusion in cultured cells.

    PubMed

    Mainali, Dipak; Smith, Emily A

    2013-04-01

    The role of ligand affinity in altering αPS2CβPS integrins' lateral mobility was studied using single particle tracking (SPT) with ligand-functionalized quantum dots (QDs) and fluorescence recovery after photobleaching (FRAP) with fluorescent protein tagged integrins. Integrins are ubiquitous transmembrane proteins that are vital for numerous cellular functions, including bidirectional signaling and cell anchorage. Wild-type and high ligand affinity mutant (αPS2CβPS-V409D) integrins were studied in S2 cells. As measured by SPT, the integrin mobile fraction decreased by 22% and had a 4× slower diffusion coefficient for αPS2CβPS-V409D compared to wild-type integrins. These differences are partially the result of αPS2CβPS-V409D integrins' increased clustering. For the wild-type integrins, the average of all diffusion coefficients measured by SPT was statistically similar to the ensemble FRAP results. A 75% slower average diffusion coefficient was measured by SPT compared to FRAP for αPS2CβPS-V409D integrins, and this may be the result of SPT measuring only ligand-bound integrins, in contrast all ligand-bound and ligand-unbound integrins are averaged in FRAP measurements. Specific binding of the ligand-functionalized QDs was 99% for integrin expressing cells. The results prove that the ligand binding affinity affects the lateral dynamics of a subset of integrins based on the complementary SPT and FRAP data.

  12. Rheostat regulation of integrin-mediated leukocyte adhesion

    PubMed Central

    Douglas, Ivor S.; Dassopoulos, Themistocles

    2007-01-01

    The homing of activated T lymphocytes to the gut in inflammatory bowel diseases is dependent on their coordinated, integrin-mediated adhesion and de-adhesion to substrates and blood vessel walls. In this issue of the JCI, Park and colleagues reveal a key modulatory role of a binding site within β integrins, known as the ADMIDAS domain, in controlling integrin de-adhesion in mice (see the related article beginning on page 2526). These observations add to our growing understanding of how integrin adhesiveness is regulated and raise the notion of the existence of a biological rheostat for lymphocyte homing. Disturbed migratory rheostat tone could account for variations in interindividual immune responses observed in patients with inflammatory bowel disease or other lymphocyte-mediated inflammatory disorders. These findings will inform future strategies to design small molecules for the treatment of a spectrum of chronic inflammatory conditions. PMID:17786236

  13. An Integrin from Shrimp Litopenaeus vannamei Mediated Microbial Agglutination and Cell Proliferation

    PubMed Central

    Zhang, Ying; Wang, Leilei; Wang, Lingling; Wu, Ning; Zhou, Zhi; Song, Linsheng

    2012-01-01

    Background Integrins are a family of adhesion receptors which regulate cell proliferation, differentiation, leukocyte migration, and complement receptor-dependent phagocytosis. In invertebrates, as a cell adhesion receptor, β integrins play an important role for the balanced activation of immune defense responses especially during the encounter of infections. The present study attempts to characterize the immune functions of shrimp integrin (LvIntegrin) to have better understanding on the immune system and its regulation mechanisms in shrimps. Methodology A shrimp integrin was identified from the Pacific white shrimp Litopenaeus vannamei (designated as LvIntegrin). Its full-length cDNA was of 2621 bp with an open reading frame (ORF) of 2439 bp encoding a polypeptide of 812 amino acids. The mRNA expression of LvIntegrin was significantly up-regulated at 3, 6 and 12 h after Listonella anguillarum challenge. The cDNA fragment encoding β integrin domains (βA and hybrid domain) of LvIntegrin was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvIntegrin) could significantly agglutinate the tested microbe including E. coli JM109, L. anguillarum, Micrococcus luteus and Candida dattiladattila in the presence of divalent cations. Moreover, when NIH3T3 cells were cultured with rLvIntegrin, the proliferation rate increased significantly in a dose-dependent manner. Conclusions LvIntegrin, a shrimp β integrin was identified from L. vannamei, shared several highly conserved features. LvIntegrin exhibited broad-spectrum agglutination activity towards both bacteria and fungi and could improve the proliferation of NIH3T3 cells, indicating that LvIntegrin is involved in the immune response against microbe challenge and regulation of cell proliferation as a cell adhesion receptor in shrimp. PMID:22792387

  14. An integrin from shrimp Litopenaeus vannamei mediated microbial agglutination and cell proliferation.

    PubMed

    Zhang, Ying; Wang, Leilei; Wang, Lingling; Wu, Ning; Zhou, Zhi; Song, Linsheng

    2012-01-01

    Integrins are a family of adhesion receptors which regulate cell proliferation, differentiation, leukocyte migration, and complement receptor-dependent phagocytosis. In invertebrates, as a cell adhesion receptor, β integrins play an important role for the balanced activation of immune defense responses especially during the encounter of infections. The present study attempts to characterize the immune functions of shrimp integrin (LvIntegrin) to have better understanding on the immune system and its regulation mechanisms in shrimps. A shrimp integrin was identified from the Pacific white shrimp Litopenaeus vannamei (designated as LvIntegrin). Its full-length cDNA was of 2621 bp with an open reading frame (ORF) of 2439 bp encoding a polypeptide of 812 amino acids. The mRNA expression of LvIntegrin was significantly up-regulated at 3, 6 and 12 h after Listonella anguillarum challenge. The cDNA fragment encoding β integrin domains (βA and hybrid domain) of LvIntegrin was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvIntegrin) could significantly agglutinate the tested microbe including E. coli JM109, L. anguillarum, Micrococcus luteus and Candida dattiladattila in the presence of divalent cations. Moreover, when NIH3T3 cells were cultured with rLvIntegrin, the proliferation rate increased significantly in a dose-dependent manner. LvIntegrin, a shrimp β integrin was identified from L. vannamei, shared several highly conserved features. LvIntegrin exhibited broad-spectrum agglutination activity towards both bacteria and fungi and could improve the proliferation of NIH3T3 cells, indicating that LvIntegrin is involved in the immune response against microbe challenge and regulation of cell proliferation as a cell adhesion receptor in shrimp.

  15. Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions.

    PubMed

    Spiess, Matthias; Hernandez-Varas, Pablo; Oddone, Anna; Olofsson, Helene; Blom, Hans; Waithe, Dominic; Lock, John G; Lakadamyali, Melike; Strömblad, Staffan

    2018-06-04

    Integrins are the core constituents of cell-matrix adhesion complexes such as focal adhesions (FAs) and play key roles in physiology and disease. Integrins fluctuate between active and inactive conformations, yet whether the activity state influences the spatial organization of integrins within FAs has remained unclear. In this study, we address this question and also ask whether integrin activity may be regulated either independently for each integrin molecule or through locally coordinated mechanisms. We used two distinct superresolution microscopy techniques, stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion microscopy (STED), to visualize active versus inactive β1 integrins. We first reveal a spatial hierarchy of integrin organization with integrin molecules arranged in nanoclusters, which align to form linear substructures that in turn build FAs. Remarkably, within FAs, active and inactive β1 integrins segregate into distinct nanoclusters, with active integrin nanoclusters being more organized. This unexpected segregation indicates synchronization of integrin activities within nanoclusters, implying the existence of a coordinate mechanism of integrin activity regulation. © 2018 Spiess et al.

  16. Substrate engagement of integrins α5β1 and αvβ3 is necessary, but not sufficient, for high directional persistence in migration on fibronectin

    PubMed Central

    Missirlis, Dimitris; Haraszti, Tamás; Scheele, Catharina v. C.; Wiegand, Tina; Diaz, Carolina; Neubauer, Stefanie; Rechenmacher, Florian; Kessler, Horst; Spatz, Joachim P.

    2016-01-01

    The interplay between specific integrin-mediated matrix adhesion and directional persistence in cell migration is not well understood. Here, we characterized fibroblast adhesion and migration on the extracellular matrix glycoproteins fibronectin and vitronectin, focusing on the role of α5β1 and αvβ3 integrins. Fibroblasts manifested high directional persistence in migration on fibronectin-, but not vitronectin-coated substrates, in a ligand density-dependent manner. Fibronectin stimulated α5β1-dependent organization of the actin cytoskeleton into oriented, ventral stress fibers, and assembly of dynamic, polarized protrusions, characterized as regions free of stress fibers and rich in nascent adhesions at their edge. Such protrusions correlated with persistent, local leading edge advancement, but were not sufficient, nor necessary for directional migration over longer times. Selective blocking of αvβ3 or α5β1 integrins using small molecule integrin antagonists reduced directional persistence on fibronectin, indicating integrin cooperativity in maintaining directionality. On the other hand, patterned substrates, designed to selectively engage either integrin, or their combination, were not sufficient to establish directional migration. Overall, our study demonstrates adhesive coating-dependent regulation of directional persistence in fibroblast migration and challenges the generality of the previously suggested role of β1 and β3 integrins in directional migration. PMID:26987342

  17. Integrins in bone metastasis formation and potential therapeutic implications.

    PubMed

    Clëzardin, P

    2009-11-01

    Integrins constitute a family of cell surface receptors that are heterodimers composed of noncovalently associated alpha and beta subunits. Integrins bind to extracellular matrix proteins and immunogobulin superfamily molecules. They exert a stringent control on cell migration, survival and proliferation. However, their expression and functions are often deregulated in cancer, and many lines of evidence implicate them as key regulators during progression from primary tumor growth to metastasis. Here, we review the role of integrins in bone metastasis formation and present evidence that the use of integrin-targeted therapeutic agents may be an efficient strategy to block tumor metastasis.

  18. Isolation of integrin-based adhesion complexes.

    PubMed

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

    2015-03-02

    The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter-receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry. Copyright © 2015 John Wiley & Sons, Inc.

  19. Integrin αIIbβ3 outside-in signaling

    PubMed Central

    2017-01-01

    Integrin αIIbβ3 is a highly abundant heterodimeric platelet receptor that can transmit information bidirectionally across the plasma membrane, and plays a critical role in hemostasis and thrombosis. Upon platelet activation, inside-out signaling pathways increase the affinity of αIIbβ3 for fibrinogen and other ligands. Ligand binding and integrin clustering subsequently stimulate outside-in signaling, which initiates and amplifies a range of cellular events driving essential platelet processes such as spreading, thrombus consolidation, and clot retraction. Integrin αIIbβ3 has served as an excellent model for the study of integrin biology, and it has become clear that integrin outside-in signaling is highly complex and involves a vast array of enzymes, signaling adaptors, and cytoskeletal components. In this review, we provide a concise but comprehensive overview of αIIbβ3 outside-in signaling, focusing on the key players involved, and how they cooperate to orchestrate this critical aspect of platelet biology. We also discuss gaps in the current understanding of αIIbβ3 outside-in signaling and highlight avenues for future investigation. PMID:28794070

  20. 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.

  1. Diverse roles of integrin receptors in articular cartilage.

    PubMed

    Shakibaei, M; Csaki, C; Mobasheri, A

    2008-01-01

    Integrins are heterodimeric integral membrane proteins made up of alpha and beta subunits. At least eighteen alpha and eight beta subunit genes have been described in mammals. Integrin family members are plasma membrane receptors involved in cell adhesion and active as intra- and extracellular signalling molecules in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic spread of tumour cells. Integrin beta 1 (beta1-integrin), the protein encoded by the ITGB1 gene (also known as CD29 and VLAB), is a multi-functional protein involved in cell-matrix adhesion, cell signalling, cellular defense, cell adhesion, protein binding, protein heterodimerisation and receptor-mediated activity. It is highly expressed in the human body (17.4 times higher than the average gene in the last updated revision of the human genome). The extracellular matrix (ECM) of articular cartilage is a unique environment. Interactions between chondrocytes and the ECM regulate many biological processes important to homeostasis and repair of articular cartilage, including cell attachment, growth, differentiation and survival. The beta1-integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these fundamental processes. Chondrocyte mechanoreceptors have been proposed to incorporate beta1-integrins and mechanosensitive ion channels which link with key ECM, cytoskeletal and signalling proteins to maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression. This review focuses on the expression and function of beta1-integrins in articular chondrocytes, its role in the unique biology of these cells and its distribution in cartilage.

  2. Evidence for the requirement of ITAM domains but not SLP-76/Gads interaction for integrin signaling in hematopoietic cells.

    PubMed

    Abtahian, Farhad; Bezman, Natalie; Clemens, Regina; Sebzda, Eric; Cheng, Lan; Shattil, Sanford J; Kahn, Mark L; Koretzky, Gary A

    2006-09-01

    Syk tyrosine kinase and Src homology 2 (SH2) domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) are signaling mediators activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-containing immunoreceptors and integrins. While the signaling cascades descending from integrins are similar to immunoreceptors, the mechanism of Syk activation and SLP-76 recruitment remains unclear. We used an in vivo structure-function approach to study the requirements for the domains of Syk and SLP-76 in immunoreceptor and integrin signaling. We found that both SH2 domains and the kinase domain of Syk are required for immunoreceptor-dependent signaling and cellular response via integrins. While the Gads-binding domain of SLP-76 is needed for immunoreceptor signaling, it appears dispensable for integrin signaling. Syk and SLP-76 also are required for initiating and/or maintaining separation between the blood and lymphatic vasculature. Therefore, we correlated the signaling requirement of the various domains of Syk and SLP-76 to their requirement in regulating vascular separation. Our data suggest ITAMs are required in Syk-dependent integrin signaling, demonstrate the separation of the structural features of SLP-76 to selectively support immunoreceptor versus integrin signaling, and provide evidence that the essential domains of SLP-76 for ITAM signals are those which most efficiently support separation between lymphatic and blood vessels.

  3. Utilizing Fibronectin Integrin-Binding Specificity to Control Cellular Responses

    PubMed Central

    Bachman, Haylee; Nicosia, John; Dysart, Marilyn; Barker, Thomas H.

    2015-01-01

    Significance: Cells communicate with the extracellular matrix (ECM) protein fibronectin (Fn) through integrin receptors on the cell surface. Controlling integrin–Fn interactions offers a promising approach to directing cell behavior, such as adhesion, migration, and differentiation, as well as coordinated tissue behaviors such as morphogenesis and wound healing. Recent Advances: Several different groups have developed recombinant fragments of Fn that can control epithelial to mesenchymal transition, sequester growth factors, and promote bone and wound healing. It is thought that these physiological responses are, in part, due to specific integrin engagement. Furthermore, it has been postulated that the integrin-binding domain of Fn is a mechanically sensitive switch that drives binding of one integrin heterodimer over another. Critical Issues: Although computational simulations have predicted the mechano-switch hypothesis and recent evidence supports the existence of varying strain states of Fn in vivo, experimental evidence of the Fn integrin switch is still lacking. Future Directions: Evidence of the integrin mechano-switch will enable the development of new Fn-based peptides in tissue engineering and wound healing, as well as deepen our understanding of ECM pathologies, such as fibrosis. PMID:26244106

  4. Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening

    NASA Technical Reports Server (NTRS)

    Lammerding, Jan; Kazarov, Alexander R.; Huang, Hayden; Lee, Richard T.; Hemler, Martin E.

    2003-01-01

    The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including alpha6beta1. To probe strength of alpha6beta1-dependent adhesion to laminin-1, defined forces (0-1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-alpha6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening alpha6beta1 integrin-mediated adhesion to laminin-1.

  5. Oxycodone Self-Administration Induces Alterations in Expression of Integrin, Semaphorin and Ephrin Genes in the Mouse Striatum.

    PubMed

    Yuferov, Vadim; Zhang, Yong; Liang, Yupu; Zhao, Connie; Randesi, Matthew; Kreek, Mary J

    2018-01-01

    Oxycodone is one a commonly used medication for pain, and is also a widely abused prescription opioid, like other short-acting MOPr agonists. Neurochemical and structural adaptations in brain following chronic MOPr-agonist administration are thought to underlie pathogenesis and persistence of opiate addiction. Many axon guidance molecules, such as integrins, semaphorins, and ephrins may contribute to oxycodone-induced neuroadaptations through alterations in axon-target connections and synaptogenesis, that may be implicated in the behaviors associated with opiate addiction. However, little is known about this important area. The aim of this study is to investigate alterations in expression of selected integrin, semaphorin, ephrins, netrin, and slit genes in the nucleus accumbens (NAc) and caudate putamen (CPu) of mice following extended 14-day oxycodone self-administration (SA), using RNAseq. Methods: Total RNA from the NAc and CPu were isolated from adult male C57BL/6J mice within 1 h after the last session of oxycodone in a 14-day self-administration paradigm (4h/day, 0.25 mg/kg/infusion, FR1) or from yoked saline controls. Gene expressions were examined using RNA sequencing (RNA-Seq) technology. RNA-Seq libraries were prepared using Illumina's TruSeq® Stranded Total RNA LT kit. The reads were aligned to the mouse reference genome (version mm10) using STAR. DESeq2 was applied to the counts of protein coding genes to estimate the fold change between the treatment groups. False Discovery Rate (FDR) q < 0.1 were used to select genes that have a significant expression change. For selection of a subset of genes related to axon guidance pathway, REACTOME was used. Results: Among 38 known genes of the integrin, semaphorin, and ephrin gene families, RNA-seq data revealed up-regulation of six genes in the NAc: heterodimer receptor, integrins Itgal, Itgb2 , and Itgam , and its ligand semaphorin Sema7a , two semaphorin receptors, plexins Plxnd1 and Plxdc1 . There was down

  6. The CXC-chemokine CXCL4 interacts with integrins implicated in angiogenesis.

    PubMed

    Aidoudi, Sallouha; Bujakowska, Kinga; Kieffer, Nelly; Bikfalvi, Andreas

    2008-07-16

    The human CXC-chemokine CXCL4 is a potent inhibitor of tumor-induced angiogenesis. Considering that CXCL4 is sequestered in platelet alpha-granules and released following platelet activation in the vicinity of vessel wall injury, we tested the hypothesis that CXCL4 might function as a ligand for integrins. Integrins are a family of adhesion receptors that play a crucial role in angiogenesis by regulating early angiogenic processes, such as endothelial cell adhesion and migration. Here, we show that CXCL4 interacts with alphavbeta3 on the surface of alphavbeta3-CHO. More importantly, human umbilical vein endothelial cells adhere to immobilized CXCL4 through alphavbeta3 integrin, and also through other integrins, such as alphavbeta5 and alpha5beta1. We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner. Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration. As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect.

  7. The CXC-Chemokine CXCL4 Interacts with Integrins Implicated in Angiogenesis

    PubMed Central

    Aidoudi, Sallouha; Bujakowska, Kinga; Kieffer, Nelly; Bikfalvi, Andreas

    2008-01-01

    The human CXC-chemokine CXCL4 is a potent inhibitor of tumor-induced angiogenesis. Considering that CXCL4 is sequestered in platelet α-granules and released following platelet activation in the vicinity of vessel wall injury, we tested the hypothesis that CXCL4 might function as a ligand for integrins. Integrins are a family of adhesion receptors that play a crucial role in angiogenesis by regulating early angiogenic processes, such as endothelial cell adhesion and migration. Here, we show that CXCL4 interacts with αvβ3 on the surface of αvβ3-CHO. More importantly, human umbilical vein endothelial cells adhere to immobilized CXCL4 through αvβ3 integrin, and also through other integrins, such as αvβ5 and α5β1. We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner. Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration. As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect. PMID:18648521

  8. Radil controls neutrophil adhesion and motility through β2-integrin activation.

    PubMed

    Liu, Lunhua; Aerbajinai, Wulin; Ahmed, Syed M; Rodgers, Griffin P; Angers, Stephane; Parent, Carole A

    2012-12-01

    Integrin activation is required to facilitate multiple adhesion-dependent functions of neutrophils, such as chemotaxis, which is critical for inflammatory responses to injury and pathogens. However, little is known about the mechanisms that mediate integrin activation in neutrophils. We show that Radil, a novel Rap1 effector, regulates β1- and β2-integrin activation and controls neutrophil chemotaxis. On activation and chemotactic migration of neutrophils, Radil quickly translocates from the cytoplasm to the plasma membrane in a Rap1a-GTP-dependent manner. Cells overexpressing Radil show a substantial increase in cell adhesion, as well as in integrin/focal adhesion kinase (FAK) activation, and exhibit an elongated morphology, with severe tail retraction defects. This phenotype is effectively rescued by treatment with either β2-integrin inhibitory antibodies or FAK inhibitors. Conversely, knockdown of Radil causes severe inhibition of cell adhesion, β2-integrin activation, and chemotaxis. Furthermore, we found that inhibition of Rap activity by RapGAP coexpression inhibits Radil-mediated integrin and FAK activation, decreases cell adhesion, and abrogates the long-tail phenotype of Radil cells. Overall, these studies establish that Radil regulates neutrophil adhesion and motility by linking Rap1 to β2-integrin activation.

  9. Talin-dependent integrin activation is required for fibrin clot retraction by platelets

    PubMed Central

    Haling, Jacob R.; Monkley, Susan J.; Critchley, David R.

    2011-01-01

    Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of talin, we show for the first time that the capacity of talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which talin functions are required for this process, we tested clot retraction in platelets expressing a talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between talin and the membrane-proximal region (MPR) in the β-integrin cytoplasmic domain. Talin-deficient and talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in talin1(L325R) platelets, but not talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that talin1(L325R) can form functional links to the actin cytoskeleton. PMID:20971947

  10. Physical and functional interaction between integrins and hERG potassium channels.

    PubMed

    Arcangeli, A; Becchetti, A; Cherubini, A; Crociani, O; Defilippi, P; Guasti, L; Hofmann, G; Pillozzi, S; Olivotto, M; Wanke, E

    2004-11-01

    Integrins are adhesion receptors capable of transmitting intracellular signals that regulate many different cellular functions. Among integrin-mediated signals, the activation of ion channels can be included. We demonstrated that a long-lasting activation of hERG (human ether-a-go-go-related gene) potassium channels occurs in both human neuroblastoma and leukaemia cells after the activation of the beta1 integrin subunit. This activation is apparently a determining factor inducing neurite extension and osteoclastic differentiation in both the cell types. More recently, we provided evidences that beta1 integrins and hERG channels co-precipitate in both the cell types. Preliminary results suggest that a macromolecular signalling complex indeed occurs between integrins and the hERG1 protein and that hERG channel activity can modulate integrin downstream signalling.

  11. Bone-Induced Expression of Tumoral Integrin beta3 Enables Targeted Nanotherapy of Breast Cancer Metastases

    NASA Astrophysics Data System (ADS)

    Ross, Michael H.

    Breast cancer is the most common cancer for women worldwide, representing approximately 25% of all new cancer cases in this population. While early detection and removal of breast cancer still confined to the primary site results in a good prognosis, approximately one- third of patients will develop distant metastases. In these patients, overall survival is markedly reduced. Of the common sites for breast cancer metastasis, the skeletal system is the most frequent. Treating breast cancer bone metastases has proven particularly difficult for several reasons, such as dissemination of metastases throughout the skeleton, poor drug localization to sites of interest, a lack of tumor-specific targets expressed across breast cancer subtypes, and the chemo-protective nature of the bone microenvironment. This dissertation is focused on investigating a potential tumor-target expressed on breast cancer bone metastases, and to improve drug treatment efficacy against tumor cells in the bone microenvironment. Integrins are heterodimeric cell surface receptors, composed of an alpha and beta subunit from a large family of selectively-compatible integrin subunits. As a heterodimeric complex, integrins can bind to components of the extracellular matrix or to other cells. One particular integrin complex, integrin alphavbeta3, is composed of the tightly regulated integrin subunit beta3 and the more widely expressed alphav subunit. I examined the expression of integrin beta3 on primary breast cancer as compared to metastases in murine cancer models, and observed that integrin expression is significantly elevated on bone metastases as compared to the primary tumors or visceral metastases. In addition, I evaluated tumor-associated integrin beta3 expression on a tissue microarray (TMA) composed of primary breast cancer and patient-matched bone metastatic tissue from 42 patients. Across nearly all patients, tumor-associated integrin beta3 expression was significantly elevated on bone

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

    PubMed Central

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

    2015-01-01

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

  13. Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis.

    PubMed

    Mahabeleshwar, Ganapati H; Feng, Weiyi; Reddy, Kumar; Plow, Edward F; Byzova, Tatiana V

    2007-09-14

    The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

  14. Radil controls neutrophil adhesion and motility through β2-integrin activation

    PubMed Central

    Liu, Lunhua; Aerbajinai, Wulin; Ahmed, Syed M.; Rodgers, Griffin P.; Angers, Stephane; Parent, Carole A.

    2012-01-01

    Integrin activation is required to facilitate multiple adhesion-dependent functions of neutrophils, such as chemotaxis, which is critical for inflammatory responses to injury and pathogens. However, little is known about the mechanisms that mediate integrin activation in neutrophils. We show that Radil, a novel Rap1 effector, regulates β1- and β2-integrin activation and controls neutrophil chemotaxis. On activation and chemotactic migration of neutrophils, Radil quickly translocates from the cytoplasm to the plasma membrane in a Rap1a-GTP–dependent manner. Cells overexpressing Radil show a substantial increase in cell adhesion, as well as in integrin/focal adhesion kinase (FAK) activation, and exhibit an elongated morphology, with severe tail retraction defects. This phenotype is effectively rescued by treatment with either β2-integrin inhibitory antibodies or FAK inhibitors. Conversely, knockdown of Radil causes severe inhibition of cell adhesion, β2-integrin activation, and chemotaxis. Furthermore, we found that inhibition of Rap activity by RapGAP coexpression inhibits Radil-mediated integrin and FAK activation, decreases cell adhesion, and abrogates the long-tail phenotype of Radil cells. Overall, these studies establish that Radil regulates neutrophil adhesion and motility by linking Rap1 to β2-integrin activation. PMID:23097489

  15. Prevention of experimental autoimmune encephalomyelitis by antibodies against α4βl integrin

    NASA Astrophysics Data System (ADS)

    Yednock, Ted A.; Cannon, Catherine; Fritz, Lawrence C.; Sanchez-Madrid, Francisco; Steinman, Lawrence; Karin, Nathan

    1992-03-01

    EXPERIMENTAL autoimmune encephalomyelitis (EAE) is an inflammatory condition of the central nervous system with similarities to multiple sclerosis1,2. In both diseases, circulating leukocytes penetrate the blood-brain barrier and damage myelin, resulting in impaired nerve conduction and paralysis3-5. We sought to identify the adhesion receptors that mediate the attachment of circulating leukocytes to inflamed brain endothelium in EAE, because this interaction is the first step in leukocyte entry into the central nervous system. Using an in vitro adhesion assay on tissue sections, we found that lymphocytes and monocytes bound selectively to inflamed EAE brain vessels. Binding was inhibited by antibodies against the integrin molecule α4βl, but not by antibodies against numerous other adhesion receptors. When tested in vivo, anti-α4 integrin effectively prevented the accumulation of leukocytes in the central nervous system and the development of EAE. Thus, therapies designed to interfere with α4βl integrin may be useful in treating inflammatory diseases of the central nervous system, such as multiple sclerosis.

  16. 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. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed Central

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

    2015-01-01

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

  18. Antihistamines modulate the integrin signaling pathway in h9c2 rat cardiomyocytes: Possible association with cardiotoxicity.

    PubMed

    Yun, J S; Kim, S Y

    2015-08-01

    The identification of biomarkers for toxicity prediction is crucial for drug development and safety evaluation. The selective and specific biomarkers for antihistamines-induced cardiotoxicity is not well identified yet. In order to evaluate the mechanism of the life-threatening effects caused by antihistamines, we used DNA microarrays to analyze genomic profiles in H9C2 rat cardiomyocytes that were treated with antihistamines. The gene expression profiles from drug-treated cells revealed changes in the integrin signaling pathway, suggesting that cardiac arrhythmias induced by antihistamine treatment may be mediated by changes in integrin-mediated signaling. It has been reported that integrin plays a role in QT prolongation that may induce cardiac arrhythmia. These results indicate that the integrin-mediated signaling pathway induced by antihistamines is involved in various biological mechanisms that lead to cardiac QT prolongation. Therefore, we suggest that genomic profiling of antihistamine-treated cardiomyocytes has the potential to reveal the mechanism of adverse drug reactions, and this signal pathway is applicable to prediction of in vitro cardiotoxicity induced by antihistamines as a biomarker candidate. © The Author(s) 2014.

  19. Conservation of the Human Integrin-Type Beta-Propeller Domain in Bacteria

    PubMed Central

    Chouhan, Bhanupratap; Denesyuk, Alexander; Heino, Jyrki; Johnson, Mark S.; Denessiouk, Konstantin

    2011-01-01

    Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca2+-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca2+-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem

  20. 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.

  1. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Integrin αvβ3 promotes infection by Japanese encephalitis virus.

    PubMed

    Fan, Wenchun; Qian, Ping; Wang, Dandan; Zhi, Xianwei; Wei, Yanming; Chen, Huanchun; Li, Xiangmin

    2017-04-01

    Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is one of the major causes of viral encephalitis diseases worldwide. The JEV envelope protein facilitates viral entry, and its domain III contains an Arg-Gly-Asp (RGD) motif, that may modulate JEV entry through the RGD-binding integrin. In this study, the roles of integrin αv and β3 on the infection of JEV were evaluated. Reduced expression of integrin αv/β3 by special shRNA confers 2 to 4-fold inhibition of JEV replication in BHK-21 cells. Meanwhile, antibodies specific for integrin αv/β3 displayed ~58% and ~33% inhibition of JEV infectivity and RGD-specific peptides produced ~36% of inhibition. Expression of E protein and JEV RNA loads were clearly increased in CHO cells transfected with cDNA encoding human integrin β3. Moreover, integrin αv mediates JEV infection in viral binding stage of life cycle. Therefore, our study suggested that integrin αv and β3 serve as a host factor associated with JEV entry into the target cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. The role of β2 integrin associated heparin-binding protein release in ARDS.

    PubMed

    Liu, Yang; Ma, Shaolin; Wang, Xuebin; Feng, Yajing; Zhang, Shouqin; Wang, Sheng; Zhang, Xiangyu

    2018-06-15

    PMNs (polymorphonuclear neutrophil) play important roles in early stage of inflammation induced ARDS (Acute Respiratory Distress Syndrome). Both HBP (Heparin-Binding Protein) released from active PMNs and β2 integrins on the surface of PMNs are involved in vascular leakage. The role and relationship of HBP and β2 integrins on ARDS still requires study. We established ARDS model using C57BL/6 mice with cecal ligation and puncture and eliminating HBP and β2 integrin with respective antibodies. The mice were also challenged with HBP endotracheal instillation. Histopathology score, lung wet/dry ratio, bronchoalveolar lavage fluid protein, plasma HBP and β2 integrin on PMNs from all groups were measured. β2 integrin and HBP were analyzed after incubated PMNs with streptococcal and pretreat with anti-CD18, anti-HBP, 1-phosphatidylinositol 3-kinase (PI3K) inhibitor and p38 mitogen-activated protein kinase (MAPK) inhibitor. All lung injury indicatrix accompanied with HBP and β2 integrin elevated in CLP group, and HBP and β2 integrin were in correlation with each other and both were in correlation with the severity of lung injury. Endotracheal instillation HBP induced lung injury in CLP mice. Inhibiting both HBP and integrin ameliorated lung injury. HBP release was suppressed by inhibiting integrin and PI3K pathway, while integrin level did not decrease after eliminating HBP. Both HBP and β2 integrin play important roles in ARDS. HBP released from PMNs is β2 integrin-PI3K signaling pathway dependent process revealing potential novel therapeutic targets for ARDS treatment. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Nanoparticle Imaging of Integrins on Tumor Cells1

    PubMed Central

    Montet, Xavier; Montet-Abou, Karin; Reynolds, Fred; Weissleder, Ralph; Josephson, Lee

    2006-01-01

    Abstract Nanoparticles 10 to 100 nm in size can deliver large payloads to molecular targets, but undergo slow diffusion and/or slow transport through delivery barriers. To examine the feasibility of nanoparticles targeting a marker expressed in tumor cells, we used the binding of cyclic arginine-glycine-aspartic acid (RGD) nanoparticle targeting integrins on BT-20 tumor as a model system. The goals of this study were: 1) to use nanoparticles to image αvβ3 integrins expressed in BT-20 tumor cells by fluorescence-based imaging and magnetic resonance imaging, and, 2) to identify factors associated with the ability of nanoparticles to target tumor cell integrins. Three factors were identified: 1) tumor cell integrin expression (the αvβ3 integrin was expressed in BT-20 cells, but not in 9L cells); 2) nanoparticle pharmacokinetics (the cyclic RGD peptide cross-linked iron oxide had a blood half-life of 180 minutes and was able to escape from the vasculature over its long circulation time); and 3) tumor vascularization (the tumor had a dense capillary bed, with distances of <100 µm between capillaries). These results suggest that nanoparticles could be targeted to the cell surface markers expressed in tumor cells, at least in the case wherein the nanoparticles and the tumor model have characteristics similar to those of the BT-20 tumor employed here. PMID:16611415

  5. Evidence that a functional fertilin-like ADAM plays a role in human sperm-oolemmal interactions.

    PubMed

    Bronson, R A; Fusi, F M; Calzi, F; Doldi, N; Ferrari, A

    1999-05-01

    Fertilin is a protein initially identified in guinea pig spermatozoa; it is the prototype of a larger family of conserved, proteins designated as a disintegrin and a metalloproteinase (ADAM). These heterodimers which consist of alpha and beta subunits, containing metalloproteinase-like and disintegrin-like domains, appear to play a role in mammalian fertilization. Peptides derived from the disintegrin domains of two ADAMs, fertilin and cyritestin, interfere with gamete adhesion and sperm-egg membrane fusion in non-human species. It has been suggested that fertilin-beta binds to an oolemmal integrin, and it is proposed that the tripeptide FEE (Phe-Glu-Glu) is the integrin recognition sequence in human fertilin-beta. We evaluated whether fertilin beta plays a role in human fertilization by studying the effects of a linear octapeptide containing the FEE sequence, SFEECDLP, and a scrambled octapeptide with the same amino acids, SFPCEDEL, on the incorporation of human spermatozoa by human zona-free eggs. The effects of G4120, a potent RGD-containing (Arg-Gly-Asp) thioether-bridged cyclic peptide which blocks both fibronectin and vitronectin receptors, and the relationship between FEE- and RGD-receptor interactions on sperm-egg interactions were also studied. The FEE-containing peptide, but not the scrampled peptide, inhibited sperm adhesion to oocytes and their penetration, over the range 1-5 microM. The inhibition induced by SFEECDLP was reversible and occurred only in the presence of peptide itself. The G4120 peptide exhibited 10-fold less inhibitory effects on sperm adhesion and penetration than did SFEECDLP. When combined, SFEECDLP and G4120 exhibited strong inhibition of both adhesion and penetration at concentrations that individually had been ineffective, suggesting co-operation between the two receptor-ligand interactions during fertilization. We propose that a fertilin-like molecule is functionally active on human spermatozoa and that its interaction with an

  6. Role of β1-Integrin in Colorectal Cancer: Case-Control Study

    PubMed Central

    Oh, Bo-Young; Kim, Kwang Ho; Chung, Soon Sup; Hong, Kyoung Sook

    2014-01-01

    Purpose In the metastatic process, interactions between circulating tumor cells (CTCs) and the extracellular matrix or surrounding cells are required. β1-Integrin may mediate these interactions. The aim of this study was to investigate whether β1-integrin is associated with the detection of CTCs in colorectal cancer. Methods We enrolled 30 patients with colorectal cancer (experimental group) and 30 patients with benign diseases (control group). Blood samples were obtained from each group, carcinoembryonic antigen (CEA) mRNA for CTCs marker and β1-integrin mRNA levels were estimated by using reverse transcription-polymerase chain reaction, and the results were compared between the two groups. In the experimental group, preoperative results were compared with postoperative results for each marker. In addition, we analyzed the correlation between the expressions of β1-integrin and CEA. Results CEA mRNA was detected more frequently in colorectal cancer patients than in control patients (P = 0.008). CEA mRNA was significantly reduced after surgery in the colorectal cancer patients (P = 0.032). β1-Integrin mRNA was detected more in colorectal cancer patients than in the patients with benign diseases (P < 0.001). In colorectal cancer patients, expression of β1-integrin mRNA was detected more for advanced-stage cancer than for early-stage cancer (P = 0.033) and was significantly decreased after surgery (P < 0.001). In addition, expression of β1-integrin mRNA was significantly associated with that of CEA mRNA in colorectal cancer patients (P = 0.001). Conclusion In conclusion, β1-integrin is a potential factor for forming a prognosis following surgical resection in colorectal cancer patients. β1-Integrin may be a candidate for use as a marker for early detection of micrometastatic tumor cells and for monitoring the therapeutic response in colorectal cancer patients. PMID:24851215

  7. 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.

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

    PubMed Central

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

    2017-01-01

    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 to hydrogel materials that promote a 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 neighboring 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. PMID:28783156

  9. Glia Maturation Factor-γ Regulates Monocyte Migration through Modulation of β1-Integrin*

    PubMed Central

    Aerbajinai, Wulin; Liu, Lunhua; Zhu, Jianqiong; Kumkhaek, Chutima; Chin, Kyung; Rodgers, Griffin P.

    2016-01-01

    Monocyte migration requires the dynamic redistribution of integrins through a regulated endo-exocytosis cycle, but the complex molecular mechanisms underlying this process have not been fully elucidated. Glia maturation factor-γ (GMFG), a novel regulator of the Arp2/3 complex, has been shown to regulate directional migration of neutrophils and T-lymphocytes. In this study, we explored the important role of GMFG in monocyte chemotaxis, adhesion, and β1-integrin turnover. We found that knockdown of GMFG in monocytes resulted in impaired chemotactic migration toward formyl-Met-Leu-Phe (fMLP) and stromal cell-derived factor 1α (SDF-1α) as well as decreased α5β1-integrin-mediated chemoattractant-stimulated adhesion. These GMFG knockdown impaired effects could be reversed by cotransfection of GFP-tagged full-length GMFG. GMFG knockdown cells reduced the cell surface and total protein levels of α5β1-integrin and increased its degradation. Importantly, we demonstrate that GMFG mediates the ubiquitination of β1-integrin through knockdown or overexpression of GMFG. Moreover, GMFG knockdown retarded the efficient recycling of β1-integrin back to the plasma membrane following normal endocytosis of α5β1-integrin, suggesting that the involvement of GMFG in maintaining α5β1-integrin stability may occur in part by preventing ubiquitin-mediated degradation and promoting β1-integrin recycling. Furthermore, we observed that GMFG interacted with syntaxin 4 (STX4) and syntaxin-binding protein 4 (STXBP4); however, only knockdown of STXBP4, but not STX4, reduced monocyte migration and decreased β1-integrin cell surface expression. Knockdown of STXBP4 also substantially inhibited β1-integrin recycling in human monocytes. These results indicate that the effects of GMFG on monocyte migration and adhesion probably occur through preventing ubiquitin-mediated proteasome degradation of α5β1-integrin and facilitating effective β1-integrin recycling back to the plasma membrane

  10. Role of Altered Sialylation of the I-Like Domain of β1 Integrin in the Binding of Fibronectin to β1 Integrin: Thermodynamics and Conformational Analyses

    PubMed Central

    Pan, Di; Song, Yuhua

    2010-01-01

    Abstract N-glycosylation of the I-like domain of β1 integrin plays an essential role in integrin structure and function, and the altered sialylation of β1 integrin regulates β1 integrin binding to fibronectin. However, the structural basis underlying the effect of altered sialylation of the β1 I-like domain on β1 integrin binding to fibronectin remains largely unknown. In this study, we used a combination of molecular dynamics simulations and binding free energy analyses to investigate changes in binding thermodynamics and in conformation of the glycosylated β1 I-like domain-FN-III9-10 complex caused by altered sialylation of the β1 I-like domain. Binding free energy analyses showed that desialylation of β1 I-like domain increased β1 integrin binding to fibronectin, consistent with experimental results. Interaction analyses showed that altered sialylation of the β1 I-like domain resulted in significant changes in the interaction of the N-glycans of the I-like domain with both the I-like domain and fibronectin, and these changes could directly affect the allosteric regulation of the interaction between the I-like domain and fibronectin. Altered sialylation of the β1 I-like domain caused significant conformational changes in key functional sites of both the β1 I-like domain and fibronectin. In addition, altered sialylation of the β1 I-like domain resulted in changes in the degree of correlated motions between residues in the I-like domain and residues in fibronectin, and in the degree of motion changes in fibronectin, which could affect β1 integrin binding to fibronectin. We believe results from this study provide thermodynamic and structural evidence for a role of altered sialylation of β1 integrin in regulating β1 integrin binding to fibronectin and it's induced cellular activities. PMID:20655849

  11. Integrin distributions in renal cell carcinomas of various grades of malignancy.

    PubMed Central

    Korhonen, M.; Laitinen, L.; Ylänne, J.; Koukoulis, G. K.; Quaranta, V.; Juusela, H.; Gould, V. E.; Virtanen, I.

    1992-01-01

    We studied 41 renal cell carcinomas, classified according to histologic grades G1 through G3, by indirect immunofluorescence microscopy using a panel of monoclonal antibodies (MAb) against various integrin subunits, and the basement membrane (BM) components laminin and collagen type IV. Selected cases also were immunostained using the avidin-biotin-complex method. The alpha 3 and beta 1 integrin subunits were detected in tumor cells of all the carcinomas. All G1 carcinomas, like normal tubular epithelial cells, expressed the alpha 6 subunit, whereas it was lacking in 20% and 40% of G2 and G3 carcinomas, respectively. Furthermore, when alpha 6 was expressed, a lack of basally polarized organization of the subunit, coupled with disorganization of the BM components, correlated with histologic grade. Another feature that appeared to characterize the more anaplastic tumors was their high level (80%) of the alpha v subunit expression as compared with its absence in the G1 carcinomas. Stromal myofibroblasts, identified by double-labeling with anti-myosin, were often characterized by the expression of the alpha 1, alpha 3, alpha 5 and beta 1 subunits. These results indicate that changes in integrin expression in renal cell carcinomas may be correlated with their degree of histologic malignancy. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1443050

  12. 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

  13. Overexpression of integrin αv correlates with poor prognosis in colorectal cancer.

    PubMed

    Ha, Sang Yun; Shin, Juyoun; Kim, Jeong Hoon; Kang, Myung Soo; Yoo, Hae-Yong; Kim, Hyeon-Ho; Um, Sung-Hee; Kim, Seok-Hyung

    2014-07-01

    Integrin αv subunits are involved in tumour angiogenesis and tumour progression in various types of cancers. Clinical trials evaluating agents targeting integrin αv are ongoing. Integrin αv expression has been reported in several cancers in association with tumour progression or poor survival. However, no study has addressed the prognostic influence of integrin αv expression on survival of patients with colorectal cancer (CRC). Immunohistochemical staining of integrin αv was performed in 198 CRC samples to evaluate its prognostic significance. High expression of integrin αv was observed in 58.1% (115/189) of colorectal adenocarcinoma samples, while only in 11.5% (3/26) of tubular adenoma samples and in none of normal mucosa or hyperplastic polyp samples. It was more frequently found in female patients and less frequently observed in well differentiated tumours. The proportion of cases with high expression of integrin αv showed an increasing trend with increased T stage (p=0.032), N stage (p=0.006) and TNM stage (p=0.001). Patients displaying exuberant expression of integrin αv showed shorter overall survival (p=0.001) and disease-free survival (p=0.004). Elevated integrin αv expression was an independent prognostic factor for overall survival (HR: 2.04, 95% CI 1.16 to 3.56; p=0.013) and disease-free survival (HR: 2.19, 95% CI 1.16 to 4.13; p=0.015). Overexpression of integrin αv is associated with advanced T and N stage and as an independent prognostic factor in CRC. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  14. Synthesis and evaluation of a radioiodinated peptide probe targeting αvβ6 integrin for the detection of pancreatic ductal adenocarcinoma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ueda, Masashi; Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501; Fukushima, Takahiro

    Highlights: • We developed a radioiodinated peptide probe targeting αvβ6 integrin ({sup 123}I-IFMDV2). • {sup 123}I-IFMDV2 had a high affinity and selectivity for αvβ6 integrin. • {sup 123}I-IFMDV2 showed a specific binding to αvβ6 integrin in vivo. • {sup 123}I-IFMDV2 enabled clear visualization of the αvβ6-integrin-positive tumor. - Abstract: Introduction: Pancreatic ductal adenocarcinoma (PDAC) remains a major cause of cancer-related death. Since significant upregulation of αvβ6 integrin has been reported in PDAC, this integrin is a promising target for PDAC detection. In this study, we aimed to develop a radioiodinated probe for the imaging of αvβ6 integrin-positive PDAC with single-photonmore » emission computed tomography (SPECT). Methods: Four peptide probes were synthesized and screened by competitive and saturation binding assays using 2 PDAC cell lines (AsPC-1, αvβ6 integrin-positive; MIA PaCa-2, αvβ6 integrin-negative). The probe showing the best affinity was used to study the biodistribution assay, an in vivo blocking study, and SPECT imaging using tumor bearing mice. Autoradiography and immunohistochemical analysis were also performed. Results: Among the 4 probes examined in this study, {sup 125}I-IFMDV2 showed the highest affinity for αvβ6 integrin expressed in AsPC-1 cells and no affinity for MIA PaCa-2 cells. The accumulation of {sup 125}I-IFMDV2 in the AsPC-1 xenograft was 3–5 times greater than that in the MIA PaCa-2 xenograft, consistent with the expression of αvβ6 integrin in each xenograft, and confirmed by immunohistochemistry. Pretreatment with excess amounts of A20FMDV2 significantly blocked the accumulation of {sup 125}I-IFMDV2 in the AsPC-1 xenograft, but not in the MIA PaCa-2 xenograft. Furthermore, {sup 123}I-IFMDV2 enabled clear visualization of the AsPC-1 xenograft. Conclusion: {sup 123}I-IFMDV2 is a potential SPECT probe for the imaging of αvβ6 integrin in PDAC.« less

  15. Design of RGD-ATWLPPR peptide conjugates for the dual targeting of αVβ3 integrin and neuropilin-1.

    PubMed

    Thoreau, Fabien; Vanwonterghem, Laetitia; Henry, Maxime; Coll, Jean-Luc; Boturyn, Didier

    2018-05-18

    Targeting the tumour microenvironment is a promising strategy to detect and/or treat cancer. The design of selective compounds that co-target several receptors frequently overexpressed in solid tumours may allow a reliable and selective detection of tumours. Here we report the modular synthesis of compounds encompassing ligands of αVβ3 integrin and neuropilin-1 that are overexpressed in the tumour microenvironment. These compounds were then evaluated through cellular experiments and imaging of tumours in mice. We observed that the peptide that displays both ligands is more specifically accumulating in the tumours than in controls. Simultaneous interaction with αVβ3 integrin and NRP1 induces NRP1 stabilization at the cell membrane surface which is not observed with the co-injection of the controls.

  16. Carbon Ion Irradiation Inhibits Glioma Cell Migration Through Downregulation of Integrin Expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rieken, Stefan, E-mail: Stefan.Rieken@med.uni-heidelberg.de; Habermehl, Daniel; Wuerth, Lena

    2012-05-01

    Purpose: To investigate the effect of carbon ion irradiation on glioma cell migration. Methods and Materials: U87 and Ln229 glioma cells were irradiated with photons and carbon ions. Migration was analyzed 24 h after irradiation. Fluorescence-activated cell sorting analysis was performed in order to quantify surface expression of integrins. Results: Single photon doses of 2 Gy and 10 Gy enhanced {alpha}{sub {nu}}{beta}{sub 3} and {alpha}{sub {nu}}{beta}{sub 5} integrin expression and caused tumor cell hypermigration on both vitronectin (Vn) and fibronectin (Fn). Compared to integrin expression in unirradiated cells, carbon ion irradiation caused decreased integrin expression and inhibited cell migration onmore » both Vn and Fn. Conclusion: Photon radiotherapy (RT) enhances the risk of tumor cell migration and subsequently promotes locoregional spread via photon induction of integrin expression. In contrast to photon RT, carbon ion RT causes decreased integrin expression and suppresses glioma cell migration on both Vn and Fn, thus promising improved local control.« less

  17. Phosphoinositide 3-Kinase p110β Regulates Integrin αIIbβ3 Avidity and the Cellular Transmission of Contractile Forces*

    PubMed Central

    Schoenwaelder, Simone M.; Ono, Akiko; Nesbitt, Warwick S.; Lim, Joanna; Jarman, Kate; Jackson, Shaun P.

    2010-01-01

    Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin αIIbβ3, necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin αIIbβ3 activation (affinity modulation) primarily occurs downstream of Gi-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin αIIbβ3 bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110β isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin αIIbβ3 activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin αIIbβ3 association with the contractile cytoskeleton. Analysis of integrin αIIbβ3 adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin αIIbβ3 bonds. These studies demonstrate an important role for PI3K p110β in regulating the avidity of high affinity integrin αIIbβ3 receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110β inhibitors. PMID:19940148

  18. EPA and DHA increased PPARγ expression and deceased integrin-linked kinase and integrin β1 expression in rat glomerular mesangial cells treated with lipopolysaccharide.

    PubMed

    Han, Wenchao; Zhao, Hui; Jiao, Bo; Liu, Fange

    2014-04-01

    Fish oil containing n-3 polyunsaturated fatty acids (n-3 PUFAs) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is known to prevent the progression of nephropathy and retard the progression of kidney disease. This study sought to investigate the underlying mechanisms of EPA and DHA in terms of peroxisome proliferator-activated receptor γ (PPARγ), integrin-linked kinase (ILK), and integrin β1 expression in glomerular mesangial cells (GMCs) because of their critical roles in the development and progression of nephropathy. Lipopolysaccharide (LPS) significantly reduced the expression of PPARγand increased the expression of ILK at the mRNA level and at the protein level in GMCs as indicated by real-time PCR and Western blotting. In addition, LPS increased integrin β1 expression in GMCs at the mRNA level. Treatment with EPA and DHA significantly increased the expression of PPARγ and decreased the expression of ILK and integrin β1 in GMCs. These data suggest that the renoprotective effects of EPA and DHA may be related to their potential to increase the expression of PPARγ and decrease the expression of ILK and integrin β1.

  19. Novel Regulation of Integrin Trafficking by Rab11-FIP5 in Aggressive Prostate Cancer.

    PubMed

    Das, Lipsa; Gard, Jaime M C; Prekeris, Rytis; Nagle, Raymond B; Morrissey, Colm; Knudsen, Beatrice S; Miranti, Cindy K; Cress, Anne E

    2018-05-14

    The laminin-binding integrins, α3β1 and α6β1, are needed for tumor metastasis and their surface expression is regulated by endocytic recycling. β1 integrins share the Rab11 recycling machinery but the trafficking of α3β1 and α6β1 are distinct by an unknown mechanism. Using a mouse PDX tumor model containing human metastatic prostate cancer, Rab11 family interacting protein 5 (Rab11-FIP5) was identified as a lead candidate for α6β1 trafficking. Rab11-FIP5 and its membrane binding domain were required for α6β1 recycling, without affecting the other laminin-binding integrin (i.e., α3β1) or unrelated membrane receptors like CD44, transferrin receptor, or E-cadherin. Depletion of Rab11-FIP5 resulted in the intracellular accumulation of α6β1 in the Rab11 recycling compartment, loss of cell migration on laminin, and an unexpected loss of α6β1 recycling in cell-cell locations. Taken together, these data demonstrate that α6β1 is distinct from α3β1 via Rab11-FIP5 recycling and recycles in an unexpected cell-cell location. Rab11-FIP5 dependent a6b1 integrin recycling may be selectively targeted to limit migration of prostate cancer cells into laminin-rich tissues. Copyright ©2018, American Association for Cancer Research.

  20. 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.

  1. SIKVAV peptide functionalized ultra-small gold nanoparticles for selective targeting of α6β1 integrin in hepatocellular carcinoma

    NASA Astrophysics Data System (ADS)

    Roskamp, M.; Coulter, T.; Ding, Y.; Perrins, R.; Espinosa Garcia, C.; Pace, A.; Hale, S.; Robinson, A.; Williams, P.; Aguilera Peral, U.; Patel, K.; Palmer, D.

    2017-04-01

    Ultra-small glycan-passivated gold nanoparticles of <2nm diameter were funtionalised with a short HS-EG(8)-COOH ligand. The nanoparticles were subsequently labelled, in a stoichiometrically controllable manner, with integrin-binding peptide SIKVAV and the maytansinoid cytotoxin DM4. In vitro assays showed significantly increased integrin-mediated uptake of SIKVAV labelled nanoparticles in HepG2 cells. SIKVAV targeted nanoparticle binding was shown to be outcompeted with free SIKVAV peptide, indicating target specific uptake. DM4 was passively attached to nanoparticles via sulfhydryl ligand exchange at the gold nanoparticle surface, which rendered them highly cytotoxic (IC50 ˜1 × 10-9M). In a rat model, pharmacokinetic studies showed that nanoparticle biodistribution was strongly altered by labelling with either peptide and DM4 moieties.

  2. Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release and coagulation.

    PubMed

    Pang, Aiming; Cui, Yujie; Chen, Yunfeng; Cheng, Ni; Delaney, M Keegan; Gu, Minyi; Stojanovic-Terpo, Aleksandra; Zhu, Cheng; Du, Xiaoping

    2018-05-31

    It is currently unclear why agonist-stimulated platelets require shear force to efficiently externalize the procoagulant phospholipid phosphatidylserine (PS) and release PS-exposed microvesicles (MVs). We reveal that integrin outside-in signaling is an important mechanism for this requirement. PS exposure and MV release were inhibited in β 3 -/- platelets or by integrin antagonists. The impaired MV release and PS exposure in β 3 -/- platelets were rescued by expressing wild type β 3 but not a Gα 13 binding-deficient β 3 mutant (E 733 EE to AAA), which blocks outside-in signaling but not ligand binding. Inhibition of Gα 13 or Src also diminished agonist/shear-dependent PS exposure and MV release, further indicating a role for integrin outside-in signaling. PS exposure in activated platelets was induced by application of pulling force via an integrin ligand, which was abolished by inhibiting Gα 13 -integrin interaction, suggesting that GGα 13 -dependent transmission of mechanical signals by integrins induces PS exposure. Inhibition of Gα 13 delayed coagulation in vitro. Furthermore, inhibition or platelet-specific knockout of Gα 13 diminished laser-induced intravascular fibrin formation in arterioles in vivo. Thus, β 3 integrins serve as a shear sensor activating the Gα 13 -dependent outside-in signaling pathway to facilitate platelet procoagulant function. Pharmacological targeting of Gα 13 -integrin interaction prevents occlusive thrombosis in vivo by inhibiting both coagulation and platelet thrombus formation. Copyright © 2018 American Society of Hematology.

  3. Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro

    NASA Technical Reports Server (NTRS)

    Moursi, A. M.; Globus, R. K.; Damsky, C. H.

    1997-01-01

    -state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes alpha5ss1, alpha3ss1 and alpha8ss1. We further conclude that the specific alpha5ss1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.

  4. Integrin beta 1 inhibition alleviates the chronic hyperproliferative dermatitis phenotype of SHARPIN-deficient mice.

    PubMed

    Peuhu, Emilia; Salomaa, Siiri I; De Franceschi, Nicola; Potter, Christopher S; Sundberg, John P; Pouwels, Jeroen

    2017-01-01

    SHARPIN (Shank-Associated RH Domain-Interacting Protein) is a component of the linear ubiquitin chain assembly complex (LUBAC), which enhances TNF-induced NF-κB activity. SHARPIN-deficient (Sharpincpdm/cpdm) mice display multi-organ inflammation and chronic proliferative dermatitis (cpdm) due to TNF-induced keratinocyte apoptosis. In cells, SHARPIN also inhibits integrins independently of LUBAC, but it has remained enigmatic whether elevated integrin activity levels in the dermis of Sharpincpdm/cpdm mice is due to increased integrin activity or is secondary to inflammation. In addition, the functional contribution of increased integrin activation to the Sharpincpdm/cpdm phenotype has not been investigated. Here, we find increased integrin activity in keratinocytes from Tnfr1-/- Sharpincpdm/cpdm double knockout mice, which do not display chronic inflammation or proliferative dermatitis, thus suggesting that SHARPIN indeed acts as an integrin inhibitor in vivo. In addition, we present evidence for a functional contribution of integrin activity to the Sharpincpdm/cpdm skin phenotype. Treatment with an integrin beta 1 function blocking antibody reduced epidermal hyperproliferation and epidermal thickness in Sharpincpdm/cpdm mice. Our data indicate that, while TNF-induced cell death triggers the chronic inflammation and proliferative dermatitis, absence of SHARPIN-dependent integrin inhibition exacerbates the epidermal hyperproliferation in Sharpincpdm/cpdm mice.

  5. Expression Profile of the Integrin Receptor Subunits in the Guinea Pig Sclera.

    PubMed

    Wang, Kevin K; Metlapally, Ravikanth; Wildsoet, Christine F

    2017-06-01

    The ocular dimensional changes in myopia reflect increased scleral remodeling, and in high myopia, loss of scleral integrity leads to biomechanical weakening and continued scleral creep. As integrins, a type of cell surface receptors, have been linked to scleral remodeling, they represent potential targets for myopia therapies. As a first step, this study aimed to characterize the integrin subunits at the messenger RNA level in the sclera of the guinea pig, a more recently added but increasingly used animal model for myopia research. Primers for α and β integrin subunits were designed using NCBI/UCSC Genome Browser and Primer3 software tools. Total RNA was extracted from normal scleral tissue and isolated cultured scleral fibroblasts, as well as liver and lung, as reference tissues, all from guinea pig. cDNA was produced by reverse transcription, PCR was used to amplify products of predetermined sizes, and products were sequenced using standard methods. Guinea pig scleral tissue expressed all known integrin alpha subunits except αD and αE. The latter integrin subunits were also not expressed by cultured guinea pig scleral fibroblasts; however, their expression was confirmed in guinea pig liver. In addition, isolated cultured fibroblasts did not express integrin subunits αL, αM, and αX. This difference between results for cultured cells and intact sclera presumably reflects the presence in the latter of additional cell types. Both guinea pig scleral tissue and isolated scleral fibroblasts expressed all known integrin beta subunits. All results were verified through sequencing. The possible contributions of integrins to scleral remodeling make them plausible targets for myopia prevention. Data from this study will help guide future ex vivo and in vitro studies directed at understanding the relationship between scleral integrins and ocular growth regulation in the guinea pig model for myopia.

  6. Loss of β1-integrin from urothelium results in overactive bladder and incontinence in mice: a mechanosensory rather than structural phenotype.

    PubMed

    Kanasaki, Keizo; Yu, Weiqun; von Bodungen, Maximilian; Larigakis, John D; Kanasaki, Megumi; Ayala de la Pena, Francisco; Kalluri, Raghu; Hill, Warren G

    2013-05-01

    Bladder urothelium senses and communicates information about bladder fullness. However, the mechanoreceptors that respond to tissue stretch are poorly defined. Integrins are mechanotransducers in other tissues. Therefore, we eliminated β1-integrin selectively in urothelium of mice using Cre-LoxP targeted gene deletion. β1-Integrin localized to basal/intermediate urothelial cells by confocal microscopy. β1-Integrin conditional-knockout (β1-cKO) mice lacking urothelial β1-integrin exhibited down-regulation and mislocalization of α3- and α5-integrins by immunohistochemistry but, surprisingly, had normal morphology, permeability, and transepithelial resistance when compared with Cre-negative littermate controls. β1-cKO mice were incontinent, as judged by random urine leakage on filter paper (4-fold higher spotting, P<0.01; 2.5-fold higher urine area percentage, P<0.05). Urodynamic function assessed by cystometry revealed bladder overfilling with 80% longer intercontractile intervals (P<0.05) and detrusor hyperactivity (3-fold more prevoid contractions, P<0.05), but smooth muscle contractility remained intact. ATP secretion into the lumen was elevated (49 vs. 22 nM, P<0.05), indicating abnormal filling-induced purinergic signaling, and short-circuit currents (measured in Ussing chambers) revealed 2-fold higher stretch-activated ion channel conductances in response to hydrostatic pressure of 1 cmH2O (P<0.05). We conclude that loss of integrin signaling from urothelium results in incontinence and overactive bladder due to abnormal mechanotransduction; more broadly, our findings indicate that urothelium itself directly modulates voiding.

  7. CLIC4 regulates cell adhesion and β1 integrin trafficking.

    PubMed

    Argenzio, Elisabetta; Margadant, Coert; Leyton-Puig, Daniela; Janssen, Hans; Jalink, Kees; Sonnenberg, Arnoud; Moolenaar, Wouter H

    2014-12-15

    Chloride intracellular channel protein 4 (CLIC4) exists in both soluble and membrane-associated forms, and is implicated in diverse cellular processes, ranging from ion channel formation to intracellular membrane remodeling. CLIC4 is rapidly recruited to the plasma membrane by lysophosphatidic acid (LPA) and serum, suggesting a possible role for CLIC4 in exocytic-endocytic trafficking. However, the function and subcellular target(s) of CLIC4 remain elusive. Here, we show that in HeLa and MDA-MB-231 cells, CLIC4 knockdown decreases cell-matrix adhesion, cell spreading and integrin signaling, whereas it increases cell motility. LPA stimulates the recruitment of CLIC4 to β1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both the internalization and the serum- or LPA-induced recycling of β1 integrin, but not for EGF receptor trafficking. Furthermore, we show that CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of β1 integrin trafficking. Our results define CLIC4 as a regulator of Rab35 activity and serum- and LPA-dependent integrin trafficking. © 2014. Published by The Company of Biologists Ltd.

  8. Molecular mechanisms of mechanotransduction in integrin-mediated cell-matrix adhesion

    PubMed Central

    Li, Zhenhai; Lee, Hyunjung; Zhu, Cheng

    2016-01-01

    Cell-matrix adhesion complexes are multi-protein structures linking the extracellular matrix (ECM) to the cytoskeleton. They are essential to both cell motility and function by bidirectionally sensing and transmitting mechanical and biochemical stimulations. Several types of cell-matrix adhesions have been identified and they share many key molecular components, such as integrins and actin-integrin linkers. Mechanochemical coupling between ECM molecules and the actin cytoskeleton has been observed from the single cell to the single molecule level and from immune cells to neuronal cells. However, the mechanisms underlying force regulation of integrin-mediated mechanotransduction still need to be elucidated. In this review article, we focus on integrin-mediated adhesions and discuss force regulation of cell-matrix adhesions and key adaptor molecules, three different force-dependent behaviors, and molecular mechanisms for mechanochemical coupling in force regulation. PMID:27720950

  9. Lebein, a snake venom disintegrin, suppresses human colon cancer cells proliferation and tumor-induced angiogenesis through cell cycle arrest, apoptosis induction and inhibition of VEGF expression.

    PubMed

    Zakraoui, Ons; Marcinkiewicz, Cezary; Aloui, Zohra; Othman, Houcemeddine; Grépin, Renaud; Haoues, Meriam; Essafi, Makram; Srairi-Abid, Najet; Gasmi, Ammar; Karoui, Habib; Pagès, Gilles; Essafi-Benkhadir, Khadija

    2017-01-01

    Lebein, is an heterodimeric disintegrin isolated from Macrovipera lebetina snake venom that was previously characterized as an inhibitor of ADP-induced platelet aggregation. In this study, we investigated the effect of Lebein on the p53-dependent growth of human colon adenocarcinoma cell lines. We found that Lebein significantly inhibited LS174 (p53wt), HCT116 (p53wt), and HT29 (p53mut) colon cancer cell viability by inducing cell cycle arrest through the modulation of expression levels of the tumor suppression factor p53, cell cycle regulating proteins cyclin D1, CDK2, CDK4, retinoblastoma (Rb), CDK1, and cyclin-dependent kinase inhibitors p21 and p27. Interestingly, Lebein-induced apoptosis of colon cancer cells was dependent on their p53 status. Thus, in LS174 cells, cell death was associated with PARP cleavage and the activation of caspases 3 and 8 while in HCT116 cells, Lebein induced caspase-independent apoptosis through increased expression of apoptosis inducing factor (AIF). In LS174 cells, Lebein triggers the activation of the MAPK ERK1/2 pathway through induction of reactive oxygen species (ROS). It also decreased cell adhesion and migration to fibronectin through down regulation of α5β1 integrin. Moreover, Lebein significantly reduced the expression of two angiogenesis stimulators, Vascular Endothelial Growth Factor (VEGF) and Neuropilin 1 (NRP1). It inhibited the VEGF-induced neovascularization process in the quail embryonic CAM system and blocked the development of human colon adenocarcinoma in nude mice. Overall, our work indicates that Lebein may be useful to design a new therapy against colon cancer. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits

    PubMed Central

    Nishimichi, Norihisa; Kawashima, Nagako; Yokosaki, Yasuyuki

    2015-01-01

    Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to “W3:34 and an top-loop”, and “solely W2:41”, accounting for 82% of published RGD-integrin-mAbs. PMID:26349930

  11. Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits.

    PubMed

    Nishimichi, Norihisa; Kawashima, Nagako; Yokosaki, Yasuyuki

    2015-09-09

    Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to "W3:34 and an top-loop", and "solely W2:41", accounting for 82% of published RGD-integrin-mAbs.

  12. β1-integrin controls cell fate specification in early lens development

    PubMed Central

    Pathania, Mallika; Wang, Yan; Simirskii, Vladimir N.; Duncan, Melinda K.

    2016-01-01

    Integrins are heterodimeric cell surface molecules that mediate cell-extracellular matrix (ECM) adhesion, ECM assembly, and regulation of both ECM and growth factor induced signaling. However, the developmental context of these diverse functions is not clear. Loss of β1-integrin from the lens vesicle (mouse E10.5) results in abnormal exit of anterior lens epithelial cells (LECs) from the cell cycle and their aberrant elongation toward the presumptive cornea by E12.5. These cells lose expression of LEC markers and initiate expression of the Maf (also known as c-Maf) and Prox1 transcription factors as well as other lens fiber cell markers, β1-integrin null LECs also upregulate the ERK, AKT and Smad1/5/8 phosphorylation indicative of BMP and FGF signaling. By E14.5, β1-integrin null lenses have undergone a complete conversion of all lens epithelial cells into fiber cells. These data suggest that shortly after lens vesicle closure, β1-integrin blocks inappropriate differentiation of the lens epithelium into fibers, potentially by inhibiting BMP and/or FGF receptor activation. Thus, β1-integrin has an important role in fine-tuning the response of the early lens to the gradient of growth factors that regulate lens fiber cell differentiation. PMID:27596755

  13. Loss of β1-integrin from urothelium results in overactive bladder and incontinence in mice: a mechanosensory rather than structural phenotype

    PubMed Central

    Kanasaki, Keizo; Yu, Weiqun; von Bodungen, Maximilian; Larigakis, John D.; Kanasaki, Megumi; Ayala de la Pena, Francisco; Kalluri, Raghu; Hill, Warren G.

    2013-01-01

    Bladder urothelium senses and communicates information about bladder fullness. However, the mechanoreceptors that respond to tissue stretch are poorly defined. Integrins are mechanotransducers in other tissues. Therefore, we eliminated β1-integrin selectively in urothelium of mice using Cre-LoxP targeted gene deletion. β1-Integrin localized to basal/intermediate urothelial cells by confocal microscopy. β1-Integrin conditional-knockout (β1-cKO) mice lacking urothelial β1-integrin exhibited down-regulation and mislocalization of α3- and α5-integrins by immunohistochemistry but, surprisingly, had normal morphology, permeability, and transepithelial resistance when compared with Cre-negative littermate controls. β1-cKO mice were incontinent, as judged by random urine leakage on filter paper (4-fold higher spotting, P<0.01; 2.5-fold higher urine area percentage, P<0.05). Urodynamic function assessed by cystometry revealed bladder overfilling with 80% longer intercontractile intervals (P<0.05) and detrusor hyperactivity (3-fold more prevoid contractions, P<0.05), but smooth muscle contractility remained intact. ATP secretion into the lumen was elevated (49 vs. 22 nM, P<0.05), indicating abnormal filling-induced purinergic signaling, and short-circuit currents (measured in Ussing chambers) revealed 2-fold higher stretch-activated ion channel conductances in response to hydrostatic pressure of 1 cmH2O (P<0.05). We conclude that loss of integrin signaling from urothelium results in incontinence and overactive bladder due to abnormal mechanotransduction; more broadly, our findings indicate that urothelium itself directly modulates voiding.—Kanasaki, K., Yu, W., von Bodungen, M., Larigakis, J. D., Kanasaki, M., Ayala de la Pena, F., Kalluri, R., Hill, W.G. Loss of β1-integrin from urothelium results in overactive bladder and incontinence in mice: a mechanosensory rather than structural phenotype. PMID:23395910

  14. Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

    PubMed Central

    Zhang, Y; Chen, M; Venugopal, S; Zhou, Y; Xiang, W; Li, Y-H; Lin, Q; Kini, R M; Chong, Y-S; Ge, R

    2011-01-01

    Isthmin (ISM) is a 60 kDa secreted-angiogenesis inhibitor that suppresses tumor growth in mouse and disrupts vessel patterning in zebrafish embryos. It selectively binds to alphavbeta5 (αvβ5) integrin on the surface of endothelial cells (ECs), but the mechanism of its antiangiogenic action remains unknown. In this work, we establish that soluble ISM suppresses in vitro angiogenesis and induces EC apoptosis by interacting with its cell surface receptor αvβ5 integrin through a novel ‘RKD' motif localized within its adhesion-associated domain in MUC4 and other proteins domain. ISM induces EC apoptosis through integrin-mediated death (IMD) by direct recruitment and activation of caspase-8 without causing anoikis. On the other hand, immobilized ISM loses its antiangiogenic function and instead promotes EC adhesion, survival and migration through αvβ5 integrin by activating focal adhesion kinase (FAK). ISM unexpectedly has both a pro-survival and death-promoting effect on ECs depending on its physical state. This dual function of a single antiangiogenic protein may impact its antiangiogenic efficacy in vivo. PMID:21544092

  15. Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state.

    PubMed

    Zhang, Y; Chen, M; Venugopal, S; Zhou, Y; Xiang, W; Li, Y-H; Lin, Q; Kini, R M; Chong, Y-S; Ge, R

    2011-05-05

    Isthmin (ISM) is a 60 kDa secreted-angiogenesis inhibitor that suppresses tumor growth in mouse and disrupts vessel patterning in zebrafish embryos. It selectively binds to alphavbeta5 (αvβ5) integrin on the surface of endothelial cells (ECs), but the mechanism of its antiangiogenic action remains unknown. In this work, we establish that soluble ISM suppresses in vitro angiogenesis and induces EC apoptosis by interacting with its cell surface receptor αvβ5 integrin through a novel 'RKD' motif localized within its adhesion-associated domain in MUC4 and other proteins domain. ISM induces EC apoptosis through integrin-mediated death (IMD) by direct recruitment and activation of caspase-8 without causing anoikis. On the other hand, immobilized ISM loses its antiangiogenic function and instead promotes EC adhesion, survival and migration through αvβ5 integrin by activating focal adhesion kinase (FAK). ISM unexpectedly has both a pro-survival and death-promoting effect on ECs depending on its physical state. This dual function of a single antiangiogenic protein may impact its antiangiogenic efficacy in vivo.

  16. Activation of c-Raf-1 kinase signal transduction pathway in alpha(7) integrin-deficient mice.

    PubMed

    Saher, G; Hildt, E

    1999-09-24

    Integrin alpha(7)-deficient mice develop a novel form of muscular dystrophy. Here we report that deficiency of alpha(7) integrin causes an activation of the c-Raf-1/mitogen-activated protein (MAP) 2 kinase signal transduction pathway in muscle cells. The observed activation of c-Raf-1/MAP2 kinases is a specific effect, because the alpha(7) integrin deficiency does not cause unspecific stress as determined by measurement of the Hsp72/73 level and activity of the JNK2 kinase. Because an increased level of activated FAK was found in muscle of alpha(7) integrin-deficient mice, the activation of c-Raf-1 kinase is triggered most likely by an integrin-dependent pathway. In accordance with this, in the integrin alpha(7)-deficient mice, part of the integrin beta(1D) variant in muscle is replaced by the beta(1A) variant, which permits the FAK activation. A recent report describes that integrin activity can be down-modulated by the c-Raf-1/MAP2 kinase pathway. Specific activation of the c-Raf-1/MAP2 kinases by cell-permeable peptides in skeletal muscle of rabbits causes degeneration of muscle fibers. Therefore, we conclude that in alpha(7) integrin-deficient mice, the continuous activation of c-Raf-1 kinase causes a permanent reduction of integrin activity diminishing integrin-dependent cell-matrix interactions and thereby contributing to the development of the dystrophic phenotype.

  17. A peptide affinity column for the identification of integrin alpha IIb-binding proteins.

    PubMed

    Daxecker, Heide; Raab, Markus; Bernard, Elise; Devocelle, Marc; Treumann, Achim; Moran, Niamh

    2008-03-01

    To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

  18. Gracilaria lemaneiformis polysaccharide as integrin-targeting surface decorator of selenium nanoparticles to achieve enhanced anticancer efficacy.

    PubMed

    Jiang, Wenting; Fu, Yuanting; Yang, Fang; Yang, Yufeng; Liu, Ting; Zheng, Wenjie; Zeng, Lilan; Chen, Tianfeng

    2014-08-27

    The poor permeability of glioma parenchyma represents a major limit for antiglioblastoma drug delivery. Gracilaria lemaneiformis polysaccharide (GLP), which has a high binding affinity to αvβ3 integrin overexpressed in glioma cells, was employed in the present study to functionalize selenium nanoparticles (SeNPs) to achieve antiglioblastoma efficacy. GLP-SeNPs showed satisfactory size distribution, high stability, and selectivity between cancer and normal cells. In U87 glioma cell membrane, which has a high integrin expression level, GLP-SeNPs exhibited significantly higher cellular uptake than unmodified SeNPs. As expected, U87 cells exhibited a greater uptake of GLP-SeNPs than C6 cells with low integrin expression level. Furthermore, the internalization of GLP-SeNPs was inhibited by cyclo-(Arg-Gly-Asp-Phe-Lys) peptides, suggesting that cellular uptake into U87 cells and C6 cells occurred via αvβ3 integrin-mediated endocytosis. For U87 cells, the cytotoxicity of SeNPs decorated by GLP was enhanced significantly because of the induction of various apoptosis signaling pathways. Internalized GLP-SeNPs triggered intracellular reactive oxygen species downregulation. Therefore, p53, MAPKs, and AKT pathways were activated to advance cell apoptosis. These findings suggest that surface decoration of nanomaterials with GLP could be an efficient strategy for design and preparation of glioblastoma targeting nanodrugs.

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

    DOEpatents

    Park, Catherine [San Francisco, CA; Bissell, Mina J [Berkeley, CA

    2009-11-17

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

  20. Prometastatic NEDD9 Regulates Individual Cell Migration via Caveolin-1-Dependent Trafficking of Integrins.

    PubMed

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

    2015-03-01

    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 the prometastatic protein, NEDD9, in breast cancer 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 colocalizes 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. Reexpression 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. This study provides valuable new insight into the potential therapeutic benefit of NEDD9 depletion to reduce dissemination of tumor cells and discovers a new regulatory role of NEDD9 in promoting migration through modulation of CAV1-dependent trafficking of integrins. ©2014 American Association for Cancer Research.

  1. Crystal Structure of the Heterotrimeric Integrin-Binding Region of Laminin-111.

    PubMed

    Pulido, David; Hussain, Sadaf-Ahmahni; Hohenester, Erhard

    2017-03-07

    Laminins are cell-adhesive glycoproteins that are essential for basement membrane assembly and function. Integrins are important laminin receptors, but their binding site on the heterotrimeric laminins is poorly defined structurally. We report the crystal structure at 2.13 Å resolution of a minimal integrin-binding fragment of mouse laminin-111, consisting of ∼50 residues of α1β1γ1 coiled coil and the first three laminin G-like (LG) domains of the α1 chain. The LG domains adopt a triangular arrangement, with the C terminus of the coiled coil situated between LG1 and LG2. The critical integrin-binding glutamic acid residue in the γ1 chain tail is surface exposed and predicted to bind to the metal ion-dependent adhesion site in the integrin β1 subunit. Additional contacts to the integrin are likely to be made by the LG1 and LG2 surfaces adjacent to the γ1 chain tail, which are notably conserved and free of obstructing glycans. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Integrin α1β1 participates in Chondrocyte Transduction of Osmotic Stress.

    PubMed Central

    Jablonski, Christina L.; Ferguson, Samuel; Pozzi, Ambra; Clark, Andrea L.

    2014-01-01

    Background/purpose: The goal of this study was to determine the role of the collagen binding receptor integrin α1β1 in regulating osmotically induced [Ca2+]i transients in chondrocytes. Methods: The [Ca2+]i transient response of chondrocytes to osmotic stress was measured using real-time confocal microscopy. Chondrocytes from wildtype and integrin α1-null mice were imaged ex vivo (in the cartilage of intact murine femora) and in vitro (isolated from the matrix, attached to glass coverslips). Immunocytochemistry was performed to detect the presence of the osmosensor, transient receptor potential vanilloid-4 (TRPV4), and the agonist GSK1016790A (GSK101) was used to test for its functionality on chondrocytes from wildtype and integrin α1-null mice. Results/interpretation: Deletion of the integrin α1 subunit inhibited the ability of chondrocytes to respond to a hypo-osmotic stress with [Ca2+]i transients ex vivo and in vitro. The percentage of chondrocytes responding ex vivo was smaller than in vitro and of the cells that responded, more single [Ca2+]i transients were observed ex vivo compared to in vitro. Immunocytochemistry confirmed the presence of TRPV4 on wildtype and integrin α1-null chondrocytes, however application of GSK101 revealed that TRPV4 could be activated on wildtype but not integrin α1-null chondrocytes. Integrin α1β1 is a key participant in chondrocyte transduction of a hypo-osmotic stress. Furthermore, the mechanism by which integrin α1β1 influences osmotransduction is independent of matrix binding, but likely dependent on the chondrocyte osmosensor TRPV4. PMID:24495803

  3. Integrin α1β1 participates in chondrocyte transduction of osmotic stress.

    PubMed

    Jablonski, Christina L; Ferguson, Samuel; Pozzi, Ambra; Clark, Andrea L

    2014-02-28

    The goal of this study was to determine the role of the collagen binding receptor integrin α1β1 in regulating osmotically induced [Ca(2+)]i transients in chondrocytes. The [Ca(2+)]i transient response of chondrocytes to osmotic stress was measured using real-time confocal microscopy. Chondrocytes from wildtype and integrin α1-null mice were imaged ex vivo (in the cartilage of intact murine femora) and in vitro (isolated from the matrix, attached to glass coverslips). Immunocytochemistry was performed to detect the presence of the osmosensor, transient receptor potential vanilloid-4 (TRPV4), and the agonist GSK1016790A (GSK101) was used to test for its functionality on chondrocytes from wildtype and integrin α1-null mice. Deletion of the integrin α1 subunit inhibited the ability of chondrocytes to respond to a hypo-osmotic stress with [Ca(2+)]i transients ex vivo and in vitro. The percentage of chondrocytes responding ex vivo was smaller than in vitro and of the cells that responded, more single [Ca(2+)]i transients were observed ex vivo compared to in vitro. Immunocytochemistry confirmed the presence of TRPV4 on wildtype and integrin α1-null chondrocytes, however application of GSK101 revealed that TRPV4 could be activated on wildtype but not integrin α1-null chondrocytes. Integrin α1β1 is a key participant in chondrocyte transduction of a hypo-osmotic stress. Furthermore, the mechanism by which integrin α1β1 influences osmotransduction is independent of matrix binding, but likely dependent on the chondrocyte osmosensor TRPV4. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. 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

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

    PubMed Central

    de Rezende, Flávia Figueiredo

    2014-01-01

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

  6. The adapter protein SLP-76 mediates "outside-in" integrin signaling and function in T cells.

    PubMed

    Baker, R G; Hsu, C J; Lee, D; Jordan, M S; Maltzman, J S; Hammer, D A; Baumgart, T; Koretzky, G A

    2009-10-01

    The adapter protein SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an essential mediator of signaling from the T-cell antigen receptor (TCR). We report here that SLP-76 also mediates signaling downstream of integrins in T cells and that SLP-76-deficient T cells fail to support adhesion to integrin ligands. In response to both TCR and integrin stimulation, SLP-76 relocalizes to surface microclusters that colocalize with phosphorylated signaling proteins. Disruption of SLP-76 recruitment to the protein named LAT (linker for activation of T cells) inhibits SLP-76 clustering downstream of the TCR but not downstream of integrins. Conversely, an SLP-76 mutant unable to bind ADAP (adhesion and degranulation-promoting adapter protein) forms clusters following TCR but not integrin engagement and fails to support T-cell adhesion to integrin ligands. These findings demonstrate that SLP-76 relocalizes to integrin-initiated signaling complexes by a mechanism different from that employed during TCR signaling and that SLP-76 relocalization corresponds to SLP-76-dependent integrin function in T cells.

  7. Phorbol esters alter alpha4 and alphad integrin usage during eosinophil adhesion to VCAM-1.

    PubMed

    Kikuchi, Matsuo; Tachimoto, Hiroshi; Nutku, Esra; Hudson, Sherry A; Bochner, Bruce S

    2003-01-01

    We examined the effect of the protein kinase C activator phorbol-12-myristate-13-acetate (PMA) on the human eosinophil adhesion molecule phenotype and attachment to VCAM-1 via alpha4 and alphad integrins under static and flow conditions. PMA increased surface expression of alphad integrins and decreased alpha4 integrin expression. Under static conditions, eosinophils bound well to VCAM-1, primarily via alpha4beta1 integrins, with a minor alphadbeta2 integrin component. Unexpectedly, PMA-stimulated eosinophils bound equally well to VCAM-1 and albumin in a temperature- and divalent cation-dependent manner, yet adhesion was independent of beta1 and beta2 integrins. Under flow conditions, eosinophils readily attached to VCAM-1, and adhesion was inhibited by both alpha4 and alphad mAbs (95 and 50% inhibition, respectively). Many fewer PMA-stimulated eosinophils bound to VCAM-1 under flow conditions, but both alpha4 and alphad mAbs inhibited adhesion equally. Thus, PMA alters eosinophil integrin expression and the relative contributions of alpha4 and alphad integrins during attachment to VCAM-1.

  8. PKD1/PKCmu promotes alphavbeta3 integrin recycling and delivery to nascent focal adhesions.

    PubMed

    Woods, Alison J; White, Dominic P; Caswell, Patrick T; Norman, Jim C

    2004-07-07

    To identify kinases that regulate integrin recycling, we have immunoprecipitated alphavbeta3 integrin from NIH 3T3 fibroblasts in the presence and absence of primaquine (a drug that inhibits receptor recycling and leads to accumulation of integrins in endosomes) and screened for co-precipitating kinases. Primaquine strongly promoted association of alphavbeta3 integrin with PKD1, and fluorescence microscopy indicated that integrin and PKD1 associate at a vesicular compartment that is downstream of a Rab4-dependent transport step. PKD1 association was mediated by the C-terminal region of the beta3 integrin cytodomain, and mutants of beta3 that were unable to recruit PKD1 did not recycle in a PDGF-dependent fashion. Furthermore, suppression of endogenous PKD1 levels by RNAi, or overexpression of catalytically inactive PKD1 inhibited PDGF-dependent recycling of alphavbeta3 from early endosomes to the plasma membrane and blocked recruitment of alphavbeta3 to newly formed focal adhesions during cell spreading. These data indicate that PKD1 influences cell migration by directing vesicular transport of the alphavbeta3 integrin heterodimer.

  9. Direct integrin alphavbeta6-ERK binding: implications for tumour growth.

    PubMed

    Ahmed, Nuzhat; Niu, Jun; Dorahy, Douglas J; Gu, Xinhua; Andrews, Sarah; Meldrum, Cliff J; Scott, Rodney J; Baker, Mark S; Macreadie, Ian G; Agrez, Michael V

    2002-02-21

    Blockade of the mitogen-activated protein (MAP) kinase pathway suppresses growth of colon cancer in vivo. Here we demonstrate a direct link between the extracellular signal-regulated kinase ERK2 and the growth-promoting cell adhesion molecule, integrin alphavbeta6, in colon cancer cells. Down-regulation of beta6 integrin subunit expression inhibits tumour growth in vivo and MAP kinase activity in response to serum stimulation. In alphavbeta6-expressing cells ERK2 is bound only to the beta6 subunit. The increase in cytosolic MAP kinase activity upon epidermal growth factor stimulation is all accounted for by beta6-bound ERK. Deletion of the ERK2 binding site on the beta6 cytoplasmic domain inhibits tumour growth and leads to an association between ERK and the beta5 subunit. The physical interaction between integrin alphavbeta6 and ERK2 defines a novel paradigm of integrin-mediated signalling and provides a therapeutic target for cancer treatment.

  10. Integrin antagonists as potential therapeutic options for the treatment of Crohn’s disease

    PubMed Central

    McLean, Leon P.; Cross, Raymond K.

    2016-01-01

    Introduction Anti-integrin therapy for the treatment of patients with Crohn’s disease is rapidly evolving. Two agents, natalizumab and vedolizumab, are approved by the United States Food and Drug Administration for the treatment of Crohn’s disease, with vedolizumab the primary anti-integrin used due to a more favorable safety profile. Several other anti-integrins are in various stages of development. Areas Covered This review discusses the current state of anti-integrin therapy as well as suggestions for positioning of these agents in clinical practice. Emerging anti-integrin therapies, their underlying mechanisms of action, and available safety and clinical data are also reviewed. Expert Opinion Anti-integrins are effective for the treatment of Crohn’s disease, even in patients refractory to other therapies. Their use should be considered in patients with Crohn’s disease who do not respond to, develop non-response to, or have contraindications to anti-TNF therapy. Anti-integrin therapies can be offered as a first biologic therapy, in particular for older patients, patients with concurrent multiple sclerosis (natalizumab only), and in patients with contraindications to anti-TNF therapy. In patients with more severe symptoms, providers should consider co-induction with corticosteroids if possible to hasten remission. PMID:26822204

  11. 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

  12. Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits adhesion of human endometriotic epithelial and stromal cells through suppression of integrin-mediated mechanisms.

    PubMed

    Lee, JeHoon; Banu, Sakhila K; Burghardt, Robert C; Starzinski-Powitz, Anna; Arosh, Joe A

    2013-03-01

    Endometriosis is a chronic gynecological disease of reproductive age women characterized by the presence of functional endometrial tissues outside the uterine cavity. Interactions between the endometriotic cells and the peritoneal extracellular matrix proteins (ECM) are crucial mechanisms that allow adhesion of the endometriotic cells into peritoneal mesothelia. Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. In previous studies, we have reported that selective inhibition of PGE2 receptors PTGER2 and PTGER4 decreases survival and invasion of human endometriotic epithelial and stromal cells through multiple mechanisms. Results of the present study indicates that selective inhibition of PTGER2- and PTGER4-mediated PGE2 signaling 1) decreases the expression and/or activity of specific integrin receptor subunits Itgb1 (beta1) and Itgb3 (beta3) but not Itgb5 (beta5), Itga1 (alpha1), Itga2 (alpha2), Itga5 (alpha5), and Itgav (alphav); 2) decreases integrin-signaling components focal adhesion kinase or protein kinase 2 (PTK2) and talin proteins; 3) inhibits interactions between Itgb1/Itgb3 subunits, PTK2, and talin and PTGER2/PTGER4 proteins through beta-arrestin-1 and Src kinase protein complex in human endometriotic epithelial cells 12Z and stromal cells 22B; and 4) decreases adhesion of 12Z and 22B cells to ECM collagen I, collagen IV, fibronectin, and vitronectin in a substrate-specific manner. These novel findings provide an important molecular framework for further evaluation of selective inhibition of PTGER2 and PTGER4 as potential nonsteroidal therapy to expand the spectrum of currently available treatment options for endometriosis in child-bearing age women.

  13. Integrins and small GTPases as modulators of phagocytosis.

    PubMed

    Sayedyahossein, Samar; Dagnino, Lina

    2013-01-01

    Phagocytosis is the mechanism whereby cells engulf large particles. This process has long been recognized as a critical component of the innate immune response, which constitutes the organism's defense against microorganisms. In addition, phagocytic internalization of apoptotic cells or cell fragments plays important roles in tissue homeostasis and remodeling. Phagocytosis requires target interactions with receptors on the plasma membrane of the phagocytic cell. Integrins have been identified as important mediators of particle clearance, in addition to their well-established roles in cell adhesion, migration and mechanotransduction. Indeed, these ubiquitously expressed proteins impart phagocytic capacity to epithelial, endothelial and mesenchymal cell types. The importance of integrins in particle internalization is emphasized by the ability of microbial and viral pathogens to exploit their signaling pathways to invade host cells, and by the wide variety of disorders that arise from abnormalities in integrin-dependent phagocytic uptake. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Disruption of integrin-fibronectin complexes by allosteric but not ligand-mimetic inhibitors.

    PubMed

    Mould, A Paul; Craig, Susan E; Byron, Sarah K; Humphries, Martin J; Jowitt, Thomas A

    2014-12-15

    Failure of Arg-Gly-Asp (RGD)-based inhibitors to reverse integrin-ligand binding has been reported, but the prevalence of this phenomenon among integrin heterodimers is currently unknown. In the present study we have investigated the interaction of four different RGD-binding integrins (α5β1, αVβ1, αVβ3 and αVβ6) with fibronectin (FN) using surface plasmon resonance. The ability of inhibitors to reverse ligand binding was assessed by their capacity to increase the dissociation rate of pre-formed integrin-FN complexes. For all four receptors we showed that RGD-based inhibitors (such as cilengitide) were completely unable to increase the dissociation rate. Formation of the non-reversible state occurred very rapidly and did not rely on the time-dependent formation of a high-affinity state of the integrin, or the integrin leg regions. In contrast with RGD-based inhibitors, Ca2+ (but not Mg2+) was able to greatly increase the dissociation rate of integrin-FN complexes, with a half-maximal response at ~0.4 mM Ca2+ for αVβ3-FN. The effect of Ca2+ was overcome by co-addition of Mn2+, but not Mg2+. A stimulatory anti-β1 monoclonal antibody (mAb) abrogated the effect of Ca2+ on α5β1-FN complexes; conversely, a function-blocking mAb mimicked the effect of Ca2+. These results imply that Ca2+ acts allosterically, probably through binding to the adjacent metal-ion-dependent adhesion site (ADMIDAS), and that the α1 helix in the β subunit I domain is the key element affected by allosteric modulators. The data suggest an explanation for the limited clinical efficacy of RGD-based integrin antagonists, and we propose that allosteric antagonists could prove to be of greater therapeutic benefit.

  15. Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

    PubMed Central

    Swaminathan, Vinay; Kalappurakkal, Joseph Mathew; Moore, Travis I.; Koga, Nobuyasu; Baker, David A.; Oldenbourg, Rudolf; Tani, Tomomi; Springer, Timothy A.; Waterman, Clare M.

    2017-01-01

    Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven “retrograde flow” of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues. PMID:29073038

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

  18. Serosal Cavities Contain Two Populations of Innate-like integrin α4highCD4+ T Cells, Integrin α4β1+α6β1+α4β7- and α4β1+α6β1-α4β7+ Cells.

    PubMed

    Yang, Jeong In; Park, Chanho; Kho, Inseong; Lee, Sujin; Suh, Kyung-Suk; Kim, Tae Jin

    2017-12-01

    We previously reported peritoneal innate-like integrin α4 (CD49d) high CD4 + T cells that provided help for B-1a cells. Here we analyzed the expression of various integrin chains on the peritoneal and pleural integrin α4 high CD4 + T cells and investigated the functional heterogeneity of the subpopulations based on the integrin expression. Pleural cavity contained a lower ratio of integrin α4 high CD4 + T cells to integrin α4 low CD4 + T cells than peritoneal cavity, but the pleural integrin α4 high CD4 + T cells have the same characteristics of the peritoneal integrin α4 high CD4 + T cells. Most of integrin α4 high CD4 + T cells were integrin β1 high β7 - , but a minor population of integrin α4 high CD4 + T cells was integrin β1 + β7 + . Interestingly, the integrin α4 high β1 high β7 - CD4 + T cells expressed high levels of integrin α4β1 and α6β1, whereas integrin α4 high β1 + β7 + CD4 + T cells expressed high levels of integrin α4β1 and α4β7, suggesting an alternative expression of integrin α6β1 or α4β7 in combination with α4β1 in respective major and minor populations of integrin α4 high CD4 + T cells. The minor population, integrin α4 high β1 + β7 + CD4 + T cells, were different from the integrin α4 high β1 high β7 - CD4 + T cells in that they secreted a smaller amount of Th1 cytokines upon stimulation and expressed lower levels of Th1-related chemokine receptors CCR5 and CXCR3 than the integrin α4 high β 1 high β 7 - CD4 + T cells. In summary, the innate-like integrin α4 high CD4 + T cells could be divided into 2 populations, integrin α4β1 + α6β1 + α4β7 - and α4β1 + α6β1 - α4β7 + cells. The functional significance of serosal integrin α4β7 + CD4 + T cells needed to be investigated especially in view of mucosal immunity.

  19. ARF6 directs axon transport and traffic of integrins and regulates axon growth in adult DRG neurons.

    PubMed

    Eva, Richard; Crisp, Sarah; Marland, Jamie R K; Norman, Jim C; Kanamarlapudi, Venkateswarlu; ffrench-Constant, Charles; Fawcett, James W

    2012-07-25

    Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of α9 integrin promotes axon regeneration, so we have investigated α9β1 trafficking and transport in axons and at the growth cone. We have previously found that α9 and β1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied α9 and β1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of β1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.

  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. EMMPRIN regulates β1 integrin-mediated adhesion through Kindlin-3 in human melanoma cells.

    PubMed

    Delyon, Julie; Khayati, Farah; Djaafri, Ibtissem; Podgorniak, Marie-Pierre; Sadoux, Aurélie; Setterblad, Niclas; Boutalbi, Zineb; Maouche, Kamel; Maskos, Uwe; Menashi, Suzanne; Lebbé, Céleste; Mourah, Samia

    2015-06-01

    EMMPRIN is known to promote tumor invasion through extracellular matrix (ECM) degradation. Here we report that EMMPRIN can regulate melanoma cell adhesion to the ECM through an interaction with β1 integrin involving kindlin-3. In this study, EMMPRIN knockdown in the human melanoma cell line M10 using siRNA decreased cell invasion and significantly increased cell adhesion and spreading. A morphological change from a round to a spread shape was observed associated with enhanced phalloidin-labelled actin staining. In situ proximity ligation assay and co-immunoprecipitation revealed that EMMPRIN silencing increased the interaction of β1 integrin with kindlin-3, a focal adhesion protein. This was associated with an increase in β1 integrin activation and a decrease in the phosphorylation of the downstream integrin kinase FAK. Moreover, the expression at both the transcript and protein level of kindlin-3 and of β1 integrin was inversely regulated by EMMPRIN. EMMPRIN did not regulate either talin expression or its interaction with β1 integrin. These results are consistent with our in vivo demonstration that EMMPRIN inhibition increased β1 integrin activation and its interaction with kindlin-3. To conclude, these findings reveal a new role of EMMPRIN in tumor cell migration through ß1 integrin/kindlin-3-mediated adhesion pathway. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils

    PubMed Central

    Yago, Tadayuki; Zhang, Nan; Zhao, Liang; Abrams, Charles S.

    2018-01-01

    Rolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)–dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins. PMID:29592875

  3. Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils.

    PubMed

    Yago, Tadayuki; Zhang, Nan; Zhao, Liang; Abrams, Charles S; McEver, Rodger P

    2018-04-10

    Rolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)-dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins. © 2018 by The American Society of Hematology.

  4. 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.

  5. High lung-metastatic variant of human osteosarcoma cells, selected by passage of lung metastasis in nude mice, is associated with increased expression of α(v)β(3) integrin.

    PubMed

    Tome, Yasunori; Kimura, Hiroaki; Maehara, Hiroki; Sugimoto, Naotoshi; Bouvet, Michael; Tsuchiya, Hiroyuki; Kanaya, Fuminori; Hoffman, Robert M

    2013-09-01

    Altered expression of αvβ3 integrin is associated with tumor progression and metastasis in several types of cancer, including metastatic osteosarcoma. In this study, we demonstrate that in vivo passaging of lung metastasis in nude mice can generate an aggressive variant of human osteosarcoma cells. Experimental metastases were established by injecting 143B human osteosarcoma cells, expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm, in the tail vein of nude mice. Lung metastases were harvested under fluorescence microscopy from nude mice to establish cell lines which were then injected via the tail vein of additional nude mice. This procedure was repeated for four passages in order to isolate highly metastatic variant sublines. When the parental and metastatic variants were transplanted orthotopically into the tibia of nude mice, the 143B-LM4 variant had the highest metastatic rate, approximately 18-fold higher than the parent (p<0.01). αvβ3 integrin expression was increased approximately 5.6-fold in 143B-LM4 compared to parental cells (p<0.05). Thus, serial passage of lung metastases created a highly metastatic variant of human osteosarcoma cells which had increased expression of αvβ3 integrin, suggesting that αvβ3 integrin plays an essential role in osteosarcoma metastasis. With this highly metastatic variant overexpressing αvβ3 integrin, it will now be possible to further investigate the mechanism by which αvβ3 integrin facilitates metastasis.

  6. Integrin expression and glycosylation patterns regulate cell-matrix adhesion and alter with breast cancer progression.

    PubMed

    Singh, Chandrajeet; Shyanti, Ritis K; Singh, Virendra; Kale, Raosaheb K; Mishra, Jai P N; Singh, Rana P

    2018-05-05

    Integrins are the major cell adhesion glycoproteins involved in cell-extracellular matrix (ECM) interaction and metastasis. Further, glycosylation on integrin is necessary for its proper folding and functionality. Herein, differential expression of integrins viz., αvβ3 and αvβ6 was examined in MDA-MB-231, MDA-MB-468 and MCF-10A cells, which signify three different stages of breast cancer development from highly metastatic to non-tumorigenic stage. The expression of αvβ3 and αvβ6 integrins at mRNA and protein levels was observed in all three cell lines and the results displayed a distinct pattern of expression. Highly metastatic cells showed enhanced expression of αvβ3 than moderate metastatic and non-tumorigenic cells. The scenario was reversed in case of αvβ6 integrin, which was strongly expressed in moderate metastatic and non-tumorigenic cells. N-glycosylation of αvβ3 and αvβ6 integrins is required for the attachment of cells to ECM proteins like fibronectin. The cell adhesion properties were found to be different in these cancer cells with respect to the type of integrins expressed. The results testify that αvβ3 integrin in highly metastatic cells, αvβ6 integrin in both moderate metastatic and non-tumorigenic cells play an important role in cell adhesion. The investigation typify that N-glycosylation on integrins is also necessary for cell-ECM interaction. Further, glycosylation inhibition by Swainsonine is found to be more detrimental to invasive property of moderate metastatic cells. Conclusively, types of integrins expressed as well as their N-glycosylation pattern alter during the course of breast cancer progression. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. 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.

  8. Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hasan, Nazarul; Hu, Chuan, E-mail: chuan.hu@louisville.edu

    2010-01-01

    Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cellmore » surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.« less

  9. Deficiency of bone marrow beta3-integrin enhances non-functional neovascularization.

    PubMed

    Watson, Alan R; Pitchford, Simon C; Reynolds, Louise E; Direkze, Natalie; Brittan, Mairi; Alison, Malcolm R; Rankin, Sara; Wright, Nicholas A; Hodivala-Dilke, Kairbaan M

    2010-03-01

    beta3-Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in beta3-integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on beta3-null endothelial cells. Here we transplanted beta3-null bone marrow (BM) into wild-type (WT) mice to dissect the role of BM beta3-integrin deficiency in pathological angiogenesis. Mice transplanted with beta3-null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow beta3-integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM beta3-integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with beta3-null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with beta3-null bone marrow a significant proportion of tumour blood vessels are non-functional when compared with tumour blood vessels in WT-transplanted controls. Furthermore, beta3-null-transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT-transplanted animals. BM beta3-integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF-induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with beta3-null bone marrow when compared with WT-transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in beta3-null-transplanted mice. In conclusion, although BM beta3-integrin is not required for pathological angiogenesis, our

  10. 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

  11. Expression of {beta}{sub 1} integrins in human endometrial stromal and decidual cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shiokawa, Shigetatsu; Yoshimura, Yasunori; Nakamura, Yukio

    The present study was undertaken to investigate the expression of {beta}{sub 1} integrins in human endometrium and decidua using flow cytometry, immunohistochemistry, and immunoprecipitation. Fluorescence-activated flow cytometry demonstrated the greater expression of the {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, and {alpha}{sub 5} subunits of the {beta}{sub 1} integrin family in cultured stromal cells from the midsecretory phase, than in those of the early proliferative phase. The addition of estradiol (E{sub 2}) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of {beta}{sub 1} integrins in vitro. Flow cytometry also demonstrated the expression of themore » {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, {alpha}{sub 3}, {alpha}{sub 5}, and {alpha}{sub 6} subunits of {beta}{sub 1} integrin family in cultured decidual cells, and the enriched-fraction of prolactin (PRL)-producing decidual cells isolated by Percoll gradients showed high levels of {beta}{sub 1} integrins expression. Immunohistochemistry confirmed the {beta}{sub 1} integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. In summary, the present study demonstrated that endometrial stromal and decidual cells expressed {beta}{sub 1} integrin subunits at their surfaces. The expression exhibited a variability throughout the menstrual cycles, being predominantly detected in the secretory phase, and was maintained highly in the decidua. Thus, {beta}{sub 1} integrins in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation. 43 refs., 7 figs., 2 tabs.« less

  12. 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

  13. Lymphocyte integrin expression differences between SIRS and sepsis patients.

    PubMed

    Heffernan, D S; Monaghan, S F; Ayala, Alfred

    2017-11-01

    Systemic Inflammatory Response Syndrome (SIRS) and sepsis remain leading causes of death. Despite many similarities, the two entities are very distinct clinically and immunologically. T-Lymphocytes play a key pivotal role in the pathogenesis and ultimately outcome following both SIRS and sepsis. Integrins are essential in the trafficking and migration of lymphocytes. They also serve vital roles in efficient wound healing and clearance of infections. Here, we investigate whether integrin expression, specifically β1 (CD29) and β2 (CD18), are disrupted in SIRS and sepsis, and assess differences in integrin expression between these two critically ill clinical categories. T-Lymphocytes were isolated from whole blood collected from ICU patients exhibiting SIRS or sepsis. Samples were analyzed for CD18 (β2) and CD29 (β1) on CD3 + T cells through flow cytometry. Septic patients were stratified into either exclusively abdominal or non-abdominal sources of sepsis. CD18 was almost ubiquitously expressed on CD3 + T cells irrespective of clinical condition. However, CD29 (β1 integrin) was lowest in SIRS patients (20.4% of CD3 + T cells) when compared with either septic patients (35.5%) or healthy volunteers (54.1%). Furthermore, there was evidence of compartmentalization in septic patients, where abdominal sources had a greater percentage of CD3 + CD29 + T cells (41.7%) when compared with those with non-abdominal sources (29.5%). Distinct differences in T-cell integrin expression exists between patients in SIRS versus sepsis, as well as relative to the source of sepsis. Further work is needed to understand cause and effect relative to the progression from SIRS into sepsis.

  14. Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes.

    PubMed

    Wiencierz, Anne Maria; Kernbach, Manuel; Ecklebe, Josephine; Monnerat, Gustavo; Tomiuk, Stefan; Raulf, Alexandra; Christalla, Peter; Malan, Daniela; Hesse, Michael; Bosio, Andreas; Fleischmann, Bernd K; Eckardt, Dominik

    2015-01-01

    Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers. In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis. Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.

  15. Role of integrin-linked kinase in regulating phosphorylation of Akt and fibroblast survival in type I collagen matrices through a beta1 integrin viability signaling pathway.

    PubMed

    Nho, Richard Seonghun; Xia, Hong; Kahm, Judy; Kleidon, Jill; Diebold, Deanna; Henke, Craig A

    2005-07-15

    A beta1 integrin phosphatidylinositol 3-kinase/Akt pathway regulates fibroblast survival in collagen matrices. When fibroblasts attach to collagen, Akt becomes phosphorylated, providing a survival signal. In contrast, in response to mechanical forces generated during collagen contraction, Akt is dephosphorylated and fibroblasts undergo apoptosis. The kinase(s) responsible for regulating Akt phosphorylation in response to matrix-derived mechanical signals are unclear. Integrin-linked kinase (ILK) is associated with the beta1 integrin in the focal adhesion complex and as such is a candidate kinase that may regulate Akt phosphorylation and fibroblast viability. Nevertheless, there is no direct evidence that matrix-derived mechanical forces regulate cell viability by modulating ILK activity. Here, we show that ILK activity decreased in response to collagen matrix contraction, which correlated with Akt dephosphorylation and induction of fibroblast apoptosis. In contrast, enforced activation of beta1 integrin by activating antibody preserved ILK and Akt activity during collagen matrix contraction, and this is associated with protection from collagen contraction-induced apoptosis. Knock-down of ILK by small, interfering RNA (siRNA) attenuated Akt phosphorylation in response to ligation of beta1 integrin by collagen or activating antibody and enhanced fibroblast apoptosis in response to collagen contraction. Kinase dead ILK attenuated Akt phosphorylation and enhanced fibroblast apoptosis, whereas hyperactive and wild type ILK augmented Akt phosphorylation and protected fibroblasts from apoptosis. Constitutively active Akt preserved Akt activity and rescued ILK siRNA-treated fibroblasts from collagen contraction-induced apoptosis. These data establish that matrix-derived mechanical forces sensed by beta1 integrin are capable of modulating ILK activity which regulates fibroblast viability via an Akt-dependent mechanism.

  16. Integrin suppresses neurogenesis and regulates brain tissue assembly in planarian regeneration.

    PubMed

    Bonar, Nicolle A; Petersen, Christian P

    2017-03-01

    Animals capable of adult regeneration require specific signaling to control injury-induced cell proliferation, specification and patterning, but comparatively little is known about how the regeneration blastema assembles differentiating cells into well-structured functional tissues. Using the planarian Schmidtea mediterranea as a model, we identify β1-integrin as a crucial regulator of blastema architecture. β1-integrin(RNAi) animals formed small head blastemas with severe tissue disorganization, including ectopic neural spheroids containing differentiated neurons normally found in distinct organs. By mimicking aspects of normal brain architecture but without normal cell-type regionalization, these spheroids bore a resemblance to mammalian tissue organoids synthesized in vitro We identified one of four planarian integrin-alpha subunits inhibition of which phenocopied these effects, suggesting that a specific receptor controls brain organization through regeneration. Neoblast stem cells and progenitor cells were mislocalized in β1-integrin(RNAi) animals without significantly altered body-wide patterning. Furthermore, tissue disorganization phenotypes were most pronounced in animals undergoing brain regeneration and not homeostatic maintenance or regeneration-induced remodeling of the brain. These results suggest that integrin signaling ensures proper progenitor recruitment after injury, enabling the generation of large-scale tissue organization within the regeneration blastema. © 2017. Published by The Company of Biologists Ltd.

  17. α4-Integrin Mediates Neutrophil-Induced Free Radical Injury to Cardiac Myocytes

    PubMed Central

    Poon, Betty Y.; Ward, Christopher A.; Cooper, Conan B.; Giles, Wayne R.; Burns, Alan R.; Kubes, Paul

    2001-01-01

    Previous work has demonstrated that circulating neutrophils (polymorphonuclear leukocytes [PMNs]) adhere to cardiac myocytes via β2-integrins and cause cellular injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. Since PMNs induced to leave the vasculature (emigrated PMNs) express the α4-integrin, we asked whether (a) these PMNs also induce myocyte injury via NADPH oxidase; (b) β2-integrins (CD18) still signal oxidant production, or if this process is now coupled to the α4-integrin; and (c) dysfunction is superoxide dependent within the myocyte or at the myocyte–PMN interface. Emigrated PMNs exposed to cardiac myocytes quickly induced significant changes in myocyte function. Myocyte shortening was decreased by 30–50% and rates of contraction and relaxation were reduced by 30% within the first 10 min. Both α4-integrin antibody (Ab)-treated PMNs and NADPH oxidase–deficient PMNs were unable to reduce myocyte shortening. An increased level of oxidative stress was detected in myocytes within 5 min of PMN adhesion. Addition of an anti–α4-integrin Ab, but not an anti-CD18 Ab, prevented oxidant production, suggesting that in emigrated PMNs the NADPH oxidase system is uncoupled from CD18 and can be activated via the α4-integrin. Addition of exogenous superoxide dismutase (SOD) inhibited all parameters of dysfunction measured, whereas overexpression of intracellular SOD within the myocytes did not inhibit the oxidative stress or the myocyte dysfunction caused by the emigrated PMNs. These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed α4-integrin functions as the ligand that signals oxidant production. The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the α4-integrin–coupled NADPH

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

    PubMed

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

    2015-11-05

    Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85-89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1β1 up regulated the level of phosphorylated Smad1, 5, 8, which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Galectin-3 modulates the polarized surface delivery of β1-integrin in epithelial cells.

    PubMed

    Hönig, Ellena; Ringer, Karina; Dewes, Jenny; von Mach, Tobias; Kamm, Natalia; Kreitzer, Geri; Jacob, Ralf

    2018-05-10

    Epithelial cells require a precise intracellular transport and sorting machinery in order to establish and maintain their polarized architecture. This machinery includes beta-galactoside binding galectins for glycoprotein targeting to the apical membrane. Galectin-3 sorts cargo destined for the apical plasma membrane into vesicular carriers. After delivery of cargo to the apical milieu, galectin-3 recycles back into sorting organelles. We analyzed the role of galectin-3 in the polarized distribution of β1-integrin in MDCK cells. Integrins are located primarily at the basolateral domain of epithelial cells. We demonstrate that a minor pool of β1-integrin interacts with galectin-3 at the apical plasma membrane. Knockdown of galectin-3 decreases apical delivery of β1-integrin. This loss is restored by supplementation with recombinant galectin-3 and galectin-3 overexpression. Our data suggest that galectin-3 targets newly synthesized β1-integrin to the apical membrane and promotes apical delivery of β1-integrin internalized from the basolateral membrane. In parallel, galectin-3 knockout results in a reduction in cell proliferation and an impairment in proper cyst development. Our results suggest that galectin-3 modulates the surface distribution of β1-integrin and affects the morphogenesis of polarized cells. © 2018. Published by The Company of Biologists Ltd.

  20. Integrin β1 activation induces an anti-melanoma host response

    PubMed Central

    Sole, Xavier; Salony; Chowdhury, Joeeta; Ross, Kenneth N.; Ramaswamy, Sridhar

    2017-01-01

    TGF-β is a cytokine thought to function as a tumor promoter in advanced malignancies. In this setting, TGF-β increases cancer cell proliferation, survival, and migration, and orchestrates complex, pro-tumorigenic changes in the tumor microenvironment. Here, we find that in melanoma, integrin β1-mediated TGF-β activation may also produce tumor suppression via an altered host response. In the A375 human melanoma cell nu/nu xenograft model, we demonstrate that cell surface integrin β1-activation increases TGF-β activity, resulting in stromal activation, neo-angiogenesis and, unexpectedly for this nude mouse model, increase in the number of intra-tumoral CD8+ T lymphocytes within the tumor microenvironment. This is associated with attenuation of tumor growth and long-term survival benefit. Correspondingly, in human melanomas, TGF-β1 correlates with integrin β1/TGF-β1 activation and the expression of markers for vasculature and stromal activation. Surprisingly, this integrin β1/TGF-β1 transcriptional footprint also correlates with the expression of markers for tumor-infiltrating lymphocytes, multiple immune checkpoints and regulatory pathways, and, importantly, better long-term survival of patients. These correlations are unique to melanoma, in that we do not observe similar associations between β1 integrin/TGF-β1 activation and better long-term survival in other human tumor types. These results suggest that activation of TGF-β1 in melanoma may be associated with the generation of an anti-tumor host response that warrants further study. PMID:28448494

  1. Galectin-3 alters the lateral mobility and clustering of β1-integrin receptors

    PubMed Central

    Yang, Esther H.; Rode, Julia; Howlader, Md. Amran; Eckermann, Marina; Santos, Jobette T.; Hernandez Armada, Daniel; Zheng, Ruixiang; Zou, Chunxia

    2017-01-01

    Glycoprotein receptors are influenced by myriad intermolecular interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examined the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the α5β1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor reductions in lateral mobility. Treatment of cells with Gal-3 increased β1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics analysis to confirm that cells expressed endogenous Gal-3, and found that addition of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3–integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins. PMID:29016609

  2. Function of the alpha 6 Integrins in Breast Carcinoma

    DTIC Science & Technology

    2001-10-01

    motility on laminin-1. Time-lapse was resistant to solubilization with Triton X-100. Cy- videomicroscopy of clone A cells on laminin-1 revealed...represent SEM. The Integrin a6/34 Participates in the Dynamic Formation of Actin-based Motility Structures: Filopodia lapse videomicroscopy in serum-free...threefold greater time-lapse videomicroscopy to understand how the cu6P34 than on an equivalent concentration of collagen type I, integrin contributes to

  3. 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

  4. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Phosphoinositide Signaling Regulates the Exocyst Complex and Polarized Integrin Trafficking in Directionally Migrating Cells

    PubMed Central

    Thapa, Narendra; Sun, Yue; Schramp, Mark; Choi, Suyoung; Ling, Kun; Anderson, Richard A

    2011-01-01

    Summary Polarized delivery of signaling and adhesion molecules to the leading edge is required for directional migration of cells. Here, we describe a role for the PIP2 synthesizing enzyme, PIPKIγi2, in regulation of exocyst complex control of cell polarity and polarized integrin trafficking during migration. Loss of PIPKIγi2 impaired directional migration, formation of cell polarity, and integrin trafficking to the leading edge. Upon initiation of directional migration PIPKIγi2 via PIP2 generation controls the integration of the exocyst complex into an integrin-containing trafficking compartment(s) that requires the talin-binding ability of PIPKIγi2, and talin for integrin recruitment to the leading edge. A PIP2 requirement is further emphasized by inhibition of PIPKIγi2-regulated directional migration by an Exo70 mutant deficient in PIP2 binding. These results reveal how phosphoinositide generation orchestrates polarized trafficking of integrin in coordination with talin that links integrins to the actin cytoskeleton, processes that are required for directional migration. PMID:22264730

  6. β4-Integrin/PI3K Signaling Promotes Tumor Progression through the Galectin-3-N-Glycan Complex.

    PubMed

    Kariya, Yukiko; Oyama, Midori; Hashimoto, Yasuhiro; Gu, Jianguo; Kariya, Yoshinobu

    2018-06-01

    Malignant transformation is associated with aberrant N -glycosylation, but the role of protein N -glycosylation in cancer progression remains poorly defined. β4-integrin is a major carrier of N -glycans and is associated with poor prognosis, tumorigenesis, and metastasis. Here, N -glycosylation of β4-integrin contributes to the activation of signaling pathways that promote β4-dependent tumor development and progression. Increased expression of β1,6GlcNAc-branched N -glycans was found to be colocalized with β4-integrin in human cutaneous squamous cell carcinoma tissues, and that the β1,6GlcNAc residue was abundant on β4-integrin in transformed keratinocytes. Interruption of β1,6GlcNAc-branching formation on β4-integrin with the introduction of bisecting GlcNAc by N -acetylglucosaminyltransferase III overexpression was correlated with suppression of cancer cell migration and tumorigenesis. N -Glycan deletion on β4-integrin impaired β4-dependent cancer cell migration, invasion, and growth in vitro and diminished tumorigenesis and proliferation in vivo The reduced abilities of β4-integrin were accompanied with decreased phosphoinositol-3 kinase (PI3K)/Akt signals and were restored by the overexpression of the constitutively active p110 PI3K subunit. Binding of galectin-3 to β4-integrin via β1,6GlcNAc-branched N -glycans promoted β4-integrin-mediated cancer cell adhesion and migration. In contrast, a neutralizing antibody against galectin-3 attenuated β4-integrin N -glycan-mediated PI3K activation and inhibited the ability of β4-integrin to promote cell motility. Furthermore, galectin-3 knockdown by shRNA suppressed β4-integrin N -glycan-mediated tumorigenesis. These findings provide a novel role for N -glycosylation of β4-integrin in tumor development and progression, and the regulatory mechanism for β4-integrin/PI3K signaling via the galectin-3- N -glycan complex. Implications: N -Glycosylation of β4-integrin plays a functional role in promoting

  7. 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

  8. 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.

  9. The role of a conserved membrane proximal cysteine in altering αPS2CβPS integrin diffusion.

    PubMed

    Syed, Aleem; Arora, Neha; Bunch, Thomas A; Smith, Emily A

    2016-11-15

    Cysteine residues (Cys) in the membrane proximal region are common post-translational modification (PTM) sites in transmembrane proteins. Herein, the effects of a highly conserved membrane proximal α-subunit Cys 1368 on the diffusion properties of αPS2CβPS integrins are reported. Sequence alignment shows that this cysteine is palmitoylated in human α3 and α6 integrin subunits. Replacing Cys 1368 in wild-type integrins with valine (Val 1368 ) putatively blocks a PTM site and alters integrins' ligand binding and diffusion characteristics. Both fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) diffusion measurements show Val 1368 integrins are more mobile compared to wild-type integrins. Approximately 33% and 8% more Val 1368 integrins are mobile as measured by FRAP and SPT, respectively. The mobile Val 1368 integrins also exhibit less time-dependent diffusion, as measured by FRAP. Tandem mass spectrometry data suggest that Cys 1368 contains a redox or palmitoylation PTM in αPS2CβPS integrins. This membrane proximal Cys may play an important role in the diffusion of other alpha subunits that contain this conserved residue.

  10. Glioma cell dispersion is driven by α5 integrin-mediated cell-matrix and cell-cell interactions.

    PubMed

    Blandin, Anne-Florence; Noulet, Fanny; Renner, Guillaume; Mercier, Marie-Cécile; Choulier, Laurence; Vauchelles, Romain; Ronde, Philippe; Carreiras, Franck; Etienne-Selloum, Nelly; Vereb, Gyorgy; Lelong-Rebel, Isabelle; Martin, Sophie; Dontenwill, Monique; Lehmann, Maxime

    2016-07-01

    Glioblastoma multiform (GBM) is the most common and most aggressive primary brain tumor. The fibronectin receptor, α5 integrin is a pertinent novel therapeutic target. Despite numerous data showing that α5 integrin support tumor cell migration and invasion, it has been reported that α5 integrin can also limit cell dispersion by increasing cell-cell interaction. In this study, we showed that α5 integrin was involved in cell-cell interaction and gliomasphere formation. α5-mediated cell-cell cohesion limited cell dispersion from spheroids in fibronectin-poor microenvironment. However, in fibronectin-rich microenvironment, α5 integrin promoted cell dispersion. Ligand-occupied α5 integrin and fibronectin were distributed in fibril-like pattern at cell-cell junction of evading cells, forming cell-cell fibrillar adhesions. Activated focal adhesion kinase was not present in these adhesions but was progressively relocalized with α5 integrin as cell migrates away from the spheroids. α5 integrin function in GBM appears to be more complex than previously suspected. As GBM overexpressed fibronectin, it is most likely that in vivo, α5-mediated dissemination from the tumor mass overrides α5-mediated tumor cell cohesion. In this respect, α5-integrin antagonists may be useful to limit GBM invasion in brain parenchyma. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Macrophage Integrins Modulate Response to Ultra-High Molecular Weight Polyethylene Particles and Direct Particle-Induced Osteolysis

    PubMed Central

    Zaveri, Toral D.; Dolgova, Natalia V.; Lewis, Jamal S.; Hamaker, Kiri; Clare-Salzler, Michael J.; Keselowsky, Benjamin G.

    2016-01-01

    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. PMID:27889664

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  13. Expression and in vitro regulation of integrins by normal human urothelial cells.

    PubMed

    Southgate, J; Kennedy, W; Hutton, K A; Trejdosiewicz, L K

    1995-08-01

    Integrins are thought to be essential adhesion receptors for the maintenance of tissue histioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed alpha 2 beta 1 and alpha 3 beta 1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, alpha 6 beta 4 and occasionally alpha v beta 4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca++ serum-free medium, the monolayer cultures expressed alpha 2 beta 1, alpha 3 beta 1 and alpha 5 beta 1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas alpha 6 beta 4 was distributed in a random pattern over the substratum. Increasing exogenous Ca++ concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, alpha 6 beta 4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of alpha 6 beta 4 in high Ca++ media, and also of alpha 3 and alpha 5, but not alpha 2, subunits. These results suggest that alpha 2 beta 1 and alpha 3 beta 1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas alpha 6 beta 4 is the principal integrin involved in substratum adhesion.

  14. Adhesive F-actin Waves: A Novel Integrin-Mediated Adhesion Complex Coupled to Ventral Actin Polymerization

    PubMed Central

    Case, Lindsay B.; Waterman, Clare M.

    2011-01-01

    At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in “ventral F-actin waves” that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These “adhesive F-actin waves” require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization. PMID:22069459

  15. Hypoxia-inducible factor regulates alphavbeta3 integrin cell surface expression.

    PubMed

    Cowden Dahl, Karen D; Robertson, Sarah E; Weaver, Valerie M; Simon, M Celeste

    2005-04-01

    Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of alpha and beta aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt(-/-) and Hifalpha(-/-) TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin alphavbeta3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O(2)). Culturing B16F0 melanoma cells at 1.5% O(2) resulted in increased alphavbeta3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O(2) tension influence placental invasion and tumor migration by increasing cell surface expression of alphavbeta3 integrin.

  16. Molecular cloning, expression and characterization of albolamin: a type P-IIa snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris).

    PubMed

    Jangprasert, Panchalee; Rojnuckarin, Ponlapat

    2014-03-01

    Snake venom metalloproteinases (SVMPs) can damage vessel wall, degrade clotting factors, inhibit integrins and block platelet functions. Studying them not only gives us deeper insights in pathogenesis of snakebites, but also potentially yields novel therapeutic agents. Here, we discovered a clone of an RGD-containing SVMP from the green pit viper (Cryptelytrops albolabris) venom gland cDNA library. Sequence analysis revealed that it belonged to the P-IIa subclass of SVMP comprising signal peptide, prodomain, metalloproteinase and disintegrin. Compared with other P-II SVMPs, it contained 2 additional conserved cysteines that were predicted to prevent the release of disintegrin from the metalloproteinase domain in the mature protein. The N-terminal histidine-tagged construct of metalloproteinase and disintegrin domains of albolamin was inserted into the pPICZαA vector and expressed in Pichia pastoris. The recombinant protein molecular weight was approximately 35 kDa on Western blot probed with anti-polyhistidine antibody. The recombinant albolamin could digest human type IV collagen starting within 15 min after incubation. In addition, it dose-dependently inhibited collagen-induced platelet aggregation with the IC50 of 1.8 μM. However, there was no effect on ADP-induced platelet aggregation. Therefore, the inhibition mechanism is probably through blocking collagen receptor(s). Albolamin activities probably contributed to pathology of green pit viper bites. Its disintegrin domain deserves further studies for the potential to be a useful agent affecting platelet functions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. High αv Integrin Level of Cancer Cells Is Associated with Development of Brain Metastasis in Athymic Rats

    PubMed Central

    WU, YINGJEN JEFFREY; PAGEL, MICHAEL A.; MULDOON, LESLIE L.; FU, RONGWEI; NEUWELT, EDWARD A.

    2018-01-01

    Background/Aim Brain metastases commonly occur in patients with malignant skin, lung and breast cancers resulting in high morbidity and poor prognosis. Integrins containing an αv subunit are cell adhesion proteins that contribute to cancer cell migration and cancer progression. We hypothesized that high expression of αv integrin cell adhesion protein promoted metastatic phenotypes in cancer cells. Materials and Methods Cancer cells from different origins were used and studied regarding their metastatic ability and intetumumab, anti-αv integrin mAb, sensitivity using in vitro cell migration assay and in vivo brain metastases animal models. Results The number of brain metastases and the rate of occurrence were positively correlated with cancer cell αv integrin levels. High αv integrin-expressing cancer cells showed significantly faster cell migration rate in vitro than low αv integrin-expressing cells. Intetumumab significantly inhibited cancer cell migration in vitro regardless of αv integrin expression level. Overexpression of αv integrin in cancer cells with low αv integrin level accelerated cell migration in vitro and increased the occurrence of brain metastases in vivo. Conclusion αv integrin promotes brain metastases in cancer cells and may mediate early steps in the metastatic cascade, such as adhesion to brain vasculature. Targeting αv integrin with intetumumab could provide clinical benefit in treating cancer patients who develop metastases. PMID:28739685

  18. The Novel α4B Murine α4 Integrin Protein Splicing Variant Inhibits α4 Protein-dependent Cell Adhesion*

    PubMed Central

    Kouro, Hitomi; Kon, Shigeyuki; Matsumoto, Naoki; Miyashita, Tomoe; Kakuchi, Ayaka; Ashitomi, Dai; Saitoh, Kodai; Nakatsuru, Takuya; Togi, Sumihito; Muromoto, Ryuta; Matsuda, Tadashi

    2014-01-01

    Integrins affect the motility of multiple cell types to control cell survival, growth, or differentiation, which are mediated by cell-cell and cell-extracellular matrix interactions. We reported previously that the α9 integrin splicing variant, SFα9, promotes WT α9 integrin-dependent adhesion. In this study, we introduced a new murine α4 integrin splicing variant, α4B, which has a novel short cytoplasmic tail. In inflamed tissues, the expression of α4B, as well as WT α4 integrin, was up-regulated. Cells expressing α4B specifically bound to VCAM-1 but not other α4 integrin ligands, such as fibronectin CS1 or osteopontin. The binding of cells expressing WT α4 integrin to α4 integrin ligands is inhibited by coexpression of α4B. Knockdown of α4B in metastatic melanoma cell lines results in a significant increase in lung metastasis. Expression levels of WT α4 integrin are unaltered by α4B, with α4B acting as a regulatory subunit for WT α4 integrin by a dominant-negative effect or inhibiting α4 integrin activation. PMID:24755217

  19. Integrin-Using Rotaviruses Bind α2β1 Integrin α2 I Domain via VP4 DGE Sequence and Recognize αXβ2 and αVβ3 by Using VP7 during Cell Entry

    PubMed Central

    Graham, Kate L.; Halasz, Peter; Tan, Yan; Hewish, Marilyn J.; Takada, Yoshikazu; Mackow, Erich R.; Robinson, Martyn K.; Coulson, Barbara S.

    2003-01-01

    Integrins α2β1, αXβ2, and αVβ3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the α2β1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the αXβ2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of α2β1, αXβ2, and αVβ3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound α2β1, and VP7 interacted with αXβ2 and αVβ3 at a postbinding stage. DGEA inhibited rotavirus binding to α2β1 and infectivity, whereas GPRP binding to αXβ2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed α2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the α2 I domain. In a novel process, integrin-using viruses bind the α2 I domain of α2β1 via DGE in VP4 and interact with αXβ2 (via GPR) and αVβ3 by using VP7 to facilitate cell entry and infection. PMID:12941907

  20. Integrin Clustering Matters: A Review of Biomaterials Functionalized with Multivalent Integrin-Binding Ligands to Improve Cell Adhesion, Migration, Differentiation, Angiogenesis, and Biomedical Device Integration.

    PubMed

    Karimi, Fatemeh; O'Connor, Andrea J; Qiao, Greg G; Heath, Daniel E

    2018-03-25

    Material systems that exhibit tailored interactions with cells are a cornerstone of biomaterial and tissue engineering technologies. One method of achieving these tailored interactions is to biofunctionalize materials with peptide ligands that bind integrin receptors present on the cell surface. However, cell biology research has illustrated that both integrin binding and integrin clustering are required to achieve a full adhesion response. This biophysical knowledge has motivated researchers to develop material systems biofunctionalized with nanoscale clusters of ligands that promote both integrin occupancy and clustering of the receptors. These materials have improved a wide variety of biological interactions in vitro including cell adhesion, proliferation, migration speed, gene expression, and stem cell differentiation; and improved in vivo outcomes including increased angiogenesis, tissue healing, and biomedical device integration. This review first introduces the techniques that enable the fabrication of these nanopatterned materials, describes the improved biological effects that have been achieved, and lastly discusses the current limitations of the technology and where future advances may occur. Although this technology is still in its nascency, it will undoubtedly play an important role in the future development of biomaterials and tissue engineering scaffolds for both in vitro and in vivo applications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Single particle tracking with sterol modulation reveals the cholesterol-mediated diffusion properties of integrin receptors.

    PubMed

    Arora, Neha; Syed, Aleem; Sander, Suzanne; Smith, Emily A

    2014-10-07

    A combination of sterol modulation with cyclodextrins plus fluorescence microscopy revealed a biophysical mechanism behind cholesterol's influence on the diffusion of a ubiquitous class of receptors called integrins. The heterogeneous diffusion of integrins bound to ligand-coated quantum dots was measured using single particle tracking (SPT), and the ensemble changes in integrin diffusion were measured by fluorescence recovery after photobleaching (FRAP). A 25 ± 1% reduction of membrane cholesterol resulted in three significant changes to the diffusion of ligand-bound αPS2CβPS integrins as measured by SPT. There was a 23% increase in ligand-bound mobile integrins; there was a statistically significant increase in the average diffusion coefficient inside zones of confined diffusion, and histograms of confined integrin trajectories showed an increased frequency in the range of 0.1-1 μm(2) s(-1) and a decreased frequency in the 0.001-0.1 μm(2) s(-1) range. No statistical change was measured in the duration of confinement nor the size of confined zones. Restoring the cholesterol-depleted cells with exogenous cholesterol or exogenous epicholesterol resulted in similar diffusion properties. Epicholesterol differs from cholesterol in the orientation of a single hydroxyl group. The ability of epicholesterol to substitute for cholesterol suggests a biophysical mechanism for cholesterol's effect on integrin diffusion. Influences of bilayer thickness, viscosity and organization are discussed as possible explanations for the measured changes in integrin diffusion when the membrane cholesterol concentration is reduced.

  2. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shih, Chun-Ho; Chiang, Tin-Bin; Wang, Wen-Jeng, E-mail: wjwang@mail.cgust.edu.tw

    Convulxin (CVX), a C-type lectin-like protein (CLPs), is a potent platelet aggregation inducer. To evaluate its potential applications in angiogenic diseases, the multimeric CVX were further explored on its mode of actions toward human coronary artery smooth muscle cells (HCASMCs). The N-terminus of β-chain of CVX (CVX-β) contains a putative disintegrin-like domain with a conserved motif upon the sequence comparison with other CLPs. Importantly, native CVX had no cytotoxic activity as examined by electrophoretic pattern. A Trp-Ala–Asp (WAD)-containing octapeptide, MTWADAEK, was thereafter synthesized and analyzed in functional assays. In the case of specific integrin antagonists as positive controls, the anti-angiogenicmore » effects of CVX on HCASMCs were investigated by series of functional analyses. CVX showed to exhibit multiple inhibitory activities toward HCASMCs proliferation, adhesion and invasion with a dose- and integrin αvβ3-dependent fashion. However, the WAD-octapeptide exerting a minor potency could also work as an active peptidomimetic. In addition, flow cytometric analysis demonstrated both the intact CVX and synthetic peptide can specifically interact with integrin-αv on HCASMCs and CVX was shown to have a down-regulatory effect on the gene expression of CXC-chemokines, such as growth-related oncogene and interleukin-8. According to nuclear factor-κB (NF-κB) p65 translocation assay and Western blotting analysis, the NF-κB activation was not involved in the signaling events of CVX-induced gene expression. In conclusion, CVX may act as a disintegrin-like protein via the interactions of WAD-motif in CVX-β with integrin-αv on HCASMCs and it also is a gene suppressor with the ability to diminish the expression of two CXC-chemokines in a NF-κB-independent manner. Indeed, more extensive investigations are needed and might create a new avenue for the development of a novel angiostatic agent. - Highlights: • The tetrameric convulxin (CVX

  3. Fibronectins containing extradomain A or B enhance osteoblast differentiation via distinct integrins

    PubMed Central

    Sens, Carla; Huck, Katrin; Pettera, Stefan; Uebel, Stephan; Wabnitz, Guido; Moser, Markus; Nakchbandi, Inaam A.

    2017-01-01

    Fibronectin is a multidomain protein secreted by various cell types. It forms a network of fibers within the extracellular matrix and impacts intracellular processes by binding to various molecules, primarily integrin receptors on the cells. Both the presence of several isoforms and the ability of the various domains and isoforms to bind to a variety of integrins result in a wide range of effects. In vivo findings suggest that fibronectin isoforms produced by the osteoblasts enhance their differentiation. Here we report that the isoform characterized by the presence of extradomain A activates α4β1 integrin and augments osteoblast differentiation. In addition, the isoform containing extradomain B enhances the binding of fibronectin through the RGD sequence to β3-containing integrin, resulting in increased mineralization by and differentiation of osteoblasts. Our study thus reveals novel functions for two fibronectin isoforms and the mediating receptors in osteoblast differentiation. PMID:28325836

  4. Persistence of fan-shaped keratocytes is a matrix-rigidity-dependent mechanism that requires α5β1 integrin engagement.

    PubMed

    Riaz, Maryam; Versaevel, Marie; Mohammed, Danahe; Glinel, Karine; Gabriele, Sylvain

    2016-09-28

    Despite the importance of matrix rigidity on cell functions, many aspects of the mechanosensing process in highly migratory cells remain elusive. Here, we studied the migration of highly motile keratocytes on culture substrates with similar biochemical properties and rigidities spanning the range between soft tissues (~kPa) and stiff culture substrates (~GPa). We show that morphology, polarization and persistence of motile keratocytes are regulated by the matrix stiffness over seven orders of magnitude, without changing the cell spreading area. Increasing the matrix rigidity leads to more F-actin in the lamellipodia and to the formation of mature contractile actomyosin fibers that control the cell rear retraction. Keratocytes remain rounded and form nascent adhesions on compliant substrates, whereas large and uniformly distributed focal adhesions are formed on fan-shaped keratocytes migrating on rigid surfaces. By combining poly-L-lysine, fibronectin and vitronectin coatings with selective blocking of α v β 3 or α 5 β 1 integrins, we show that α V β 3 integrins permit the spreading of keratocytes but are not sufficient for polarization and rigidity sensing that require the engagement of α 5 β 1 integrins. Our study demonstrates a matrix rigidity-dependent regulation of the directional persistence in motile keratocytes and refines the role of α v β 3 and α 5 β 1 integrins in the molecular clutch model.

  5. Research on the expression of integrin β3 and leukaemia inhibitory factor in the decidua of women with cesarean scar pregnancy.

    PubMed

    Qian, Zhi-Da; Weng, Yue; Wang, Chun-Fen; Huang, Li-Li; Zhu, Xiao-Ming

    2017-03-11

    Cesarean scar pregnancy (CSP) is a late serious complication of cesarean section. There has been an increase in the incidence of CSP worldwide in recent years. It's a life-threatening condition because of the high risk of uncontrolled hemorrhage and uterine rupture. The mechanism of CSP is still unclear. The endometrial receptivity might be different in the cesarean scar between CSP and normal pregnancies. Endometrial expression of integrin β3 and LIF positively correlates with endometrial receptivity and embryo implantation. The purpose of the study is to explore the mechanism of CSP. The EnVision two-step immunohistochemical staining technique was used to detect the expression of integrin β3 and LIF in the decidua of women with CSP (20 cases) and normal pregnancies (20 cases). The distribution and staining intensity of integrin β3 and LIF in the two groups were observed. Observation of the staining were done using microscope within five randomly selected high-power fields (HPF, 10 × 40). All data analyses were conducted with SPSS 17.0 and the statistical significance was set at P <0.05. The decidua in the different parts of both two groups that stained with the anti-integrin β3 and anti-LIF antibody: most of the integrin β3 and LIF positive cells were located in glandular epithelium. The expression intensity of integrin β3 in the cesarean scar in CSP group was significant higher than the uterine cavity in CSP group and the cesarean scar in normal pregnancy group. It's similar with the uterine cavity in normal pregnancy group. The expression intensity of LIF in the cesarean scar in CSP group was significant higher than the uterine cavity in CSP group and the cesarean scar in normal pregnancy group. It's significant lower than the uterine cavity in normal pregnancy group. The decidual integrin β3 and LIF might play an important role in the mechanism of CSP. The increase expression of integrin β3 and LIF in the cesarean scar decidua might be associated

  6. Insulin resistance in striated muscle-specific integrin receptor beta1-deficient mice.

    PubMed

    Zong, Haihong; Bastie, Claire C; Xu, Jun; Fassler, Reinhard; Campbell, Kevin P; Kurland, Irwin J; Pessin, Jeffrey E

    2009-02-13

    Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin beta1 subunit in striated muscle results in a near complete loss of integrin beta1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin beta1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR.Rictor.LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin beta1 signaling events in mediating cross-talk to that of insulin action.

  7. High αv Integrin Level of Cancer Cells Is Associated with Development of Brain Metastasis in Athymic Rats.

    PubMed

    Wu, Yingjen Jeffrey; Pagel, Michael A; Muldoon, Leslie L; Fu, Rongwei; Neuwelt, Edward A

    2017-08-01

    Brain metastases commonly occur in patients with malignant skin, lung and breast cancers resulting in high morbidity and poor prognosis. Integrins containing an αv subunit are cell adhesion proteins that contribute to cancer cell migration and cancer progression. We hypothesized that high expression of αv integrin cell adhesion protein promoted metastatic phenotypes in cancer cells. Cancer cells from different origins were used and studied regarding their metastatic ability and intetumumab, anti-αv integrin mAb, sensitivity using in vitro cell migration assay and in vivo brain metastases animal models. The number of brain metastases and the rate of occurrence were positively correlated with cancer cell αv integrin levels. High αv integrin-expressing cancer cells showed significantly faster cell migration rate in vitro than low αv integrin-expressing cells. Intetumumab significantly inhibited cancer cell migration in vitro regardless of αv integrin expression level. Overexpression of αv integrin in cancer cells with low αv integrin level accelerated cell migration in vitro and increased the occurrence of brain metastases in vivo. αv integrin promotes brain metastases in cancer cells and may mediate early steps in the metastatic cascade, such as adhesion to brain vasculature. Targeting αv integrin with intetumumab could provide clinical benefit in treating cancer patients who develop metastases. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  8. β1-Integrin Deletion From the Lens Activates Cellular Stress Responses Leading to Apoptosis and Fibrosis

    PubMed Central

    Wang, Yichen; Terrell, Anne M.; Riggio, Brittany A.; Anand, Deepti; Lachke, Salil A.; Duncan, Melinda K.

    2017-01-01

    Purpose Previous research showed that the absence of β1-integrin from the mouse lens after embryonic day (E) 13.5 (β1MLR10) leads to the perinatal apoptosis of lens epithelial cells (LECs) resulting in severe microphthalmia. This study focuses on elucidating the molecular connections between β1-integrin deletion and this phenotype. Methods RNA sequencing was performed to identify differentially regulated genes (DRGs) in β1MLR10 lenses at E15.5. By using bioinformatics analysis and literature searching, Egr1 (early growth response 1) was selected for further study. The activation status of certain signaling pathways (focal adhesion kinase [FAK]/Erk, TGF-β, and Akt signaling) was studied via Western blot and immunohistochemistry. Mice lacking both β1-integrin and Egr1 genes from the lenses were created (β1MLR10/Egr1−/−) to study their relationship. Results RNA sequencing identified 120 DRGs that include candidates involved in the cellular stress response, fibrosis, and/or apoptosis. Egr1 was investigated in detail, as it mediates cellular stress responses in various cell types, and is recognized as an upstream regulator of numerous other β1MLR10 lens DRGs. In β1MLR10 mice, Egr1 levels are elevated shortly after β1-integrin loss from the lens. Further, pErk1/2 and pAkt are elevated in β1MLR10 LECs, thus providing the potential signaling mechanism that causes Egr1 upregulation in the mutant. Indeed, deletion of Egr1 from β1MLR10 lenses partially rescues the microphthalmia phenotype. Conclusions β1-integrin regulates the appropriate levels of Erk1/2 and Akt phosphorylation in LECs, whereas its deficiency results in the overexpression of Egr1, culminating in reduced cell survival. These findings provide insight into the molecular mechanism underlying the microphthalmia observed in β1MLR10 mice. PMID:28763805

  9. Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

    PubMed Central

    Wiencierz, Anne Maria; Kernbach, Manuel; Ecklebe, Josephine; Monnerat, Gustavo; Tomiuk, Stefan; Raulf, Alexandra; Christalla, Peter; Malan, Daniela; Hesse, Michael; Bosio, Andreas; Fleischmann, Bernd K.; Eckardt, Dominik

    2015-01-01

    Rationale Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers. Methods and Results In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis. Conclusion Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications. PMID:26618511

  10. Direct Interactions with the Integrin β1 Cytoplasmic Tail Activate the Abl2/Arg Kinase*

    PubMed Central

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

    2015-01-01

    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. PMID:25694433

  11. The role of a conserved membrane proximal cysteine in altering αPS2CβPS integrin diffusion

    NASA Astrophysics Data System (ADS)

    Syed, Aleem; Arora, Neha; Bunch, Thomas A.; Smith, Emily A.

    2016-12-01

    Cysteine residues (Cys) in the membrane proximal region are common post-translational modification (PTM) sites in transmembrane proteins. Herein, the effects of a highly conserved membrane proximal α-subunit Cys1368 on the diffusion properties of αPS2CβPS integrins are reported. Sequence alignment shows that this cysteine is palmitoylated in human α3 and α6 integrin subunits. Replacing Cys1368 in wild-type integrins with valine (Val1368) putatively blocks a PTM site and alters integrins’ ligand binding and diffusion characteristics. Both fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) diffusion measurements show Val1368 integrins are more mobile compared to wild-type integrins. Approximately 33% and 8% more Val1368 integrins are mobile as measured by FRAP and SPT, respectively. The mobile Val1368 integrins also exhibit less time-dependent diffusion, as measured by FRAP. Tandem mass spectrometry data suggest that Cys1368 contains a redox or palmitoylation PTM in αPS2CβPS integrins. This membrane proximal Cys may play an important role in the diffusion of other alpha subunits that contain this conserved residue.

  12. Genetic ablation of the alpha 6-integrin subunit in Tie1Cre mice enhances tumour angiogenesis.

    PubMed

    Germain, Mitchel; De Arcangelis, Adèle; Robinson, Stephen D; Baker, Marianne; Tavora, Bernardo; D'Amico, Gabriela; Silva, Rita; Kostourou, Vassiliki; Reynolds, Louise E; Watson, Alan; Jones, J Louise; Georges-Labouesse, Elisabeth; Hodivala-Dilke, Kairbaan

    2010-02-01

    Laminins are expressed highly in blood vessel basement membranes and have been implicated in angiogenesis. alpha6beta1- and alpha6beta4-integrins are major receptors for laminins in endothelial cells, but the precise role of endothelial alpha6-integrin in tumour angiogenesis is not clear. We show that blood vessels in human invasive ductal carcinoma of the breast have decreased expression of the alpha6-integrin-subunit when compared with normal breast tissue. These data suggest that a decrease in alpha6-integrin-subunit expression in endothelial cells is associated with tumour angiogenesis. To test whether the loss of the endothelial alpha6-integrin subunit affects tumour growth and angiogenesis, we generated alpha6fl/fl-Tie1Cre+ mice and showed that endothelial deletion of alpha6-integrin is sufficient to enhance tumour size and tumour angiogenesis in both murine B16F0 melanoma and Lewis cell lung carcinoma. Mechanistically, endothelial alpha6-integrin deficiency elevated significantly VEGF-mediated angiogenesis both in vivo and ex vivo. In particular, alpha6-integrin-deficient endothelial cells displayed increased levels of VEGF-receptor 2 (VEGFR2) and VEGF-mediated downstream ERK1/2 activation. By developing the first endothelial-specific alpha6-knockout mice, we show that the expression of the alpha6-integrin subunit in endothelial cells acts as a negative regulator of angiogenesis both in vivo and ex vivo. Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  13. PET-radioimmunodetection of integrins: imaging acute colitis using a ⁶⁴Cu-labeled anti-β₇ integrin antibody.

    PubMed

    Dearling, Jason L J; Packard, Alan B

    2012-01-01

    Integrins are involved in a wide range of cell interactions. Imaging their distribution using high-resolution noninvasive techniques that are directly translatable to the clinic can provide new insights into disease processes and presents the opportunity to directly monitor new therapies. In this chapter, we describe a protocol to image, the in vivo distribution of the integrin β(7), expressed by lymphocytes recruited to and retained by the inflamed gut, using a radiolabeled whole antibody. The antibody is purified, conjugated with a bifunctional chelator for labeling with a radiometal, labeled with the positron-emitting radionuclide (64)Cu, and injected into mice for microPET studies. Mice with DSS-induced colitis were found to have higher uptake of the (64)Cu-labeled antibody in the gut than control groups.

  14. Leukocyte integrin activation mediates transient neutropenia after G-CSF administration

    PubMed Central

    Tuschong, Laura; Bauer, Thomas R.; Yau, Yu Ying; Leitman, Susan F.; Hickstein, Dennis D.

    2011-01-01

    After administration of granulocyte colony-stimulating factor (G-CSF), there is a marked, albeit transient, drop in circulating neutrophils. To determine the role of leukocyte integrins in this disappearance, a dog having canine leukocyte adhesion deficiency (CLAD) or CLAD dogs who had undergone gene correction either by matched littermate allogeneic transplant or autologous gene therapy were evaluated. Shortly after G-CSF administration, a dramatic, yet transient, neutropenia was observed in the control littermates. This neutropenia was not as marked in the CLAD dogs. In all instances, it was CD18+ neutrophils that preferentially egressed from the circulation. The association of CD18 with this rapid loss suggested leukocyte integrin activation after G-CSF administration. To determine the activation status of the integrin, a monoclonal antibody recognizing the activated α-subunit cation binding domain (mAb24) was used to evaluate human leukocytes after G-CSF administration. Mirroring the dramatic decrease in circulating neutrophil numbers, there was a dramatic and specific increase in the activation of the α-subunit after G-CSF expression on polymorphonuclear leukocytes. This activation, like the drop in neutrophil count, was transient. These results demonstrate that the leukocyte integrin on circulating neutrophils is transiently activated after G-CSF administration and mediates the transient neutropenia observed after G-CSF administration. PMID:21844566

  15. Absence of αvβ6 Integrin Is Linked to Initiation and Progression of Periodontal Disease

    PubMed Central

    Ghannad, Farzin; Nica, Daniela; Garcia Fulle, Maria I.; Grenier, Daniel; Putnins, Edward E.; Johnston, Sarah; Eslami, Ameneh; Koivisto, Leeni; Jiang, Guoqiao; McKee, Marc D.; Häkkinen, Lari; Larjava, Hannu

    2008-01-01

    Integrin αvβ6 is generally not expressed in adult epithelia but is induced in wound healing, cancer, and certain fibrotic disorders. Despite this generalized absence, we observed that αvβ6 integrin is constitutively expressed in the healthy junctional epithelium linking the gingiva to tooth enamel. Moreover, expression of αvβ6 integrin was down-regulated in human periodontal disease, a common medical condition causing tooth loss and also contributing to the development of cardiovascular diseases by increasing the total systemic inflammatory burden. Remarkably, integrin β6 knockout mice developed classic signs of spontaneous, chronic periodontal disease with characteristic inflammation, epithelial down-growth, pocket formation, and bone loss around the teeth. Integrin αvβ6 acts as a major activator of transforming growth factor-β1 (TGF-β1), a key anti-inflammatory regulator in the immune system. Co-expression of TGF-β1 and αvβ6 integrin was observed in the healthy junctional epithelium. Moreover, an antibody that blocks αvβ6 integrin-mediated activation of TGF-β1 initiated inflammatory periodontal disease in a rat model of gingival inflammation. Thus, αvβ6 integrin is constitutively expressed in the epithelium sealing the gingiva to the tooth and plays a central role in protection against inflammatory periodontal disease through activation of TGF-β1. PMID:18385522

  16. Monitoring of integrin-mediated adhesion of human ovarian cancer cells to model protein surfaces by quartz crystal resonators: evaluation in the impedance analysis mode.

    PubMed

    Li, Jing; Thielemann, Christiane; Reuning, Ute; Johannsmann, Diethelm

    2005-01-15

    The quartz crystal microbalance (QCM) was used to monitor specific, integrin-mediated adhesion of human ovarian cancer cells to distinct extracellular matrix (ECM) proteins immobilized on gold-coated quartz crystal resonators. The QCM was operated in the impedance analysis mode, where frequency shift as well as bandwidth are accessible on a broad range of overtones. The increase in bandwidth caused by covering the quartz resonator with cells was reproducible and largely independent of overtone order, whereas the frequency shift displayed some variability. Thus the bandwidth proved to be the more robust parameter for sensing cell adhesive events. The bandwidth increased in proportion to the number of seeded cells to the quartz crystal as long as the number was below 150,000 cells/ml. Comparing the resonance parameters on different harmonics, one finds that viscoelastic modeling with homogeneous layer systems cannot reproduce the results: lateral heterogeneity has to be taken into account. The differences in adhesive strength of human ovarian cancer cells towards selected ECM proteins monitored by QCM was in good agreement with data obtained by conventional cell adhesion assays. Strong cell adhesion was observed to the ECM proteins vitronectin (VN) and fibronectin (FN), while only weak attachment occurred on laminin. In order to prove specific, integrin alphavbeta3-mediated cell adhesion to its ligands FN and VN, the cyclic integrin alphavbeta3-directed peptide c(RGDfV) was used as competitor and significantly reversed cell adhesion. Since integrin interaction with ECM proteins is dependent on the presence of bivalent cations, cell detachment was also seen after treatment of cell monolayers with the chelator ethylene-dinitro-tetra-acetic acid (EDTA). The QCM technique is a reliable method to monitor cell adsorption to ECM-pretreated surfaces in real time. It may be an alternative tool for screening specific and selective antagonists of integrin/ECM interaction.

  17. Identification of integrin heterodimers functioning on the surface of undifferentiated porcine primed embryonic stem cells.

    PubMed

    Kim, Hwa-Young; Baek, Song; Han, Na Rae; Lee, Eunsong; Park, Choon-Keun; Lee, Seung Tae

    2018-05-29

    In vitro expansion of undifferentiated porcine primed embryonic stem (ES) cells is facilitated by use of non-cellular niches that mimic three-dimensional (3D) microenvironments enclosing an inner cell mass of porcine blastocysts. Therefore, we investigated the integrin heterodimers on the surface of undifferentiated porcine primed ES cells for the purpose of developing a non-cellular niche to support in vitro maintenance of the self-renewal ability of porcine primed ES cells. Immunocytochemistry and a fluorescence immunoassay were performed to assess integrin α and β subunit levels, and attachment and antibody inhibition assays were used to evaluate the function of integrin heterodimers. The integrin α 3 , α 5 , α 6 , α 9 , α V , and β 1 subunits, but not the α 1 , α 2 , α 4 , α 7 , and α 8 subunits, were identified on the surface of undifferentiated porcine primed ES cells. Subsequently, significant increase of their adhesion to fibronectin, tenascin C and vitronectin were observed and functional blocking of integrin heterodimer α 5 β 1 , α 9 β 1 , or α V β 1 showed significantly inhibited adhesion to fibronectin, tenascin C, or vitronectin. No integrin α 6 β 1 heterodimer?mediated adhesion to laminin was detected. These results demonstrate that active α 5 β 1 , α 9 β 1 , and α V β 1 integrin heterodimers are present on the surface of undifferentiated porcine primed ES cells, together with inactive integrin α 3 (presumed) and α 6 subunits. This article is protected by copyright. All rights reserved.

  18. Suppression of Eosinophil Integrins Prevents Remodeling of Airway Smooth Muscle in Asthma

    PubMed Central

    Januskevicius, Andrius; Gosens, Reinoud; Sakalauskas, Raimundas; Vaitkiene, Simona; Janulaityte, Ieva; Halayko, Andrew J.; Hoppenot, Deimante; Malakauskas, Kestutis

    2017-01-01

    Background: Airway smooth muscle (ASM) remodeling is an important component of the structural changes to airways seen in asthma. Eosinophils are the prominent inflammatory cells in asthma, and there is some evidence that they contribute to ASM remodeling via released mediators and direct contact through integrin–ligand interactions. Eosinophils express several types of outer membrane integrin, which are responsible for cell–cell and cell–extracellular matrix interactions. In our previous study we demonstrated that asthmatic eosinophils show increased adhesion to ASM cells and it may be important factor contributing to ASM remodeling in asthma. According to these findings, in the present study we investigated the effects of suppression of eosinophil integrin on eosinophil-induced ASM remodeling in asthma. Materials and Methods: Individual combined cell cultures of immortalized human ASM cells and eosinophils from peripheral blood of 22 asthmatic patients and 17 healthy controls were prepared. Eosinophil adhesion was evaluated using eosinophil peroxidase activity assay. Genes expression levels in ASM cells and eosinophils were measured using quantitative real-time PCR. ASM cell proliferation was measured using alamarBlue® solution. Eosinophil integrins were blocked by incubating with Arg-Gly-Asp-Ser peptide. Results: Eosinophils from the asthma group showed increased outer membrane α4β1 and αMβ2 integrin expression, increased adhesion to ASM cells, and overexpression of TGF-β1 compared with eosinophils from the healthy control group. Blockade of eosinophil RGD-binding integrins by Arg-Gly-Asp-Ser peptide significantly reduced adhesion of eosinophils to ASM cells in both groups. Integrin-blocking decreased the effects of eosinophils on TGF-β1, WNT-5a, and extracellular matrix protein gene expression in ASM cells and ASM cell proliferation in both groups. These effects were more pronounced in the asthma group compared with the control group. Conclusion

  19. Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.

    PubMed

    Vespa, Alisa; Darmon, Alison J; Turner, Christopher E; D'Souza, Sudhir J A; Dagnino, Lina

    2003-03-28

    Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.

  20. Increasing α7β1-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression

    PubMed Central

    Liu, Jianming; Burkin, Dean J.; Kaufman, Stephen J.

    2008-01-01

    The dystrophin-glycoprotein complex maintains the integrity of skeletal muscle by associating laminin in the extracellular matrix with the actin cytoskeleton. Several human muscular dystrophies arise from defects in the components of this complex. The α7β1-integrin also binds laminin and links the extracellular matrix with the cytoskeleton. Enhancement of α7-integrin levels alleviates pathology in mdx/utrn−/− mice, a model of Duchenne muscular dystrophy, and thus the integrin may functionally compensate for the absence of dystrophin. To test whether increasing α7-integrin levels affects transcription and cellular functions, we generated α7-integrin-inducible C2C12 cells and transgenic mice that overexpress the integrin in skeletal muscle. C2C12 myoblasts with elevated levels of integrin exhibited increased adhesion to laminin, faster proliferation when serum was limited, resistance to staurosporine-induced apoptosis, and normal differentiation. Transgenic expression of eightfold more integrin in skeletal muscle did not result in notable toxic effects in vivo. Moreover, high levels of α7-integrin in both myoblasts and in skeletal muscle did not disrupt global gene expression profiles. Thus increasing integrin levels can compensate for defects in the extracellular matrix and cytoskeleton linkage caused by compromises in the dystrophin-glycoprotein complex without triggering apparent overt negative side effects. These results support the use of integrin enhancement as a therapy for muscular dystrophy. PMID:18045857

  1. 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.

  2. 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.

  3. Dual mechanism of integrin αIIbβ3 closure in procoagulant platelets.

    PubMed

    Mattheij, Nadine J A; Gilio, Karen; van Kruchten, Roger; Jobe, Shawn M; Wieschhaus, Adam J; Chishti, Athar H; Collins, Peter; Heemskerk, Johan W M; Cosemans, Judith M E M

    2013-05-10

    Inactivation of integrin αIIbβ3 reverses platelet aggregate formation upon coagulation. Platelets from patient (Scott) and mouse (Capn1(-/-) and Ppif(-/-)) blood reveal a dual mechanism of αIIbβ3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling. These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets. Aggregation of platelets via activated integrin αIIbβ3 is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that αIIbβ3 inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the β3 chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed αIIbβ3 inactivation. Integrin αIIbβ3 inactivation was unchanged in platelets from Capn1(-/-) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced αIIbβ3 inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(-/-) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and αIIbβ3 inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of αIIbβ3 inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation.

  4. Colorectal Cancer Metastases Settle in the Hepatic Microenvironment Through α5β1 Integrin.

    PubMed

    Pelillo, Chiara; Bergamo, Alberta; Mollica, Hilaria; Bestagno, Marco; Sava, Gianni

    2015-10-01

    Colorectal cancer (CRC) metastasis dissemination to secondary sites represents the critical point for the patient's survival. The microenvironment is crucial to cancer progression, influencing tumour cell behaviour by modulating the expression and activation of molecules such as integrins, the cell-extracellular matrix interacting proteins participating in different steps of the tumour metastatic process. In this work, we investigated the role of α5β1 integrin and how the microenvironment influences this adhesion molecule, in a model of colon cancer progression to the liver. The culture medium conditioned by the IHH hepatic cell line, and the extracellular matrix (ECM) proteins, modulate the activation of α5β1 integrin in the colon cancer cell line HCT-116, and drives FAK phosphorylation during the process of cell adhesion to fibronectin, one of the main components of liver ECM. In these conditions, α5β1 modulates the expression/activity of another integrin, α2β1, involved in the cell adhesion to collagen I. These results suggest that α5β1 integrin holds a leading role in HCT-116 colorectal cancer cells adhesion to the ECM through the modulation of the intracellular focal adhesion kinase FAK and the α2β1 integrin activity. The driving role of the tumour microenvironment on CRC dissemination, here detected, and described, strengthens and adds new value to the concept that α5β1 integrin can be an appropriate and relevant therapeutic target for the control of CRC metastases. © 2015 Wiley Periodicals, Inc.

  5. The integrin-binding motif RGDS induces protein tyrosine phosphorylation without activation in Bufo arenarum (Amphibia) oocytes.

    PubMed

    Mouguelar, Valeria S; Cabada, Marcelo O; Coux, Gabriela

    2011-05-01

    Integrins are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction. Nevertheless, their function in mammalian fertilization is still controversial, as different species behave differently. In amphibians, their role is mainly supported by Xenopus laevis studies, where RGDS peptide induces oocyte activation. We recently provided evidence suggesting the presence and involvement of integrins in the interaction of the oocyte plasma membrane (PM) with sperm in the amphibian Bufo arenarum. In order to understand the role of integrin homologs in oocytes and their possible contribution to egg activation mechanisms, we examined the presence of integrin subunits and the effect of RGDS peptide on oocytes and during fertilization. Western blot studies detected integrin subunits α5, αV and β1 in oocytes. In sperm, we could detect only the αV integrin subunit. We found that RGDS peptide was unable to elicit egg activation or MAPK dephosphorylation, but can induce reversible inhibition of fertilization. A similar partial inhibition was produced by an anti-β1 integrin antibody. Using an anti-phosphotyrosine antibody we found major changes in phosphotyrosine-containing proteins in egg extracts minutes after fertilization. Cytosol and PMs isolated from oocytes and fertilized eggs showed additional fertilization-induced phosphorylated proteins. Some of these were also present in cytosol and PMs from RGDS-treated oocytes (partially mimicking fertilization). These findings suggest that B. arenarum fertilization involves integrins (e.g. β1 subunit) as adhesion proteins. Our data support the view that RGDS-binding receptors may function as signaling receptors in B. arenarum oocytes, but integrin engagement by RGDS is not sufficient for oocyte activation.

  6. Peripheral myelin protein-22 (PMP22) modulates alpha 6 integrin expression in the human endometrium.

    PubMed

    Rao, Rajiv G; Sudhakar, Deepthi; Hogue, Claire P; Amici, Stephanie; Gordon, Lynn K; Braun, Jonathan; Notterpek, Lucia; Goodglick, Lee; Wadehra, Madhuri

    2011-04-25

    PMP22, a member of the GAS3 family of tetraspan proteins, is associated with a variety of neurological diseases. Previous studies have shown that PMP22 is expressed in proliferative endometrium, but its function within this tissue is poorly understood. In this study, we first characterized the expression of PMP22 in the human menstrual cycle and began to characterize its function in the endometrium. Using a combination of immunohistochemistry and quantitative PCR, we characterized the expression of PMP22 in both proliferative and secretory endometrium. Differences in PMP22 expression between proliferative and secretory endometrium were determined using a Mann-Whitney U test. In order to investigate the influence of PMP22 on α6 integrin expression, cells were created that ectopically overexpressed PMP22 or expressed a siRNA to inhibit its expression. These cells were analyzed for changes in integrins and binding to extracellular matrices. In this study, we show that PMP22 expression is higher in proliferative phase than secretory phase. Functionally, we have begun to characterize the functional significance of this expression. Previous studies have suggested a link between PMP22 and α6 integrin, and therefore we asked whether PMP22 could associate or potentially modulate the expression of α6 integrin. Expression of both PMP22 and α6 integrin were detectable in endometrial epithelial and stromal cells, and we show that both proteins can associate and colocalize with each other. To understand if PMP22 directly altered the expression of a6 integrin, we examined cell lines with modulated levels of the protein. Overexpression of PMP22 was sufficient to increase α6 integrin surface expression with a concominant increase in binding to the extracellular matrix laminin, while a reduction in PMP22 suppressed α6 integrin surface expression. These findings suggest a physiologic role for PMP22 on the expression of α6 integrin. We predict that this may be important for the

  7. β1 integrin is a crucial regulator of pancreatic β-cell expansion

    PubMed Central

    Diaferia, Giuseppe R.; Jimenez-Caliani, Antonio J.; Ranjitkar, Prerana; Yang, Wendy; Hardiman, Gary; Rhodes, Christopher J.; Crisa, Laura; Cirulli, Vincenzo

    2013-01-01

    Development of the endocrine compartment of the pancreas, as represented by the islets of Langerhans, occurs through a series of highly regulated events encompassing branching of the pancreatic epithelium, delamination and differentiation of islet progenitors from ductal domains, followed by expansion and three-dimensional organization into islet clusters. Cellular interactions with the extracellular matrix (ECM) mediated by receptors of the integrin family are postulated to regulate key functions in these processes. Yet, specific events regulated by these receptors in the developing pancreas remain unknown. Here, we show that ablation of the β1 integrin gene in developing pancreatic β-cells reduces their ability to expand during embryonic life, during the first week of postnatal life, and thereafter. Mice lacking β1 integrin in insulin-producing cells exhibit a dramatic reduction of the number of β-cells to only ∼18% of wild-type levels. Despite the significant reduction in β-cell mass, these mutant mice are not diabetic. A thorough phenotypic analysis of β-cells lacking β1 integrin revealed a normal expression repertoire of β-cell markers, normal architectural organization within islet clusters, and a normal ultrastructure. Global gene expression analysis revealed that ablation of this ECM receptor in β-cells inhibits the expression of genes regulating cell cycle progression. Collectively, our results demonstrate that β1 integrin receptors function as crucial positive regulators of β-cell expansion. PMID:23863477

  8. Cigarette smoke induces β2-integrin-dependent neutrophil migration across human endothelium

    PubMed Central

    2011-01-01

    Background Cigarette smoking induces peripheral inflammatory responses in all smokers and is the major risk factor for neutrophilic lung disease such as chronic obstructive pulmonary disease. The aim of this study was to investigate the effect of cigarette smoke on neutrophil migration and on β2-integrin activation and function in neutrophilic transmigration through endothelium. Methods and results Utilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and β2-integrin activation and function in neutrophilic transmigration was studied. In this report, we demonstrated that cigarette smoke extract (CSE) dose dependently induced migration of neutrophils in vitro. Moreover, CSE promoted neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that Mac-1 (CD11b/CD18) is responsible for the cigarette smoke-induced firm adhesion of neutrophils to fibrinogen. Furthermore, neutrophils transmigrated through endothelium by cigarette smoke due to the activation of β2-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration. Conclusion This is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of β2-integrins on the cell surface. Blocking this activation of β2-integrins might be an important target in cigarette smoke induced neutrophilic diseases. PMID:21651795

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

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

    PubMed Central

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

    2017-01-01

    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 arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y2R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αIIbβ3 integrins and (RGD)P2Y2R 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. PMID:28835374

  11. Upregulated Expression of Integrin α1 in Mesangial Cells and Integrin α3 and Vimentin in Podocytes of Col4a3-Null (Alport) Mice

    PubMed Central

    Steenhard, Brooke M.; Vanacore, Roberto; Friedman, David; Zelenchuk, Adrian; Stroganova, Larysa; Isom, Kathryn; St. John, Patricia L.; Hudson, Billy G.; Abrahamson, Dale R.

    2012-01-01

    Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of

  12. 9-cis-Retinoic Acid Promotes Cell Adhesion Through Integrin Dependent and Independent Mechanisms Across Immune Lineages

    PubMed Central

    Whelan, Jarrett T.; Chen, Jianming; Miller, Jabin; Morrow, Rebekah L.; Lingo, Joshuah D.; Merrell, Kaitlin; Shaikh, Saame Raza; Bridges, Lance C.

    2012-01-01

    Retinoids are essential in the proper establishment and maintenance of immunity. Although retinoids are implicated in immune related processes, their role in immune cell adhesion has not been well established. In this study, the effect of 9-cis-retinoic acid (9-cis-RA) on human hematopoietic cell adhesion was investigated. 9-cis-RA treatment specifically induced cell adhesion of the human immune cell lines HuT-78, NB4, RPMI 8866, and U937. Due to the prominent role of integrin receptors in mediating immune cell adhesion, we sought to evaluate if cell adhesion was integrin-dependent. By employing a variety of integrin antagonist including function-blocking antibodies and EDTA, we establish that 9-cis-RA prompts immune cell adhesion through established integrin receptors in addition to a novel integrin-independent process. The novel integrin-independent adhesion required the presence of retinoid and was attenuated by treatment with synthetic corticosteroids. Finally, we demonstrate that 9-cis-RA treatment of primary murine B-cells induces ex vivo adhesion that persists in the absence of integrin function. Our study is the first to demonstrate that 9-cis-retinoic acid influences immune cell adhesion through at least two functionally distinct mechanisms. PMID:22925918

  13. Multiple alpha subunits of integrin are involved in cell-mediated responses of the Manduca immune system.

    PubMed

    Zhuang, Shufei; Kelo, Lisha; Nardi, James B; Kanost, Michael R

    2008-01-01

    The cell-mediated responses of the insect innate immune system-phagocytosis, nodulation, encapsulation-involve multiple cell adhesion molecules of hemocyte surfaces. A hemocyte-specific (HS) integrin and a member of the immunoglobulin (Ig) superfamily (neuroglian) are involved in the encapsulation response of hemocytes in Manduca sexta. In addition, two new integrin alpha (alpha) subunits have been found on these hemocytes. The alpha2 subunit is mainly expressed in epidermis and Malphigian tubules, whereas the alpha3 subunit is primarily expressed on hemocytes and fat body cells. Of the three known alpha subunits, the alpha1 subunit found in HS integrin is the predominant subunit of hemocytes. Cell adhesion assays indicate that alpha2 belongs to the integrin family with RGD-binding motifs, confirming the phylogenetic analysis of alpha subunits based on the amino-acid sequence alignment of different alpha subunits. Double-stranded RNAs (dsRNAs) targeting each of these three integrin alpha subunits not only specifically decreased transcript expression of each alpha subunit in hemocytes, but also abolished the cell-mediated encapsulation response of hemocytes to foreign surfaces. The individual alpha subunits of M. sexta integrins, like their integrin counterparts in mammalian immune systems, have critical, individual roles in cell-substrate and cell-cell interactions during immune responses.

  14. 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

  15. Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface

    PubMed Central

    Priglinger, Claudia S.; Szober, Christoph M.; Priglinger, Siegfried G.; Merl, Juliane; Euler, Kerstin N.; Kernt, Marcus; Gondi, Gabor; Behler, Jennifer; Geerlof, Arie; Kampik, Anselm; Ueffing, Marius; Hauck, Stefanie M.

    2013-01-01

    Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and

  16. Sulfur mustard disrupts human α3β1-integrin receptors in concert with α6β4-integrin receptors and collapse of the keratin K5/K14 cytoskeleton

    NASA Astrophysics Data System (ADS)

    Werrlein, Robert J.; Braue, Catherine R.

    2004-06-01

    Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a chemical warfare agent that produces persistent, incapacitating blisters of the skin. The lesions inducing vesication remain elusive, and there is no completely effective treatment. Using mulitphoton microscopy and immunofluorescent staining, we found that exposing human epidermal keratinocytes (HEK) and intact epidermis to SM (400 μm for 5 min) caused progressive collapse of the keratin (K5/K14) cytoskeleton and depletion of α6β integrins. We now report that SM causes concomitant disruption nad collapse of the basal cell's α3β1-integrin receptors. At 1 h postexposure, images of Alexa488-conjugated HEK/α3β1 integrins showed almost complete withdrawal and disappearance of retraction fibers and a progressive loss of polarized mobility. With stero imaging, in vitro expression of this SM effect was characterized by collapse and abutment of adjacent cell membranes. At 2 h postexposure, there was an average 13% dorso-ventral collapse of HEK membranes that paralleled progressive collapse of the K5/K14 cytoskeleton. α3β1 integrin, like α6β4 integrin, is a regulator of cytoskeletal assembly, a receptor for laminin 5 and a mediator of HEK attachment to the basement membrane. Our images indicate that SM disrupts these receptors. We suggest that the progressive disruption destabilizes and potentiates blistering of the epidermal-dermal junction.

  17. Lebein, a Snake Venom Disintegrin, Induces Apoptosis in Human Melanoma Cells

    PubMed Central

    Hammouda, Manel B.; Montenegro, María F.; Sánchez-del-Campo, Luis; Zakraoui, Ons; Aloui, Zohra; Riahi-Chebbi, Ichrak; Karoui, Habib; Rodríguez-López, José Neptuno; Essafi-Benkhadir, Khadija

    2016-01-01

    Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterized by its very invasive and chemoresistant properties. Despite the recent promising news from the field of immunotherapy, there is an urgent need for new therapeutic approaches that are free of resistance mechanisms and side effects. Anti-neoplasic properties have been highlighted for different disintegrins from snake venom including Lebein; however, the exact effect of Lebein on melanoma has not yet been defined. In this study, we showed that Lebein blocks melanoma cell proliferation and induces a more differentiated phenotype with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and microphthalmia-associated transcription factor (MITF) overexpression. Melanoma cells became detached but were less invasive with upregulation of E-cadherin after Lebein exposure. Lebein induced a caspase-independent apoptotic program with apoptosis inducing factor (AIF), BCL-2-associated X protein (BAX) and Bim overexpression together with downregulation of B-cell lymphoma-2 (BCL-2). It generated a distinct response in reactive oxygen species (ROS) generation and p53 levels depending on the p53 cell line status (wild type or mutant). Therefore, we propose Lebein as a new candidate for development of potential therapies for melanoma. PMID:27399772

  18. Integrin β6 serves as an immunohistochemical marker for lymph node metastasis and promotes cell invasiveness in cholangiocarcinoma

    PubMed Central

    Li, Zequn; Biswas, Siddhartha; Liang, Benjia; Zou, Xueqing; Shan, Liqun; Li, Yang; Fang, Ruliang; Niu, Jun

    2016-01-01

    Cholangiocarcinoma is a devastating malignancy that is notoriously difficult to diagnose and is associated with a high mortality. Despite extensive efforts to improve the diagnosis and treatment of this neoplasm, limited progress has been made. Integrin β6 is a subtype of integrin that is expressed exclusively on the surfaces of epithelial cells and is associated with a variety of tumors. In the present study, we investigated the expression and roles of integrin β6 in cholangiocarcinoma. β6 upregulation in cholangiocarcinoma was correlated with lymph node metastasis and distant metastasis. Moreover, integrin β6 was identified as a biomarker for the diagnosis of cholangiocarcinoma and an indicator of lymph node metastasis. Integrin β6 significantly promoted the proliferation, migration and invasion of cholangiocarcinoma cells. Furthermore, integrin β6 increased Rac1-GTPase, resulting in the upregulation of metalloproteinase-9 (MMP9) and F-actin polymerization. Taken together, our results indicate that integrin β6 promotes tumor invasiveness in a Rac1-dependent manner and is a potential biomarker for tumor metastasis. Integrin β6 may help to improve the diagnostic accuracy, and targeting β6 may be a novel strategy for the treatment of cholangiocarcinoma. PMID:27440504

  19. Integrin β6 serves as an immunohistochemical marker for lymph node metastasis and promotes cell invasiveness in cholangiocarcinoma.

    PubMed

    Li, Zequn; Biswas, Siddhartha; Liang, Benjia; Zou, Xueqing; Shan, Liqun; Li, Yang; Fang, Ruliang; Niu, Jun

    2016-07-21

    Cholangiocarcinoma is a devastating malignancy that is notoriously difficult to diagnose and is associated with a high mortality. Despite extensive efforts to improve the diagnosis and treatment of this neoplasm, limited progress has been made. Integrin β6 is a subtype of integrin that is expressed exclusively on the surfaces of epithelial cells and is associated with a variety of tumors. In the present study, we investigated the expression and roles of integrin β6 in cholangiocarcinoma. β6 upregulation in cholangiocarcinoma was correlated with lymph node metastasis and distant metastasis. Moreover, integrin β6 was identified as a biomarker for the diagnosis of cholangiocarcinoma and an indicator of lymph node metastasis. Integrin β6 significantly promoted the proliferation, migration and invasion of cholangiocarcinoma cells. Furthermore, integrin β6 increased Rac1-GTPase, resulting in the upregulation of metalloproteinase-9 (MMP9) and F-actin polymerization. Taken together, our results indicate that integrin β6 promotes tumor invasiveness in a Rac1-dependent manner and is a potential biomarker for tumor metastasis. Integrin β6 may help to improve the diagnostic accuracy, and targeting β6 may be a novel strategy for the treatment of cholangiocarcinoma.

  20. Integrins β1 and β3 are biomarkers of uterine condition for embryo transfer.

    PubMed

    Chen, Guowu; Xin, Aijie; Liu, Yulin; Shi, Changgen; Chen, Junling; Tang, Xiaofeng; Chen, Ying; Yu, Min; Peng, Xiandong; Li, Lu; Sun, Xiaoxi

    2016-10-26

    Clinical ovulation induction induces blood estrogen (E 2 ) in excess of physiological levels, which can hinder uterine receptivity. In contrast, progesterone produces the opposite clinical effect, suggesting that it might be capable of recovering the lost receptivity resulting from exposure to high estrogen levels. Integrins are the most widely used biological markers for monitoring uterine conditions. We studied progesterone-induced changes in integrin β expression patterns as biomarkers for changes in uterine receptivity in response to increased estrogen levels. Endometrial biopsy samples from patients were screened for their estrogen (E 2 ) and progesterone (P4) content and expressing levels of integrin β1 and β3. Uterine receptivity was evaluated using human endometrial adenocarcinoma cells in an embryo attachment model. The respective and concatenated effects of embryo attachment and changes in the integrin β1 and β3 expression patterns on the adenocarcinoma cell plasma membranes in response to 100 nM concentrations of E 2 and P4 were evaluated. Increased blood E 2 concentrations were associated with significantly decreased the levels of integrin β3 expression in uterine biopsy samples. In vitro experiments revealed that a 100 nM E 2 concentration inhibited the distribution of integrin β3 on the plasma membranes of human endometrial adenocarcinoma cells used in the embryo attachment model, and resulted in decreased rates of embryo attachment. In contrast, P4 enhanced the expression of integrin β1 and promoted its distribution on the plasma membranes. Furthermore, P4 recovered the embryo attachment efficiency that was lost by exposure to 100 nM E 2 . Blood E2 and P4 levels and integrin β3 and β1 expression levels in uterine biopsy samples should be considered as biomarkers for evaluating uterine receptivity and determining the optimal time for embryo transfer. Trial registration Trial number: ChiCTR-TRC-13003777; Name of registry: Chinese Clinical

  1. Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

    PubMed

    Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu

    2013-08-01

    Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility.

    PubMed

    Strachan, Lauren R; Condic, Maureen L

    2004-11-08

    Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin alpha subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.

  3. Dominant Suppression of β1 Integrin by Ectopic CD98-ICD Inhibits Hepatocellular Carcinoma Progression

    PubMed Central

    Wu, Bo; Zhou, Yang; Wang, Yu; Yang, Xiang-Min; Liu, Zhen-Yu; Li, Jiang-Hua; Feng, Fei; Chen, Zhi-Nan; Jiang, Jian-Li

    2016-01-01

    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 involves β1-integrin suppression. Moreover, the expression levels of CD98, β1-integrin-A (the activated form of β1-integrin) and Ki-67 were significantly increased in HCC tissues relative to those of normal liver tissues. Therefore, our preliminary study indicates that ectopic CD98-ICD has an inhibitory role in the malignant development of HCC, and shows that CD98-ICD acts as a dominant negative mutant of CD98 that attenuates β1-integrin activation. CD98-ICD may emerge as a promising candidate for antitumor treatment. PMID:27834933

  4. Antibodies causing thrombocytopenia in patients treated with RGD-mimetic platelet inhibitors recognize ligand-specific conformers of αIIb/β3 integrin

    PubMed Central

    Rasmussen, Mark; Zhu, Jieqing; Aster, Richard H.

    2012-01-01

    Arginine-glycine-aspartic acid (RGD)–mimetic platelet inhibitors act by occupying the RGD recognition site of αIIb/β3 integrin (GPIIb/IIIa), thereby preventing the activated integrin from reacting with fibrinogen. Thrombocytopenia is a well-known side effect of treatment with this class of drugs and is caused by Abs, often naturally occurring, that recognize αIIb/β3 in a complex with the drug being administered. RGD peptide and RGD-mimetic drugs are known to induce epitopes (ligand-induced binding sites [LIBS]) in αIIb/β3 that are recognized by certain mAbs. It has been speculated, but not shown experimentally, that Abs from patients who develop thrombocytopenia when treated with an RGD-mimetic inhibitor similarly recognize LIBS determinants. We addressed this question by comparing the reactions of patient Abs and LIBS-specific mAbs against αIIb/β3 in a complex with RGD and RGD-mimetic drugs, and by examining the ability of selected non-LIBS mAbs to block binding of patient Abs to the liganded integrin. Findings made provide evidence that the patient Abs recognize subtle, drug-induced structural changes in the integrin head region that are clustered about the RGD recognition site. The target epitopes differ from classic LIBS determinants, however, both in their location and by virtue of being largely drug-specific. PMID:22490676

  5. Two conformations of the integrin A-domain (I-domain): a pathway for activation?

    PubMed

    Lee, J O; Bankston, L A; Arnaout, M A; Liddington, R C

    1995-12-15

    Integrins are plasma membrane proteins that mediate adhesion to other cells and to components of the extracellular matrix. Most integrins are constitutively inactive in resting cells, but are rapidly and reversibly activated in response to agonists, leading to highly regulated cell adhesion. This activation is associated with conformational changes in their extracellular portions, but the nature of the structural changes that lead to a change in adhesiveness is not understood. The interactions of several integrins with their extracellular ligands are mediated by an A-type domain (generally called the I-domain in integrins). Binding of the I-domain to protein ligands is dependent on divalent cations. We have described previously the structure of the I-domain from complement receptor 3 with bound Mg2+, in which the glutamate side chain from a second I-domain completes the octahedral coordination sphere of the metal, acting as a ligand mimetic. We now describe a new crystal form of the I-domain with bound Mn2+, in which water completes the metal coordination sphere and there is no equivalent of the glutamate ligand. Comparison of the two crystal forms reveals a change in metal coordination which is linked to a large (10 A) shift of the C-terminal helix and the burial of two phenylalanine residues into the hydrophobic core of the Mn2+ form. These structural changes, analogous to those seen in the signal-transducing G-proteins, alter the electrophilicity of the metal, reducing its ability to bind ligand-associated acidic residues, and dramatically alter the surface of the protein implicated in binding ligand. Our observations provide the first atomic resolution view of conformational changes in an integrin domain, and suggest how these changes are linked to a change in integrin adhesiveness. We propose that the Mg2+ form represents the conformation of the domain in the active state and the Mn2+ form the conformation in the inactive state of the integrin.

  6. ß3 integrin modulates transforming growth factor beta induced (TGFBI) function and paclitaxel response in ovarian cancer cells.

    PubMed

    Tumbarello, David A; Temple, Jillian; Brenton, James D

    2012-05-28

    The extracellular matrix (ECM) has a key role in facilitating the progression of ovarian cancer and we have shown recently that the secreted ECM protein TGFBI modulates the response of ovarian cancer to paclitaxel-induced cell death. We have determined TGFBI signaling from the extracellular environment is preferential for the cell surface αvß3 integrin heterodimer, in contrast to periostin, a TGFBI paralogue, which signals primarily via a ß1 integrin-mediated pathway. We demonstrate that suppression of ß1 integrin expression, in ß3 integrin-expressing ovarian cancer cells, increases adhesion to rTGFBI. In addition, Syndecan-1 and -4 expression is dispensable for adhesion to rTGFBI and loss of Syndecan-1 cooperates with the loss of ß1 integrin to further enhance adhesion to rTGFBI. The RGD motif present in the carboxy-terminus of TGFBI is necessary, but not sufficient, for SKOV3 cell adhesion and is dispensable for adhesion of ovarian cancer cells lacking ß3 integrin expression. In contrast to TGFBI, the carboxy-terminus of periostin, lacking a RGD motif, is unable to support adhesion of ovarian cancer cells. Suppression of ß3 integrin in SKOV3 cells increases resistance to paclitaxel-induced cell death while suppression of ß1 integrin has no effect. Furthermore, suppression of TGFBI expression stimulates a paclitaxel resistant phenotype while suppression of fibronectin expression, which primarily signals through a ß1 integrin-mediated pathway, increases paclitaxel sensitivity. Therefore, different ECM components use distinct signaling mechanisms in ovarian cancer cells and in particular, TGFBI preferentially interacts through a ß3 integrin receptor mediated mechanism to regulate the response of cells to paclitaxel-induced cell death.

  7. Differential Influence of Components Resulting from Atmospheric-Pressure Plasma on Integrin Expression of Human HaCaT Keratinocytes

    PubMed Central

    Haertel, Beate; Straßenburg, Susanne; Wende, Kristian; von Woedtke, Thomas

    2013-01-01

    Adequate chronic wound healing is a major problem in medicine. A new solution might be non-thermal atmospheric-pressure plasma effectively inactivating microorganisms and influencing cells in wound healing. Plasma components as, for example, radicals can affect cells differently. HaCaT keratinocytes were treated with Dielectric Barrier Discharge plasma (DBD/air, DBD/argon), ozone or hydrogen peroxide to find the components responsible for changes in integrin expression, intracellular ROS formation or apoptosis induction. Dependent on plasma treatment time reduction of recovered cells was observed with no increase of apoptotic cells, but breakdown of mitochondrial membrane potential. DBD/air plasma increased integrins and intracellular ROS. DBD/argon caused minor changes. About 100 ppm ozone did not influence integrins. Hydrogen peroxide caused similar effects compared to DBD/air plasma. In conclusion, effects depended on working gas and exposure time to plasma. Short treatment cycles did neither change integrins nor induce apoptosis or ROS. Longer treatments changed integrins as important for influencing wound healing. Plasma effects on integrins are rather attributed to induction of other ROS than to generation of ozone. Changes of integrins by plasma may provide new solutions of improving wound healing, however, conditions are needed which allow initiating the relevant influence on integrins without being cytotoxic to cells. PMID:23936843

  8. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ozeki, Nobuaki; Kawai, Rie; Yamaguchi, Hideyuki

    2014-04-15

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7{sup +}hSMSC)-derived osteoblast-like (α7{sup +}hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations,more » however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7{sup +}hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7{sup +}hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7{sup +}hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form

  9. The Calcium-Sensing Receptor and Integrins in Cellular Differentiation and Migration

    PubMed Central

    Tharmalingam, Sujeenthar; Hampson, David R.

    2016-01-01

    The calcium-sensing receptor (CaSR) is a widely expressed homodimeric G-protein coupled receptor structurally related to the metabotropic glutamate receptors and GPRC6A. In addition to its well characterized role in maintaining calcium homeostasis and regulating parathyroid hormone release, evidence has accumulated linking the CaSR with cellular differentiation and migration, brain development, stem cell engraftment, wound healing, and tumor growth and metastasis. Elevated expression of the CaSR in aggressive metastatic tumors has been suggested as a potential novel prognostic marker for predicting metastasis, especially to bone tissue where extracellular calcium concentrations may be sufficiently high to activate the receptor. Recent evidence supports a model whereby CaSR-mediated activation of integrins promotes cellular migration. Integrins are single transmembrane spanning heterodimeric adhesion receptors that mediate cell migration by binding to extracellular matrix proteins. The CaSR has been shown to form signaling complexes with the integrins to facilitate both the movement and differentiation of cells, such as neurons during normal brain development and tumor cells under pathological circumstances. Thus, CaSR/integrin complexes may function as a universal cell migration or homing complex. Manipulation of this complex may be of potential interest for treating metastatic cancers, and for developmental disorders pertaining to aberrant neuronal migration. PMID:27303307

  10. The role of integrin α8β1 in fetal lung morphogenesis and injury

    PubMed Central

    Benjamin, John T.; Gaston, David C.; Halloran, Brian A.; Schnapp, Lynn M.; Zent, Roy; Prince, Lawrence S.

    2009-01-01

    Prenatal inflammation prevents normal lung morphogenesis and leads to bronchopulmonary dysplasia (BPD), a common complication of preterm birth. We previously demonstrated in a bacterial endotoxin mouse model of BPD that disrupting fibronectin localization in the fetal lung mesenchyme causes arrested saccular airway branching. In this study we show that expression of the fibronectin receptor, integrin α8β1, is decreased in the lung mesenchyme in the same inflammation model suggesting it is required for normal lung development. We verified a role for integrin α8β1 in lung development using integrin α8-null mice, which develop fusion of the medial and caudal lobes as well as abnormalities in airway division. We further show in vivo and vitro that α8-null fetal lung mesenchymal cells fail to form stable adhesions and have increased migration. Thus we propose that integrin α8β1 plays a critical role in lung morphogenesis by regulating mesenchymal cell adhesion and migration. Furthermore, our data suggests that disruption of the interactions between extracellular matrix and integrin α8β1 may contribute to the pathogenesis of BPD. PMID:19769957

  11. Protein expression and purification of integrin I domains and IgSF ligands for crystallography.

    PubMed

    Zhang, Hongmin; Wang, Jia-Huai

    2012-01-01

    Cell adhesion depends on combinational expression and interactions of a large number of adhesion molecules at cell-to-cell or cell-to-matrix contact sites. Integrins and their immunoglobulin superfamily (IgSF) ligands represent foremost classes of cell adhesion molecules in immune system. Structural study is critical for a better understanding of the interactions between integrins and their IgSF ligands. Here we describe protocols for protein expression of integrin αL I domain and its IgSF ligand ICAM-5 D1D2 fragment for crystallography.

  12. The Effect of Conditional Inactivation of Beta 1 Integrins using Twist 2 Cre, Osterix Cre and Osteocalcin Cre Lines on Skeletal Phenotype

    PubMed Central

    Shekaran, Asha; Shoemaker, James T.; Kavanaugh, Taylor E.; Lin, Angela S.; LaPlaca, Michelle C.; Fan, Yuhong; Guldberg, Robert E.; García, Andrés J.

    2014-01-01

    Skeletal development and growth are complex processes regulated by multiple microenvironmental cues, including integrin-ECM interactions. The β1 sub-family of integrins is the largest integrin sub-family and constitutes the main integrin binding partners of collagen I, the major ECM component of bone. As complete β1 integrin integrin knockout results in embryonic lethality, studies of β1 integrin function in vivo rely on tissue-specific gene deletions. While multiple in vitro studies indicate that β1 integrins are crucial regulators of osteogenesis and mineralization, in vivo osteoblast-specific perturbations of β1 integrins have resulted in mild and sometimes contradictory skeletal phenotypes. To further investigate the role of β1 integrins on skeletal phenotype, we used the Twist2-Cre, Osterix-Cre and Osteocalcin-Cre lines to generate conditional β1 integrin deletions, where cre is expressed primarily in mesenchymal condensation, pre-osteoblast, and mature osteoblast lineage cells respectively within these lines. Mice with Twist2-specific β1 integrin disruption were smaller, had impaired skeletal development, especially in the craniofacial and vertebral tissues at E19.5, and did not survive beyond birth. Osterix-specific β1 integrin deficiency resulted in viable mice which were normal at birth but displayed early defects in calvarial ossification, incisor eruption and growth as well as femoral bone mineral density, structure, and mechanical properties. Although these defects persisted into adulthood, they became milder with age. Finally, a lack of β1 integrins in mature osteoblasts and osteocytes resulted in minor alterations to femur structure but had no effect on mineral density, biomechanics or fracture healing. Taken together, our data indicate that β1 integrin expression in early mesenchymal condensations play an important role in skeletal ossification, while β1 integrin-ECM interactions in pre-osteoblast, odontoblast- and hypertrophic

  13. microPET Imaging of Glioma Integrin (alpha-v, beta-3) Expression Using Cu-64-Labeled Tetrameric RGD Peptide

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wu, Yun; Zhang, , Xianzhong; Xiong, , Zhengming

    2005-10-01

    Integrins ?v?3 and ?v?5 play a critical role in tumor-induced angiogenesis and metastasis, and have become promising diagnostic indicators and therapeutic targets of tumors. Radiolabeled RGD peptides that are integrin-specific may be used for non-invasive imaging of integrin expression level as well as for integrin-targeted radionuclide therapy. We previously conjugated a series of mono- and dimeric RGD peptides with 1,4,7,10-tetraazacyclododecane-N, N?,N??,N???-tetraacetic acid (DOTA) and labeled these with copper-64 for microPET imaging in various mouse xenograft models. The copper-64 tracers showed ?v?3-selective tumor uptake, but the magnitude of tumor uptake was relatively low, the tumor washout was rapid, and non-target organ/tissuemore » retention was high. In this study we developed a tetrameric RGD peptide tracer 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 for positron emission tomography (PET) imaging of integrin ?v?3 expression in a subcutaneous U87MG glioma xenograft model in female athymic nude mice. The RGD tetramer showed significantly higher integrin binding affinity than the corresponding mono- and dimeric RGD analogs, most likely due to polyvalency effect. The radiolabeled peptide showed rapid blood clearance (0.61 ? 0.01%ID/g at 30 min and 0.21 ? 0.01 %ID/g at 4 h postinjection (p.i.), respectively) and predominantly renal excretion. Tumor uptake was rapid and high and the tumor washout was slow (9.93 ? 1.05 %ID/g at 30 min p.i. and 4.56 ? 0.51 %ID/g at 24 h post-injection). The metabolic stability of 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 was determined in mouse blood, urine, and liver and kidney homogenates at different times after tracer injection. The average fractions of intact tracer in these organs at 1 h were approximately 70, 58, 51 and 26 percent, respectively. Non-invasive microPET imaging studies showed significant tumor uptake and good contrast in the subcutaneous tumor-bearing mice, which agreed well with the biodistribution

  14. αMβ₂ integrin activation prevents alternative activation of human and murine macrophages and impedes foam cell formation.

    PubMed

    Yakubenko, Valentin P; Bhattacharjee, Ashish; Pluskota, Elzbieta; Cathcart, Martha K

    2011-03-04

    The alternative activation of monocytes by interleukin (IL)-13 and IL-4 is a significant component of the inflammatory response. The consequences of alternative activation in inflammatory diseases remain to be determined. In this report, we explored how integrins, receptors important for monocyte migration to inflammatory sites, regulate IL-13-mediated monocyte activation. We focused on the analysis of 2 proteins, which are upregulated during the alternative activation and are important for the development of atherosclerosis, an oxidative enzyme 15-lipoxygenase (15-LO) and a scavenger receptor CD36. We found that adhesion of resting monocytes through β(2) integrins and inside-out activation of β(2) integrins by monocyte chemoattractant protein-1 did not change IL-13-stimulated 15-LO upregulation; however, preincubation of monocytes with the antibody MEM48, which generates full activation of β(2) integrins, significantly inhibited 15-LO mRNA and protein expression. In contrast, activation of β(1) integrins had no effect on 15-LO expression. Analysis of integrin clustering through α(M), α(L), α(X), and α(D) subunits demonstrated the pivotal role for integrin α(M)β(2) in inhibiting 15-LO expression. IL-13 treatment upregulates 15-LO-dependent CD36 expression on human monocytes; our studies showed that β(2) integrin activation and α(M) integrin clustering significantly inhibited IL-13-dependent CD36 mRNA and protein expression, as well as CD36-related foam cell formation. Moreover, IL-13 stimulation of α(M)-deficient peritoneal macrophages demonstrated an upregulated level of 15-LO induction, CD36 expression, and lipid accumulation as compared with wild-type controls. The adhesion of monocytes/macrophages through activated integrin α(M)β(2) has a regulatory and potential atheroprotective function during the alternative activation of macrophages.

  15. αMβ2 Integrin Activation Prevents Alternative Activation of Human and Murine Macrophages and Impedes Foam Cell Formation

    PubMed Central

    Yakubenko, Valentin P.; Bhattacharjee, Ashish; Pluskota, Elzbieta; Cathcart, Martha K.

    2011-01-01

    Rationale The alternative activation of monocytes by IL-13 and IL-4 is a significant component of the inflammatory response. The consequences of alternative activation in inflammatory diseases remain to be determined. Objective In this paper we explored how integrins, receptors important for monocyte migration to inflammatory sites, regulate IL-13-mediated monocyte activation. We focused on the analysis of two proteins, which are upregulated during the alternative activation and are important for the development of atherosclerosis - an oxidative enzyme 15-lipoxygenase (15-LO) and a scavenger receptor CD36. Methods and Results We found that adhesion of resting monocytes through β2 integrins and inside-out activation of β2 integrins by MCP-1 did not change IL-13-stimulated 15-LO upregulation; however, preincubation of monocytes with the antibody MEM48, which generates full activation of β2 integrins, significantly inhibited 15-LO mRNA and protein expression. In contrast, activation of β1 integrins had no effect on 15-LO expression. Analysis of integrin clustering through αM, αL, αX and αD subunits demonstrated the pivotal role for integrin αMβ2 in inhibiting 15-LO expression. IL-13 treatment upregulates 15-LO-dependent CD36 expression on human monocytes, our studies showed that β2 integrin activation and αM integrin clustering significantly inhibited IL-13-dependent CD36 mRNA and protein expression as well as CD36-related foam cell formation. Moreover, IL-13 stimulation of αM-deficient peritoneal macrophages demonstrated an upregulated level of 15-LO induction, CD36 expression and lipid accumulation as compared to wild type controls. Conclusions The adhesion of monocytes/macrophages through activated integrin αMβ2 has a regulatory and potential athero-protective function during the alternative activation of macrophages. PMID:21252155

  16. Gfi1b controls integrin signaling-dependent cytoskeleton dynamics and organization in megakaryocytes.

    PubMed

    Beauchemin, Hugues; Shooshtarizadeh, Peiman; Vadnais, Charles; Vassen, Lothar; Pastore, Yves D; Möröy, Tarik

    2017-03-01

    Mutations in GFI1B are associated with inherited bleeding disorders called GFI1B -related thrombocytopenias. We show here that mice with a megakaryocyte-specific Gfi1b deletion exhibit a macrothrombocytopenic phenotype along a megakaryocytic dysplasia reminiscent of GFI1B -related thrombocytopenia. GFI1B deficiency increases megakaryocyte proliferation and affects their ploidy, but also abrogates their responsiveness towards integrin signaling and their ability to spread and reorganize their cytoskeleton. Gfi1b -null megakaryocytes are also unable to form proplatelets, a process independent of integrin signaling. GFI1B-deficient megakaryocytes exhibit aberrant expression of several components of both the actin and microtubule cytoskeleton, with a dramatic reduction of α-tubulin. Inhibition of FAK or ROCK, both important for actin cytoskeleton organization and integrin signaling, only partially restored their response to integrin ligands, but the inhibition of PAK, a regulator of the actin cytoskeleton, completely rescued the responsiveness of Gfi1b -null megakaryocytes to ligands, but not their ability to form proplatelets. We conclude that Gfi1b controls major functions of megakaryocytes such as integrin-dependent cytoskeleton organization, spreading and migration through the regulation of PAK activity whereas the proplatelet formation defect in GFI1B-deficient megakaryocytes is due, at least partially, to an insufficient α-tubulin content. Copyright© Ferrata Storti Foundation.

  17. Inhibition of Focal Adhesion Kinase Signaling by Integrin α6β1 Supports Human Pluripotent Stem Cell Self-Renewal.

    PubMed

    Villa-Diaz, Luis G; Kim, Jin Koo; Laperle, Alex; Palecek, Sean P; Krebsbach, Paul H

    2016-07-01

    Self-renewal of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs)-known as pluripotent stem cells (PSC)-is influenced by culture conditions, including the substrate on which they are grown. However, details of the molecular mechanisms interconnecting the substrate and self-renewal of these cells remain unclear. We describe a signaling pathway in hPSCs linking self-renewal and expression of pluripotency transcription factors to integrin α6β1 and inactivation of focal adhesion kinase (FAK). Disruption of this pathway results in hPSC differentiation. In hPSCs, α6β1 is the dominant integrin and FAK is not phosphorylated at Y397, and thus, it is inactive. During differentiation, integrin α6 levels diminish and Y397 FAK is phosphorylated and activated. During reprogramming of fibroblasts into iPSCs, integrin α6 is upregulated and FAK is inactivated. Knockdown of integrin α6 and activation of β1 integrin lead to FAK phosphorylation and reduction of Nanog, Oct4, and Sox2, suggesting that integrin α6 functions in inactivation of integrin β1 and FAK signaling and prevention of hPSC differentiation. The N-terminal domain of FAK, where Y397 is localized, is in the nuclei of hPSCs interacting with Oct4 and Sox2, and this immunolocalization is regulated by Oct4. hPSCs remodel the extracellular microenvironment and deposit laminin α5, the primary ligand of integrin α6β1. Knockdown of laminin α5 resulted in reduction of integrin α6 expression, phosphorylation of FAK and decreased Oct4. In conclusion, hPSCs promote the expression of integrin α6β1, and nuclear localization and inactivation of FAK to supports stem cell self-renewal. Stem Cells 2016;34:1753-1764. © 2016 AlphaMed Press.

  18. Role of Integrin Subunits in Mesenchymal Stem Cell Differentiation and Osteoblast Maturation on Graphitic Carbon-coated Microstructured Surfaces

    PubMed Central

    Olivares-Navarrete, Rene; Rodil, Sandra E.; Hyzy, Sharon L.; Dunn, Ginger R.; Almaguer-Flores, Argelia; Schwartz, Zvi; Boyan, Barbara D.

    2015-01-01

    Surface roughness, topography, chemistry, and energy promote osteoblast differentiation and increase osteogenic local factor production in vitro and bone-to-implant contact in vivo, but the mechanisms involved are not well understood. Knockdown of integrin heterodimer alpha2beta1 (α2β1) blocks the osteogenic effects of the surface, suggesting signaling by this integrin homodimer is required. The purpose of the present study was to separate effects of surface chemistry and surface structure on integrin expression by coating smooth or rough titanium (Ti) substrates with graphitic carbon, retaining surface morphology but altering surface chemistry. Ti surfaces (smooth [Ra<0.4μm], rough [Ra≥3.4μm]) were sputter-coated using a magnetron sputtering system with an ultrapure graphite target, producing a graphitic carbon thin film. Human mesenchymal stem cells and MG63 osteoblast-like cells had higher mRNA for integrin subunits α1, α2, αv, and β1 on rough surfaces in comparison to smooth, and integrin αv on graphitic-carbon-coated rough surfaces in comparison to Ti. Osteogenic differentiation was greater on rough surfaces in comparison to smooth, regardless of chemistry. Silencing integrins β1, α1, or α2 decreased osteoblast maturation on rough surfaces independent of surface chemistry. Silencing integrin αv decreased maturation only on graphitic carbon-coated surfaces, not on Ti. These results suggest a major role of the integrin β1 subunit in roughness recognition, and that integrin alpha subunits play a major role in surface chemistry recognition. PMID:25770999

  19. Hypoxia-inducible Factor Regulates αvβ3 Integrin Cell Surface Expression

    PubMed Central

    Cowden Dahl, Karen D.; Robertson, Sarah E.; Weaver, Valerie M.; Simon, M. Celeste

    2005-01-01

    Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of α and β aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt-/- and Hifα-/- TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin αvβ3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O2). Culturing B16F0 melanoma cells at 1.5% O2 resulted in increased αvβ3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O2 tension influence placental invasion and tumor migration by increasing cell surface expression of αvβ3 integrin. PMID:15689487

  20. Signal Regulatory Protein α Negatively Regulates β2 Integrin-Mediated Monocyte Adhesion, Transendothelial Migration and Phagocytosis

    PubMed Central

    Liu, Dan-Qing; Li, Li-Min; Guo, Ya-Lan; Bai, Rui; Wang, Chen; Bian, Zhen; Zhang, Chen-Yu; Zen, Ke

    2008-01-01

    Background Signal regulate protein α (SIRPα) is involved in many functional aspects of monocytes. Here we investigate the role of SIRPα in regulating β2 integrin-mediated monocyte adhesion, transendothelial migration (TEM) and phagocytosis. Methodology/Principal Findings THP-1 monocytes/macropahges treated with advanced glycation end products (AGEs) resulted in a decrease of SIRPα expression but an increase of β2 integrin cell surface expression and β2 integrin-mediated adhesion to tumor necrosis factor-α (TNFα)–stimulated human microvascular endothelial cell (HMEC-1) monolayers. In contrast, SIRPα overexpression in THP-1 cells showed a significant less monocyte chemotactic protein-1 (MCP-1)–triggered cell surface expression of β2 integrins, in particular CD11b/CD18. SIRPα overexpression reduced β2 integrin-mediated firm adhesion of THP-1 cells to either TNFα–stimulated HMEC-1 monolayers or to immobilized intercellular adhesion molecule-1 (ICAM-1). SIRPα overexpression also reduced MCP-1–initiated migration of THP-1 cells across TNFα–stimulated HMEC-1 monolayers. Furthermore, β2 integrin-mediated THP-1 cell spreading and actin polymerization in response to MCP-1, and phagocytosis of bacteria were both inhibited by SIRPα overexpression. Conclusions/Significance SIRPα negatively regulates β2 integrin-mediated monocyte adhesion, transendothelial migration and phagocytosis, thus may serve as a critical molecule in preventing excessive activation and accumulation of monocytes in the arterial wall during early stage of atherosclerosis. PMID:18820737

  1. Interaction between Fibronectin and β1 Integrin Is Essential for Tooth Development

    PubMed Central

    Yamada, Aya; Yuasa, Kenji; Yoshizaki, Keigo; Iwamoto, Tsutomu; Saito, Masahiro; Nakamura, Takashi; Fukumoto, Satoshi

    2015-01-01

    The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fibronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ1lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation. PMID:25830530

  2. Integrin-mediated interactions between B cells and follicular dendritic cells influence germinal center B cell fitness1

    PubMed Central

    Wang, Xiaoming; Rodda, Lauren; Bannard, Oliver; Cyster, Jason G.

    2014-01-01

    Integrin-ligand interactions between germinal center (GC) B cells and antigen-presenting follicular dendritic cells (FDCs) have been suggested to play central roles during GC responses but their in vivo requirement has not been directly tested. Here we show that while integrins αLβ2 and α4β1 are highly expressed and functional on mouse GC B cells, removal of single integrins or their ligands had little effect on B cell participation in the GC response. Combined β2-integrin deficiency and α4-integrin blockade also did not affect the GC response against a particulate antigen. However, the combined integrin deficiency did cause B cells to be outcompeted in splenic GC responses against a soluble protein antigen and in mesenteric lymph node GC responses against gut-derived antigens. Similar findings were made for β2-deficient B cells in mice lacking VCAM1 on FDCs. The reduced fitness of the GC B cells did not appear to be due to decreased antigen acquisition, proliferation rates or pAKT levels. In summary, our findings provide evidence that αLβ2 and α4β1 play overlapping and context-dependent roles in supporting interactions with FDCs that can augment the fitness of responding GC B cells. We also find that mouse GC B cells upregulate αvβ3 and adhere to vitronectin and milk fat globule EGF-factor-8 protein. Integrin β3-deficient B cells contributed in a slightly exaggerated manner to GC responses suggesting this integrin has a regulatory function in GC B cells. PMID:24740506

  3. Dynamin 2 regulation of integrin endocytosis, but not VEGF signaling, is crucial for developmental angiogenesis

    PubMed Central

    Lee, Monica Y.; Skoura, Athanasia; Park, Eon Joo; Landskroner-Eiger, Shira; Jozsef, Levente; Luciano, Amelia K.; Murata, Takahisa; Pasula, Satish; Dong, Yunzhou; Bouaouina, Mohamed; Calderwood, David A.; Ferguson, Shawn M.; De Camilli, Pietro; Sessa, William C.

    2014-01-01

    Here we show that dynamin 2 (Dnm2) is essential for angiogenesis in vitro and in vivo. In cultured endothelial cells lacking Dnm2, vascular endothelial growth factor (VEGF) signaling and receptor levels are augmented whereas cell migration and morphogenesis are impaired. Mechanistically, the loss of Dnm2 increases focal adhesion size and the surface levels of multiple integrins and reduces the activation state of β1 integrin. In vivo, the constitutive or inducible loss of Dnm2 in endothelium impairs branching morphogenesis and promotes the accumulation of β1 integrin at sites of failed angiogenic sprouting. Collectively, our data show that Dnm2 uncouples VEGF signaling from function and coordinates the endocytic turnover of integrins in a manner that is crucially important for angiogenesis in vitro and in vivo. PMID:24598168

  4. The role of α9β1 integrin and its ligands in the development of autoimmune diseases.

    PubMed

    Kon, Shigeyuki; Uede, Toshimitsu

    2018-03-01

    Adhesion of cells to extracellular matrix proteins through integrins expressed on the cell surface is important for cell adhesion/motility, survival, and differentiation. Recently, α9β1 integrin was reported to be important for the development of autoimmune diseases including rheumatoid arthritis, multiple sclerosis, and their murine models. In addition, ligands for α9β1 integrin, such as osteopontin and tenascin-C, are well established as key regulators of autoimmune diseases. Therefore, this review focused on the role of interactions between α9β1 integrin and its ligands in the development of autoimmune diseases.

  5. Simultaneous Targeting of RGD-Integrins and Dual Murine Double Minute Proteins in Glioblastoma Multiforme.

    PubMed

    Merlino, Francesco; Daniele, Simona; La Pietra, Valeria; Di Maro, Salvatore; Di Leva, Francesco Saverio; Brancaccio, Diego; Tomassi, Stefano; Giuntini, Stefano; Cerofolini, Linda; Fragai, Marco; Luchinat, Claudio; Reichart, Florian; Cavallini, Chiara; Costa, Barbara; Piccarducci, Rebecca; Taliani, Sabrina; Da Settimo, Federico; Martini, Claudia; Kessler, Horst; Novellino, Ettore; Marinelli, Luciana

    2018-05-18

    In the fight against Glioblastoma Multiforme, recent literature data have highlighted that integrin α5β1 and p53 are part of convergent pathways in the control of glioma apoptosis. This observation prompted us to seek a molecule able to simultaneously modulate both target families. Analyzing the results of a previous virtual screening against murine double minute 2 protein (MDM2), we envisaged that Arg-Gly-Asp (RGD)-mimetic molecules could be inhibitors of MDM2/4. Herein we present the discovery of compound 7, which inhibits both MDM2/4 and α5β1/αvβ3 integrins. A lead optimization campaign was carried out on 7 with aim to preserve the activities on integrins while improving those on MDM proteins. Compound 9 turned out to be a potent MDM2/4, and α5β1/αvβ3 blocker. In p53-wild type glioma cells, 9 arrested cell cycle and proliferation and strongly reduced cell invasiveness, emerging as the first molecule of a novel class of integrin/MDM inhibitors, which might be especially useful in subpopulations of patients with glioblastoma expressing a functional p53 concomitantly with a high level of α5β1 integrin.

  6. β3 integrin promotes chemoresistance to epirubicin in MDA-MB-231 through repression of the pro-apoptotic protein, BAD

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nair, Madhumathy G.; Desai, Krisha; Prabhu, Jyothi S.

    Resistance to anthracycline based chemotherapy is a major limitation in the treatment of breast cancer, particularly of the triple negative sub-type that lacks targeted therapies. Resistance that arises from tumor-stromal interaction facilitated by integrins provides the possibility of targeted disruption. In the present study, we demonstrate that integrin β3 signaling inhibits apoptosis induced by a DNA-damaging chemotherapeutic agent, epirubicin, in MDA-MB-231 breast cancer cells. Drug efflux based mechanisms do not contribute to this effect. We show that integrin β3 employs the PI3K-Akt and the MAPK pathway for enabling cell survival and proliferation. Further, our results indicate that integrin β3 helpsmore » inhibit epirubicin induced cytotoxicity by repression of the pro-apoptotic protein BAD, thus promoting an anti-apoptotic response. Myristoylated RGT peptide and a monoclonal antibody against integrin β3 brought about a reversal of this effect and chemosensitized the cells. These results identify β3 integrin signaling via repression of BAD as an important survival pathway used by breast cancer cells to evade chemotherapy induced stress. - Highlights: • Integrin β3 signaling promotes chemoresistance to epirubicin in breast cancer cells. • Integrin β3 promotes cell survival and proliferation in drug treated cells through the PI3K and MAPK pathways. • Integrin signaling helps evade drug induced cytotoxicity by repression of pro-apoptotic molecule; BAD.« less

  7. Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation

    PubMed Central

    Goult, Benjamin T; Bouaouina, Mohamed; Elliott, Paul R; Bate, Neil; Patel, Bipin; Gingras, Alexandre R; Grossmann, J Günter; Roberts, Gordon C K; Calderwood, David A; Critchley, David R; Barsukov, Igor L

    2010-01-01

    Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for β-integrin tails, F0 and F1 are also required for activation of β1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of β1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins. PMID:20150896

  8. Bone marrow-derived mesenchymal stem cells enhance angiogenesis via their α6β1 integrin receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Carrion, Bita; Kong, Yen P.; Kaigler, Darnell

    Bone marrow-derived mesenchymal stem cells (BMSCs) facilitate the angiogenic response of endothelial cells (ECs) within three-dimensional (3D) matrices in vivo and in engineered tissues in vitro in part through paracrine mediators and by acting as stabilizing pericytes. However, the molecular interactions between BMSCs and nascent tubules during the process of angiogenesis are not fully understood. In this study, we have used a tractable 3D co-culture model to explore the functional role of the α6β1 integrin adhesion receptor on BMSCs in sprouting angiogenesis. We report that knockdown of the α6 integrin subunit in BMSCs significantly reduces capillary sprouting, and causes theirmore » failure to associate with the nascent vessels. Furthermore, we demonstrate that the BMSCs with attenuated α6 integrin proliferate at a significantly lower rate relative to either control cells expressing non-targeting shRNA or wild type BMSCs; however, despite adding more cells to compensate for this deficit in proliferation, deficient sprouting persists. Collectively, our findings demonstrate that the α6 integrin subunit in BMSCs is important for their ability to stimulate vessel morphogenesis. This conclusion may have important implications in the optimization of cell-based strategies to promote angiogenesis. Highlights: • BMSCs stimulate angiogenesis, but the mechanisms remain unclear. • We silenced the expression of the α6 integrin subunit in BMSCs. • Silencing this receptor subunit significantly inhibited angiogenic sprouting. • Knocking down α6 integrin affected laminin and αSMA expression. • Silencing α6 integrin expression also reduced BMSC proliferation.« less

  9. Combating resistance to anti-IGFR antibody by targeting the integrin β3-Src pathway.

    PubMed

    Shin, Dong Hoon; Lee, Hyo-Jong; Min, Hye-Young; Choi, Sun Phil; Lee, Mi-Sook; Lee, Jung Weon; Johnson, Faye M; Mehta, Kapil; Lippman, Scott M; Glisson, Bonnie S; Lee, Ho-Young

    2013-10-16

    Several phase II/III trials of anti-insulin-like growth factor 1 receptor (IGF-1R) monoclonal antibodies (mAbs) have shown limited efficacy. The mechanisms of resistance to IGF-1R mAb-based therapies and clinically applicable strategies for overcoming drug resistance are still undefined. IGF-1R mAb cixutumumab efficacy, alone or in combination with Src inhibitors, was evaluated in 10 human head and neck squamous cell carcinoma (HNSCC) and six non-small cell lung cancer (NSCLC) cell lines in vitro in two- or three-dimensional culture systems and in vivo in cell line- or patient-derived xenograft tumors in athymic nude mice (n = 6-9 per group). Cixutumumab-induced changes in cell signaling and IGF-1 binding to integrin β3 were determined by Western or ligand blotting, immunoprecipitation, immunofluorescence, and cell adhesion analyses and enzyme-linked immunosorbent assay. Data were analyzed by the two-sided Student t test or one-way analysis of variance. Integrin β3-Src signaling cascade was activated by IGF-1 in HNSCC and NSCLC cells, when IGF-1 binding to IGF-1R was hampered by cixutumumab, resulting in Akt activation and cixutumumab resistance. Targeting integrin β3 or Src enhanced antitumor activity of cixutumumab in multiple cixutumumab-resistant cell lines and patient-derived tumors in vitro and in vivo. Mean tumor volume of mice cotreated with cixutumumab and integrin β3 siRNA was 133.7 mm(3) (95% confidence interval [CI] = 57.6 to 209.8 mm(3)) compared with those treated with cixutumumab (1472.5 mm(3); 95% CI = 1150.7 to 1794.3 mm(3); P < .001) or integrin β3 siRNA (903.2 mm(3); 95% CI = 636.1 to 1170.3 mm(3); P < .001) alone. Increased Src activation through integrin ανβ3 confers considerable resistance against anti-IGF-1R mAb-based therapies in HNSCC and NSCLC cells. Dual targeting of the IGF-1R pathway and collateral integrin β3-Src signaling module may override this resistance.

  10. 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

  11. Oxidative stress reduces trophoblast FOXO1 and integrin β3 expression that inhibits cell motility.

    PubMed

    Chen, Chie-Pein; Chen, Cheng-Yi; Wu, Yi-Hsin; Chen, Chia-Yu

    2018-06-08

    Preeclampsia is a serious pregnancy complication associated with placental oxidative stress and impaired trophoblast migration. The mechanism of defective trophoblast migration remains unknown. Forkhead box O1 (FOXO1) is a transcription factor. Integrin β3 is involved in cell motility. We hypothesized that FOXO1 mediates expression of trophoblast integrin β3, which could be impaired by oxidative stress and have implications in preeclampsia. The expressions of FOXO1 and integrin β3 were significantly reduced in preeclamptic placentas (n = 15) compared to that of controls (n = 15; p < 0.01). HTR-8/SVneo and JEG-3 trophoblasts were transfected to express wild-type FOXO1-WT or constitutively-expressed nuclear mutant form, FOXO1-AAA. The FOXO1 in HTR-8/SVneo and 3A-Sub-E trophoblasts was silenced by small interfering RNA. AKT-mediated phosphorylation inactivated FOXO1, but FOXO1-AAA was not phosphorylated. The expression of trophoblast integrin β3 was significantly elevated by FOXO1 overexpression and inhibited by FOXO1 knockdown. FOXO1 regulates integrin β3 at the transcriptional level via binding to the putative FOXO1 response element site between position -1154 to -1139 (TGAGATGTTTTGAAAG) in HTR-8/SVneo trophoblasts. The level of phosphorylated FOXO1 was decreased, and the FOXO1 level was increased in trophoblasts treated with AKT inhibitor MK2206, leading to upregulation of integrin β3. The capabilities of trophoblast adhesion and migration were enhanced by FOXO1-overexpression or MK2206, and inhibited by silencing FOXO1 or oxidative stress with H 2 O 2 . These results suggest that FOXO1 enhances trophoblast integrin β3 expression, and mediates cell adhesion and migration. By affecting the expression of FOXO1 and cell motility in trophoblasts, oxidative stress plays a role in the development of preeclampsia. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. β1D chain increases α7β1 integrin and laminin and protects against sarcolemmal damage in mdx mice

    PubMed Central

    Liu, Jianming; Milner, Derek J.; Boppart, Marni D.; Ross, Robert S.; Kaufman, Stephen J.

    2012-01-01

    The dystrophin–glycoprotein complex connects myofibers with extracellular matrix laminin. In Duchenne muscular dystrophy, this linkage system is absent and the integrity of muscle fibers is compromised. One potential therapy for addressing muscular dystrophy is to augment the amount of α7β1 integrin, the major laminin-binding integrin in skeletal muscle. Whereas transgenic over-expression of α7 chain may alleviate development of muscular dystrophy and extend the lifespan of severely dystrophic mdx/utrn−/− mice, further enhancing levels of α7 chain provided little additional membrane integrin and negligible additional improvement in mdx mice. We demonstrate here that normal levels of β1 chain limit formation of integrin heterodimer and that increasing β1D chain in mdx mice results in more functional integrin at the sarcolemma, more matrix laminin and decreased damage of muscle fibers. Moreover, increasing the amount of β1D chain in vitro enhances transcription of α7 integrin and α2 laminin genes and the amounts of these proteins. Thus manipulation of β1D integrin expression offers a novel approach to enhance integrin-mediated therapy for muscular dystrophy. PMID:22180459

  13. Selectin catch-bonds mechanotransduce integrin activation and neutrophil arrest on inflamed endothelium under shear flow.

    PubMed

    Morikis, Vasilios A; Chase, Shannon; Wun, Ted; Chaikof, Elliot L; Magnani, John L; Simon, Scott I

    2017-11-09

    E-selectin extends from the plasma membrane of inflamed endothelium and serves to capture leukocytes from flowing blood via long-lived catch-bonds that support slow leukocyte rolling under shear stress. Its ligands are glycosylated with the tetrasaccharide sialyl Lewis x (sLe x ), which contributes to bond affinity and specificity. E-selectin-mediated rolling transmits signals into neutrophils that trigger activation of high-affinity β 2 -integrins necessary for transition to shear-resistant adhesion and transendothelial migration. Rivipansel is a glycomimetic drug that inhibits E-selectin-mediated vaso-occlusion induced by integrin-dependent sickle-red blood cell-leukocyte adhesion. How Rivipansel antagonizes ligand recognition by E-selectin and blocks outside-in signaling of integrin-mediated neutrophil arrest while maintaining rolling immune-surveillance is unknown. Here, we demonstrate that sLe x expressed on human L-selectin is preferentially bound by E-selectin and, on ligation, initiates secretion of MRP8/14 that binds TLR4 to elicit the extension of β 2 -integrin to an intermediate affinity state. Neutrophil rolling over E-selectin at precise shear stress transmits tension and catch-bond formation with L-selectin via sLe x , resulting in focal clusters that deliver a distinct signal to upshift β 2 -integrins to a high-affinity state. Rivipansel effectively blocked formation of selectin catch-bonds, revealing a novel mechanotransduction circuit that rapidly converts extended β 2 -integrins to high-affinity shear-resistant bond clusters with intracellular adhesion molecule 1 on inflamed endothelium.

  14. Vedolizumab: A novel anti-integrin drug for treatment of inflammatory bowel disease

    PubMed Central

    Singh, Harmanjit; Grewal, Nipunjot; Arora, Ekta; Kumar, Harish; Kakkar, Ashish Kumar

    2016-01-01

    Inflammatory bowel disease (IBD) is the chronic inflammatory disorder of gastrointestinal tract consisting of two subtypes: Ulcerative colitis and Crohn's disease. IBD occurs due to infiltration of leukocytes in intestinal mucosa and derangements in intestinal barrier function. One of the most important steps in pathogenesis of IBD is the interactions between integrins on the surface of leukocyte. The α4β7 integrin expressing T-cell is an important leukocyte involved in pathogenesis and represents a new drug target for the treatment of IBD. Vedolizumab is a humanized monoclonal antibody, which acts against α4β7 integrin heterodimer and blocks the interaction of α4β7 integrin with MAdCAM-1. It prevents leukocyte binding to endothelial surface and its extravasation into affected tissue. The efficacy and safety of the vedolizumab have been established in many clinical studies. It has shown promising results in various clinical studies where a greater percentage of patients as compared to a placebo achieved and maintained clinical response, clinical remission, and corticosteroid-free clinical remission. Vedolizumab has been shown to be well tolerated with slightly higher risk of infections, headache, naspharyngitis as compared to placebo. This review focuses on the potential role of vedolizumab for the treatment of IBD. PMID:27003961

  15. Physical and functional interactions between a glioma cation channel and integrin-β1 require α-actinin

    PubMed Central

    Rooj, Arun K.; Liu, Zhiyong; McNicholas, Carmel M.

    2015-01-01

    Major plasma membrane components of the tumor cell, ion channels, and integrins play crucial roles in metastasis. Glioma cells express an amiloride-sensitive nonselective cation channel composed of acid-sensing ion channel (ASIC)-1 and epithelial Na+ channel (ENaC) α- and γ-subunits. Inhibition of this channel is associated with reduced cell migration and proliferation. Using the ASIC-1 subunit as a reporter for the channel complex, we found a physical and functional interaction between this channel and integrin-β1. Short hairpin RNA knockdown of integrin-β1 attenuated the amiloride-sensitive current, which was due to loss of surface expression of ASIC-1. In contrast, upregulation of membrane expression of integrin-β1 increased the surface expression of ASIC-1. The link between the amiloride-sensitive channel and integrin-β1 was mediated by α-actinin. Downregulation of α-actinin-1 or -4 attenuated the amiloride-sensitive current. Mutation of the putative binding site for α-actinin on the COOH terminus of ASIC-1 reduced the membrane localization of ASIC-1 and also resulted in attenuation of the amiloride-sensitive current. Our data suggest a novel interaction between the amiloride-sensitive glioma cation channel and integrin-β1, mediated by α-actinin. This interaction may form a mechanism by which channel activity can regulate glioma cell proliferation and migration. PMID:26108662

  16. Epithelial cell integrin β1 is required for developmental angiogenesis in the pituitary gland

    PubMed Central

    Scully, Kathleen M.; Skowronska-Krawczyk, Dorota; Krawczyk, Michal; Merkurjev, Daria; Taylor, Havilah; Livolsi, Antonia; Tollkuhn, Jessica; Stan, Radu V.; Rosenfeld, Michael G.

    2016-01-01

    As a key component of the vertebrate neuroendocrine system, the pituitary gland relies on the progressive and coordinated development of distinct hormone-producing cell types and an invading vascular network. The molecular mechanisms that drive formation of the pituitary vasculature, which is necessary for regulated synthesis and secretion of hormones that maintain homeostasis, metabolism, and endocrine function, remain poorly understood. Here, we report that expression of integrin β1 in embryonic pituitary epithelial cells is required for angiogenesis in the developing mouse pituitary gland. Deletion of pituitary epithelial integrin β1 before the onset of angiogenesis resulted in failure of invading endothelial cells to recruit pericytes efficiently, whereas deletion later in embryogenesis led to decreased vascular density and lumen formation. In both cases, lack of epithelial integrin β1 was associated with a complete absence of vasculature in the pituitary gland at birth. Within pituitary epithelial cells, integrin β1 directs a large transcriptional program that includes components of the extracellular matrix and associated signaling factors that are linked to the observed non–cell-autonomous effects on angiogenesis. We conclude that epithelial integrin β1 functions as a critical and canonical regulator of developmental angiogenesis in the pituitary gland, thus providing insight into the long-standing systems biology conundrum of how vascular invasion is coordinated with tissue development. PMID:27810956

  17. The impact of quercetin on wound healing relates to changes in αV and β1 integrin expression.

    PubMed

    Doersch, Karen M; Newell-Rogers, M Karen

    2017-08-01

    Overly fibrotic wound healing can lead to excess scar formation, causing functional impairment and undesirable cosmetic results. However, there are few successful treatments available to prevent or remediate scars. This study sought to explore the molecular mechanisms by which quercetin, a naturally-occurring antifibrotic agent, diminishes scar formation. Using both mice and fibroblast cells, we examined quercetin's impact on fibrosis and the wound healing rate, and potential molecular mechanisms underlying the quercetin-mediated reduction of fibrosis. While cultured fibroblasts demonstrated normal growth in response to quercetin, quercetin increased surface αV integrin and decreased β1 integrin. These changes in surface integrin expression may impact factors that contribute to fibrosis including cell migration, proliferation, and extracellular matrix production. In both quercetin-treated and control mice, wounds healed in about 14 days. Masson's trichrome stain revealed diminished fibrosis at the wound site in quercetin-treated animals despite the normal healing rate, indicating the potential for better cosmetic results without delaying healing. An in vitro scratch wound model using cells plated on an artificial extracellular matrix demonstrated delayed closure following quercetin treatment. The extracellular matrix also ameliorated quercetin's effect on αV integrin. Thus, αV integrin recruitment in response to quercetin treatment may promote the quercetin-mediated decrease extracellular matrix because cells require less extracellular matrix to migrate into a wound. With added extracellular matrix, β1 integrin remained diminished in response to quercetin, indicating that quercetin's effect on β1 integrin expression is independent of extracellular matrix -mediated signaling and is likely driven by inhibition of the intracellular mechanisms driving β1 expression. These findings suggest that quercetin could alter the cells' interactions with the extracellular

  18. Relating conformation to function in integrin α5β1.

    PubMed

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

    2016-07-05

    Whether β1 integrin ectodomains visit conformational states similarly to β2 and β3 integrins has not been characterized. Furthermore, despite a wealth of activating and inhibitory antibodies to β1 integrins, the conformational states that these antibodies stabilize, and the relation of these conformations to function, remain incompletely characterized. Using negative-stain electron microscopy, we show that the integrin α5β1 ectodomain adopts extended-closed and extended-open conformations as well as a bent conformation. Antibodies SNAKA51, 8E3, N29, and 9EG7 bind to different domains in the α5 or β1 legs, activate, and stabilize extended ectodomain conformations. Antibodies 12G10 and HUTS-4 bind to the β1 βI domain and hybrid domains, respectively, activate, and stabilize the open headpiece conformation. Antibody TS2/16 binds a similar epitope as 12G10, activates, and appears to stabilize an open βI domain conformation without requiring extension or hybrid domain swing-out. mAb13 and SG/19 bind to the βI domain and βI-hybrid domain interface, respectively, inhibit, and stabilize the closed conformation of the headpiece. The effects of the antibodies on cell adhesion to fibronectin substrates suggest that the extended-open conformation of α5β1 is adhesive and that the extended-closed and bent-closed conformations are nonadhesive. The functional effects and binding sites of antibodies and fibronectin were consistent with their ability in binding to α5β1 on cell surfaces to cross-enhance or inhibit one another by competitive or noncompetitive (allosteric) mechanisms.

  19. The CD157-integrin partnership controls transendothelial migration and adhesion of human monocytes.

    PubMed

    Lo Buono, Nicola; Parrotta, Rossella; Morone, Simona; Bovino, Paola; Nacci, Giulia; Ortolan, Erika; Horenstein, Alberto L; Inzhutova, Alona; Ferrero, Enza; Funaro, Ada

    2011-05-27

    CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β(1) and β(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.

  20. Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding

    PubMed Central

    Iturri, Jagoba; García-Fernández, Luis; Reuning, Ute; García, Andrés J.; Campo, Aránzazu del; Salierno, Marcelo J.

    2015-01-01

    The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies. PMID:25825012

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

    NASA Technical Reports Server (NTRS)

    Alenghat, Francis J.; Ingber, Donald E.

    2002-01-01

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

  2. Combination of X-ray crystallography, SAXS and DEER to obtain the structure of the FnIII-3, 4 domains of integrin α6β4

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alonso-García, Noelia; García-Rubio, Inés; Academia General Militar, Carretera de Huesca s/n, 50090 Zaragoza

    The structure of the FnIII-3, 4 region of integrin β4 was solved using a hybrid approach that combines crystallographic structures, SAXS, DEER and molecular modelling. The structure helps in understanding how integrin β4 might bind to other hemidesmosomal proteins and mediate signalling. Integrin α6β4 is a major component of hemidesmosomes that mediate the stable anchorage of epithelial cells to the underlying basement membrane. Integrin α6β4 has also been implicated in cell proliferation and migration and in carcinoma progression. The third and fourth fibronectin type III domains (FnIII-3, 4) of integrin β4 mediate binding to the hemidesmosomal proteins BPAG1e and BPAG2,more » and participate in signalling. Here, it is demonstrated that X-ray crystallography, small-angle X-ray scattering and double electron–electron resonance (DEER) complement each other to solve the structure of the FnIII-3, 4 region. The crystal structures of the individual FnIII-3 and FnIII-4 domains were solved and the relative arrangement of the FnIII domains was elucidated by combining DEER with site-directed spin labelling. Multiple structures of the interdomain linker were modelled by Monte Carlo methods complying with DEER constraints, and the final structures were selected against experimental scattering data. FnIII-3, 4 has a compact and cambered flat structure with an evolutionary conserved surface that is likely to correspond to a protein-interaction site. Finally, this hybrid method is of general application for the study of other macromolecules and complexes.« less

  3. N-Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4

    PubMed Central

    Hang, Qinglei; Isaji, Tomoya; Hou, Sicong; Zhou, Ying; Fukuda, Tomohiko; Gu, Jianguo

    2016-01-01

    N-Glycosylation of integrin α5β1 is involved in multiple cell behaviors. We previously reported that the N-glycosylations of the calf domain on integrin α5 (S3–5,10–14) are essential for its inhibitory effect on EGFR signaling in regulating cell proliferation. However, the importance of the individual N-glycosylation and the underlying mechanisms of inhibition remain unclear. Here, we characterize the S3–5,10–14 mutants in detail and found that the N-glycosylation of site-11 (Asn712) is key for cell growth. The restoration of site-11, unlike the other individual sites, significantly suppressed cell growth and EGFR signaling in a manner that was similar to that of wild-type (WT). Mechanistically, this N-glycosylation inhibited the response abilities upon EGF stimulation and EGFR dimerization. Interestingly, we found this N-glycosylation controlled the EGFR complex formation with integrin α5β1 or α6β4; i.e., the loss of site-11 switched EGFR-α5β1 to EGFR-α6β4, which is well known to promote cellular signaling for cell growth. Moreover, the site-11 N-glycan exhibited a more branching structure compared with other sites, which may be required for EGFR-α5β1 formation. Taken together, these data clearly demonstrate that the site-11 N-glycosylation on α5 is most important for its inhibitory effect on EGFR signaling, which may provide a novel regulatory mechanism for crosstalks between integrins and EGFR. PMID:27641064

  4. Mesodermal expression of integrin α5β1 regulates neural crest development and cardiovascular morphogenesis

    PubMed Central

    Liang, Dong; Wang, Xia; Mittal, Ashok; Dhiman, Sonam; Hou, Shuan-Yu; Degenhardt, Karl; Astrof, Sophie

    2014-01-01

    Integrin α5-null embryos die in mid-gestation from severe defects in cardiovascular morphogenesis, which stem from defective development of the neural crest, heart and vasculature. To investigate the role of integrin α5β1 in cardiovascular development, we used the Mesp1Cre knock-in strain of mice to ablate integrin α5 in the anterior mesoderm, which gives rise to all of the cardiac and many of the vascular and muscle lineages in the anterior portion of the embryo. Surprisingly, we found that mutant embryos displayed numerous defects related to the abnormal development of the neural crest such as cleft palate, ventricular septal defect, abnormal development of hypoglossal nerves, and defective remodeling of the aortic arch arteries. We found that defects in arch artery remodeling stem from the role of mesodermal integrin α5β1 in neural crest proliferation and differentiation into vascular smooth muscle cells, while proliferation of pharyngeal mesoderm and differentiation of mesodermal derivatives into vascular smooth muscle cells was not defective. Taken together our studies demonstrate a requisite role for mesodermal integrin α5β1 in signaling between the mesoderm and the neural crest, thereby regulating neural crest-dependent morphogenesis of essential embryonic structures. PMID:25242040

  5. Integrin αIIb (CD41) plays a role in the maintenance of hematopoietic stem cell activity in the mouse embryonic aorta

    PubMed Central

    Boisset, Jean-Charles; Clapes, Thomas; Van Der Linden, Reinier; Dzierzak, Elaine; Robin, Catherine

    2013-01-01

    Summary Integrins are transmembrane receptors that play important roles as modulators of cell behaviour through their adhesion properties and the initiation of signaling cascades. The αIIb integrin subunit (CD41) is one of the first cell surface markers indicative of hematopoietic commitment. αIIb pairs exclusively with β3 to form the αIIbβ3 integrin. β3 (CD61) also pairs with αv (CD51) to form the αvβ3 integrin. The expression and putative role of these integrins during mouse hematopoietic development is as yet unknown. We show here that hematopoietic stem cells (HSCs) differentially express αIIbβ3 and αvβ3 integrins throughout development. Whereas the first HSCs generated in the aorta at mid-gestation express both integrins, HSCs from the placenta only express αvβ3, and most fetal liver HSCs do not express either integrin. By using αIIb deficient embryos, we show that αIIb is not only a reliable HSC marker but it also plays an important and specific function in maintaining the HSC activity in the mouse embryonic aorta. PMID:23789102

  6. The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms

    PubMed Central

    Ophir, Michael J.; Liu, Beiyun C.

    2013-01-01

    The T cell receptor (TCR) triggers the assembly of “SLP-76 microclusters,” which mediate signals required for T cell activation. In addition to regulating integrin activation, we show that Src kinase–associated phosphoprotein of 55 kD (SKAP55) is required for microcluster persistence and movement, junctional stabilization, and integrin-independent adhesion via the TCR. These functions require the dimerization of SKAP55 and its interaction with the adaptor adhesion and degranulation-promoting adaptor protein (ADAP). A “tandem dimer” containing two ADAP-binding SKAP55 Src homology 3 (SH3) domains stabilized SLP-76 microclusters and promoted T cell adhesion via the TCR, but could not support adhesion to integrin ligands. Finally, the SKAP55 dimerization motif (DM) enabled the coimmunoprecipitation of the Rap1-dependent integrin regulator Rap1-GTP–interacting adaptor molecule (RIAM), the recruitment of talin into TCR-induced adhesive junctions, and “inside-out” signaling to β1 integrins. Our data indicate that SKAP55 dimers stabilize SLP-76 microclusters, couple SLP-76 to the force-generating systems responsible for microcluster movement, and enable adhesion via the TCR by mechanisms independent of RIAM, talin, and β1 integrins. PMID:24368808

  7. Combating Resistance to Anti-IGFR Antibody by Targeting the Integrin β3-Src Pathway

    PubMed Central

    2013-01-01

    Background Several phase II/III trials of anti–insulin-like growth factor 1 receptor (IGF-1R) monoclonal antibodies (mAbs) have shown limited efficacy. The mechanisms of resistance to IGF-1R mAb-based therapies and clinically applicable strategies for overcoming drug resistance are still undefined. Methods IGF-1R mAb cixutumumab efficacy, alone or in combination with Src inhibitors, was evaluated in 10 human head and neck squamous cell carcinoma (HNSCC) and six non–small cell lung cancer (NSCLC) cell lines in vitro in two- or three-dimensional culture systems and in vivo in cell line– or patient-derived xenograft tumors in athymic nude mice (n = 6–9 per group). Cixutumumab-induced changes in cell signaling and IGF-1 binding to integrin β3 were determined by Western or ligand blotting, immunoprecipitation, immunofluorescence, and cell adhesion analyses and enzyme-linked immunosorbent assay. Data were analyzed by the two-sided Student t test or one-way analysis of variance. Results Integrin β3–Src signaling cascade was activated by IGF-1 in HNSCC and NSCLC cells, when IGF-1 binding to IGF-1R was hampered by cixutumumab, resulting in Akt activation and cixutumumab resistance. Targeting integrin β3 or Src enhanced antitumor activity of cixutumumab in multiple cixutumumab-resistant cell lines and patient-derived tumors in vitro and in vivo. Mean tumor volume of mice cotreated with cixutumumab and integrin β3 siRNA was 133.7mm3 (95% confidence interval [CI] = 57.6 to 209.8mm3) compared with those treated with cixutumumab (1472.5mm3; 95% CI = 1150.7 to 1794.3mm3; P < .001) or integrin β3 siRNA (903.2mm3; 95% CI = 636.1 to 1170.3mm3; P < .001) alone. Conclusions Increased Src activation through integrin ανβ3 confers considerable resistance against anti–IGF-1R mAb-based therapies in HNSCC and NSCLC cells. Dual targeting of the IGF-1R pathway and collateral integrin β3–Src signaling module may override this resistance. PMID:24092920

  8. Does endometrial integrin expression in endometriosis patients predict enhanced in vitro fertilization cycle outcomes after prolonged GnRH agonist therapy?

    PubMed

    Surrey, Eric S; Lietz, Annette K; Gustofson, Robert L; Minjarez, Debra A; Schoolcraft, William B

    2010-02-01

    To determine whether endometrial expression of the integrin alpha(v)beta(3) vitronectin can predict which endometriosis patient subgroup will benefit from pre-IVF cycle prolonged GnRH agonist (GnRHa) therapy. Prospective randomized institutional review board approved pilot trial. Private assisted reproductive technology program. IVF candidates with regular menses, surgically confirmed endometriosis, and normal ovarian reserve. All patients underwent endometrial biopsy 9 to 11 days post-LH surge to evaluate alpha(v)beta(3) integrin expression. Patients were randomized either to receive depot leuprolide acetate 3.75 mg every 28 days for three doses before controlled ovarian hyperstimulation (COH) or to proceed directly to COH and IVF. Group 1: integrin-positive controls (N = 12); group 2: integrin-positive administered prolonged GnRHa (N = 8). Group A: integrin-negative controls (N = 7); group B: integrin-negative administered prolonged GnRHa (N = 9). COH responses, ongoing pregnancy and implantation rates. There were no significant effects of GnRH agonist treatment in either of the integrin expression strata regarding ongoing pregnancy or implantation rates, although these outcomes were more frequent in group 2 vs. 1 (62.5% vs. 41.6% and 35% vs. 20.6%, respectively). This effect may have because of limited sample size. The value of a negative integrin biopsy in predicting an ongoing pregnancy after prolonged GnRH agonist therapy was only 44.4%. Endometrial alpha(v)beta(3) integrin expression did not predict which endometriosis patients would benefit from prolonged GnRHa therapy before IVF. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  9. α6-Integrin alternative splicing: distinct cytoplasmic variants in stem cell fate specification and niche interaction.

    PubMed

    Zhou, Zijing; Qu, Jing; He, Li; Peng, Hong; Chen, Ping; Zhou, Yong

    2018-05-02

    α6-Integrin subunit (also known as CD49f) is a stemness signature that has been found on the plasma membrane of more than 30 stem cell populations. A growing body of studies have focused on the critical role of α6-containing integrins (α6β1 and α6β4) in the regulation of stem cell properties, lineage-specific differentiation, and niche interaction. α6-Integrin subunit can be alternatively spliced at the post-transcriptional level, giving rise to divergent isoforms which differ in the cytoplasmic and/or extracellular domains. The cytoplasmic domain of integrins is an important functional part of integrin-mediated signals. Structural changes in the cytoplasmic domain of α6 provide an efficient means for the regulation of stem cell responses to biochemical stimuli and/or biophysical cues in the stem cell niche, thus impacting stem cell fate determination. In this review, we summarize the current knowledge on the structural variants of the α6-integrin subunit and spatiotemporal expression of α6 cytoplasmic variants in embryonic and adult stem/progenitor cells. We highlight the roles of α6 cytoplasmic variants in stem cell fate decision and niche interaction, and discuss the potential mechanisms involved. Understanding of the distinct functions of α6 splicing variants in stem cell biology may inform the rational design of novel stem cell-based therapies for a range of human diseases.

  10. Platelets lacking PIP5KIγ have normal integrin activation but impaired cytoskeletal-membrane integrity and adhesion

    PubMed Central

    Wang, Yanfeng; Zhao, Liang; Suzuki, Aae; Lian, Lurong; Min, Sang H.; Wang, Ziqian; Litvinov, Rustem I.; Stalker, Timothy J.; Yago, Tadayuki; Klopocki, Arkadiusz G.; Schmidtke, David W.; Yin, Helen; Choi, John K.; McEver, Rodger P.; Weisel, John W.; Hartwig, John H.; Abrams, Charles S.

    2013-01-01

    Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIβ, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion. PMID:23372168

  11. β2 integrin mediates hantavirus-induced release of neutrophil extracellular traps

    PubMed Central

    Raftery, Martin J.; Lalwani, Pritesh; Krautkrӓmer, Ellen; Peters, Thorsten; Scharffetter-Kochanek, Karin; Krüger, Renate; Hofmann, Jörg; Seeger, Karl; Krüger, Detlev H.

    2014-01-01

    Rodent-borne hantaviruses are emerging human pathogens that cause severe human disease. The underlying mechanisms are not well understood, as hantaviruses replicate in endothelial and epithelial cells without causing any cytopathic effect. We demonstrate that hantaviruses strongly stimulated neutrophils to release neutrophil extracellular traps (NETs). Hantavirus infection induced high systemic levels of circulating NETs in patients and this systemic NET overflow was accompanied by production of autoantibodies to nuclear antigens. Analysis of the responsible mechanism using neutrophils from β2 null mice identified β2 integrin receptors as a master switch for NET induction. Further experiments suggested that β2 integrin receptors such as complement receptor 3 (CR3) and 4 (CR4) may act as novel hantavirus entry receptors. Using adenoviruses, we confirmed that viral interaction with β2 integrin induced strong NET formation. Collectively, β2 integrin–mediated systemic NET overflow is a novel viral mechanism of immunopathology that may be responsible for characteristic aspects of hantavirus-associated disease such as kidney and lung damage. PMID:24889201

  12. Prostaglandin E2 suppresses beta1-integrin expression via E-prostanoid receptor in human monocytes/macrophages.

    PubMed

    Hasegawa, Shunji; Ichiyama, Takashi; Kohno, Fumitaka; Korenaga, Yuno; Ohsaki, Ayami; Hirano, Reiji; Haneda, Yasuhiro; Fukano, Reiji; Furukawa, Susumu

    2010-01-01

    Beta1-integrins mediate cell attachment to different extracellular matrix proteins, intracellular proteins, and intercellular adhesions. Recently, it has been reported that prostaglandin E2 (PGE2) has anti-inflammatory properties such as inhibition of the expression of adhesion molecules or production of chemokines. However, the effect of PGE2 on the expression of beta1-integrin remains unknown. In this study, we investigated the effects of PGE2 on the expression of beta1-integrin in the human monocytic cell line THP-1 and in CD14+ monocytes/macrophages in human peripheral blood. For this, we examined the role of four subtypes of PGE2 receptors and E-prostanoid (EP) receptors on PGE2-mediated inhibition. We found that PGE2 significantly inhibited the expression of beta1-integrin, mainly through EP4 receptors in THP-1 cells and CD14+ monocytes/macrophages in human peripheral blood. We suggest that PGE2 has anti-inflammatory effects, leading to the inhibited expression of beta1-integrin in human monocytes/macrophages, and that the EP4 receptor may play an important role in PGE2-mediated inhibition. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  13. Collagen Gly missense mutations: Effect of residue identity on collagen structure and integrin binding.

    PubMed

    Qiu, Yimin; Mekkat, Arya; Yu, Hongtao; Yigit, Sezin; Hamaia, Samir; Farndale, Richard W; Kaplan, David L; Lin, Yu-Shan; Brodsky, Barbara

    2018-05-11

    Gly missense mutations in type I collagen, which replace a conserved Gly in the repeating (Gly-Xaa-Yaa) n sequence with a larger residue, are known to cause Osteogenesis Imperfecta (OI). The clinical consequences of such mutations range from mild to lethal, with more serious clinical severity associated with larger Gly replacement residues. Here, we investigate the influence of the identity of the residue replacing Gly within and adjacent to the integrin binding 502 GFPGER 507 sequence on triple-helix structure, stability and integrin binding using a recombinant bacterial collagen system. Recombinant collagens were constructed with Gly substituted by Ala, Ser or Val at four positions within the integrin binding region. All constructs formed a stable triple-helix structure with a small decrease in melting temperature. Trypsin was used to probe local disruption of the triple helix, and Gly to Val replacements made the triple helix trypsin sensitive at three of the four sites. Any mutation at Gly505, eliminated integrin binding, while decreased integrin binding affinity was observed in the replacement of Gly residues at Gly502 following the order Val > Ser > Ala. Molecular dynamics simulations indicated that all Gly replacements led to transient disruption of triple-helix interchain hydrogen bonds in the region of the Gly replacement. These computational and experimental results lend insight into the complex molecular basis of the varying clinical severity of OI. Copyright © 2018. Published by Elsevier Inc.

  14. Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

    PubMed

    Coopman, P J; Thomas, D M; Gehlsen, K R; Mueller, S C

    1996-11-01

    The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3

  15. Multifaced Roles of the αvβ3 Integrin in Ehlers–Danlos and Arterial Tortuosity Syndromes’ Dermal Fibroblasts

    PubMed Central

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

    2018-01-01

    The αvβ3 integrin, an endothelial cells’ receptor-binding fibronectin (FN) in the extracellular matrix (ECM) of blood vessels, regulates ECM remodeling during migration, invasion, angiogenesis, wound healing and inflammation, and is also involved in the epithelial mesenchymal transition. In vitro-grown human control fibroblasts organize a fibrillar network of FN, which is preferentially bound on the entire cell surface to its canonical α5β1 integrin receptor, whereas the αvβ3 integrin is present only in rare patches in focal contacts. We report on the preferential recruitment of the αvβ3 integrin, due to the lack of FN–ECM and its canonical integrin receptor, in dermal fibroblasts from Ehlers–Danlos syndromes (EDS) and arterial tortuosity syndrome (ATS), which are rare multisystem connective tissue disorders. We review our previous findings that unraveled different biological mechanisms elicited by the αvβ3 integrin in fibroblasts derived from patients affected with classical (cEDS), vascular (vEDS), hypermobile EDS (hEDS), hypermobility spectrum disorders (HSD), and ATS. In cEDS and vEDS, respectively, due to defective type V and type III collagens, αvβ3 rescues patients’ fibroblasts from anoikis through a paxillin-p60Src-mediated cross-talk with the EGF receptor. In hEDS and HSD, without a defined molecular basis, the αvβ3 integrin transduces to the ILK-Snail1-axis inducing a fibroblast-to-myofibroblast-transition. In ATS cells, the deficiency of the dehydroascorbic acid transporter GLUT10 leads to redox imbalance, ECM disarray together with the activation of a non-canonical αvβ3 integrin-TGFBRII signaling, involving p125FAK/p60Src/p38MAPK. The characterization of these different biological functions triggered by αvβ3 provides insights into the multifaced nature of this integrin, at least in cultured dermal fibroblasts, offering future perspectives for research in this field. PMID:29587413

  16. Matrix Metalloproteinase-2 Cleavage of the β1 Integrin Ectodomain Facilitates Colon Cancer Cell Motility*

    PubMed Central

    Kryczka, Jakub; Stasiak, Marta; Dziki, Lukasz; Mik, Michał; Dziki, Adam; Cierniewski, Czesław S.

    2012-01-01

    Cancer cell invasion is a key element in metastasis that requires integrins for adhesion/de-adhesion, as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Herein we show that MMP-2 is up-regulated in resected colorectal tumors and degrades β1 integrins with the release of fragments containing the β1 I-domain. The β1 cleavage pattern is similar to that produced by digestion of α5β1 and α2β1 with MMP-2. Two such fragments, at 25 and 75 kDa, were identified after immunoprecipitation, with monoclonal antibody BD610468 reacting with the NH2-terminal I-like ectodomain followed by SDS-PAGE and microsequencing using electrospray (ISI-Q-TOF-Micromass) spectrometry. Cleavage of the β1 integrin can be abolished by inhibition of MMP-2 activity; it can be induced by up-regulation of MMP-2 expression, as exemplified by HT29 colon cancer cells transfected with pCMV6-XL5-MMP-2. Co-immunoprecipitation studies of colon cancer cells showed that the β1 integrin subunit is associated with MMP-2. The MMP-2-mediated shedding of the I-like domain from β1 integrins resulted in decreased adhesion of colon cancer cells to collagen and fibronectin, thus abolishing their receptivity. Furthermore, such cells showed enhanced motility as evaluated by a “wound healing-like” assay and time-lapse microscopy, indicating their increased invasiveness. Altogether, our data demonstrate that MMP-2 amplifies the motility of colon cancer cells, not only by digesting the extracellular matrix components in the vicinity of cancer cells but also by inactivating their major β1 integrin receptors. PMID:22898815

  17. β1 integrin- and JNK-dependent tumor growth upon hypofractionated radiation.

    PubMed

    Sayeed, Aejaz; Lu, Huimin; Liu, Qin; Deming, David; Duffy, Alexander; McCue, Peter; Dicker, Adam P; Davis, Roger J; Gabrilovich, Dmitry; Rodeck, Ulrich; Altieri, Dario C; Languino, Lucia R

    2016-08-16

    Radiation therapy is an effective cancer treatment modality although tumors invariably become resistant. Using the transgenic adenocarcinoma of mouse prostate (TRAMP) model system, we report that a hypofractionated radiation schedule (10 Gy/day for 5 consecutive days) effectively blocks prostate tumor growth in wild type (β1wt /TRAMP) mice as well as in mice carrying a conditional ablation of β1 integrins in the prostatic epithelium (β1pc-/- /TRAMP). Since JNK is known to be suppressed by β1 integrins and mediates radiation-induced apoptosis, we tested the effect of SP600125, an inhibitor of c-Jun amino-terminal kinase (JNK) in the TRAMP model system. Our results show that SP600125 negates the effect of radiation on tumor growth in β1pc-/- /TRAMP mice and leads to invasive adenocarcinoma. These effects are associated with increased focal adhesion kinase (FAK) expression and phosphorylation in prostate tumors in β1pc-/- /TRAMP mice. In marked contrast, radiation-induced tumor growth suppression, FAK expression and phosphorylation are not altered by SP600125 treatment of β1wt /TRAMP mice. Furthermore, we have reported earlier that abrogation of insulin-like growth factor receptor (IGF-IR) in prostate cancer cells enhances the sensitivity to radiation. Here we further explore the β1/IGF-IR crosstalk and report that β1 integrins promote cell proliferation partly by enhancing the expression of IGF-IR. In conclusion, we demonstrate that β1 integrin-mediated inhibition of JNK signaling modulates tumor growth rate upon hypofractionated radiation.

  18. Do Integrins Mediate the Skeletal Response to Altered Loading?

    NASA Technical Reports Server (NTRS)

    vanderMeulen, Marjolein C. H.

    2004-01-01

    In vivo experiments were performed to examine the role of B1 integrin in skeletal adaptation to reduced and increased loading. Transgenic mice were generated with a dominant negative form of the B1 integrin cytoplasmic domain with expression driven by the osteocalcin promoter (pOCb1DN). This fragment consists of the transmembrane and intracellular domains and interferes with endogenous integrin signalling in vitro. This promoter targets expression of the transgene to mature bone cells. Expression of the transgene was confirmed by immunoprecipitation and western blotting. Reduced loading was generated by hindlimb suspension and increased loading the resumption of normal loading following hindlimb suspension. Two groups of female 35-day old mice were examined: poCb1DN transgenic mice (TG) and wild-type littermate controls (WT). Animals were hindlimb suspended for 1 week (HU, n = l0/gp) or 4 weeks (HU, n = 4 - 7/gp) or suspended for 4 weeks followed by reloading by normal ambulation for 4 weeks (RL, n = l0/gp). Age-matched controls (CT) were pairfed based on the HU food intake. The protocols were approved by the NASA Ames Research Center IACUC. Upon completion of the experimental protocol, body mass was recorded and tissues of interest removed and analyzed following standard procedures. Femoral whole bone structural behavior was measured in torsion to failure to obtain whole bone strength (failure torque) and torsional rigidity. Ash content (ash) and fraction (% ash) were determined for the tibia. Total ash is indicative of bone size whereas %ash is a material property. Tibial curvature was measured from microradiographs. For each experiment, the effects of genotype (TG, WT) and treatment (CT, HU/RL) were assessed by two-factor ANOVA followed by the Tukey-Kramer posthoc to identify significant differences at an alpha level of 0.05. Our goal was to understand differences resulting from altered integrin function in the adaptation to altered loading.

  19. Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients.

    PubMed

    Haller, H; Ziegler, E M; Homuth, V; Drab, M; Eichhorn, J; Nagy, Z; Busjahn, A; Vetter, K; Luft, F C

    1997-01-01

    Endothelial cell activation is important in the pathogenesis of preeclampsia; however, the nature of the activation is unknown. We investigated 22 patients with preeclampsia. 29 normotensive pregnancies, and 18 nonpregnant women to test the hypothesis that serum from preeclamptic patients induces expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) and stimulates intracellular free calcium concentrations [Ca2+]i in cultured endothelial cells. We then asked whether the corresponding integrin adhesive counter receptors lymphocyte function-associated antigen-1 (CD11a/CD18), macrophage-1 antigen (CD11b/CD18), p150,95 (CD11c/CD18), and very late activation antigen-4 (CD49/CD29) are increased in patients with preeclampsia. In the pregnant women, the measurements were conducted both before and after delivery. Integrin expression was measured by fluorescent antibody cell sorting analysis using monoclonal antibodies. ICAM-1 and VCAM-1 were analyzed on endothelial cells by enzyme-linked immunosorbent assay. [Ca2+]i was measured with fura 2. Serum from preeclamptic patients increased endothelial cell ICAM-1 expression but not VCAM-1 expression. Preeclamptic patients' serum also increased [Ca2+]i in endothelial cells compared with serum from normal nonpregnant or normal pregnant women. Endothelial cell [Ca2+]i concentrations were correlated with the ICAM-1 expression in preeclamptic patients (r = .80, P < .001) before but not after delivery. Expression of the integrin counter receptors on leukocytes was similarly increased in preclampsia and normal pregnancy compared with the nonpregnant state. The expression decreased significantly after delivery in both groups. Our results demonstrate that serum from preeclamptic women induces increased ICAM-1 surface expression on endothelial cells, while the expression of the integrin counterreceptors was not different. The effect on endothelial cells may be related to an increase in [Ca2+]i

  20. Urokinase-type plasminogen activator receptor (uPAR) ligation induces a raft-localized integrin signaling switch that mediates the hypermotile phenotype of fibrotic fibroblasts.

    PubMed

    Grove, Lisa M; Southern, Brian D; Jin, Tong H; White, Kimberly E; Paruchuri, Sailaja; Harel, Efrat; Wei, Ying; Rahaman, Shaik O; Gladson, Candece L; Ding, Qiang; Craik, Charles S; Chapman, Harold A; Olman, Mitchell A

    2014-05-02

    The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

  1. Effects and mechanism of integrin-β1 gene expression inhibited by shRNA in invasion of pancreatic carcinoma PANC-1 cells.

    PubMed

    Yu, Feng; Li, Hua; Bu, Xuefeng; Zhang, Yongjun

    2012-01-01

    To investigate the effects of integrin-β1 gene expression inhibited by shRNA on invasion of pancreatic carcinoma PANC-1 cells in vitro. The eukaryotic expression plasmid of short hairpin RNA (shRNA) targeting integrin-β1 gene (integrin-β1-shRNA) was constructed and transfected into PANC-1 cells. The expressions of integrin-β1 mRNA and protein were detected by real-time quantitative polymerase chain reaction (PCR) and western blot assay, respectively. The invasive ability of PANC-1 cells was observed with a transwell cell culture chamber and the expressions of MMP-2 and MMP-9 were assayed. Compared to the untransfected group, recombinant expression plasmid integrin-β1-shRNA resulted in reduction of integrin-β1 mRNA and protein by 78.58%±7.24% and 92.88%±3.18%, respectively and the average number of invading PANC-1 cells were decreased from 52±5 to 21±4 (p<0.01) and the expressions of MMP-2 and MMP-9 were inhibited. Recombinant expression plasmid integrin-β1- shRNA can inhibit the expression of integrin-β1 gene and suppress the invasion of PANC-1 cells in vitro significantly.

  2. The CD157-Integrin Partnership Controls Transendothelial Migration and Adhesion of Human Monocytes*

    PubMed Central

    Lo Buono, Nicola; Parrotta, Rossella; Morone, Simona; Bovino, Paola; Nacci, Giulia; Ortolan, Erika; Horenstein, Alberto L.; Inzhutova, Alona; Ferrero, Enza; Funaro, Ada

    2011-01-01

    CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β1 and β2 integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes. PMID:21478153

  3. Cytokinesis failure due to derailed integrin traffic induces aneuploidy and oncogenic transformation in vitro and in vivo

    PubMed Central

    Högnäs, G; Tuomi, S; Veltel, S; Mattila, E; Murumägi, A; Edgren, H; Kallioniemi, O; Ivaska, J

    2012-01-01

    Aneuploidy is frequently detected in solid tumors but the mechanisms regulating the generation of aneuploidy and their relevance in cancer initiation remain under debate and are incompletely characterized. Spatial and temporal regulation of integrin traffic is critical for cell migration and cytokinesis. Impaired integrin endocytosis, because of the loss of Rab21 small GTPase or mutations in the integrin β-subunit cytoplasmic tail, induces failure of cytokinesis in vitro. Here, we describe that repeatedly failed cytokinesis, because of impaired traffic, is sufficient to trigger the generation of aneuploid cells, which display characteristics of oncogenic transformation in vitro and are tumorigenic in vivo. Furthermore, in an in vivo mouse xenograft model, non-transformed cells with impaired integrin traffic formed tumors with a long latency. More detailed investigation of these tumors revealed that the tumor cells were aneuploid. Therefore, abnormal integrin traffic was linked with generation of aneuploidy and cell transformation also in vivo. In human prostate and ovarian cancer samples, downregulation of Rab21 correlates with increased malignancy. Loss-of-function experiments demonstrate that long-term depletion of Rab21 is sufficient to induce chromosome number aberrations in normal human epithelial cells. These data are the first to demonstrate that impaired integrin traffic is sufficient to induce conversion of non-transformed cells to tumorigenic cells in vitro and in vivo. PMID:22120710

  4. Identification of an Endogenously Generated Cryptic Collagen Epitope (XL313) That May Selectively Regulate Angiogenesis by an Integrin Yes-associated Protein (YAP) Mechano-transduction Pathway*

    PubMed Central

    Ames, Jacquelyn J.; Contois, Liangru; Caron, Jennifer M.; Tweedie, Eric; Yang, Xuehui; Friesel, Robert; Vary, Calvin; Brooks, Peter C.

    2016-01-01

    Extracellular matrix (ECM) remodeling regulates angiogenesis. However, the precise mechanisms by which structural changes in ECM proteins contribute to angiogenesis are not fully understood. Integrins are molecules with the ability to detect compositional and structural changes within the ECM and integrate this information into a network of signaling circuits that coordinate context-dependent cell behavior. The role of integrin αvβ3 in angiogenesis is complex, as evidence exists for both positive and negative functions. The precise downstream signaling events initiated by αvβ3 may depend on the molecular characteristics of its ligands. Here, we identified an RGD-containing cryptic collagen epitope that is generated in vivo. Surprisingly, rather than inhibiting αvβ3 signaling, this collagen epitope promoted αvβ3 activation and stimulated angiogenesis and inflammation. An antibody directed to this RGDKGE epitope but not other RGD collagen epitopes inhibited angiogenesis and inflammation in vivo. The selective ability of this RGD epitope to promote angiogenesis and inflammation depends in part on its flanking KGE motif. Interestingly, a subset of macrophages may represent a physiologically relevant source of this collagen epitope. Here, we define an endothelial cell mechano-signaling pathway in which a cryptic collagen epitope activates αvβ3 leading to an Src and p38 MAPK-dependent cascade that leads to nuclear accumulation of Yes-associated protein (YAP) and stimulation of endothelial cell growth. Collectively, our findings not only provide evidence for a novel mechano-signaling pathway, but also define a possible therapeutic strategy to control αvβ3 signaling by targeting a pro-angiogenic and inflammatory ligand of αvβ3 rather than the receptor itself. PMID:26668310

  5. Focal complex formation in adult cardiomyocytes is accompanied by the activation of beta3 integrin and c-Src.

    PubMed

    Willey, Christopher D; Balasubramanian, Sundaravadivel; Rodríguez Rosas, María C; Ross, Robert S; Kuppuswamy, Dhandapani

    2003-06-01

    In pressure-overloaded myocardium, our recent study demonstrated cytoskeletal assembly of c-Src and other signaling proteins which was partially mimicked in vitro using adult feline cardiomyocytes embedded in three-dimensional (3D) collagen matrix and stimulated with an integrin-binding Arg-Gly-Asp (RGD) peptide. In the present study, we improved this model further to activate c-Src and obtain a full assembly of the focal adhesion complex (FAC), and characterized c-Src localization and integrin subtype(s) involved. RGD dose response experiments revealed that c-Src activation occurs subsequent to its cytoskeletal recruitment and is accompanied by p130Cas cytoskeletal binding and focal adhesion kinase (FAK) Tyr925 phosphorylation. When cardiomyocytes expressing hexahistidine-tagged c-Src via adenoviral gene delivery were used for RGD stimulation, the expressed c-Src exhibited relocation: (i) biochemical analysis revealed c-Src movement from the detergent-soluble to the -insoluble cytoskeletal fraction and (ii) confocal microscopic analysis showed c-Src movement from a nuclear/perinuclear to a sarcolemmal region. RGD treatment also caused sarcolemmal co-localization of FAK and vinculin. Characterization of integrin subtypes revealed that beta3, but not beta1, integrin plays a predominant role: (i) expression of cytoplasmic domain of beta1A integrin did not affect the RGD-stimulated FAC formation and (ii) both pressure-overloaded myocardium and RGD-stimulated cardiomyocytes exhibited phosphorylation of beta3 integrin at Tyr773/785 sites but not beta1 integrin at Thr788/789 sites. Together these data indicate that RGD treatment in cardiomyocytes causes beta3 integrin activation and c-Src sarcolemmal localization, that subsequent c-Src activation is accompanied by p130Cas binding and FAK Tyr925 phosphorylation, and that these events might be crucial for growth and remodeling of hypertrophying adult cardiomyocytes.

  6. [Expression of integrin alpha5 and actin in the cells of intervertebral disc under cyclic hydrostatic pressure in vitro].

    PubMed

    Yu, Sheng-ji; Qiu, Gui-xing; Burton, Yang; Sandra, Roth; Cari, Whyne; Albert, Yee

    2005-12-15

    To investigate the expression of integrin alpha5 and actin in the cells of intervertebral disc under cyclic hydrostatic pressure in vitro. The porcine lumbar intervertebral disc cells were isolated and cultured in vitro, and the cells underwent cyclic hydrostatic loading. After that, the expression of integrin alpha5 and actin in intervertebral disc cells were studied by means of morphology observing, Western blot and immunohistochemistry staining. The morphology of intervertebral disc cells were changed into smaller and flatten shape, and the expression of integrin alpha5 and actin were decreased after loading. The expression of integrin alpha5 decreases under cyclic hydrostatic pressure, and the actin is affected at the same time when signals are transferred into the cells by integrin alpha5. That may be one of the important mechanisms of the mechanotransduction in the cells of intervertebral disc.

  7. Requirement of Vascular Integrin α_vβ_3 for Angiogenesis

    NASA Astrophysics Data System (ADS)

    Brooks, Peter C.; Clark, Richard A. F.; Cheresh, David A.

    1994-04-01

    Angiogenesis depends on the adhesive interactions of vascular cells. The adhesion receptor integrin α_vβ_3 was identified as a marker of angiogenic vascular tissue. Integrin α_vβ_3 was expressed on blood vessels in human wound granulation tissue but not in normal skin, and it showed a fourfold increase in expression during angiogenesis on the chick chorioallantoic membrane. In the latter assay, a monoclonal antibody to α_vβ_3 blocked angiogenesis induced by basic fibroblast growth factor, tumor necrosis factor-α, and human melanoma fragments but had no effect on preexisting vessels. These findings suggest that α_vβ_3 may be a useful therapeutic target for diseases characterized by neovascularization.

  8. The integrin expression profile modulates orientation and dynamics of force transmission at cell-matrix adhesions.

    PubMed

    Balcioglu, Hayri E; van Hoorn, Hedde; Donato, Dominique M; Schmidt, Thomas; Danen, Erik H J

    2015-04-01

    Integrin adhesion receptors connect the extracellular matrix (ECM) to the cytoskeleton and serve as bidirectional mechanotransducers. During development, angiogenesis, wound healing and cancer progression, the relative abundance of fibronectin receptors, including integrins α5β1 and αvβ3, changes, thus altering the integrin composition of cell-matrix adhesions. Here, we show that enhanced αvβ3 expression can fully compensate for loss of α5β1 and other β1 integrins to support outside-in and inside-out force transmission. α5β1 and αvβ3 each mediate actin cytoskeletal remodeling in response to stiffening or cyclic stretching of the ECM. Likewise, α5β1 and αvβ3 support cellular traction forces of comparable magnitudes and similarly increase these forces in response to ECM stiffening. However, cells using αvβ3 respond to lower stiffness ranges, reorganize their actin cytoskeleton more substantially in response to stretch, and show more randomly oriented traction forces. Centripetal traction force orientation requires long stress fibers that are formed through the action of Rho kinase (ROCK) and myosin II, and that are supported by α5β1. Thus, altering the relative abundance of fibronectin-binding integrins in cell-matrix adhesions affects the spatiotemporal organization of force transmission. © 2015. Published by The Company of Biologists Ltd.

  9. Very late antigen integrins are involved in the adhesive interaction of lymphoid cells to human gingival fibroblasts.

    PubMed Central

    Murakami, S; Saho, T; Shimabukuro, Y; Isoda, R; Miki, Y; Okada, H

    1993-01-01

    To date, it is still unclear how the trafficking and retention of activated lymphocytes in periodontal lesions are regulated. In this study, we investigated the molecular basis for the adhesive interactions between lymphocytes and human gingival fibroblasts (HGF). Peripheral blood T lymphocytes (PBT) exhibited binding ability, but only when the calls were activated with phorbol 12-myristate 13-acetate (PMA). Among several human cell lines tested, PMA-stimulated Molt-4, a human T-cell leukaemia line, also displayed significant binding ability to HGF. In order to clarify the molecule(s) involved in this cell-cell interaction, a panel of monoclonal antibodies (mAb) was prepared to PMA-activated Molt-4 and one clone, 4-145, was selected on the basis of its ability to block the binding of PMA-activated Molt-4 to HGF. Moreover, 4-145 inhibited the binding of not only activated Molt-4 but also activated PBT and other cell types to HGF. Biochemical and flow cytometric analyses revealed that 4-145 probably recognizes the beta 1 chain of very late antigen (VLA) integrins. Blocking experiments using mAb specific for the alpha-chain of VLA integrins demonstrated the involvement of alpha 4 (VLA-4) and, to a lesser extent, alpha 5 (VLA-5) chains in the adhesive interactions between T cells and HGF. Despite the significant involvement of VLA integrins in the adhesive interaction between PBT and HGF, the binding of PBT to human dermal fibroblasts (HDF) was not abrogated by 4-145, suggesting that HGF and HDF differ in their requirement of VLA integrins for adhesion to activated PBT. Furthermore, the fact that vascular cell adhesion molecule-1 (VCAM-1), one of the ligands of VLA-4, was not detected on HGF by flow cytometry and anti-fibronectin (FN) Ab did not block the adhesive interaction to HGF suggests that not-yet-identified ligand(s) for VLA-4 might be present on HGF. Images Figure 4 PMID:8406571

  10. A dual role for Integrin α6β4 in modulating hereditary neuropathy with liability to pressure palsies.

    PubMed

    Poitelon, Yannick; Matafora, Vittoria; Silvestri, Nicholas; Zambroni, Desirée; McGarry, Claire; Serghany, Nora; Rush, Thomas; Vizzuso, Domenica; Court, Felipe A; Bachi, Angela; Wrabetz, Lawrence; Feltri, Maria Laura

    2018-05-01

    Peripheral myelin protein 22 (PMP22) is a component of compact myelin in the peripheral nervous system. The amount of PMP22 in myelin is tightly regulated, and PMP22 over or under-expression cause Charcot-Marie-Tooth 1A (CMT1A) and Hereditary Neuropathy with Pressure Palsies (HNPP). Despite the importance of PMP22, its function remains largely unknown. It was reported that PMP22 interacts with the β4 subunit of the laminin receptor α6β4 integrin, suggesting that α6β4 integrin and laminins may contribute to the pathogenesis of CMT1A or HNPP. Here we asked if the lack of α6β4 integrin in Schwann cells influences myelin stability in the HNPP mouse model. Our data indicate that PMP22 and β4 integrin may not interact directly in myelinating Schwann cells, however, ablating β4 integrin delays the formation of tomacula, a characteristic feature of HNPP. In contrast, ablation of integrin β4 worsens nerve conduction velocities and non-compact myelin organization in HNPP animals. This study demonstrates that indirect interactions between an extracellular matrix receptor and a myelin protein influence the stability and function of myelinated fibers. © 2018 International Society for Neurochemistry.

  11. Integrins engage mitochondrial function for signal transduction by a mechanism dependent on Rho GTPases

    PubMed Central

    Werner, Erica; Werb, Zena

    2002-01-01

    We show here the transient activation of the small GTPase Rac, followed by a rise in reactive oxygen species (ROS), as necessary early steps in a signal transduction cascade that lead to NFκB activation and collagenase-1 (CL-1)/matrix metalloproteinase-1 production after integrin-mediated cell shape changes. We show evidence indicating that this constitutes a new mechanism for ROS production mediated by small GTPases. Activated RhoA also induced ROS production and up-regulated CL-1 expression. A Rac mutant (L37) that prevents reorganization of the actin cytoskeleton prevented integrin-induced CL-1 expression, whereas mutations that abrogate Rac binding to the neutrophil NADPH membrane oxidase in vitro (H26 and N130) did not. Instead, ROS were produced by integrin-induced changes in mitochondrial function, which were inhibited by Bcl-2 and involved transient membrane potential loss. The cells showing this transient decrease in mitochondrial membrane potential were already committed to CL-1 expression. These results unveil a new molecular mechanism of signal transduction triggered by integrin engagement where a global mitochondrial metabolic response leads to gene expression rather than apoptosis. PMID:12119354

  12. Integrins engage mitochondrial function for signal transduction by a mechanism dependent on Rho GTPases.

    PubMed

    Werner, Erica; Werb, Zena

    2002-07-22

    We show here the transient activation of the small GTPase Rac, followed by a rise in reactive oxygen species (ROS), as necessary early steps in a signal transduction cascade that lead to NFkappaB activation and collagenase-1 (CL-1)/matrix metalloproteinase-1 production after integrin-mediated cell shape changes. We show evidence indicating that this constitutes a new mechanism for ROS production mediated by small GTPases. Activated RhoA also induced ROS production and up-regulated CL-1 expression. A Rac mutant (L37) that prevents reorganization of the actin cytoskeleton prevented integrin-induced CL-1 expression, whereas mutations that abrogate Rac binding to the neutrophil NADPH membrane oxidase in vitro (H26 and N130) did not. Instead, ROS were produced by integrin-induced changes in mitochondrial function, which were inhibited by Bcl-2 and involved transient membrane potential loss. The cells showing this transient decrease in mitochondrial membrane potential were already committed to CL-1 expression. These results unveil a new molecular mechanism of signal transduction triggered by integrin engagement where a global mitochondrial metabolic response leads to gene expression rather than apoptosis.

  13. Integrin αvβ6 critically regulates hepatic progenitor cell function and promotes ductular reaction, fibrosis, and tumorigenesis.

    PubMed

    Peng, Zhen-Wei; Ikenaga, Naoki; Liu, Susan B; Sverdlov, Deanna Y; Vaid, Kahini A; Dixit, Richa; Weinreb, Paul H; Violette, Shelia; Sheppard, Dean; Schuppan, Detlef; Popov, Yury

    2016-01-01

    Integrin αvβ6 is rapidly up-regulated on cells of epithelial lineage during tissue injury, where one of its primary functions is activation of latent transforming growth factor beta 1 (TGFβ1). In human liver cirrhosis, αvβ6 is overexpressed by cells comprising the ductular reaction, and its inhibition suppresses experimental biliary fibrosis in rodents. Here, we show that αvβ6 is expressed on the actively proliferating subset of hepatic progenitor cells and is required for their progenitor function in vivo and in vitro through integrin αvβ6-dependent TGFβ1 activation. Freshly isolated αvβ6(+) liver cells demonstrate clonogenic potential and differentiate into cholangiocytes and functional hepatocytes in vitro, whereas colony formation by epithelial cell adhesion molecule-positive progenitor cells is blocked by αvβ6-neutralizing antibody and in integrin beta 6-deficient cells. Inhibition of progenitors by anti-αvβ6 antibody is recapitulated by TGFβ1 neutralization and rescued by addition of bioactive TGFβ1. Genetic disruption or selective targeting of αvβ6 with 3G9 antibody potently inhibits progenitor cell responses in mouse models of chronic biliary injury and protects from liver fibrosis and tumorigenesis, two conditions clinically associated with exacerbated ductular reaction. These results suggest that αvβ6 is a promising target for chronic fibrotic liver diseases and associated cancers. © 2015 by the American Association for the Study of Liver Diseases.

  14. Anti-IL-5 attenuates activation and surface density of β(2) -integrins on circulating eosinophils after segmental antigen challenge.

    PubMed

    Johansson, M W; Gunderson, K A; Kelly, E A B; Denlinger, L C; Jarjour, N N; Mosher, D F

    2013-03-01

    IL-5 activates α(M) β(2) integrin on blood eosinophils in vitro. Eosinophils in bronchoalveolar lavage (BAL) following segmental antigen challenge have activated β(2) -integrins. To identify roles for IL-5 in regulating human eosinophil integrins in vivo. Blood and BAL eosinophils were analysed by flow cytometry in ten subjects with allergic asthma who underwent a segmental antigen challenge protocol before and after anti-IL-5 administration. Blood eosinophil reactivity with monoclonal antibody (mAb) KIM-127, which recognizes partially activated β(2) -integrins, was decreased after anti-IL-5. Before anti-IL-5, surface densities of blood eosinophil β(2) , α(M) and α(L) integrin subunits increased modestly post challenge. After anti-IL-5, such increases did not occur. Before or after anti-IL-5, surface densities of β(2) , α(M) , α(L) and α(D) and reactivity with KIM-127 and mAb CBRM1/5, which recognizes high-activity α(M) β(2) , were similarly high on BAL eosinophils 48 h post-challenge. Density and activation state of β(1) -integrins on blood and BAL eosinophils were not impacted by anti-IL-5, even though anti-IL-5 ablated a modest post-challenge increase on blood or BAL eosinophils of P-selectin glycoprotein ligand-1 (PSGL-1), a receptor for P-selectin that causes activation of β(1) -integrins. Forward scatter of blood eosinophils post-challenge was less heterogeneous and on the average decreased after anti-IL-5; however, anti-IL-5 had no effect on the decreased forward scatter of eosinophils in post-challenge BAL compared with eosinophils in blood. Blood eosinophil KIM-127 reactivity at the time of challenge correlated with the percentage of eosinophils in BAL post-challenge. IL-5 supports a heterogeneous population of circulating eosinophils with partially activated β(2) -integrins and is responsible for up-regulation of β(2) -integrins and PSGL-1 on circulating eosinophils following segmental antigen challenge but has minimal effects on

  15. Foot-and-Mouth Disease Virus Receptors: Comparison of Bovine αV Integrin Utilization by Type A and O Viruses

    PubMed Central

    Duque, Hernando; Baxt, Barry

    2003-01-01

    Three members of the αV integrin family of cellular receptors, αVβ1, αVβ3, and αVβ6, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid (RGD) amino acid sequence motif located within the βG-βH (G-H) loop of VP1. Other αV integrins, as well as several other integrins, recognize and bind to RGD motifs on their natural ligands and also may be candidate receptors for FMDV. To analyze the roles of the αV integrins from a susceptible species as viral receptors, we molecularly cloned the bovine β1, β5, and β6 integrin subunits. Using these subunits, along with previously cloned bovine αV and β3 subunits, in a transient expression assay system, we compared the efficiencies of infection mediated by αVβ1, αVβ3, αVβ5, and αVβ6 among three strains of FMDV serotype A and two strains of serotype O. While all the viruses could infect cells expressing these integrins, they exhibited different efficiencies of integrin utilization. All the type A viruses used αVβ3 and αVβ6 with relatively high efficiency, while only one virus utilized αVβ1 with moderate efficiency. In contrast, both type O viruses utilized αVβ6 and αVβ1 with higher efficiency than αVβ3. Only low levels of viral replication were detected in αVβ5-expressing cells infected with either serotype. Experiments in which the ligand-binding domains among the β subunits were exchanged indicated that this region of the integrin subunit appears to contribute to the differences in integrin utilizations among strains. In contrast, the G-H loops of the different viruses do not appear to be involved in this phenomenon. Thus, the ability of the virus to utilize multiple integrins in vitro may be a reflection of the use of multiple receptors during the course of infection within the susceptible host. PMID:12551988

  16. Anti-IL-5 attenuates activation and surface density of β2-integrins on circulating eosinophils after segmental antigen challenge

    PubMed Central

    Johansson, Mats W.; Gunderson, Kristin A.; Kelly, Elizabeth A. B.; Denlinger, Loren C.; Jarjour, Nizar N.; Mosher, Deane F.

    2013-01-01

    Background IL-5 activates αMβ2 integrin on blood eosinophils in vitro. Eosinophils in bronchoalveolar lavage (BAL) following segmental antigen challenge have activated β2-integrins. Objective To identify roles for IL-5 in regulating human eosinophil integrins in vivo. Methods Blood and BAL eosinophils were analyzed by flow cytometry in ten subjects with allergic asthma who underwent a segmental antigen challenge protocol before and after anti-IL-5 administration. Results Blood eosinophil reactivity with monoclonal antibody (mAb) KIM-127, which recognizes partially activated β2-integrins, was decreased after anti-IL-5. Before anti-IL-5, surface densities of blood eosinophil β2, αM, and αL integrin subunits increased modestly post-challenge. After anti-IL-5, such increases did not occur. Before or after anti-IL-5, surface densities of β2,αM, αL, and αD and reactivity with KIM-127 and mAb CBRM1/5, which recognizes high-activity αMβ2, were similarly high on BAL eosinophils 48 h post-challenge. Density and activation state of β1-integrins on blood and BAL eosinophils were not impacted by anti-IL-5, even though anti-IL-5 ablated a modest post-challenge increase on blood or BAL eosinophils of P-selectin glycoprotein ligand-1 (PSGL-1), a receptor for P-selectin that causes activation of β1-integrins. Forward scatter of blood eosinophils post-challenge was less heterogeneous and on the average decreased after anti-IL-5; however, anti-IL-5 had no effect on the decreased forward scatter of eosinophils in post-challenge BAL compared to eosinophils in blood. Blood eosinophil KIM-127 reactivity at the time of challenge correlated with the percentage of eosinophils in BAL post-challenge. Conclusion and Clinical Relevance IL-5 supports a heterogeneous population of circulating eosinophils with partially activated β2-integrins and is responsible for upregulation of β2-integrins and PSGL-1 on circulating eosinophils following segmental antigen challenge but has

  17. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

    PubMed

    Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P

    The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

  18. EGFRvIII/integrin β3 interaction in hypoxic and vitronectinenriching microenvironment promote GBM progression and metastasis.

    PubMed

    Liu, Zhaoyu; Han, Lei; Dong, Yucui; Tan, Yanli; Li, Yongsheng; Zhao, Manli; Xie, Hui; Ju, Huanyu; Wang, He; Zhao, Yu; Zheng, Qifan; Wang, Qixue; Su, Jun; Fang, Chuan; Fu, Songbin; Jiang, Tao; Liu, Jiaren; Li, Xia; Kang, Chunsheng; Ren, Huan

    2016-01-26

    Glioblastoma (GBM) is one of the most lethal brain tumors with a short survival time. EGFR amplification and mutation is the most significant genetic signature in GBM. About half of the GBMs with EGFR amplification express a constitutively autophosphorylated variant of EGFR, known as EGFRvIII. Our in vitro data demonstrated further enhanced EGFRvIII activity and tumor cell invasion in the tumor microenvironment of hypoxia plus extracellular matrix (ECM) vitronectin, in which EGFRvIII and integrin β3 tended to form complexes. The treatment with ITGB3 siRNA or the integrin antagonist cilengetide preferentially interrupted the EGFRvIII/integrin β3 complex, effectively reduced tumor cell invasion and activation of downstream signaling effectors. Cilengitide is recently failed in Phase III CENTRIC trial in unselected patients with GBM. However, we found that cilengitide demonstrated efficacious tumor regression via inhibition of tumor growth and angiogenesis in EGFRvIII orthotopic xenografts. Bioinformatics analysis emphasized key roles of integrin β3, hypoxia and vitronectin and their strong correlations with EGFRvIII expression in malignant glioma patient samples in vivo. In conclusion, we demonstrate that EGFRvIII/integrin β3 complexes promote GBM progression and metastasis in the environment of hypoxia and vitronectin-enrichment, and cilengitide may serve as a promising therapeutics for EGFRvIII-positive GBMs.

  19. WAVE2 Regulates High-Affinity Integrin Binding by Recruiting Vinculin and Talin to the Immunological Synapse▿

    PubMed Central

    Nolz, Jeffrey C.; Medeiros, Ricardo B.; Mitchell, Jason S.; Zhu, Peimin; Freedman, Bruce D.; Shimizu, Yoji; Billadeau, Daniel D.

    2007-01-01

    T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin. PMID:17591693

  20. WAVE2 regulates high-affinity integrin binding by recruiting vinculin and talin to the immunological synapse.

    PubMed

    Nolz, Jeffrey C; Medeiros, Ricardo B; Mitchell, Jason S; Zhu, Peimin; Freedman, Bruce D; Shimizu, Yoji; Billadeau, Daniel D

    2007-09-01

    T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin.

  1. Selecting the correct cellular model for assessing of the biological response of collagen-based biomaterials.

    PubMed

    Davidenko, Natalia; Hamaia, Samir; Bax, Daniel V; Malcor, Jean-Daniel; Schuster, Carlos F; Gullberg, Donald; Farndale, Richard W; Best, Serena M; Cameron, Ruth E

    2018-01-01

    the cell adhesion results, showing receptor class- and species-specificities. The understanding of the physiologically relevant cell anchorage characteristics of bio-constructs may assist in the selection of (1) the optimum collagen source for cellular supports and (2) the correct cellular model for their biological assessment. This, in turn, may allow reliable prediction of the biological performance of bio-scaffolds in vivo for specific TE applications. Integrins play a vital role in cellular responses to environmental cues during early-stage cell-substrate interaction. We describe physiologically relevant cell anchorage to collagen substrates that present different affinity cell-recognition motifs, to provide experimental tools to assist in understanding integrin binding. Using different cell types and recombinant integrin α1-I-domains, we found that cellular response was highly dependent on collagen type, origin and EDC-crosslinking status, as well as on the integrin class and species of origin. This comprehensive study establishes selectivity amongst the four collagen-binding integrins and species-specific properties that together may influence choice of cell type and receptor in different experimental settings. This work offers key guidance in selecting of the correct cellular model for the biological testing of collagen-based biomaterials. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  2. Oroxylin A reverses CAM-DR of HepG2 cells by suppressing Integrinβ1 and its related pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhu, Binbin; Zhao, Li; Zhu, Litao

    Oroxylin A, a naturally occurring monoflavonoid extracted from Scutellariae radix, shows effective anticancer activities and low toxicities both in vivo and in vitro in previous studies. In this study, we investigated whether the CAM-DR model of HepG2 cells showed resistance to cytotoxic agents compared with normally cultured HepG2 cells. Furthermore, after the treatment of Paclitaxel, less inhibitory effects and decreased apoptosis rate were detected in the model. Data also revealed increased expression of Integrinβ1 might be responsible for the resistance ability. Moreover, Integrinβ1-siRNA-transfected CAM-DR HepG2 cells exhibited more inhibitory effects and higher levels of apoptosis than the non-transfected CAM-DR cells.more » The data corroborated that Integrinβ1 played a significant role in CAM-DR. After the treatment of weakly-toxic concentrations of Oroxylin A, the apoptosis induced by Paclitaxel in the CAM-DR model increased dramatically. Western blot assay revealed Oroxylin A markedly down-regulated the expression of Integrinβ1 and the activity of related pathway. As a conclusion, Oroxylin A can reverse the resistance of CAM-DR via inhibition of Integrinβ1 and its related pathway. Oroxylin A may be a potential candidate of a CAM-DR reversal agent. Highlights: ► Adhesion of HepG2 cells to fibronectin exhibited resistance to Paclitaxel. ► The resistance was associated with the increased expression of Integrinβ1. ► Knocking down Integrinβ1 can increase the toxicity of Paclitaxel on CAM-DR model. ► Oroxylin A reversed the resistance by suppressing Integrinβ1 and related pathway.« less

  3. Epigenetic Regulation of Galectin-3 Expression by β1 Integrins Promotes Cell Adhesion and Migration*

    PubMed Central

    Margadant, Coert; van den Bout, Iman; van Boxtel, Antonius L.; Thijssen, Victor L.; Sonnenberg, Arnoud

    2012-01-01

    Introduction of the integrin β1- but not the β3-subunit in GE11 cells induces an epithelial-to-mesenchymal-transition (EMT)-like phenomenon that is characterized by the loss of cell-cell contacts, cell scattering, increased cell migration and RhoA activity, and fibronectin fibrillogenesis. Because galactose-binding lectins (galectins) have been implicated in these phenomena, we investigated whether galectins are involved in the β1-induced phenotype. We examined 9 galectins and, intriguingly, found that the expression of galectin-3 (Gal-3) is specifically induced by β1 but not by β3. Using β1-β3 chimeric integrins, we show that the induction of Gal-3 expression requires the hypervariable region in the extracellular domain of β1, but not its cytoplasmic tail. Furthermore, Gal-3 expression does not depend on RhoA signaling, serum factors, or any of the major signal transduction pathways involving protein kinase C (PKC), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase-1/-2 (ERK-1/2), phosphatidylinositol-3-OH kinase (PI3-K), or Src kinases. Instead, Gal-3 expression is controlled in an epigenetic manner. Whereas DNA methylation of the Lgals3 promoter maintains Gal-3 silencing in GE11 cells, expression of β1 causes its demethylation, leading to transcriptional activation of the Lgals3 gene. In turn, Gal-3 expression enhances β1 integrin-mediated cell adhesion to fibronectin (FN) and laminin (LN), as well as cell migration. Gal-3 also promotes β1-mediated cell adhesion to LN and Collagen-1 (Col)-1 in cells that endogenously express Gal-3 and β1 integrins. In conclusion, we identify a functional feedback-loop between β1 integrins and Gal-3 that involves the epigenetic induction of Gal-3 expression during integrin-induced EMT and cell scattering. PMID:23118221

  4. Bombesin-dependent pro-MMP-9 activation in prostatic cancer cells requires beta1 integrin engagement.

    PubMed

    Festuccia, Claudio; Angelucci, Adriano; Gravina, Giovanni; Eleuterio, Enrica; Vicentini, Carlo; Bologna, Mauro

    2002-10-15

    Bombesin-like peptides, including the mammalian homologue gastrin-releasing peptide, are highly expressed and secreted by neuroendocrine cells in prostate carcinoma tissues and are likely to be related to the progression of this neoplastic disease. Previously, we demonstrated that bombesin increased migration and protease expression in androgen-independent cells. In this work we show that bombesin is able to activate pro-MMP-9 through a mechanism involving the beta1 integrin subunit. In fact, MMP-9 processing was evident only when beta1 integrin was engaged with specific adhesive substrates, such as type I collagen, or when cells were seeded on dishes coated with antibodies against beta1 integrin, resulting in activation of the surface ligand. When exogenous pro-MMP-9 was added to PC3 cells, MMP-9 active forms were produced within 30 min by bombesin-treated cultures while control cultures expressed activated forms only after a longer time and at lower levels. MMP-9 activation required cytoskeleton integrity since this effect was abolished by cytochalasin D. Engagement of beta1 integrin caused an increased membrane-linked uPA activity which was required for MMP-9 activation. The cross talk between bombesin- and beta1-integrin-engaged signals seems to be crucial for the modulation of both membrane-linked uPA activity and MMP-9 activation and triggers complex intracellular signaling pathways requiring activation of tyrosine kinase activity, including that of src and PI3K. The beta1 integrin may be considered an important mechanism by which bombesin induces MMP-9 activation. This finding supports the idea that cellular responses to growth factors may be driven by cell-matrix interactions and stresses the role of neuroendocrine factors in prostate carcinoma progression.

  5. Cross-activating c-Met/β1 integrin complex drives metastasis and invasive resistance in cancer

    PubMed Central

    Jahangiri, Arman; Nguyen, Alan; Sidorov, Maxim K.; Yagnik, Garima; Rick, Jonathan; Han, Sung Won; Chen, William; Flanigan, Patrick M.; Schneidman-Duhovny, Dina; Mascharak, Smita; De Lay, Michael; Imber, Brandon; Park, Catherine C.; Matsumoto, Kunio; Lu, Kan; Bergers, Gabriele; Sali, Andrej; Weiss, William A.

    2017-01-01

    The molecular underpinnings of invasion, a hallmark of cancer, have been defined in terms of individual mediators but crucial interactions between these mediators remain undefined. In xenograft models and patient specimens, we identified a c-Met/β1 integrin complex that formed during significant invasive oncologic processes: breast cancer metastases and glioblastoma invasive resistance to antiangiogenic VEGF neutralizing antibody, bevacizumab. Inducing c-Met/β1 complex formation through an engineered inducible heterodimerization system promoted features crucial to overcoming stressors during metastases or antiangiogenic therapy: migration in the primary site, survival under hypoxia, and extravasation out of circulation. c-Met/β1 complex formation was up-regulated by hypoxia, while VEGF binding VEGFR2 sequestered c-Met and β1 integrin, preventing their binding. Complex formation promoted ligand-independent receptor activation, with integrin-linked kinase phosphorylating c-Met and crystallography revealing the c-Met/β1 complex to maintain the high-affinity β1 integrin conformation. Site-directed mutagenesis verified the necessity for c-Met/β1 binding of amino acids predicted by crystallography to mediate their extracellular interaction. Far-Western blotting and sequential immunoprecipitation revealed that c-Met displaced α5 integrin from β1 integrin, creating a complex with much greater affinity for fibronectin (FN) than α5β1. Thus, tumor cells adapt to microenvironmental stressors induced by metastases or bevacizumab by coopting receptors, which normally promote both cell migration modes: chemotaxis, movement toward concentrations of environmental chemoattractants, and haptotaxis, movement controlled by the relative strengths of peripheral adhesions. Tumor cells then redirect these receptors away from their conventional binding partners, forming a powerful structural c-Met/β1 complex whose ligand-independent cross-activation and robust affinity for FN

  6. Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin β1

    PubMed Central

    Klockenbusch, Cordula; Kast, Juergen

    2010-01-01

    Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β1, α4 and α6 or β1, α6, α2, and α5, respectively. PMID:20634879

  7. Identification, Characterization, and Epitope Mapping of Human Monoclonal Antibody J19 That Specifically Recognizes Activated Integrin α4β7*

    PubMed Central

    Qi, JunPeng; Zhang, Kun; Zhang, Qiao; Sun, Yi; Fu, Ting; Li, GuoHui; Chen, JianFeng

    2012-01-01

    Integrin α4β7 is a lymphocyte homing receptor that mediates both rolling and firm adhesion of lymphocytes on vascular endothelium, two of the critical steps in lymphocyte migration and tissue-specific homing. The rolling and firm adhesions of lymphocytes rely on the dynamic shift between the inactive and active states of integrin α4β7, which is associated with the conformational rearrangement of integrin molecules. Activation-specific antibodies, which specifically recognize the activated integrins, have been used as powerful tools in integrin studies, whereas there is no well characterized activation-specific antibody to integrin α4β7. Here, we report the identification, characterization, and epitope mapping of an activation-specific human mAb J19 against integrin α4β7. J19 was discovered by screening a human single-chain variable fragment phage library using an activated α4β7 mutant as target. J19 IgG specifically bound to the high affinity α4β7 induced by Mn2+, DTT, ADP, or CXCL12, but not to the low affinity integrin. Moreover, J19 IgG did not interfere with α4β7-MAdCAM-1 interaction. The epitope of J19 IgG was mapped to Ser-331, Ala-332, and Ala-333 of β7 I domain and a seven-residue segment from 184 to 190 of α4 β-propeller domain, which are buried in low affinity integrin with bent conformation and only exposed in the high affinity extended conformation. Taken together, J19 is a potentially powerful tool for both studies on α4β7 activation mechanism and development of novel therapeutics targeting the activated lymphocyte expressing high affinity α4β7. PMID:22418441

  8. Prognostic role of integrin β1, E-cadherin, and rac1 expression in small cell lung cancer.

    PubMed

    Chang, Myung Hee; Lee, Kyungji; Lee, Kyo-Young; Kim, Yeon Sil; Kim, Young Kyoon; Kang, Jin-Hyoung

    2012-01-01

    Integrin β(1) mediates cellular adhesion to the extracellular matrix (ECM) and is correlated with highly invasive and metastatic behavior in small cell lung cancer (SCLC). E-cadherin (ECAD) is a calcium-dependent cell-cell adhesion receptor that restricts invasion of cells and reduces metastasis. Rac1 is involved in the regulation of the actin cytoskeleton, adhesion, migration, invasion, and tumor metastasis. The aim of this study was to examine integrin β(1) , ECAD and rac1 expression in SCLC and to analyze the prognostic value of these markers in patients with SCLC. We analyzed integrin β(1) , ECAD, and rac1 expression in 112 SCLC tissues by immunohistochemical staining. Correlative analyses between integrin β(1) , ECAD, and rac1 expression and cliniopathological factors were performed. A total of 65 patients had extensive disease (ED) (58%), and 47 had limited disease (LD) (42%). The median follow-up duration was 61 months (range: 14-117 months), and the median progression free survival (PFS) and overall survival (OS) were 6.1 months (range: 4.8-7.4 months) and 9.7 months (range: 8.1-11.3 months), respectively. The expression of integrin β(1) , ECAD, and rac1 protein was observed in 64, 73, and 99 of SCLC tissues, respectively. The correlative analyses between integrin β(1) , ECAD, or rac1 expression and various clinical parameters did not show any statistical significance. However, the ECAD expression was associated with OS in the entire cohort. In contrast, the expression of integrin β(1) and rac1 was not associated with PFS or OS. In a subgroup analysis, patients with less than two metastasis had significantly longer OS (p = 0.047) if their tumors expressed integrin β(1) compared to those without integrin β(1) expression. In addition, OS was longer for patients with ECAD positive tumors compared to those whose tumors did not express ECAD in males (p = 0.032) and patients who never smoked (p < 0.001). Multivariate analysis showed that LD (p = 0

  9. Integrin and GPCR Crosstalk in the Regulation of ASM Contraction Signaling in Asthma.

    PubMed

    Teoh, Chun Ming; Tam, John Kit Chung; Tran, Thai

    2012-01-01

    Airway hyperresponsiveness (AHR) is one of the cardinal features of asthma. Contraction of airway smooth muscle (ASM) cells that line the airway wall is thought to influence aspects of AHR, resulting in excessive narrowing or occlusion of the airway. ASM contraction is primarily controlled by agonists that bind G protein-coupled receptor (GPCR), which are expressed on ASM. Integrins also play a role in regulating ASM contraction signaling. As therapies for asthma are based on symptom relief, better understanding of the crosstalk between GPCRs and integrins holds good promise for the design of more effective therapies that target the underlying cellular and molecular mechanism that governs AHR. In this paper, we will review current knowledge about integrins and GPCRs in their regulation of ASM contraction signaling and discuss the emerging concept of crosstalk between the two and the implication of this crosstalk on the development of agents that target AHR.

  10. A Signaling Network Induced by β2 Integrin Controls the Polarization of Lytic Granulesin Cytotoxic Cells

    PubMed Central

    Zhang, Minggang; March, Michael E.; Lane, William S.; Long, Eric O.

    2014-01-01

    Cytotoxic lymphocyte skill target cells by polarized release of the content of perforin-containing granules. In natural killer cells, the binding of β2 integrin to its ligand ICAM-1 is sufficient to promote not only adhesion but also lytic granule polarization. This provided a unique opportunity to study polarization in the absence of degranulation, and β2 integrin signaling independently of inside-out signals from other receptors. Using an unbiased proteomics approach we identified a signaling network centered on an integrin-linked kinase (ILK)–Pyk2–Paxillin core that was required for granule polarization. Downstream of ILK, the highly conserved Cdc42–Par6 signaling pathway that controls cell polarity was activated and required for granule polarization. These results delineate two connected signaling networks induced upon β2 integrin engagement alone, which are integrated to control polarization of the microtubule organizing center and associated lytic granules toward the site of contact with target cells during cellular cytotoxicity. PMID:25292215

  11. Pathogenetic role of Arg-Gly-Asp-recognizing integrins in acute renal failure. off.

    PubMed Central

    Goligorsky, M S; DiBona, G F

    1993-01-01

    Reorientation of the alpha 3 subunit of integrins from predominantly basal to the apical cell surface of cultured renal tubular epithelial cells subjected to oxidant stress has previously been demonstrated. The present study was designed to assess functional competence of ectopically expressed apical integrins. Cell-cell adhesion assay revealed enhanced cytoatractant properties of stressed cells. Stressed epithelial cells exhibited specific recognition and binding of laminin-coated latex beads. These processes were inhibited with the peptide Gly-Arg-Gly-Asp-Asn-Pro (GRGDNP) suggesting a role of RGD-recognizing integrins in augmented adhesion to stressed cells. Given that such enhanced adhesion in in vivo acute renal failure may govern tubular obstruction by desquamated epithelium, a physiological marker of patency of tubular lumen, proximal tubular pressure, was monitored in rats subjected to 60 min of renal ischemia followed by reperfusion. Proximal tubular pressure increased 2-fold after 2 hr of reperfusion in animals that had undergone 60 min of ischemia. Infusion of GRGDNP into the renal artery during reperfusion period virtually abolished an increase in proximal tubular pressure observed in ischemic acute renal failure. These in vitro and in vivo findings are consistent with the hypothesis that RGD-recognizing integrins play an important role in the pathogenesis of tubular obstruction in ischemic acute renal failure. Images Fig. 2 Fig. 3 PMID:8516318

  12. NFκB-Induced Periostin Activates Integrin-β3 Signaling to Promote Renal Injury in GN

    PubMed Central

    Prakoura, Niki; Kavvadas, Panagiotis; Kormann, Raphaёl; Dussaule, Jean-Claude; Chadjichristos, Christos E.

    2017-01-01

    De novo expression in the kidney of periostin, a protein involved in odontogenesis and osteogenesis, has been suggested as a biomarker of renal disease. In this study, we investigated the mechanism(s) of induction and the role of periostin in renal disease. Using a combination of bioinformatics, reporter assay, and chromatin immunoprecipitation analyses, we found that NFκB and other proinflammatory transcription factors induce periostin expression in vitro and that binding of these factors on the periostin promoter is enriched in glomeruli during experimental GN. Mice lacking expression of periostin displayed preserved renal function and structure during GN. Furthermore, delayed administration of periostin antisense oligonucleotides in wild-type animals with GN reversed already established proteinuria, diminished tissue inflammation, and improved renal structure. Lack of periostin expression also blunted the de novo renal expression of integrin-β3 and phosphorylation of focal adhesion kinase and AKT, known mediators of integrin-β3 signaling that affect cell motility and survival, observed during GN in wild-type animals. In vitro, recombinant periostin increased the expression of integrin-β3 and the concomitant phosphorylation of focal adhesion kinase and AKT in podocytes. Notably, periostin and integrin-β3 were highly colocalized in biopsy specimens from patients with inflammatory GN. These results demonstrate that interplay between periostin and renal inflammation orchestrates inflammatory and fibrotic responses, driving podocyte damage through downstream activation of integrin-β3 signaling. Targeting periostin may be a novel therapeutic strategy for treating CKD. PMID:27920156

  13. Internal relationship between symptomatic venous thromboembolism and risk factors: up-regulation of integrin β1, β2 and β3 levels

    PubMed Central

    Duan, Qianglin; Wang, Lemin; Yang, Fan; Li, Jue; Song, Yanli; Gong, Zhu; Li, Guiyuan; Song, Haoming; Zhang, Xiaoyu; Shen, Zugang; Dart, Anthony

    2015-01-01

    Background: To compare different expression of core proteins among venous thromboembolism (VTE) and those with risk factor groups and analyze the relative risk for VTE after integrating integrin β1, β2 and β3 expression. Methods: A total of 1006 subjects were recruited and divided into VTE group, risk factor groups and control (non- risk factor) group. Flow cytometry was performed to detect the expression of integrin β1, β2 and β3. The relative risk for VTE was evaluated with independent, parallel and serial methods. Results: The expression of integrin β1 increased markedly in VTE patients, and those with risk factors (acute infection, malignancy, and autoimmune diseases), respectively (P < 0.001 or 0.01). The expression of integrin β1 in trauma/surgery group was not significantly different with control group (P > 0.05). The expression of integrin β2 or β3 significantly increased in VTE group, but that in risk factor groups was not significantly increased (P > 0.05). Multivariate analysis revealed the trauma/surgery groups had no significantly increased risk for VTE. Conclusions: VTE group patients have significantly increased expression of integrin β1, β2 and β3, and risk factor groups (acute infection, malignancy and autoimmune disease) have significantly increased expression of integrin β1. The significant increase in integrin β2, β3 expression is a marker differentiating of VTE group patients with other risk factor groups. Trauma/surgery group has no increased expression of integrin β1, β2 and β3 as other risk factors. Thus, that trauma/surgery may be not the “true” risk factor for VTE. PMID:26045901

  14. Osteoblast mineralization requires β1 integrin/ICAP-1–dependent fibronectin deposition

    PubMed Central

    Brunner, Molly; Millon-Frémillon, Angélique; Chevalier, Genevieve; Nakchbandi, Inaam A.; Mosher, Deane; Block, Marc R.

    2011-01-01

    The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1–null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin–containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization. PMID:21768292

  15. The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.

    PubMed

    deHart, Gregory W; Healy, Kevin E; Jones, Jonathan C R

    2003-02-01

    Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the

  16. The Gain-of-Function Integrin β3 Pro33 Variant Alters the Serotonin System in the Mouse Brain.

    PubMed

    Dohn, Michael R; Kooker, Christopher G; Bastarache, Lisa; Jessen, Tammy; Rinaldi, Capria; Varney, Seth; Mazalouskas, Matthew D; Pan, Hope; Oliver, Kendra H; Velez Edwards, Digna R; Sutcliffe, James S; Denny, Joshua C; Carneiro, Ana M D

    2017-11-15

    Engagement of integrins by the extracellular matrix initiates signaling cascades that drive a variety of cellular functions, including neuronal migration and axonal pathfinding in the brain. Multiple lines of evidence link the ITGB3 gene encoding the integrin β3 subunit with the serotonin (5-HT) system, likely via its modulation of the 5-HT transporter (SERT). The ITGB3 coding polymorphism Leu33Pro (rs5918, Pl A2 ) produces hyperactive αvβ3 receptors that influence whole-blood 5-HT levels and may influence the risk for autism spectrum disorder (ASD). Using a phenome-wide scan of psychiatric diagnoses, we found significant, male-specific associations between the Pro33 allele and attention-deficit hyperactivity disorder and ASDs. Here, we used knock-in (KI) mice expressing an Itgb3 variant that phenocopies the human Pro33 variant to elucidate the consequences of constitutively enhanced αvβ3 signaling to the 5-HT system in the brain. KI mice displayed deficits in multiple behaviors, including anxiety, repetitive, and social behaviors. Anatomical studies revealed a significant decrease in 5-HT synapses in the midbrain, accompanied by decreases in SERT activity and reduced localization of SERTs to integrin adhesion complexes in synapses of KI mice. Inhibition of focal adhesion kinase (FAK) rescued SERT function in synapses of KI mice, demonstrating that constitutive active FAK signaling downstream of the Pro32Pro33 integrin αvβ3 suppresses SERT activity. Our studies identify a complex regulation of 5-HT homeostasis and behaviors by integrin αvβ3, revealing an important role for integrins in modulating risk for neuropsychiatric disorders. SIGNIFICANCE STATEMENT The integrin β3 Leu33Pro coding polymorphism has been associated with autism spectrum disorders (ASDs) within a subgroup of patients with elevated blood 5-HT levels, linking integrin β3, 5-HT, and ASD risk. We capitalized on these interactions to demonstrate that the Pro33 coding variation in the murine

  17. Integrins are required for tissue organization and restriction of neurogenesis in regenerating planarians

    PubMed Central

    Seebeck, Florian; März, Martin; Meyer, Anna-Wiebke; Reuter, Hanna; Vogg, Matthias C.; Stehling, Martin; Mildner, Karina; Zeuschner, Dagmar; Rabert, Franziska

    2017-01-01

    Tissue regeneration depends on proliferative cells and on cues that regulate cell division, differentiation, patterning and the restriction of these processes once regeneration is complete. In planarians, flatworms with high regenerative potential, muscle cells express some of these instructive cues. Here, we show that members of the integrin family of adhesion molecules are required for the integrity of regenerating tissues, including the musculature. Remarkably, in regenerating β1-integrin RNAi planarians, we detected increased numbers of mitotic cells and progenitor cell types, as well as a reduced ability of stem cells and lineage-restricted progenitor cells to accumulate at wound sites. These animals also formed ectopic spheroid structures of neural identity in regenerating heads. Interestingly, those polarized assemblies comprised a variety of neural cells and underwent continuous growth. Our study indicates that integrin-mediated cell adhesion is required for the regenerative formation of organized tissues and for restricting neurogenesis during planarian regeneration. PMID:28137894

  18. 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

  19. Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes.

    PubMed

    Terpstra, Leonieke; Prud'homme, Josée; Arabian, Alice; Takeda, Shu; Karsenty, Gérard; Dedhar, Shoukat; St-Arnaud, René

    2003-07-07

    Chondrocyte proliferation and differentiation requires their attachment to the collagen type II-rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.

  20. Expression, purification and renaturation of truncated human integrin β1 from inclusion bodies of Escherichia coli.

    PubMed

    Shi, Tonglin; Zhang, Lichao; Li, Zhuoyu; Newton, Ian P; Zhang, Quanbin

    2015-03-01

    Integrins are a family of transmembrane receptors and among their members, integrin β1 is one of the best known. It plays a very important role in cell adhesion/migration and in cancer metastasis. Preparation of integrin β1 has a great potential value especially in studies focused on its function. To this end, recombinant plasmids were constructed containing DNA segments representing 454 amino acids of the N-terminal of integrin β1. The recombinant plasmid was transformed into Escherichiacoli BL21 (DE3) cells and after induction by isopropyl-β-D-thiogalactopyranoside (IPTG), the recombinant protein (molecular weight: 53 kD) was expressed, mainly in the form of inclusion bodies. The inclusion bodies were solubilized by 8M urea solution then purified by nickel affinity chromatography. The recombinant protein was renatured by a stepwise dialysis and finally dissolved in phosphate buffered saline. The final yield was approximately 5.4 mg/L of culture and the purity of the renatured recombinant protein was greater than 98% as assessed by SDS-PAGE. The integrity of the protein was shown by Western blot using monoclonal antibodies against his-tag and integrin β1. Its secondary structure was verified as native by circular dichroism spectra and the bioactivity of the recombinant protein was displayed through the conformation switch under Mn(2+) stimulation. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Kon-tiki enhances PS2 integrin adhesion and localizes its ligand, Thrombospondin, in the myotendinous junction.

    PubMed

    Pérez-Moreno, Juan J; Espina-Zambrano, Agueda G; García-Calderón, Clara B; Estrada, Beatriz

    2017-03-01

    Cell-extracellular-matrix adhesion is mediated by cell receptors, mainly integrins and transmembrane proteoglycans, which can functionally interact. How these receptors are regulated and coordinated is largely unknown. We show that the conserved transmembrane Drosophila proteoglycan Kon-tiki (Kon, also known as Perdido) interacts with the αPS2βPS integrin (αPS2 is encoded by inflated and βPS by myospheroid ) to mediate muscle-tendon adhesion. kon and inflated double mutant embryos show a synergistic increase in muscle detachment. Furthermore, Kon modulates αPS2βPS signaling at the muscle attachment, since phosphorylated Fak is reduced in kon mutants. This reduction in integrin signaling can be rescued by the expression of a truncated Kon protein containing its transmembrane and extracellular domains, suggesting that these domains are sufficient to mediate this signaling. We show that these domains are sufficient to properly localize the αPS2βPS ligand, Thrombospondin, to the muscle attachment, and to partially rescue Kon-dependent muscle-tendon adhesion. We propose that Kon can engage in a protein complex with αPS2βPS and enhance integrin-mediated signaling and adhesion by recruiting its ligand, which would increase integrin-binding affinity to the extracellular matrix, resulting in the consolidation of the myotendinous junction. © 2017. Published by The Company of Biologists Ltd.

  2. Absence of integrin alpha 7 causes a novel form of muscular dystrophy.

    PubMed

    Mayer, U; Saher, G; Fässler, R; Bornemann, A; Echtermeyer, F; von der Mark, H; Miosge, N; Pöschl, E; von der Mark, K

    1997-11-01

    Integrin alpha 7 beta 1 is a specific cellular receptor for the basement membrane protein laminin-1 (refs 1,2), as well as for the laminin isoforms -2 and -4 (ref. 3). The alpha 7 subunit is expressed mainly in skeletal and cardiac muscle and has been suggested to be involved in differentiation and migration processes during myogenesis. Three cytoplasmic and two extracellular splice variants that have been described are developmentally regulated and expressed in different sites in the muscle. In adult muscle, the alpha 7A and alpha 7B subunits are concentrated in myotendinous junctions but can also be detected in neuromuscular junctions and along the sarcolemmal membrane. To study the potential involvement of alpha 7 integrin, during myogenesis and its role in muscle integrity and function, we generated a null allele of the alpha 7 gene (Itga7) in the germline of mice by homologous recombination in embryonic stem (ES) cells. Surprisingly, mice homozygous for the mutation are viable and fertile, indicating that the alpha 7 beta 1 integrin is not essential for myogenesis. However, histological analysis of skeletal muscle revealed typical symptoms of a progressive muscular dystrophy starting soon after birth, but with a distinct variability in different muscle types. The observed histopathological changes strongly indicate an impairment of function of the myotendinous junctions. These findings demonstrate that alpha 7 beta 1 integrin represents an indispensable linkage between the muscle fibre and the extracellular matrix that is independent of the dystrophin-dystroglycan complex-mediated interaction of the cytoskeleton with the muscle basement membrane.

  3. β1 Integrins Mediate Attachment of Mesenchymal Stem Cells to Cartilage Lesions

    PubMed Central

    Zwolanek, Daniela; Flicker, Magdalena; Kirstätter, Elisabeth; Zaucke, Frank; van Osch, Gerjo J.V.M.; Erben, Reinhold G.

    2015-01-01

    Abstract Mesenchymal stem cells (MSC) may have great potential for cell-based therapies of osteoarthritis. However, after injection in the joint, only few cells adhere to defective articular cartilage and contribute to cartilage regeneration. Little is known about the molecular mechanisms of MSC attachment to defective articular cartilage. Here, we developed an ex vivo attachment system, using rat osteochondral explants with artificially created full-thickness cartilage defects in combination with genetically labeled MSC isolated from bone marrow of human placental alkaline phosphatase transgenic rats. Binding of MSC to full-thickness cartilage lesions was improved by serum, but not hyaluronic acid, and was dependent on the presence of divalent cations. Additional in vitro tests showed that rat MSC attach, in a divalent cation-dependent manner, to collagen I, collagen II, and fibronectin, but not to collagen XXII or cartilage oligomeric matrix protein (COMP). RGD peptides partially blocked the adhesion of MSC to fibronectin in vitro and to cartilage lesions ex vivo. Furthermore, the attachment of MSC to collagen I and II in vitro and to cartilage lesions ex vivo was almost completely abolished in the presence of a β1 integrin blocking antibody. In conclusion, our data suggest that attachment of MSC to ex vivo full-thickness cartilage lesions is almost entirely β1 integrin-mediated, whereby both RGD- and collagen-binding integrins are involved. These findings suggest a key role of integrins during MSC attachment to defective cartilage and may pave the way for improved MSC-based therapies in the future. PMID:26309781

  4. Variation in One Residue Associated with the Metal Ion-Dependent Adhesion Site Regulates αIIbβ3 Integrin Ligand Binding Affinity

    PubMed Central

    Wu, Xue; Xiu, Zhilong; Li, Guohui; Luo, Bing-Hao

    2013-01-01

    The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS) divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala252 and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS) of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala252 to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion. PMID:24116162

  5. Phoyunnanin E inhibits migration of non-small cell lung cancer cells via suppression of epithelial-to-mesenchymal transition and integrin αv and integrin β3.

    PubMed

    Petpiroon, Nareerat; Sritularak, Boonchoo; Chanvorachote, Pithi

    2017-12-29

    The conversion of the epithelial phenotype of cancer cells into cells with a mesenchymal phenotype-so-called epithelial-mesenchymal transition (EMT)-has been shown to enhance the capacity of the cells to disseminate throughout the body. EMT is therefore becoming a potential target for anti-cancer drug discovery. Here, we showed that phoyunnanin E, a compound isolated from Dendrobium venustum, possesses anti-migration activity and addressed its mechanism of action. The cytotoxic and proliferative effects of phoyunnanin E on human non-small cell lung cancer-derived H460, H292, and A549 cells and human keratinocyte HaCaT cells were investigated by MTT assay. The effect of phoyunnanin E on EMT was evaluated by determining the colony formation and EMT markers. The migration and invasion of H460, H292, A549 and HaCaT cells was evaluated by wound healing assay and transwell invasion assay, respectively. EMT markers, integrins and migration-associated proteins were examined by western blot analysis. Phoyunnanin E at the concentrations of 5 and 10 μM, which are non-toxic to H460, H292, A549 and HaCaT cells showed good potential to inhibit the migratory activity of three types of human lung cancer cells. The anti-migration effect of phoyunnanin E was shown to relate to the suppressed EMT phenotypes, including growth in anchorage-independent condition, cell motility, and EMT-specific protein markers (N-cadherin, vimentin, slug, and snail). In addition to EMT suppression, we found that phoyunnanin E treatment with 5 and 10 μM could decrease the cellular level of integrin αv and integrin β3, these integrins are frequently up-regulated in highly metastatic tumor cells. We further characterized the regulatory proteins in cell migration and found that the cells treated with phoyunnanin E exhibited a significantly lower level of phosphorylated focal adhesion kinase (p-FAK) and phosphorylated ATP-dependent tyrosine kinase (p-AKT), and their downstream effectors (including

  6. Hydrostatic Compress Force Enhances the Viability and Decreases the Apoptosis of Condylar Chondrocytes through Integrin-FAK-ERK/PI3K Pathway.

    PubMed

    Ma, Dandan; Kou, Xiaoxing; Jin, Jing; Xu, Taotao; Wu, Mengjie; Deng, Liquan; Fu, Lusi; Liu, Yi; Wu, Gang; Lu, Haiping

    2016-11-07

    Reduced mechanical stimuli in many pathological cases, such as hemimastication and limited masticatory movements, can significantly affect the metabolic activity of mandibular condylar chondrocytes and the growth of mandibles. However, the molecular mechanisms for these phenomena remain unclear. In this study, we hypothesized that integrin-focal adhesion kinase (FAK)-ERK (extracellular signal-regulated kinase)/PI3K (phosphatidylinositol-3-kinase) signaling pathway mediated the cellular response of condylar chondrocytes to mechanical loading. Primary condylar chondrocytes were exposed to hydrostatic compressive forces (HCFs) of different magnitudes (0, 50, 100, 150, 200, and 250 kPa) for 2 h. We measured the viability, morphology, and apoptosis of the chondrocytes with different treatments as well as the gene, protein expression, and phosphorylation of mechanosensitivity-related molecules, such as integrin α2, integrin α5, integrin β1, FAK, ERK, and PI3K. HCFs could significantly increase the viability and surface area of condylar chondrocytes and decrease their apoptosis in a dose-dependent manner. HCF of 250 kPa resulted in a 1.51 ± 0.02-fold increase of cell viability and reduced the ratio of apoptotic cells from 18.10% ± 0.56% to 7.30% ± 1.43%. HCFs could significantly enhance the mRNA and protein expression of integrin α2, integrin α5, and integrin β1 in a dose-dependent manner, but not ERK1, ERK2, or PI3K. Instead, HCF could significantly increase phosphorylation levels of FAK, ERK1/2, and PI3K in a dose-dependent manner. Cilengitide, the potent integrin inhibitor, could dose-dependently block such effects of HCFs. HCFs enhances the viability and decreases the apoptosis of condylar chondrocytes through the integrin-FAK-ERK/PI3K pathway.

  7. Hydrostatic Compress Force Enhances the Viability and Decreases the Apoptosis of Condylar Chondrocytes through Integrin-FAK-ERK/PI3K Pathway

    PubMed Central

    Ma, Dandan; Kou, Xiaoxing; Jin, Jing; Xu, Taotao; Wu, Mengjie; Deng, Liquan; Fu, Lusi; Liu, Yi; Wu, Gang; Lu, Haiping

    2016-01-01

    Reduced mechanical stimuli in many pathological cases, such as hemimastication and limited masticatory movements, can significantly affect the metabolic activity of mandibular condylar chondrocytes and the growth of mandibles. However, the molecular mechanisms for these phenomena remain unclear. In this study, we hypothesized that integrin-focal adhesion kinase (FAK)-ERK (extracellular signal–regulated kinase)/PI3K (phosphatidylinositol-3-kinase) signaling pathway mediated the cellular response of condylar chondrocytes to mechanical loading. Primary condylar chondrocytes were exposed to hydrostatic compressive forces (HCFs) of different magnitudes (0, 50, 100, 150, 200, and 250 kPa) for 2 h. We measured the viability, morphology, and apoptosis of the chondrocytes with different treatments as well as the gene, protein expression, and phosphorylation of mechanosensitivity-related molecules, such as integrin α2, integrin α5, integrin β1, FAK, ERK, and PI3K. HCFs could significantly increase the viability and surface area of condylar chondrocytes and decrease their apoptosis in a dose-dependent manner. HCF of 250 kPa resulted in a 1.51 ± 0.02-fold increase of cell viability and reduced the ratio of apoptotic cells from 18.10% ± 0.56% to 7.30% ± 1.43%. HCFs could significantly enhance the mRNA and protein expression of integrin α2, integrin α5, and integrin β1 in a dose-dependent manner, but not ERK1, ERK2, or PI3K. Instead, HCF could significantly increase phosphorylation levels of FAK, ERK1/2, and PI3K in a dose-dependent manner. Cilengitide, the potent integrin inhibitor, could dose-dependently block such effects of HCFs. HCFs enhances the viability and decreases the apoptosis of condylar chondrocytes through the integrin-FAK-ERK/PI3K pathway. PMID:27827993

  8. Proteolytic processing of endogenous and recombinant beta 4 integrin subunit

    PubMed Central

    1992-01-01

    The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the

  9. Epidermal growth factor receptor and integrins control force-dependent vinculin recruitment to E-cadherin junctions.

    PubMed

    Sehgal, Poonam; Kong, Xinyu; Wu, Jun; Sunyer, Raimon; Trepat, Xavier; Leckband, Deborah

    2018-03-20

    This study reports novel findings that link E-cadherin (also known as CDH1)-mediated force-transduction signaling to vinculin targeting to intercellular junctions via epidermal growth factor receptor (EGFR) and integrins. These results build on previous findings that demonstrated that mechanically perturbed E-cadherin receptors activate phosphoinositide 3-kinase and downstream integrins in an EGFR-dependent manner. Results of this study show that this EGFR-mediated kinase cascade controls the force-dependent recruitment of vinculin to stressed E-cadherin complexes - a key early signature of cadherin-based mechanotransduction. Vinculin targeting requires its phosphorylation at tyrosine 822 by Abl family kinases (hereafter Abl), but the origin of force-dependent Abl activation had not been identified. We now present evidence that integrin activation, which is downstream of EGFR signaling, controls Abl activation, thus linking E-cadherin to Abl through a mechanosensitive signaling network. These findings place EGFR and integrins at the center of a positive-feedback loop, through which force-activated E-cadherin signals regulate vinculin recruitment to cadherin complexes in response to increased intercellular tension.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.

  10. The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes

    PubMed Central

    Benito-Jardón, Maria; Klapproth, Sarah; Gimeno-LLuch, Irene; Petzold, Tobias; Bharadwaj, Mitasha; Müller, Daniel J; Zuchtriegel, Gabriele; Reichel, Christoph A; Costell, Mercedes

    2017-01-01

    Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5β1, αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1syn/syn) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of β3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5β1 with the RGD, but essential to re-enforce the binding of α5β1/αIIbβ3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvβ3 integrin levels decrease below a critical level. DOI: http://dx.doi.org/10.7554/eLife.22264.001 PMID:28092265

  11. Mechanotransduction molecules in the plant gravisensory response: amyloplast/statolith membranes contain a beta 1 integrin-like protein

    NASA Technical Reports Server (NTRS)

    Lynch, T. M.; Lintilhac, P. M.; Domozych, D.

    1998-01-01

    It has been hypothesized that the sedimentation of amyloplasts within root cap cells is the primary event in the plant gravisensory-signal transduction cascade. Statolith sedimentation, with its ability to generate weighty mechanical signals, is a legitimate means for organisms to discriminate the direction of the gravity vector. However, it has been demonstrated that starchless mutants with reduced statolith densities maintain some ability to sense gravity, calling into question the statolith sedimentation hypothesis. Here we report on the presence of a beta 1 integrin-like protein localized inside amyloplasts of tobacco NT-1 suspension culture, callus cells, and whole-root caps. Two different antibodies to the beta 1 integrin, one to the cytoplasmic domain and one to the extracellular domain, localize in the vicinity of the starch grains within amyloplasts of NT-1. Biochemical data reveals a 110-kDa protein immunoprecipitated from membrane fractions of NT-1 suspension culture indicating size homology to known beta 1 integrin in animals. This study provides the first direct evidence for the possibility of integrin-mediated signal transduction in the perception of gravity by higher plants. An integrin-mediated pathway, initiated by starch grain sedimentation within the amyloplast, may provide the signal amplification necessary to explain the gravitropic response in starch-depleted cultivars.

  12. Clustered Integrin Ligands as a Novel Approach for the Targeting of Non-Viral Vectors

    NASA Astrophysics Data System (ADS)

    Ng, Quinn Kwan Tai

    Gene transfer or gene delivery is described as the process in which foreign DNA is introduced into cells. Over the years, gene delivery has gained the attention of many researchers and has been developed as powerful tools for use in biotechnology and medicine. With the completion of the Human Genome Project, such advances in technology allowed for the identification of diseases ranging from hereditary disorders to acquired ones (cancer) which were thought to be incurable. Gene therapy provides the means necessary to treat or eliminate genetic diseases from its origin, unlike traditional medicine which only treat symptoms. With ongoing clinical trials for gene therapy increasing, the greatest difficulty still lies in developing safe systems which can target cells of interest to provide efficient delivery. Nature, over millions of years of evolution, has provided an example of one of the most efficient delivery systems: viruses. Although the use of viruses for gene delivery has been well studied, the safety issues involving immunogenicity, insertional mutagenesis, high cost, and poor reproducibility has provided problems for their clinical application. From understanding viruses, we gain insight to designing new systems for non-viral gene delivery. One of these techniques utilized by adenoviruses is the clustering of ligands on its surface through the use of a protein called a penton base. Through the use of nanotechnology we can mimic this basic concept in non-viral gene delivery systems. This dissertation research is focused on developing and applying a novel system for displaying the integrin binding ligand (RGD) in a constrained manner to form a clustered integrin ligand binding platform to be used to enhance the targeting and efficiency of non-viral gene delivery vectors. Peptide mixed monolayer protected gold nanoparticles provides a suitable surface for ligand clustering. A relationship between the peptide ratios in the reaction solution used to form these

  13. Simulated Microgravity Alters Actin Cytoskeleton and Integrin-Mediated Focal Adhesions of Cultured Human Mesenchymal Stromal Cells

    NASA Astrophysics Data System (ADS)

    Gershovich, P. M.; Gershovic, J. G.; Buravkova, L. B.

    2008-06-01

    Cytoskeletal alterations occur in several cell types including lymphocytes, glial cells, and osteoblasts, during spaceflight and under simulated microgravity (SMG) (3, 4). One potential mechanism for cytoskeletal gravisensitivity is disruption of extracellular matrix (ECM) and integrin interactions. Focal adhesions are specialized sites of cell-matrix interaction composed of integrins and the diversity of focal adhesion-associated cytoplasmic proteins including vinculin, talin, α-actinin, and actin filaments (4, 5). Integrins produce signals essential for proper cellular function, survival and differentiation. Therefore, we investigated the effects of SMG on F-actin cytoskeleton structure, vinculin focal adhesions, expression of some integrin subtypes and cellular adhesion molecules (CAMs) in mesenchymal stem cells derived from human bone marrow (hMSCs). Simulated microgravity was produced by 3D-clinostat (Dutch Space, Netherlands). Staining of actin fibers with TRITC-phalloidin showed reorganization even after 30 minutes of simulated microgravity. The increasing of cells number with abnormal F-actin was observed after subsequent terms of 3D-clinorotation (6, 24, 48, 120 hours). Randomization of gravity vector altered dimensional structure of stress fibers and resulted in remodeling of actin fibers inside the cells. In addition, we observed vinculin redistribution inside the cells after 6 hours and prolonged terms of clinorotation. Tubulin fibers in a contrast with F-actin and vinculin didn't show any reorganization even after long 3Dclinorotation (120 hours). The expression of integrin α2 increased 1,5-6-fold in clinorotated hMSCs. Also we observed decrease in number of VCAM-1-positive cells and changes in expression of ICAM-1. Taken together, our findings indicate that SMG leads to microfilament and adhesion alterations of hMSCs most probably associated with involvement of some integrin subtypes.

  14. Signal transduction by beta1 integrin receptors in human chondrocytes in vitro: collaboration with the insulin-like growth factor-I receptor.

    PubMed

    Shakibaei, M; John, T; De Souza, P; Rahmanzadeh, R; Merker, H J

    1999-09-15

    We have examined the mechanism by which collagen-binding integrins co-operate with insulin-like growth factor-I (IGF-I) receptors (IGF-IR) to regulate chondrocyte phenotype and differentiation. Adhesion of chondrocytes to anti-beta1 integrin antibodies or collagen type II leads to phosphorylation of cytoskeletal and signalling proteins localized at focal adhesions, including alpha-actinin, vinculin, paxillin and focal adhesion kinase (FAK). These stimulate docking proteins such as Shc (Src-homology collagen). Moreover, exposure of collagen type II-cultured chondrocytes to IGF-I leads to co-immunoprecipitation of Shc protein with the IGF-IR and with beta1, alpha1 and alpha5 integrins, but not with alpha3 integrin. Shc then associates with growth factor receptor-bound protein 2 (Grb2), an adaptor protein and extracellular signal-regulated kinase. The expression of the docking protein Shc occurs only when chondrocytes are bound to collagen type II or integrin antibodies and increases when IGF-I is added, suggesting a collaboration between integrins and growth factors in a common/shared biochemical signalling pathway. Furthermore, these results indicate that focal adhesion assembly may facilitate signalling via Shc, a potential common target for signal integration between integrin and growth-factor signalling regulatory pathways. Thus, the collagen-binding integrins and IGF-IR co-operate to regulate focal adhesion components and these signalling pathways have common targets (Shc-Grb2 complex) in subcellular compartments, thereby linking to the Ras-mitogen-activated protein kinase signalling pathway. These events may play a role during chondrocyte differentiation.

  15. 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

  16. Integrins (alpha7beta1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy.

    PubMed Central

    Vachon, P H; Xu, H; Liu, L; Loechel, F; Hayashi, Y; Arahata, K; Reed, J C; Wewer, U M; Engvall, E

    1997-01-01

    Mutations in genes coding for dystrophin, for alpha, beta, gamma, and delta-sarcoglycans, or for the alpha2 chain of the basement membrane component merosin (laminin-2/4) cause various forms of muscular dystrophy. Analyses of integrins showed an abnormal expression and localization of alpha7beta1 isoforms in myofibers of merosin-deficient human patients and mice, but not in dystrophin-deficient or sarcoglycan-deficient humans and animals. It was shown previously that skeletal muscle fibers require merosin for survival and function (Vachon, P.H., F. Loechel, H. Xu, U.M. Wewer, and E. Engvall. 1996. J. Cell Biol. 134:1483-1497). Correction of merosin deficiency in vitro through cell transfection with the merosin alpha2 chain restored the normal localization of alpha7beta1D integrins as well as myotube survival. Overexpression of the apoptosis-suppressing molecule Bcl-2 also promoted the survival of merosin-deficient myotubes, but did not restore a normal expression of alpha7beta1D integrins. Blocking of beta1 integrins in normal myotubes induced apoptosis and severely reduced their survival. These findings (a) identify alpha7beta1D integrins as the de facto receptors for merosin in skeletal muscle; (b) indicate a merosin dependence for the accurate expression and membrane localization of alpha7beta1D integrins in myofibers; (c) provide a molecular basis for the critical role of merosin in myofiber survival; and (d) add new insights to the pathogenesis of neuromuscular disorders. PMID:9312189

  17. Lubrol-RAFTs in melanoma cells: a molecular platform for tumor-promoting ephrin-B2-integrin-beta1 interaction.

    PubMed

    Meyer, Stefanie; Orsó, Evelyn; Schmitz, Gerd; Landthaler, Michael; Vogt, Thomas

    2007-07-01

    Ephrins control cell motility and matrix adhesion. These functions play a pivotal role in cancer progression, for example, in malignant melanomas. We have previously shown that the ephrin-B2-tumor-promoting action is partly mediated by integrin-beta1 interaction. However, the subcellular prerequisites for molecular interaction like molecular proximity and co-compartmentalization have not been elucidated yet. Specific cholesterol-rich microdomains, termed lipid rafts (RAFTs), are known to be essential for functional ephrin-B2 signalling and integrin-mediated effects. Therefore, we addressed the question whether RAFT co-compartmentalization of both molecules could provide the molecular platform for their tumor-promoting interaction. In this study, we show that overexpressed ephrin-B2 is not only compartmentalized to classical Triton X-100 RAFTs in B16 melanoma cells, but also to the recently defined Lubrol-RAFTs. Interestingly, in the melanoma cells investigated, integrin-beta1 is also preferentially detected in such Lubrol-RAFTs. Accordingly, the presence of ephrin-B2 and integrin-beta1 in RAFTs and their function in cell migration and matrix attachment are highly sensitive to RAFT disruption by cholesterol depletion. Confocal fluorescence microscopy analyses also support the concept of a close molecular proximity and functional interplay of ephrin-B2 and integrin-beta1 in the plasma membrane. We conclude that Lubrol-RAFTs probably represent the platform for tumor-promoting ephrin-B2-integrin-beta1 interaction, which could become an interesting target for future antitumoral therapies.

  18. EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces.

    PubMed

    Saeki, Noritaka; Nishino, Shingo; Shimizu, Tomohiro; Ogawa, Kazushige

    2015-01-01

    Eph signaling, which arises following stimulation by ephrins, is known to induce opposite cell behaviors such as promoting and inhibiting cell adhesion as well as promoting cell-cell adhesion and repulsion by altering the organization of the actin cytoskeleton and influencing the adhesion activities of integrins. However, crosstalk between Eph/ephrin with integrin signaling has not been fully elucidated in leukocytes, including monocytes and their related cells. Using a cell attachment stripe assay, we have shown that, following stimulation with ephrin-A1, kinase-independent EphA2 promoted cell spreading/elongation as well as adhesion to integrin ligand-coated surfaces in cultured U937 (monocyte) and J774.1 (monocyte/macrophage) cells as well as sublines of these cells expressing dominant negative EphA2 that lacks most of the intracellular region. Moreover, a pull-down assay showed that dominant negative EphA2 is recruited to the β2 integrin/ICAM1 and β2 integrin/VCAM1 molecular complexes in the subline cells following stimulation with ephrin-A1-Fc. Notably, this study is the first comprehensive analysis of the effects of EphA2 receptors on integrin-mediated cell adhesion in monocytic cells. Based on these findings we propose that EphA2 promotes cell adhesion by an unknown signaling pathway that largely depends on the extracellular region of EphA2 and the activation of outside-in integrin signaling.

  19. Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro

    NASA Technical Reports Server (NTRS)

    Kieffer, J. D.; Plopper, G.; Ingber, D. E.; Hartwig, J. H.; Kupper, T. S.

    1995-01-01

    The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

  20. PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

    PubMed

    Roberts, M; Barry, S; Woods, A; van der Sluijs, P; Norman, J

    2001-09-18

    It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

  1. Covisualization by computational optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts

    NASA Technical Reports Server (NTRS)

    Gens, J. S.; Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal beta 1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protoplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not beta 1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specifity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105-110 and 115-125 kDa. These bands are again recognized by the visualization antibody, which was raised against the extracellular domain of chicken beta 1 integrin, and are also recognized by an antibody against the intracellular domain of chicken beta 1 integrin. Because beta 1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronection are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesion sites in animals.

  2. RGD-containing peptides activate S6K1 through beta3 integrin in adult cardiac muscle cells.

    PubMed

    Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

    2003-10-24

    The enzyme p70S6 kinase (S6K1) is critical for cell growth, and we have reported its activation during cardiac hypertrophy. Because cardiac hypertrophy also involves integrin activation, we analyzed whether integrins could contribute to S6K1 activation. Using adult feline cardiomyocytes, here we report that integrin-interacting Arg-Gly-Asp (RGD) peptides activate S6K1 as observed by band shifting, kinase activity and phosphorylation at Thr-389 and Thr-421/Ser-424 of S6K1, and S6 protein phosphorylation. Perturbation of specific integrin function with blocking antibodies and by overexpressing the beta1A cytoplasmic tail revealed that beta3 but not beta1 integrin mediates the RGD-induced S6K1 activation. This activation is focal adhesion complex-independent and is accompanied by the activation of extracellular signal-regulated kinases 1/2 (ERK) and mammalian target of rapamycin (mTOR). Studies using specific inhibitors and dominant negative c-Raf expression in cardiomyocytes indicate that the S6K1 activation involves mTOR, MEK/ERK, and phosphatidylinositol 3-kinase pathways and is independent of protein kinase C and c-Raf. Finally, addition of fluorescent-labeled RGD peptide to cardiomyocytes exhibits its internalization and localization to the endocytic vesicles, and pretreatment of cardiomyocytes with endocytic inhibitors reduced the S6K1 activation. These data suggest that RGD interaction with beta3 integrin and its subsequent endocytosis trigger specific signaling pathway(s) for S6K1 activation in cardiomyocytes and that this process may contribute to hypertrophic growth and remodeling of myocardium.

  3. Oxidation-induced Structural Changes of Ceruloplasmin Foster NGR Motif Deamidation That Promotes Integrin Binding and Signaling

    PubMed Central

    Barbariga, Marco; Curnis, Flavio; Spitaleri, Andrea; Andolfo, Annapaola; Zucchelli, Chiara; Lazzaro, Massimo; Magnani, Giuseppe; Musco, Giovanna; Corti, Angelo; Alessio, Massimo

    2014-01-01

    Asparagine deamidation occurs spontaneously in proteins during aging; deamidation of Asn-Gly-Arg (NGR) sites can lead to the formation of isoAsp-Gly-Arg (isoDGR), a motif that can recognize the RGD-binding site of integrins. Ceruloplasmin (Cp), a ferroxidase present in the cerebrospinal fluid (CSF), contains two NGR sites in its sequence: one exposed on the protein surface (568NGR) and the other buried in the tertiary structure (962NGR). Considering that Cp can undergo oxidative modifications in the CSF of neurodegenerative diseases, we investigated the effect of oxidation on the deamidation of both NGR motifs and, consequently, on the acquisition of integrin binding properties. We observed that the exposed 568NGR site can deamidate under conditions mimicking accelerated Asn aging. In contrast, the hidden 962NGR site can deamidate exclusively when aging occurs under oxidative conditions, suggesting that oxidation-induced structural changes foster deamidation at this site. NGR deamidation in Cp was associated with gain of integrin-binding function, intracellular signaling, and cell pro-adhesive activity. Finally, Cp aging in the CSF from Alzheimer disease patients, but not in control CSF, causes Cp deamidation with gain of integrin-binding function, suggesting that this transition might also occur in pathological conditions. In conclusion, both Cp NGR sites can deamidate during aging under oxidative conditions, likely as a consequence of oxidative-induced structural changes, thereby promoting a gain of function in integrin binding, signaling, and cell adhesion. PMID:24366863

  4. Clinical Response to Vedolizumab in Ulcerative Colitis Patients Is Associated with Changes in Integrin Expression Profiles.

    PubMed

    Fuchs, Friederike; Schillinger, Daniela; Atreya, Raja; Hirschmann, Simon; Fischer, Sarah; Neufert, Clemens; Atreya, Imke; Neurath, Markus F; Zundler, Sebastian

    2017-01-01

    Despite large clinical success, deeper insights into the immunological effects of vedolizumab therapy for inflammatory bowel diseases are scarce. In particular, the reasons for differential clinical response in individual patients, the precise impact on the equilibrium of integrin-expressing T cell subsets, and possible associations between these issues are not clear. Blood samples from patients receiving clinical vedolizumab therapy were sequentially collected and analyzed for expression of integrins and chemokine receptors on T cells. Moreover, clinical and laboratory data from the patients were collected, and changes between homing marker expression and clinical parameters were analyzed for possible correlations. While no significant correlation of changes in integrin expression and changes in outcome parameters were identified in Crohn's disease (CD), increasing α4β7 levels in ulcerative colitis (UC) seemed to be associated with favorable clinical development, whereas increasing α4β1 and αEβ7 correlated with negative changes in outcome parameters. Changes in α4β1 integrin expression after 6 weeks were significantly different in responders and non-responders to vedolizumab therapy as assessed after 16 weeks with a cutoff of +4.2% yielding 100% sensitivity and 100% specificity in receiver-operator-characteristic analysis. Our data show that clinical response to vedolizumab therapy in UC but not in CD is associated with specific changes in integrin expression profiles opening novel avenues for mechanistic research and possibly prediction of response to therapy.

  5. Long residence time of ultrasound microbubbles targeted to integrin in murine tumor model.

    PubMed

    Jun, Hong Young; Park, Seong Hoon; Kim, Hun Soo; Yoon, Kwon-Ha

    2010-01-01

    The aim of this study was to evaluate the intratumoral residence time of microbubbles (MBs) targeted to alpha(v)beta(3) integrin expressed in the endothelial cells of mice during the process of tumor angiogenesis. For the preparation of MBs, decafluorobutane gas was sonically dispersed in phosphate buffer saline containing L-A-phosphatidylcholine-distearoyl, polyethylene glycol 40 stearate, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)2000] in a 77:15:8 molar ratio. Avidin-fluorescein isothiocyanate and biotin-cyclic arginine-glycine-aspartate-D-tyrosine-lysine (cRGD) or biotin-alanine-glycine-aspartate (AGD) conjugates were added to the reaction mixture. Adhesion testing of the targeting MBs was performed for the MS-1 cell line expressing alpha(v)beta(3) integrin in vitro. The in vivo acoustic properties of the MBs were assessed by clinical ultrasound on the HT1080 fibrosarcoma model (n = 8) for 1 hour. Cryosections were stained with hematoxylin and eosin and by immunohistochemical staining to identify expression of alpha(v)beta(3) integrin in the HT1080 tumor. The adherence of the MBs conjugated to cRGD was significantly greater than the adherence of the MBs conjugated to biotin-AGD (P < .01) for the MS-1 endothelial cell line. The acoustic enhancement on ultrasound was observed as a stable imaging window until 1 hour after injection of the MB conjugates in the mice. The MBs targeted via cRGD preferentially adhered to the vascular endothelium of the HT-1080 tumors. The findings of ultrasound imaging were correlated with immunohistochemical findings for the expression of alpha(v)beta(3) integrin on the vascular endothelium of the tumors. The prepared MBs conjugated with cRGD demonstrated a sufficient residence time to attach to the target integrin of tumor tissues. This finding suggests that the MBs are a potential molecular contrast agent that enables characterization of tumor angiogenesis and the monitoring of antitumor and

  6. α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ozeki, Nobuaki; Kawai, Rie; Hase, Naoko

    We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blotmore » and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells. - Highlights: • Odontoblast differentiation requires activation of α2 integrin, Emmprin and MMP-3. • α2 integrin, Emmprin and MMP-3 form a sequential signaling cascade. • β1 integrin acts a specific trigger for odontoblast differentiation. • The role of these effectors is highly novel and unanticipated.« less

  7. Amplification and oscillations in the FAK/Src kinase system during integrin signaling.

    PubMed

    Caron-Lormier, G; Berry, H

    2005-01-21

    Integrin signaling is a major pathway of cell adhesion to extracellular matrices that regulates many physiological cell behaviors such as cell proliferation, migration or differentiation and is implied in pathologies such as tumor invasion. In this paper, we focused on the molecular system formed by the two kinases FAK (focal adhesion kinase) and Src, which undergo auto- and co-activation during early steps of integrin signaling. The system is modelled using classical kinetic equations and yields a set of three nonlinear ordinary differential equations describing the dynamics of the different phosphorylation forms of FAK. Analytical and numerical analysis of these equations show that this system may in certain cases amplify incoming signals from the integrins. A quantitative condition is obtained, which indicates that the total FAK charge in the system acts as a critical mass that must be exceeded for amplification to be effective. Furthermore, we show that when FAK activity is lower than Src activity, spontaneous oscillations of FAK phosphorylation forms may appear. The oscillatory behavior is studied using bifurcation and stability diagrams. We finally discuss the significance of this behavior with respect to recent experimental results evidencing FAK dynamics.

  8. The integrin alphav beta3 increases cellular stiffness and cytoskeletal remodeling dynamics to facilitate cancer cell invasion

    NASA Astrophysics Data System (ADS)

    Mierke, Claudia Tanja

    2013-01-01

    The process of cancer cell invasion through the extracellular matrix (ECM) of connective tissue plays a prominent role in tumor progression and is based fundamentally on biomechanics. Cancer cell invasion usually requires cell adhesion to the ECM through the cell-matrix adhesion receptors integrins. The expression of the αvβ3 integrin is increased in several tumor types and is consistently associated with increased metastasis formation in patients. The hypothesis was that the αvβ3 integrin expression increases the invasiveness of cancer cells through increased cellular stiffness, and increased cytoskeletal remodeling dynamics. Here, the invasion of cancer cells with different αvβ3 integrin expression levels into dense three-dimensional (3D) ECMs has been studied. Using a cell sorter, two subcell lines expressing either high or low amounts of αvβ3 integrins (αvβ3high or αvβ3low cells, respectively) have been isolated from parental MDA-MB-231 breast cancer cells. αvβ3high cells showed a threefold increased cell invasion compared to αvβ3low cells. Similar results were obtained for A375 melanoma, 786-O kidney and T24 bladder carcinoma cells, and cells in which the β3 integrin subunit was knocked down using specific siRNA. To investigate whether contractile forces are essential for αvβ3 integrin-mediated increased cellular stiffness and subsequently enhanced cancer cell invasion, invasion assays were performed in the presence of myosin light chain kinase inhibitor ML-7 and Rho kinase inhibitor Y27632. Indeed, cancer cell invasiveness was reduced after addition of ML-7 and Y27632 in αvβ3high cells but not in αvβ3low cells. Moreover, after addition of the contractility enhancer calyculin A, an increase in pre-stress in αvβ3low cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase, STAT3 or Rac1 strongly reduced the invasiveness of αvβ3high cells, whereas the invasiveness of β3 specific knock

  9. Structural Characterization of the Ectodomain of a Disintegrin and Metalloproteinase-22 (ADAM22), a Neural Adhesion Receptor Instead of Metalloproteinase INSIGHTS ON ADAM FUNCTION

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Heli; Shim, Ann H.R.; He, Xiaolin

    2009-12-01

    ADAMs (adisintegrin and metalloproteinases) are a family of multidomain transmembrane glycoproteins with diverse roles in physiology and diseases, with several members being drug targets for cancer and inflammation therapies. The spatial organization of the ADAM extracellular segment and its influence on the function of ADAMs have been unclear. Although most members of the ADAM family are active zinc metalloproteinases, 8 of 21 ADAMs lack functional metalloproteinase domains and are implicated in protein-protein interactions instead of membrane protein ectodomain shedding. One of such non-proteinase ADAMs, ADAM22, acts as a receptor on the surface of the postsynaptic neuron to regulate synaptic signalmore » transmission. The crystal structure of the full ectodomain of mature human ADAM22 shows that it is a compact four-leaf clover with the metalloproteinase-like domain held in the concave face of a rigid module formed by the disintegrin, cysteine-rich, and epidermal growth factor-like domains. The loss of metalloproteinase activity is ensured by the absence of critical catalytic residues, the filling of the substrate groove, and the steric hindrance by the cysteine-rich domain. The structure, combined with calorimetric experiments, suggests distinct roles of three putative calcium ions bound to ADAM22, with one in the metalloproteinase-like domain being regulatory and two in the disintegrin domain being structural. The metalloproteinase-like domain contacts the rest of ADAM22 with discontinuous, hydrophilic, and poorly complemented interactions, suggesting the possibility of modular movement of ADAM22 and other ADAMs. The ADAM22 structure provides a framework for understanding how different ADAMs exert their adhesive function and shedding activities.« less

  10. Integrin alphaIIb-subunit cytoplasmic domain mutations demonstrate a requirement for tyrosine phosphorylation of beta3-subunits in actin cytoskeletal organization.

    PubMed

    Yamodo, Innocent H; Blystone, Scott D

    2004-01-01

    Using truncated or mutated alphaIIb integrin cytoplasmic domains fused to the alphaV extracellular domain and expressed with the beta3 integrin subunit, we demonstrate that the double mutation of proline residues 998 and 999 to alanine (PP998/999AA), previously shown to disturb the C-terminal conformation of the alphaIIb integrin cytoplasmic domain, prevents tyrosine phosphorylation of beta3 integrin induced by Arg-Gly-Asp peptide ligation. This mutation also inhibits integrin mediated actin assembly and cell adhesion to vitronectin. In contrast, progressive truncation of the alphaIIb-subunit cytoplasmic domain did not reproduce these effects. Interestingly, the PP998/999AA mutations of alphaIIb did not affect beta3 tyrosine phosphorylation, cell adhesion, or actin polymerization induced by manganese. Exogenous addition of manganese was sufficient to rescue beta3 phosphorylation, cell adhesion, and actin assembly in cells expressing the PP998/999AA mutation when presented with a vitronectin substrate. Further, induction of the high affinity conformation of this mutant beta3 integrin by incubation with either Arg-Gly-Asp peptide or exogenous manganese was equivalent. These results suggest that the extracellular structure of beta3 integrins in the high affinity conformation is not directly related to the structure of the cytoplasmic face of the integrin. Moreover, the requirement for beta3 phosphorylation is demonstrated without mutation of the beta3 subunit. In support of our previous hypothesis of a role for beta3 phosphorylation in adhesion, these studies demonstrate a strong correlation between beta3 tyrosine phosphorylation and assembly of a cytoskeleton competent to support firm cell adhesion.

  11. Functional single-walled carbon nanotubes based on an integrin αvβ3 monoclonal antibody for highly efficient cancer cell targeting

    NASA Astrophysics Data System (ADS)

    Ou, Zhongmin; Wu, Baoyan; Xing, Da; Zhou, Feifan; Wang, Huiying; Tang, Yonghong

    2009-03-01

    The application of single-walled carbon nanotubes (SWNTs) in the field of biomedicine is becoming an entirely new and exciting topic. In this study, a novel functional SWNT based on an integrin αvβ3 monoclonal antibody was developed and was used for cancer cell targeting in vitro. SWNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). The PL-PEG functionalized SWNTs were then conjugated with protein A. A SWNT-integrin αvβ3 monoclonal antibody system (SWNT-PEG-mAb) was thus constructed by conjugating protein A with the fluorescein labeled integrin αvβ3 monoclonal antibody. In vitro study revealed that SWNT-PEG-mAb presented a high targeting efficiency on integrin αvβ3-positive U87MG cells with low cellular toxicity, while for integrin αvβ3-negative MCF-7 cells, the system had a low targeting efficiency, indicating that the high targeting to U87MG cells was due to the specific integrin targeting of the monoclonal antibody. In conclusion, SWNT-PEG-mAb developed in this research is a potential candidate for cancer imaging and drug delivery in cancer targeting therapy.

  12. Inhibition of integrin-linked kinase blocks podocyte epithelial-mesenchymal transition and ameliorates proteinuria.

    PubMed

    Kang, Young Sun; Li, Yingjian; Dai, Chunsun; Kiss, Lawrence P; Wu, Chuanyue; Liu, Youhua

    2010-08-01

    Proteinuria is a primary clinical symptom of a large number of glomerular diseases that progress to end-stage renal failure. Podocyte dysfunctions play a fundamental role in defective glomerular filtration in many common forms of proteinuric kidney disorders. Since binding of these cells to the basement membrane is mediated by integrins, we determined the role of integrin-linked kinase (ILK) in podocyte dysfunction and proteinuria. ILK expression was induced in mouse podocytes by various injurious stimuli known to cause proteinuria including TGF-beta1, adriamycin, puromycin, and high ambient glucose. Podocyte ILK was also found to be upregulated in human proteinuric glomerular diseases. Ectopic expression of ILK in podocytes decreased levels of the epithelial markers nephrin and ZO-1, induced mesenchymal markers such as desmin, fibronectin, matrix metalloproteinase-9 (MMP-9), and alpha-smooth muscle actin (alpha-SMA), promoted cell migration, and increased the paracellular albumin flux across podocyte monolayers. ILK also induced Snail, a key transcription factor mediating epithelial-mesenchymal transition (EMT). Blockade of ILK activity with a highly selective small molecule inhibitor reduced Snail induction and preserved podocyte phenotypes following TGF-beta1 or adriamycin stimulation. In vivo, this ILK inhibitor ameliorated albuminuria, repressed glomerular induction of MMP-9 and alpha-SMA, and preserved nephrin expression in murine adriamycin nephropathy. Our results show that upregulation of ILK is a convergent pathway leading to podocyte EMT, migration, and dysfunction. ILK may be an attractive target for therapeutic intervention of proteinuric kidney diseases.

  13. DR-nm23 expression affects neuroblastoma cell differentiation, integrin expression, and adhesion characteristics.

    PubMed

    Amendola, R; Martinez, R; Negroni, A; Venturelli, D; Tanno, B; Calabretta, B; Raschellà, G

    2001-01-01

    Nm23 gene family has been associated with metastasis suppression and differentiation. We studied DR-nm23 during neuroblastoma cells differentiation. DR-nm23 expression increased after retinoic acid induction of differentiation in human cell lines SK-N-SH and LAN-5. In several cell lines, overexpression of DR-nm23 was associated with more differentiated phenotypes. SK-N-SH cells increased vimentin expression, increased deposition of collagen type IV, modulated integrin expression, and underwent growth arrest; the murine neuroblastoma cell line N1E-115 showed neurite outgrowth and a striking enhancement of beta1 integrin expression. Up-regulation of beta1 integrin was specifically responsible for the increase in the adhesion to collagen type I-coated plates. Finally, cells overexpressing DR-nm23 were unable to growth in soft agar. In conclusion, DR-nm23 expression is directly involved in differentiation of neuroblastoma cells, and its ability to affects the adhesion to extracellular substrates and to inhibit growth in soft agar suggests an involvement in the metastatic potential of neuroblastoma.

  14. Epithelial Membrane Protein 2 and β1 integrin signaling regulate APC-mediated processes.

    PubMed

    Lesko, Alyssa C; Prosperi, Jenifer R

    2017-01-01

    Adenomatous Polyposis Coli (APC) plays a critical role in cell motility, maintenance of apical-basal polarity, and epithelial morphogenesis. We previously demonstrated that APC loss in Madin Darby Canine Kidney (MDCK) cells increases cyst size and inverts polarity independent of Wnt signaling, and upregulates the tetraspan protein, Epithelial Membrane Protein 2 (EMP2). Herein, we show that APC loss increases β1 integrin expression and migration of MDCK cells. Through 3D in vitro model systems and 2D migration analysis, we have depicted the molecular mechanism(s) by which APC influences polarity and cell motility. EMP2 knockdown in APC shRNA cells revealed that APC regulates apical-basal polarity and cyst size through EMP2. Chemical inhibition of β1 integrin and its signaling components, FAK and Src, indicated that APC controls cyst size and migration, but not polarity, through β1 integrin and its downstream targets. Combined, the current studies have identified two distinct and novel mechanisms required for APC to regulate polarity, cyst size, and cell migration independent of Wnt signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Interaction between integrin α5 and PDE4D regulates endothelial inflammatory signalling

    PubMed Central

    Yun, Sanguk; Budatha, Madhusudhan; Dahlman, James E.; Coon, Brian G.; Cameron, Ryan T.; Langer, Robert; Anderson, Daniel G.; Baillie, George; Schwartz, Martin A.

    2016-01-01

    Atherosclerosis is primarily a disease of lipid metabolism and inflammation; however, it is also closely associated with endothelial extracellular matrix (ECM) remodelling, with fibronectin accumulating in the laminin–collagen basement membrane. To investigate how fibronectin modulates inflammation in arteries, we replaced the cytoplasmic tail of the fibronectin receptor integrin α5 with that of the collagen/laminin receptor integrin α2. This chimaera suppressed inflammatory signalling in endothelial cells on fibronectin and in knock-in mice. Fibronectin promoted inflammation by suppressing anti-inflammatory cAMP. cAMP was activated through endothelial prostacyclin secretion; however, this was ECM-independent. Instead, cells on fibronectin suppressed cAMP via enhanced phosphodiesterase (PDE) activity, through direct binding of integrin α5 to phosphodiesterase-4D5 (PDE4D5), which induced PP2A-dependent dephosphorylation of PDE4D5 on the inhibitory site Ser651. In vivo knockdown of PDE4D5 inhibited inflammation at athero-prone sites. These data elucidate a molecular mechanism linking ECM remodelling and inflammation, thereby identifying a new class of therapeutic targets. PMID:27595237

  16. The α5β1 Integrin Mediates Elimination of Amyloid-β Peptide and Protects Against Apoptosis

    PubMed Central

    Matter, Michelle L.; Zhang, Zhuohua; Nordstedt, Christer; Ruoslahti, Erkki

    1998-01-01

    The amyloid-β peptide (Aβ) can mediate cell attachment by binding to β1 integrins through an arg-his-asp sequence. We show here that the α5β1 integrin, a fibronectin receptor, is an efficient binder of Aβ, and mediates cell attachment to nonfibrillar Aβ. Cells engineered to express α5β1 internalized and degraded more added Aβ1-40 than did α5β1-negative control cells. Deposition of an insoluble Aβ1-40 matrix around the α5β1-expressing cells was reduced, and the cells showed less apoptosis than the control cells. Thus, the α5β1 integrin may protect against Aβ deposition and toxicity, which is a course of Alzheimer's disease lesions. PMID:9585419

  17. Chondrocyte heterogeneity: immunohistologically defined variation of integrin expression at different sites in human fetal knees.

    PubMed

    Salter, D M; Godolphin, J L; Gourlay, M S

    1995-04-01

    During development and at maturity different forms of cartilage vary in morphology and macromolecular content. This reflects heterogeneity of chondrocyte activity, in part involving differential interactions with the adjacent extracellular matrix via specialized cell surface receptors such as integrins. We undertook an immunohistological study on a series of human fetal knee joints to assess variation in the expression of integrins by chondrocytes and potential matrix ligands in articular, epiphyseal, growth plate, and meniscal cartilage. The results show that articular chondrocytes (beta 1+, beta 5 alpha V+, alpha 1+, alpha 2+/-, alpha 5+, weakly alpha 6+, alpha V+) differed from epiphyseal (beta 1+, beta 5 alpha V+, alpha 1+/-, alpha 2+/-, alpha 5+, alpha 6+, alpha V+) growth plate (beta 1+, beta 5 alpha V+, alpha 1-, alpha 2-, alpha 5+, alpha 6+, alpha V+), and meniscal cells (beta 1+, beta 5 alpha V+, alpha 1+, strongly alpha 2+, alpha 5+, alpha 6+, alpha V+ in expression of integrin subunits. There was no expression of beta 3, beta 4, beta 6, or alpha 3 by chondrocytes. These results differ from previous reports on the expression of integrins by adult articular cartilage, where alpha 2 and alpha 6 are not seen. Variation in distribution of matrix ligands was also seen. Fibronectin, laminin and Type VI collagen were expressed in all cartilages but there was restricted expression of tenascin, ED-A and ED-B fibronectin isoforms (articular cartilage and meniscus), and vitronectin (absent from growth plate cartilage). Regulated expression of integrins by chondrocytes, associated with changes in the pericellular matrix composition, is of potential importance in control of cartilage differentiation and function in health and disease.

  18. Cellular Interaction of Integrin α3β1 with Laminin 5 Promotes Gap Junctional Communication

    PubMed Central

    Lampe, Paul D.; Nguyen, Beth P.; Gil, Susana; Usui, Marcia; Olerud, John; Takada, Yoshikazu; Carter, William G.

    1998-01-01

    Wounding of skin activates epidermal cell migration over exposed dermal collagen and fibronectin and over laminin 5 secreted into the provisional basement membrane. Gap junctional intercellular communication (GJIC) has been proposed to integrate the individual motile cells into a synchronized colony. We found that outgrowths of human keratinocytes in wounds or epibole cultures display parallel changes in the expression of laminin 5, integrin α3β1, E-cadherin, and the gap junctional protein connexin 43. Adhesion of keratinocytes on laminin 5, collagen, and fibronectin was found to differentially regulate GJIC. When keratinocytes were adhered on laminin 5, both structural (assembly of connexin 43 in gap junctions) and functional (dye transfer) assays showed a two- to threefold increase compared with collagen and five- to eightfold over fibronectin. Based on studies with immobilized integrin antibody and integrin-transfected Chinese hamster ovary cells, the interaction of integrin α3β1 with laminin 5 was sufficient to promote GJIC. Mapping of intermediate steps in the pathway linking α3β1–laminin 5 interactions to GJIC indicated that protein trafficking and Rho signaling were both required. We suggest that adhesion of epithelial cells to laminin 5 in the basement membrane via α3β1 promotes GJIC that integrates individual cells into synchronized epiboles. PMID:9852164

  19. Skeletal Phenotype of Transgenic Mice Expressing the Beta1 Integrin Cytoplasmic Tail In Osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; vanderMeulen, M. C. H.; Damsky, D.; Kim, J.-B.; Amblard, D.; Amblard, D.; Nishimura, Y.; Almeida, E.; Iwaniec, U. T.; Wronski, T. J.; hide

    2002-01-01

    tibia and humerus mass were less obvious in TG than in WT mice. Since hindlimb unloading caused skeletal changes in both loaded and unloaded bones, systemic changes may contribute to bone responses observed using this animal model. In conclusion, transgene expression resulted in marked metabolic changes during growth and in the aged female. Our results demonstrate that expression of the Beta1 integrin cytoplasmic tail in vivo causes gender- and age-specific changes in select morphometric parameters, bone length, and bone mass.

  20. Electric Signals Regulate the Directional Migration of Oligodendrocyte Progenitor Cells (OPCs) via β1 Integrin.

    PubMed

    Zhu, Bangfu; Nicholls, Matthew; Gu, Yu; Zhang, Gaofeng; Zhao, Chao; Franklin, Robin J M; Song, Bing

    2016-11-22

    The guided migration of neural cells is essential for repair in the central nervous system (CNS). Oligodendrocyte progenitor cells (OPCs) will normally migrate towards an injury site to re-sheath demyelinated axons; however the mechanisms underlying this process are not well understood. Endogenous electric fields (EFs) are known to influence cell migration in vivo, and have been utilised in this study to direct the migration of OPCs isolated from neonatal Sprague-Dawley rats. The OPCs were exposed to physiological levels of electrical stimulation, and displayed a marked electrotactic response that was dependent on β1 integrin, one of the key subunits of integrin receptors. We also observed that F-actin, an important component of the cytoskeleton, was re-distributed towards the leading edge of the migrating cells, and that this asymmetric rearrangement was associated with β1 integrin function.

  1. Compression force sensing regulates integrin αIIbβ3 adhesive function on diabetic platelets.

    PubMed

    Ju, Lining; McFadyen, James D; Al-Daher, Saheb; Alwis, Imala; Chen, Yunfeng; Tønnesen, Lotte L; Maiocchi, Sophie; Coulter, Brianna; Calkin, Anna C; Felner, Eric I; Cohen, Neale; Yuan, Yuping; Schoenwaelder, Simone M; Cooper, Mark E; Zhu, Cheng; Jackson, Shaun P

    2018-03-14

    Diabetes is associated with an exaggerated platelet thrombotic response at sites of vascular injury. Biomechanical forces regulate platelet activation, although the impact of diabetes on this process remains ill-defined. Using a biomembrane force probe (BFP), we demonstrate that compressive force activates integrin α IIb β 3 on discoid diabetic platelets, increasing its association rate with immobilized fibrinogen. This compressive force-induced integrin activation is calcium and PI 3-kinase dependent, resulting in enhanced integrin affinity maturation and exaggerated shear-dependent platelet adhesion. Analysis of discoid platelet aggregation in the mesenteric circulation of mice confirmed that diabetes leads to a marked enhancement in the formation and stability of discoid platelet aggregates, via a mechanism that is not inhibited by therapeutic doses of aspirin and clopidogrel, but is eliminated by PI 3-kinase inhibition. These studies demonstrate the existence of a compression force sensing mechanism linked to α IIb β 3 adhesive function that leads to a distinct prothrombotic phenotype in diabetes.

  2. Vedolizumab: an α4β7 integrin antagonist for ulcerative colitis and Crohn’s disease

    PubMed Central

    Cherry, Lauren N.; Yunker, Nancy S.; Lambert, Erika R.; Vaughan, DaleMarie

    2015-01-01

    Ulcerative colitis (UC) and Crohn’s disease (CD) are chronic, relapsing inflammatory bowel diseases associated with significant morbidity. Conventional therapies for these diseases include corticosteroids, aminosalicylates, immunomodulators, and monoclonal antibodies. Over the years tumor necrosis factor (TNF)-α antagonists alone or in combination with other therapies have emerged as the cornerstone of treatment for induction and maintenance of remission of moderate to severe UC and CD. Unfortunately, some patients with moderate to severe UC and CD are unable to attain or maintain remission with TNF-α antagonist treatment. Vedolizumab, a humanized monoclonal antibody, is the first integrin receptor antagonist approved that selectively antagonizes α4β7 gastrointestinal integrin receptors. US Food and Drug Administration approval is for treatment of patients with moderate to severe active UC and CD who have inadequate response with, lost response to, or are intolerant to a TNF-α antagonist or an immunomodulator; or have inadequate response with, are intolerant to, or demonstrate dependence on corticosteroids. When administered according to approved dosing in patients with moderate to severe CD and UC, vedolizumab induces clinical response rates up to 31.4% and 47.1% at week 6, and clinical remission rates up to 39% and 41.8% at week 52, respectively. Serious adverse events reported with vedolizumab include serious infections, malignancies, and anaphylaxis. Since vedolizumab is gastrointestinal selective, to date, it has not shown evidence of causing progressive multifocal leukoencephalopathy; however, postmarketing studies monitoring for this adverse effect are ongoing. Further assessment of vedolizumab earlier in the course of these diseases and in combination with other therapies is warranted. PMID:26336591

  3. The adaptor molecule RIAM integrates signaling events critical for integrin-mediated control of immune function and cancer progression.

    PubMed

    Patsoukis, Nikolaos; Bardhan, Kankana; Weaver, Jessica D; Sari, Duygu; Torres-Gomez, Alvaro; Li, Lequn; Strauss, Laura; Lafuente, Esther M; Boussiotis, Vassiliki A

    2017-08-22

    Lymphocyte activation requires adhesion to antigen-presenting cells. This is a critical event linking innate and adaptive immunity. Lymphocyte adhesion is accomplished through LFA-1, which must be activated by a process referred to as inside-out integrin signaling. Among the few signaling molecules that have been implicated in inside-out integrin activation in hematopoietic cells are the small guanosine triphosphatase (GTPase) Rap1 and its downstream effector Rap1-interacting molecule (RIAM), a multidomain protein that defined the Mig10-RIAM-lamellipodin (MRL) class of adaptor molecules. Through its various domains, RIAM is a critical node of signal integration for activation of T cells, recruits monomeric and polymerized actin to drive actin remodeling and cytoskeletal reorganization, and promotes inside-out integrin signaling in T cells. As a regulator of inside-out integrin activation, RIAM affects multiple functions of innate and adaptive immunity. The effects of RIAM on cytoskeletal reorganization and integrin activation have implications in cell migration and trafficking of cancer cells. We provide an overview of the structure and interactions of RIAM, and we discuss the implications of RIAM functions in innate and adaptive immunity and cancer. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  4. Evidence that β7 Integrin Regulates Hematopoietic Stem Cell Homing and Engraftment Through Interaction with MAdCAM-1.

    PubMed

    Murakami, Jodi L; Xu, Baohui; Franco, Christopher B; Hu, Xingbin; Galli, Stephen J; Weissman, Irving L; Chen, Ching-Cheng

    2016-01-01

    α4β7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis is controversial. We identified a subset of hematopoietic stem cells (HSCs) in the bone marrow (BM) that expressed β7 integrin. These β7(+) HSCs were capable of multilineage, long-term reconstitution and had an inherent competitive advantage over β7(-) HSCs. On the other hand, HSCs that lacked β7 integrin (β7KO) had reduced engraftment potential. Interestingly, quantitative RT-PCR and flow cytometry revealed that β7KO HSCs expressed lower levels of the chemokine receptor CXCR4. Accordingly, β7KO HSCs exhibited impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the α4β7 integrin ligand-mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on BM endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that β7 integrin, when expressed by HSCs, interacted with its endothelial ligand MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment.

  5. Crystal structure of the complete integrin αVβ3 ectodomain plus an α/β transmembrane fragment

    PubMed Central

    Xiong, Jian-Ping; Mahalingham, Bhuvaneshwari; Alonso, Jose Luis; Borrelli, Laura Ann; Rui, Xianliang; Anand, Saurabh; Hyman, Bradley T.; Rysiok, Thomas; Müller-Pompalla, Dirk; Goodman, Simon L.

    2009-01-01

    We determined the crystal structure of 1TM-αVβ3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the αV and β3 subunits. 1TM-αVβ3 is more compact and less active in solution when compared with ΔTM-αVβ3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the α–β interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length αVβ3 on live cells yields a donor–membrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2–thigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state. PMID:19704023

  6. Optimized multimodal nanoplatforms for targeting αvβ3 integrins

    NASA Astrophysics Data System (ADS)

    Bolley, Julie; Lalatonne, Yoann; Haddad, Oualid; Letourneur, Didier; Soussan, Michael; Pérard-Viret, Joelle; Motte, Laurence

    2013-11-01

    Magnetic Resonance Imaging (MRI) using contrast agents is a very powerful technique for diagnosis in clinical medicine and biomedical research. The synthesis of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles targeting αvβ3 integrins and acting as new MRI contrast agents seems to be a promising way for cancer diagnosis. Indeed, it is well established that αvβ3 integrin plays a key role in tumor angiogenesis acting like a receptor for the extracellular matrix proteins like vitronectin, fibronectin through the arginine-glycine-aspartic acid (RGD) sequence. Up-regulation of αvβ3 has been found to be associated with a wide range of cancers, making it a broad-spectrum tumor-marker. In this study, USPIO nanocrystals were synthesized and surface passivated with caffeic acid. The large number of the carboxylic acid functions at the outer surface of the nanoplatforms was used for the covalent coupling of Rhodamine123, polyethylene glycol (PEG) and cyclic RGD. Soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to crosslink carboxylic acid with the amino group of the ligands. We examined the design of the nanoplatforms with each individual entity and then the combination of two and three of them. Several methods were used to characterize the nanoparticle surface functionalization and the magnetic properties of these contrast agents were studied using a 1.5 T clinical MRI scanner. The affinity towards integrins was evidenced by surface plasmon resonance and solid-phase receptor-binding assay.Magnetic Resonance Imaging (MRI) using contrast agents is a very powerful technique for diagnosis in clinical medicine and biomedical research. The synthesis of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles targeting αvβ3 integrins and acting as new MRI contrast agents seems to be a promising way for cancer diagnosis. Indeed, it is well established that αvβ3 integrin plays a key role in tumor angiogenesis acting like a receptor for

  7. Distinct Effects of RGD-glycoproteins on Integrin-Mediated Adhesion and Osteogenic Differentiation of Human Mesenchymal Stem Cells

    PubMed Central

    Schwab, Elisabeth H.; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A.

    2013-01-01

    The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α5-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β3-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation. PMID:24324361

  8. Distinct effects of RGD-glycoproteins on Integrin-mediated adhesion and osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Schwab, Elisabeth H; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A

    2013-01-01

    The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α₅-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β₃-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation.

  9. Deoxycholic acid promotes development of gastroesophageal reflux disease and Barrett's oesophagus by modulating integrin-αv trafficking.

    PubMed

    Prichard, David O; Byrne, Anne Marie; Murphy, James O; Reynolds, John V; O'Sullivan, Jacintha; Feighery, Ronan; Doyle, Brendan; Eldin, Osama Sharaf; Finn, Stephen P; Maguire, Aoife; Duff, Deirdre; Kelleher, Dermot P; Long, Aideen

    2017-12-01

    The fundamental mechanisms underlying erosive oesophagitis and subsequent development of Barrett's oesophagus (BO) are poorly understood. Here, we investigated the contribution of specific components of the gastric refluxate on adhesion molecules involved in epithelial barrier maintenance. Cell line models of squamous epithelium (HET-1A) and BO (QH) were used to examine the effects of bile acids on cell adhesion to extracellular matrix proteins (Collagen, laminin, vitronectin, fibronectin) and expression of integrin ligands (α 3 , α 4, α 5 , α 6 and α ν ). Experimental findings were validated in human explant oesophageal biopsies, a rat model of gastroesophageal reflux disease (GORD) and in patient tissue microarrays. The bile acid deoxycholic acid (DCA) specifically reduced adhesion of HET-1A cells to vitronectin and reduced cell-surface expression of integrin-α ν via effects on endocytic recycling processes. Increased expression of integrin-α v was observed in ulcerated tissue in a rat model of GORD and in oesophagitis and Barrett's intestinal metaplasia patient tissue compared to normal squamous epithelium. Increased expression of integrin-α ν was observed in QH BO cells compared to HET-1A cells. QH cells were resistant to DCA-mediated loss of adhesion and reduction in cell-surface expression of integrin-α ν . We demonstrated that a specific component of the gastric refluxate, DCA, affects the epithelial barrier through modulation of integrin α ν expression, providing a novel mechanism for bile acid-mediated erosion of oesophageal squamous epithelium and promotion of BO. Strategies aimed at preventing bile acid-mediated erosion should be considered in the clinical management of patients with GORD. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  10. Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells

    PubMed Central

    Storm, Rickard J.; Persson, B. David; Skalman, Lars Nygård; Frängsmyr, Lars; Lindström, Mona; Rankin, Greg; Lundmark, Richard; Domellöf, Fatima Pedrosa

    2016-01-01

    ABSTRACT Epidemic keratoconjunctivitis (EKC) is a severe, contagious ocular disease that affects 20 to 40 million individuals worldwide every year. EKC is mainly caused by six types of human adenovirus (HAdV): HAdV-8, -19, -37, -53, -54, and -56. Of these, HAdV-8, -19, and -37 use sialic acid-containing glycans as cellular receptors. αVβ3, αVβ5, and a few additional integrins facilitate entry and endosomal release of other HAdVs. With the exception of a few biochemical analyses indicating that HAdV-37 can interact physically with αVβ5, little is known about the integrins used by EKC-causing HAdVs. Here, we investigated the overall integrin expression on human corneal cells and found expression of α2, α3, α6, αV, β1, and β4 subunits in human corneal in situ epithelium and/or in a human corneal epithelial (HCE) cell line but no or less accessible expression of α4, α5, β3, or β5. We also identified the integrins used by HAdV-37 through a series of binding and infection competition experiments and different biochemical approaches. Together, our data suggest that HAdV-37 uses αVβ1 and α3β1 integrins for infection of human corneal epithelial cells. Furthermore, to confirm the relevance of these integrins in the HAdV-37 life cycle, we developed a corneal multilayer tissue system and found that HAdV-37 infection correlated well with the patterns of αV, α3, and β1 integrin expression. These results provide further insight into the tropism and pathogenesis of EKC-causing HAdVs and may be of importance for future development of new antiviral drugs. IMPORTANCE Keratitis is a hallmark of EKC, which is caused by six HAdV types (HAdV-8, -19, -37, -53, -54, and -56). HAdV-37 and some other HAdV types interact with integrin αVβ5 in order to enter nonocular human cells. In this study, we found that αVβ5 is not expressed on human corneal epithelial cells, thus proposing other host factors mediate corneal infection. Here, we first characterized integrin

  11. Differential expression of extracellular matrix molecules and the alpha 6-integrins in the normal and neoplastic prostate.

    PubMed Central

    Knox, J. D.; Cress, A. E.; Clark, V.; Manriquez, L.; Affinito, K. S.; Dalkin, B. L.; Nagle, R. B.

    1994-01-01

    The epithelial basal lamina composition and integrin expression profile of normal and neoplastic human prostate was characterized using immunohistochemical analysis of frozen samples. The major components of the basal lamina surrounding normal acini were laminin, type IV collagen, entactin, and type VII collagen with variable amounts of tenascin. The basal lamina of neoplastic acini had a similar composition, except for the loss of type VII collagen, which was observed in all grades of carcinoma. The basal cells of the normal prostate express the alpha 6-, beta 1-, and beta 4-integrin subunits, suggesting that both the alpha 6 beta 1- and alpha 6 beta 4-integrin complexes are formed. In prostate carcinoma there is a complete loss of beta 4 expression and the alpha 6- and beta 1-integrin subunits, which are restricted to the basal and basal lateral surfaces of basal cells, are distributed diffusely throughout the cytoplasmic membrane. The differential expression of type VII collagen and beta 4 are discussed in relationship to their possible role in tumor progression. Images Figure 1 Figure 2 Figure 3 PMID:8030747

  12. Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αVβ3 and Inhibits Osteoclast Differentiation.

    PubMed

    Miyazaki, Tatsuya; Miyauchi, Satoshi; Anada, Takahisa; Tawada, Akira; Suzuki, Osamu

    2015-10-01

    Integrins and their ligands have been suggested to be associated with osteoclast-mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS-E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin-labeled CS-E, and the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase staining and a pit formation assay. CS-E as well as CS-B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow-derived macrophages. Pre-coating of OA to synthetic calcium phosphate-coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS-E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVβ3, and inhibited the direct binding of OA to integrin αVβ3. Furthermore, CS-E blocked the binding of OA to cells and inhibited OA-induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS-E is capable of inhibiting OA-induced osteoclast differentiation by blocking the interaction of OA to integrin αVβ3 and HSPG. © 2015 Wiley Periodicals, Inc.

  13. A Novel Role for Integrin-linked Kinase in Epithelial Sheet Morphogenesis

    PubMed Central

    Vespa, Alisa; D'Souza, Sudhir J.A.; Dagnino, Lina

    2005-01-01

    Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca2+ triggers formation of cell–cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca2+ treatment of keratinocytes induces rapid (≤1 h) translocation to the cell membrane of the adherens junction (AJ) proteins E-cadherin and β-catenin. This is followed by slower (>6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell–cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/β-catenin to cell borders, precluding Ca2+-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell–cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell–cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration. PMID:15975904

  14. A novel role for integrin-linked kinase in epithelial sheet morphogenesis.

    PubMed

    Vespa, Alisa; D'Souza, Sudhir J A; Dagnino, Lina

    2005-09-01

    Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.

  15. Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus.

    PubMed

    Kanasaki, Keizo; Kanda, Yoshiko; Palmsten, Kristin; Tanjore, Harikrishna; Lee, Soo Bong; Lebleu, Valerie S; Gattone, Vincent H; Kalluri, Raghu

    2008-01-15

    The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte beta1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin beta1 (podocin-Cre beta1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre beta1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin beta1 activity in epithelial cells. To further explore whether integrin beta1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte beta1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.

  16. Exosome release of ADAM15 and the functional implications of human macrophage-derived ADAM15 exosomes.

    PubMed

    Lee, Hee Doo; Koo, Bon-Hun; Kim, Yeon Hyang; Jeon, Ok-Hee; Kim, Doo-Sik

    2012-07-01

    A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15-mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12-myristate 13-acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane-associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin αvβ3 in an RGD-dependent manner and suppress vitronectin- and fibronectin-induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage-derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome-mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity.

  17. Structural basis of substrate discrimination and integrin binding by autotaxin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos

    2013-09-25

    Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates.more » We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.« less

  18. Usage of heparan sulfate, integrins, and FAK in HPV16 infection

    PubMed Central

    Abban, Cynthia Y.; Meneses, Patricio I.

    2010-01-01

    Human Papillomavirus Type 16 (HPV16) is the major causative agent of cervical cancer. Studies regarding the early binding and signaling molecules that play a significant role in infection are still lacking. The current study analyses the role of heparan sulfate, integrins, and the signaling molecule FAK in HPV16 infection of human adult keratinocytes cell line (HaCaTs). Our data demonstrate that infection requires the binding of viral particles to heparan sulfate followed by activation of focal adhesion kinase through an integrin. Infections were reduced in the presence of the FAK inhibitor, TAE226. TAE226 was observed to inhibit viral entry to the early endosome a known infectious route. These findings suggest that FAK can serve as a novel target for antiviral therapy. PMID:20441998

  19. Secondary reduction of alpha7B integrin in laminin alpha2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle.

    PubMed

    Cohn, R D; Mayer, U; Saher, G; Herrmann, R; van der Flier, A; Sonnenberg, A; Sorokin, L; Voit, T

    1999-03-01

    The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The alpha7beta1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the alpha7 integrin are laminin-1 (alpha1-beta1-gamma1), laminin-2 (alpha2-beta1-gamma1) and laminin-4 (alpha2-beta2-gamma1). Targeted deletion of the gene for the alpha7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the alpha7B and beta1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the alpha7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the beta1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of alpha7B integrin was significantly reduced at the sarcolemma in six patients with laminin alpha2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin alpha2 chain expressed. In contrast, the expression of the laminin alpha2 chain was not altered in the skeletal muscle of the alpha7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the alpha7 integrin subunit and that of the laminin alpha2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of alpha7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even

  20. Oncofetal Chondroitin Sulfate Glycosaminoglycans Are Key Players in Integrin Signaling and Tumor Cell Motility.

    PubMed

    Clausen, Thomas Mandel; Pereira, Marina Ayres; Al Nakouzi, Nader; Oo, Htoo Zarni; Agerbæk, Mette Ø; Lee, Sherry; Ørum-Madsen, Maj Sofie; Kristensen, Anders Riis; El-Naggar, Amal; Grandgenett, Paul M; Grem, Jean L; Hollingsworth, Michael A; Holst, Peter J; Theander, Thor; Sorensen, Poul H; Daugaard, Mads; Salanti, Ali

    2016-12-01

    Many tumors express proteoglycans modified with oncofetal chondroitin sulfate glycosaminoglycan chains (ofCS), which are normally restricted to the placenta. However, the role of ofCS in cancer is largely unknown. The function of ofCS in cancer was analyzed using the recombinant ofCS-binding VAR2CSA protein (rVAR2) derived from the malaria parasite, Plasmodium falciparum We demonstrate that ofCS plays a key role in tumor cell motility by affecting canonical integrin signaling pathways. Binding of rVAR2 to tumor cells inhibited the interaction of cells with extracellular matrix (ECM) components, which correlated with decreased phosphorylation of Src kinase. Moreover, rVAR2 binding decreased migration, invasion, and anchorage-independent growth of tumor cells in vitro Mass spectrometry of ofCS-modified proteoglycan complexes affinity purified from tumor cell lines on rVAR2 columns revealed an overrepresentation of proteins involved in cell motility and integrin signaling, such as integrin-β1 (ITGB1) and integrin-α4 (ITGA4). Saturating concentrations of rVAR2 inhibited downstream integrin signaling, which was mimicked by knockdown of the core chondroitin sulfate synthesis enzymes β-1,3-glucuronyltransferase 1 (B3GAT1) and chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1). The ofCS modification was highly expressed in both human and murine metastatic lesions in situ and preincubation or early intravenous treatment of tumor cells with rVAR2 inhibited seeding and spreading of tumor cells in mice. This was associated with a significant increase in survival of the animals. These data functionally link ofCS modifications with cancer cell motility and further highlights ofCS as a novel therapeutic cancer target. The cancer-specific expression of ofCS aids in metastatic phenotypes and is a candidate target for therapy. Mol Cancer Res; 14(12); 1288-99. ©2016 AACR. ©2016 American Association for Cancer Research.

  1. Tunable ultrasmall visible-to-extended near-infrared emitting silver sulfide quantum dots for integrin-targeted cancer imaging.

    PubMed

    Tang, Rui; Xue, Jianpeng; Xu, Baogang; Shen, Duanwen; Sudlow, Gail P; Achilefu, Samuel

    2015-01-27

    The large size of many near-infrared (NIR) fluorescent nanoparticles prevents rapid extravasation from blood vessels and subsequent diffusion to tumors. This confines in vivo uptake to the peritumoral space and results in high liver retention. In this study, we developed a viscosity modulated approach to synthesize ultrasmall silver sulfide quantum dots (QDs) with distinct tunable light emission from 500 to 1200 nm and a QD core diameter between 1.5 and 9 nm. Conjugation of a tumor-avid cyclic pentapeptide (Arg-Gly-Asp-DPhe-Lys) resulted in monodisperse, water-soluble QDs (hydrodynamic diameter < 10 nm) without loss of the peptide's high binding affinity to tumor-associated integrins (KI = 1.8 nM/peptide). Fluorescence and electron microscopy showed that selective integrin-mediated internalization was observed only in cancer cells treated with the peptide-labeled QDs, demonstrating that the unlabeled hydrophilic nanoparticles exhibit characteristics of negatively charged fluorescent dye molecules, which typically do not internalize in cells. The biodistribution profiles of intravenously administered QDs in different mouse models of cancer reveal an exceptionally high tumor-to-liver uptake ratio, suggesting that the small sized QDs evaded conventional opsonization and subsequent high uptake in the liver and spleen. The seamless tunability of the QDs over a wide spectral range with only a small increase in size, as well as the ease of labeling the bright and noncytotoxic QDs with biomolecules, provides a platform for multiplexing information, tracking the trafficking of single molecules in cells, and selectively targeting disease biomarkers in living organisms without premature QD opsonization in circulating blood.

  2. Discovery, SAR, and Radiolabeling of Halogenated Benzimidazole Carboxamide Antagonists as Useful Tools for (alpha)4(beta)1 Integrin Expressed on T- and B-cell Lymphomas

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Carpenter, R D; Natarajan, A; Lau, E Y

    2010-02-08

    The cell surface receptor {alpha}{sub 4}{beta}{sub 1} integrin is an attractive yet poorly understood target for selective diagnosis and treatment of T- and B-cell lymphomas. This report focuses on the rapid microwave preparation of medicinally pertinent benzimidazole heterocycles, structure-activity relationships (SAR) of novel halobenzimidazole carboxamide antagonists 3-6, and preliminary biological evaluation of radioiodinated agents 7, 8, and 18. The I-125 derivative 18 had good tumor uptake (12 {+-} 1% ID/g at 24 h; 4.5 {+-} 1% ID/g at 48 h) and tumor:kidney ratio ({approx}4:1 at 24 h; 2.5:1 at 48 h) in xenograft murine models of B-cell lymphoma. Molecular homologymore » models of {alpha}{sub 4}{beta}{sub 1} integrin have predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. These high affinity ({approx} pM binding) halogenated ligands are attractive tools for medicinal and biological use; the fluoro and iodo derivatives are potential radiodiagnostic ({sup 18}F) or radiotherapeutic ({sup 131}I) agents, whereas the chloro and bromo analogues could provide structural insight into integrin-ligand interactions through photoaffinity cross-linking/mass spectroscopy experiments, as well as co-crystallization X-ray studies.« less

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  4. A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins

    PubMed Central

    1995-01-01

    To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail- less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4- mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin. PMID:7721947

  5. Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK–dependent integrin outside-in retractile signaling pathway

    PubMed Central

    Flevaris, Panagiotis; Li, Zhenyu; Zhang, Guoying; Zheng, Yi; Liu, Junling

    2009-01-01

    Mitogen-activated protein kinases (MAPK), p38, and extracellular stimuli-responsive kinase (ERK), are acutely but transiently activated in platelets by platelet agonists, and the agonist-induced platelet MAPK activation is inhibited by ligand binding to the integrin αIIbβ3. Here we show that, although the activation of MAPK, as indicated by MAPK phosphorylation, is initially inhibited after ligand binding to integrin αIIbβ3, integrin outside-insignaling results in a late but sustained activation of MAPKs in platelets. Furthermore, we show that the early agonist-induced MAPK activation and the late integrin-mediated MAPK activation play distinct roles in different stages of platelet activation. Agonist-induced MAPK activation primarily plays an important role in stimulating secretion of platelet granules, while integrin-mediated MAPK activation is important in facilitating clot retraction. The stimulatory role of MAPK in clot retraction is mediated by stimulating myosin light chain (MLC) phosphorylation. Importantly, integrin-dependent MAPK activation, MAPK-dependent MLC phosphorylation, and clot retraction are inhibited by a Rac1 inhibitor and in Rac1 knockout platelets, indicating that integrin-induced activation of MAPK and MLC and subsequent clot retraction is Rac1-dependent. Thus, our results reveal 2 different activation mechanisms of MAPKs that are involved in distinct aspects of platelet function and a novel Rac1-MAPK–dependent cell retractile signaling pathway. PMID:18957688

  6. Bone Marrow-Derived Mesenchymal Stem Cells Enhance Angiogenesis via their α6β1 Integrin Receptor

    PubMed Central

    Carrion, Bita; Kong, Yen P.; Kaigler, Darnell; Putnam, Andrew J

    2013-01-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) facilitate the angiogenic response of endothelial cells (ECs) within three-dimensional (3D) matrices in vivo and in engineered tissues in vitro in part through paracrine mediators and by acting as stabilizing pericytes. However, the molecular interactions between BMSCs and nascent tubules during the process of angiogenesis are not fully understood. In this study, we have used a tractable 3D co-culture model to explore the functional role of the α6β1 integrin adhesion receptor on BMSCs in sprouting angiogenesis. We report that knockdown of the α6 integrin subunit in BMSCs significantly reduces capillary sprouting, and causes their failure to associate with the nascent vessels. Furthermore, we demonstrate that the BMSCs with attenuated α6 integrin proliferate at a significantly lower rate relative to either control cells expressing non-targeting shRNA or wild type BMSCs; however, despite adding more cells to compensate for this deficit in proliferation, deficient sprouting persists. Collectively, our findings demonstrate that the α6 integrin subunit in BMSCs is important for their ability to stimulate vessel morphogenesis. This conclusion may have important implications in the optimization of cell-based strategies to promote angiogenesis. PMID:24056178

  7. Glioblastoma stem cells exploit the αvβ8 integrin-TGFβ1 signaling axis to drive tumor initiation and progression

    PubMed Central

    Guerrero, PA; Tchaicha, JH; Chen, Z; Morales, JE; McCarty, N; Wang, Q; Sulman, EP; Fuller, G; Lang, FF; Rao, G; McCarty, JH

    2018-01-01

    Glioblastoma (GBM) is a primary brain cancer that contains populations of stem-like cancer cells (GSCs) that home to specialized perivascular niches. GSC interactions with their niche influence self-renewal, differentiation and drug resistance, although the pathways underlying these events remain largely unknown. Here, we report that the integrin αvβ8 and its latent transforming growth factor β1 (TGFβ1) protein ligand have central roles in promoting niche co-option and GBM initiation. αvβ8 integrin is highly expressed in GSCs and is essential for self-renewal and lineage commitment in vitro. Fractionation of β8high cells from freshly resected human GBM samples also reveals a requirement for this integrin in tumorigenesis in vivo. Whole-transcriptome sequencing reveals that αvβ8 integrin regulates tumor development, in part, by driving TGFβ1-induced DNA replication and mitotic checkpoint progression. Collectively, these data identify the αvβ8 integrin-TGFβ1 signaling axis as crucial for exploitation of the perivascular niche and identify potential therapeutic targets for inhibiting tumor growth and progression in patients with GBM. PMID:28783169

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ohkawa, Yuki; Miyazaki, Sayaka; Miyata, Maiko

    2008-08-15

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

  9. Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

    PubMed

    Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

    1996-07-01

    Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

  10. Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting.

    PubMed

    Vehlow, Anne; Klapproth, Erik; Storch, Katja; Dickreuter, Ellen; Seifert, Michael; Dietrich, Antje; Bütof, Rebecca; Temme, Achim; Cordes, Nils

    2017-07-25

    Resistance of cancer stem-like and cancer tumor bulk cells to radiochemotherapy and destructive infiltration of the brain fundamentally influence the treatment efficiency to cure of patients suffering from Glioblastoma (GBM). The interplay of adhesion and stress-related signaling and activation of bypass cascades that counteract therapeutic approaches remain to be identified in GBM cells. We here show that combined inhibition of the adhesion receptor β1 integrin and the stress-mediator c-Jun N-terminal kinase (JNK) induces radiosensitization and blocks invasion in stem-like and patient-derived GBM cultures as well as in GBM cell lines. In vivo, this treatment approach not only significantly delays tumor growth but also increases median survival of orthotopic, radiochemotherapy-treated GBM mice. Both, in vitro and in vivo, effects seen with β1 integrin/JNK co-inhibition are superior to the monotherapy. Mechanistically, the in vitro radiosensitization provoked by β1 integrin/JNK targeting is caused by defective DNA repair associated with chromatin changes, enhanced ATM phosphorylation and prolonged G2/M cell cycle arrest. Our findings identify a β1 integrin/JNK co-dependent bypass signaling for GBM therapy resistance, which might be therapeutically exploitable.

  11. Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and β1.

    PubMed

    Staunstrup, Nicklas Heine; Stenderup, Karin; Mortensen, Sidsel; Primo, Maria Nascimento; Rosada, Cecilia; Steiniche, Torben; Liu, Ying; Li, Rong; Schmidt, Mette; Purup, Stig; Dagnæs-Hansen, Frederik; Schrøder, Lisbeth Dahl; Svensson, Lars; Petersen, Thomas Kongstad; Callesen, Henrik; Bolund, Lars; Mikkelsen, Jacob Giehm

    2017-07-01

    Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and β1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology. © 2017. Published by The Company of Biologists Ltd.

  12. IL-32γ promotes integrin αvβ6 expression through the activation of NF-κB in HSCs

    PubMed Central

    Liu, Hongcan; Pan, Xingfei; Cao, Hong; Shu, Xin; Sun, Haixia; Lu, Jianxi; Liang, Jiayin; Zhang, Ka; Zhu, Fengqin; Li, Gang; Zhang, Qi

    2017-01-01

    Hepatic stellate cell (HSC) activation is important in the pathogenesis of liver fibrosis. However, the molecular mechanism of HSC activation is not completely understood. In the present study, it was demonstrated that interleukin-32γ (IL-32γ) is capable of enhancing intefgrin αvβ6 expression by inducing integrin αvβ6 promoter activity in a dose-dependent manner in HSCs. Furthermore, it was determined that nuclear factor κB (NF-κB) activation is required for IL-32γ-induced integrin αvβ6 expression. Increased integrin αvβ6 expression is then able to activate HSCs. These results indicate that NF-κB activation is required for IL-32γ to induce integrin αvβ6 expression and consequently promote HSC activation. Therefore, IL-32γ activates HSCs and therefore may be associated with hepatic fibrogenesis. These results may enable the development of novel effective strategies to treat hepatic fibrosis. PMID:29042996

  13. Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and β1

    PubMed Central

    Staunstrup, Nicklas Heine; Stenderup, Karin; Mortensen, Sidsel; Primo, Maria Nascimento; Steiniche, Torben; Liu, Ying; Li, Rong; Schmidt, Mette; Purup, Stig; Dagnæs-Hansen, Frederik; Schrøder, Lisbeth Dahl; Svensson, Lars; Petersen, Thomas Kongstad; Callesen, Henrik; Bolund, Lars

    2017-01-01

    ABSTRACT Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and β1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology. PMID:28679670

  14. Functional blockade of α5β1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

    PubMed Central

    2010-01-01

    Background Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration. Here, we investigated the genomic events occurring during this process induced by functional blockade of α5β1 integrin in liver progenitor cells. Results Cells treated with a specific antibody against α5β1 integrin exhibited cell spreading and scattering, over-expression of liver stem/progenitor cell markers and activation of the ERK1/2 and p38 MAPKs signaling cascades, in a similar manner to the process triggered by HGF/SF1 stimulation. Gene expression profiling revealed marked transcriptional changes of genes involved in cell adhesion and migration, as well as genes encoding chromatin remodeling factors. These responses were accompanied by conspicuous spatial reorganization of centromeres, while integrin genes conserved their spatial positioning in the interphase nucleus. Conclusion Collectively, our results demonstrate that α5β1 integrin functional blockade induces cell migration of hepatic progenitor cells, and that this involves a dramatic remodeling of the nuclear landscape. PMID:20958983

  15. Characterization of white shrimp Litopenaeus vannamei integrin β and its role in immunomodulation by dsRNA-mediated gene silencing.

    PubMed

    Lin, Yong-Chin; Chen, Jiann-Chu; Chen, Yu-Yuan; Liu, Chun-Hung; Cheng, Winton; Hsu, Chih-Hung; Tsui, Wen-Ching

    2013-06-01

    The full sequence of white shrimp Litopenaeus vannamei integrin β (LV-B) is 2879bp which encodes 787 amino acids (aa) of the open reading frame (ORF). The mature protein (764 aa) contains (1) an extracellular domain (ED) of 692 aa, (2) a transmembrane domain (TD) of 23 aa, and (3) a cytoplasmic domain (CD) of 49 aa. The cloned LV-B grouped together with crayfish Pacifastacus leniusculus integrin β (PL-B1), but was far away from vertebrate integrin β1, β3, β5, β6, β7, and β8, and another L. vannamei integrin β (LV). A Southern blot analysis indicated that the cloned LV-B was a single copy of genomic DNA. LV-B mRNA was expressed in all tissues, and was highly expressed in haemocytes. LV-B was downregulated in shrimp 24 and 96h after having received white spot syndrome virus (WSSV). LV-B expression by haemocytes of shrimp was higher in the postmoult (A and B) stage, and lower in the premoult (D2/D3) stage. LV-B expression was significantly higher by shrimp reared in 2.5‰ and 5‰ salinities. Shrimp injected with integrin β dsRNA showed gene silencing of integrin β after 36h. LV-B-silenced shrimp showed decreased hyaline cells (HCs), granular cells (GCs, including semi-granular cells), the total haemocyte count (THC), respiratory bursts (RBs), and lysozyme activity, but showed increased RB/HC, superoxide dismutase (SOD) activity/HC, and the phenoloxidase (PO) activity/GC. LV-B-silenced shrimp showed upregulated expressions of lipopolysaccharide- and β-glucan-binding protein (LGBP), peroxinectin (PX), prophenoloxidase I (proPO I), proPO II, proPO-activating enzyme (ppA), α2-macroglobulin (α2-M), cytMnSOD, mtMnSOD, and heat shock protein 70 (HSP70). It was concluded that integrin β plays important roles in proPO activation, phagocytosis, and the antioxidant system for immunomodulation in shrimp. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. The Plasma Membrane Sialidase NEU3 Regulates the Malignancy of Renal Carcinoma Cells by Controlling β1 Integrin Internalization and Recycling*

    PubMed Central

    Tringali, Cristina; Lupo, Barbara; Silvestri, Ilaria; Papini, Nadia; Anastasia, Luigi; Tettamanti, Guido; Venerando, Bruno

    2012-01-01

    The human plasma membrane sialidase NEU3 is a key enzyme in the catabolism of membrane gangliosides, is crucial in the regulation of cell surface processes, and has been demonstrated to be significantly up-regulated in renal cell carcinomas (RCCs). In this report, we show that NEU3 regulates β1 integrin trafficking in RCC cells by controlling β1 integrin recycling to the plasma membrane and controlling activation of the epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK)/protein kinase B (AKT) signaling. NEU3 silencing in RCC cells increased the membrane ganglioside content, in particular the GD1a content, and changed the expression of key regulators of the integrin recycling pathway. In addition, NEU3 silencing up-regulated the Ras-related protein RAB25, which directs internalized integrins to lysosomes, and down-regulated the chloride intracellular channel protein 3 (CLIC3), which induces the recycling of internalized integrins to the plasma membrane. In this manner, NEU3 silencing enhanced the caveolar endocytosis of β1 integrin, blocked its recycling and reduced its levels at the plasma membrane, and, consequently, inhibited EGFR and FAK/AKT. These events had the following effects on the behavior of RCC cells: they (a) decreased drug resistance mediated by the block of autophagy and the induction of apoptosis; (b) decreased metastatic potential mediated by down-regulation of the metalloproteinases MMP1 and MMP7; and (c) decreased adhesion to collagen and fibronectin. Therefore, our data identify NEU3 as a key regulator of the β1 integrin-recycling pathway and FAK/AKT signaling and demonstrate its crucial role in RCC malignancy. PMID:23139422

  17. Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and α7 integrin

    PubMed Central

    Marshall, Jamie L.; Oh, Jennifer; Chou, Eric; Lee, Joy A.; Holmberg, Johan; Burkin, Dean J.; Crosbie-Watson, Rachelle H.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of the dystrophin–glycoprotein complex, a laminin receptor that connects the myofiber to its surrounding extracellular matrix. Utrophin, a dystrophin ortholog that is normally localized to the neuromuscular junction, is naturally upregulated in DMD muscle, which partially compensates for the loss of dystrophin. Transgenic overexpression of utrophin causes broad sarcolemma localization of utrophin, restoration of laminin binding and amelioration of disease in the mdx mouse model of DMD. We previously demonstrated that overexpression of sarcospan, a dystrophin- and utrophin-binding protein, ameliorates mdx muscular dystrophy. Sarcospan boosts levels of utrophin to therapeutic levels at the sarcolemma, where attachment to laminin is restored. However, understanding the compensatory mechanism is complicated by concomitant upregulation of α7β1 integrin, which also binds laminin. Similar to the effects of utrophin, transgenic overexpression of α7 integrin prevents DMD disease in mice and is accompanied by increased abundance of utrophin around the extra-synaptic sarcolemma. In order to investigate the mechanisms underlying sarcospan ‘rescue’ of muscular dystrophy, we created double-knockout mice to test the contributions of utrophin or α7 integrin. We show that sarcospan-mediated amelioration of muscular dystrophy in DMD mice is dependent on the presence of both utrophin and α7β1 integrin, even when they are individually expressed at therapeutic levels. Furthermore, we found that association of sarcospan into laminin-binding complexes is dependent on utrophin and α7β1 integrin. PMID:25504048

  18. α5β1 Integrin-Fibronectin Interactions Specify Liquid to Solid Phase Transition of 3D Cellular Aggregates

    PubMed Central

    Caicedo-Carvajal, Carlos E.; Shinbrot, Troy; Foty, Ramsey A.

    2010-01-01

    Background Tissue organization during embryonic development and wound healing depends on the ability of cells on the one hand to exchange adhesive bonds during active rearrangement and on the other to become fixed in place as tissue homeostasis is reached. Cells achieve these contradictory tasks by regulating either cell-cell adhesive bonds, mediated by cadherins, or cell-extracellular matrix (ECM) connections, regulated by integrins. Integrin α5β1 and soluble fibronectin (sFN) are key players in cell-ECM force generation and in ECM polymerization. Here, we explore the interplay between integrin α5β1 and sFN and its influence on tissue mechanical properties and cell sorting behavior. Methodology/Principal Findings We generated a series of cell lines varying in α5β1 receptor density. We then systematically explored the effects of different sFN concentrations on aggregate biomechanical properties using tissue surface tensiometry. We found previously unreported complex behaviors including the observation that interactions between fibronectin and integrin α5β1 generates biphasic tissue cohesion profiles. Specifically, we show that at constant sFn concentration, aggregate cohesion increases linearly as α5β1 receptor density is increased from low to moderate levels, producing a transition from viscoelastic-liquid to pseudo viscoelastic-solid behavior. However, further increase in receptor density causes an abrupt drop in tissue cohesion and a transition back to viscoelastic-liquid properties. We propose that this may be due to depletion of sFn below a critical value in the aggregate microenvironment at high α5β1 levels. We also show that differential expression of α5β1 integrin can promote phase-separation between cells. Conclusions/Significance The interplay between α5-integrin and sFn contributes significantly to tissue cohesion and, depending on their level of expression, can mediate a shift from liquid to elastic behavior. This interplay represents a

  19. Immunohistochemical study of integrin α₅β₁, fibronectin, and Bcl-2 in normal oral mucosa, inflammatory fibroepithelial hyperplasia, oral epithelial dysplasia, and oral squamous cell carcinoma.

    PubMed

    Núñez, Manuel Antonio Gordón; de Matos, Felipe Rodrigues; Freitas, Roseana de Almeida; Galvão, Hébel Cavalcanti

    2013-07-01

    The objective of this study was to compare the immunoexpression of integrin α₅β₁, fibronectin, and the Bcl-2 protein in normal oral mucosa (NOM), inflammatory fibroepithelial hyperplasia (IFH), oral epithelial dysplasia (OED), and oral squamous cell carcinoma (OSCC). Eleven cases of NOM, 16 IFH, 20 OED, and 27 OSCC were selected for analysis of the immunoexpression of integrin α₅β₁, fibronectin, and bcl-2 protein. There was an association between the intensity and location of the integrin α₅β₁ expression, especially in the OSCC, that 48.1% of cases showed weak immunoreactivity and 40.7% in the suprabasal layer (P < 0.05). There was an association between the pattern and distribution of fibronectin expression in basement membrane, where 90% of NOM showed a pattern of linear continuous and 80% of OED exhibited focal distribution (P < 0.05). The fibronectin expression in connective tissue was predominantly intense with an association of staining pattern among the different specimens, where 37% of OSCC showed a reticular pattern (P < 0.05). There was an association of bcl-2 protein among the types of specimens, especially in IFH and OSCC, where 100% of the cases exhibited scores 1 of staining (P < 0.05). Within this context, the interaction of integrin α₅β₁ with its main ligand in the extracellular matrix, fibronectin, is suggested to influence the survival of tumor cells and to favor their proliferation by modulating apoptosis through the upregulation of antiapoptotic proteins or the suppression of apoptotic mediators.

  20. Clustering T cell GM1 Lipid Rafts Increases Cellular Resistance to Shear on Fibronectin through Changes in Integrin Affinity and Cytoskeletal Dynamics

    PubMed Central

    Mitchell, Jason S.; Brown, Wells S.; Woodside, Darren G.; Vanderslice, Peter; McIntyre, Bradley W.

    2008-01-01

    Lipid rafts are small laterally mobile microdomains that are highly enriched in lymphocyte signaling molecules. GM1 gangliosides are a common lipid raft component and have been shown to be important in many T cell functions. The aggregation of specific GM1 lipid rafts can control many T cell activation events, including their novel association with T cell integrins. We found that clustering GM1 lipid rafts can regulate β1 integrin function. This was apparent through increased resistance to shear flow dependent detachment of T cells adherent to the α4β1 and α5β1 integrin ligand fibronectin (FN). Adhesion strengthening as a result of clustering GM1 enriched lipid rafts correlated with increased cellular rigidity and morphology through the localization of cortical F-actin, the resistance to shear induced cell stretching, and an increase in the surface area and symmetry of the contact area between the cell surface and adhesive substrate. Furthermore, clustering GM1 lipid rafts could initiate integrin “inside-out” signaling mechanisms. This was seen through increased integrin-cytoskeleton associations and enhanced soluble binding of FN and VCAM-1 suggesting the induction of high affinity integrin conformations. The activation of these adhesion strengthening characteristics appear to be specific for the aggregation of GM1 lipid rafts as the aggregation of the heterogeneous raft associated molecule CD59 failed to activate these functions. These findings indicate a novel mechanism to signal to β1 integrins and to activate adhesion strengthening processes. PMID:19139760

  1. Amygdalin blocks the in vitro adhesion and invasion of renal cell carcinoma cells by an integrin-dependent mechanism.

    PubMed

    Juengel, Eva; Afschar, Masud; Makarević, Jasmina; Rutz, Jochen; Tsaur, Igor; Mani, Jens; Nelson, Karen; Haferkamp, Axel; Blaheta, Roman A

    2016-03-01

    Information about the natural compound amygdalin, which is employed as an antitumor agent, is sparse and thus its efficacy remains controversial. In this study, to determine whether amygdalin exerts antitumor effects on renal cell carcinoma (RCC) cells, its impact on RCC metastatic activity was investigated. The RCC cell lines, Caki-1, KTC-26 and A498, were exposed to amygdalin from apricot kernels, and adhesion to human vascular endothelium, immobilized collagen or fibronectin was investigated. The influence of amygdalin on chemotactic and invasive activity was also determined, as was the influence of amygdalin on surface and total cellular α and β integrin expression, which are involved in metastasis. We noted that amygdalin caused significant reductions in chemotactic activity, invasion and adhesion to endothelium, collagen and fibronectin. Using FACScan analysis, we noted that amygdalin also induced reductions, particularly in integrins α5 and α6, in all three cell lines. Functional blocking of α5 resulted in significantly diminished adhesion of KTC-26 and A498 to collagen and also in decreased chemotactic behavior in all three cell lines. Blocking α6 integrin significantly reduced chemotactic activity in all three cell lines. Thus, we suggest that exposing RCC cells to amygdalin inhibits metastatic spread and is associated with downregulation of α5 and α6 integrins. Therefore, we posit that amygdalin exerts antitumor activity in vitro, and this may be linked to integrin regulation.

  2. Clearance of apoptotic photoreceptors: elimination of apoptotic debris into the subretinal space and macrophage-mediated phagocytosis via phosphatidylserine receptor and integrin alphavbeta3.

    PubMed

    Hisatomi, Toshio; Sakamoto, Taiji; Sonoda, Koh-Hei; Tsutsumi, Chikako; Qiao, Hong; Enaida, Hiroshi; Yamanaka, Ichiro; Kubota, Toshiaki; Ishibashi, Tatsuro; Kura, Shinobu; Susin, Santos A; Kroemer, Guido

    2003-06-01

    The effective phagocytotic clearance of apoptotic debris is fundamental to the maintenance of neural tissues during apoptosis. Retinal photoreceptors undergo apoptosis after retinal detachment. Although their induction phase of apoptosis has been well discussed, their phagocytotic process remains quite unclear. We herein demonstrate that apoptotic photoreceptors are selectively eliminated from their physiological localization, the outer nuclear layer, to the subretinal space, and then phagocytosed by monocyte-derived macrophages. This could be shown by an ultrastructural and immunophenotypic analysis. Moreover, in chimera mice expressing transgenic green fluorescent protein in bone marrow-derived cells, the local infiltration of macrophages could be detected after retinal detachment-induced photoreceptor apoptosis. The local injection of an antibody blocking the phosphatidylserine receptor (PSR) or a peptide (GRGDSP)-blocking integrin alphavbeta3 revealed that phagocytotic clearance involves the PSR as well as integrin alphavbeta3 in vivo. Importantly, the level of blockade obtained with these reagents was different. Although anti-PSR increased the frequency of apoptotic cells that fail to bind to macrophages, GRGDSP prevented the engulfment (but not the recognition) of apoptotic photoreceptor cells by macrophages. To our knowledge, this is the first report describing the mechanisms through which apoptotic photoreceptors are selectively eliminated via a directional process in the subretinal space.

  3. Essential role of integrin-linked kinase in regulation of phagocytosis in keratinocytes.

    PubMed

    Sayedyahossein, Samar; Nini, Lylia; Irvine, Timothy S; Dagnino, Lina

    2012-10-01

    Phagocytic melanosome uptake by epidermal keratinocytes is a central protective mechanism against damage induced by ultraviolet radiation. Phagocytosis requires formation of pseudopodia via actin cytoskeleton rearrangements. Integrin-linked kinase (ILK) is an important modulator of actin cytoskeletal dynamics. We have examined the role of ILK in regulation of phagocytosis, using epidermal keratinocytes isolated from mice with epidermis-restricted Ilk gene inactivation. ILK-deficient cells exhibited severely impaired capacity to engulf fluorescent microspheres in response to stimulation of the keratinocyte growth factor (KGF) receptor or the protease-activated receptor-2. KGF induced ERK phosphorylation in ILK-expressing and ILK-deficient cells, suggesting that ILK is not essential for KGF receptor signaling. In contrast, KGF promoted activation of Rac1 and formation of pseudopodia in ILK-expressing, but not in ILK-deficient cells. Rac1-deficient keratinocytes also showed substantially impaired phagocytic ability, underlining the importance of ILK-dependent Rac1 function for particle engulfment. Finally, cross-modulation of KGF receptors by integrins may be another important element, as integrin β1-deficient keratinocytes also fail to show significant phagocytosis in response to KGF. Thus, we have identified a novel signaling pathway essential for phagocytosis in keratinocytes, which involves ILK-dependent activation of Rac1 in response to KGF, resulting in the formation of pseudopodia and particle uptake.

  4. α5β1-Integrin inhibitor (CLT-28643) effective in rabbit trabeculectomy model.

    PubMed

    Schultheiss, Maximilian; Schnichels, Sven; Konrad, Eva-Maria; Bartz-Schmidt, Karl U; Zahn, Grit; Caldirola, Patrizia; Fsadni, Mario G; Caram-Lelham, Ninus; Spitzer, Martin S

    2017-02-01

    Glaucoma filtration surgery (GFS) fails due to fibrosis. The α5β1-integrin plays a pivotal role in fibrosis, angiogenesis and inflammation. This is the first experiment evaluating the prevention of fibrosis after GFS by a specific small molecule α5β1-integrin inhibitor (CLT-28643). Twenty-four rabbits received trabeculectomy on their right eyes. The rabbits were randomized into three groups of eight eyes each. CLT-28643 was given as a single subconjunctival injection intraoperatively to two of the right eye groups followed by postoperative vehicle eye drops (CLT+ group) or CLT-28643 eye drops 4 times daily (CLT++ group). A third group received mitomycin-C (MMC) intraoperatively (sponge application, 0.04%, 2 min) followed by vehicle eye drops postoperatively. The control-surgery group consisted of 12 left eyes having trabeculectomy with no adjunctive therapy. The remaining 12 left eyes formed the untreated group. Clinical assessment included intraocular pressure (IOP) measurement, slit-lamp examination (including bleb survival and morphology) and bleb photography. The rabbits were killed after four weeks for histology. Both CLT-28643-treated groups showed significantly prolonged bleb survival, and better bleb score compared to the control-surgery group. At end of the study, most functioning blebs were found in the MMC group (MMC group 75%; CLT+ group 12.5%, CLT++ group 25%; CLT+ group 12.5%, control-surgery group 0%). CLT-28643 was non-toxic and well tolerated. This rabbit GFS study indicates that inhibition of α5β1-integrin by the novel α5β1-integrin antagonist CLT-28643 significantly improved the outcome. The effect of a single intro-operative application of CLT-28643 seems to be inferior to 0.04% MMC. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  5. Top surface blade residues and the central channel water molecules are conserved in every repeat of the integrin-like β-propeller structures.

    PubMed

    Denesyuk, Alexander; Denessiouk, Konstantin; Johnson, Mark S

    2018-02-01

    An integrin-like β-propeller domain contains seven repeats of a four-stranded antiparallel β-sheet motif (blades). Previously we described a 3D structural motif within each blade of the integrin-type β-propeller. Here, we show unique structural links that join different blades of the β-propeller structure, which together with the structural motif for a single blade are repeated in a β-propeller to provide the functional top face of the barrel, found to be involved in protein-protein interactions and substrate recognition. We compare functional top face diagrams of the integrin-type β-propeller domain and two non-integrin type β-propeller domains of virginiamycin B lyase and WD Repeat-Containing Protein 5. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Altered cell-matrix associated ADAM proteins in Alzheimer disease.

    PubMed

    Gerst, J L; Raina, A K; Pirim, I; McShea, A; Harris, P L; Siedlak, S L; Takeda, A; Petersen, R B; Smith, M A

    2000-03-01

    Alterations in cell-matrix 'contact' are often related to a disruption of cell cycle regulation and, as such, occur variously in neoplasia. Given the recent findings showing cell cycle alterations in Alzheimer disease, we undertook a study of ADAM-1 and 2 (A Disintegrin And Metalloprotease), developmentally-regulated, integrin-binding, membrane-bound metalloproteases. Our results show that whereas ADAM-1 and 2 are found in susceptible hippocampal neurons in Alzheimer disease, these proteins were not generally increased in similar neuronal populations in younger or age-matched controls except in association with age-related neurofibrillary alterations. This increase in both ADAM-1 and 2 in cases of Alzheimer disease was verified by immunoblot analysis (P < 0.05). An ADAM-induced loss of matrix integration would effectively "reset" the mitotic clock and thereby stimulate re-entry into the cell cycle in neurons in Alzheimer disease. Furthermore, given the importance of integrins in maintaining short-term memory, alterations in ADAM proteins or their proteolytic activity could also play a proximal role in the clinico-pathological manifestations of Alzheimer disease. Copyright 2000 Wiley-Liss, Inc.

  7. Bartonella henselae engages inside-out and outside-in signaling by integrin β1 and talin1 during invasome-mediated bacterial uptake.

    PubMed

    Truttmann, Matthias C; Misselwitz, Benjamin; Huser, Sonja; Hardt, Wolf-Dietrich; Critchley, David R; Dehio, Christoph

    2011-11-01

    The VirB/D4 type IV secretion system (T4SS) of the bacterial pathogen Bartonella henselae (Bhe) translocates seven effector proteins (BepA-BepG) into human cells that subvert host cellular functions. Two redundant pathways dependent on BepG or the combination of BepC and BepF trigger the formation of a bacterial uptake structure termed the invasome. Invasome formation is a multi-step process consisting of bacterial adherence, effector translocation, aggregation of bacteria on the cell surface and engulfment, and eventually, complete internalization of the bacterial aggregate occurs in an F-actin-dependent manner. In the present study, we show that Bhe-triggered invasome formation depends on integrin-β1-mediated signaling cascades that enable assembly of the F-actin invasome structure. We demonstrate that Bhe interacts with integrin β1 in a fibronectin- and VirB/D4 T4SS-independent manner and that activated integrin β1 is essential for both effector translocation and the actin rearrangements leading to invasome formation. Furthermore, we show that talin1, but not talin2, is required for inside-out activation of integrin β1 during invasome formation. Finally, integrin-β1-mediated outside-in signaling by FAK, Src, paxillin and vinculin is necessary for invasome formation. This is the first example of a bacterial entry process that fully exploits the bi-directional signaling capacity of integrin receptors in a talin1-specific manner.

  8. Alpha-v Integrin Targeted PET Imaging of Breast Cancer Angiogenesis and Low-Dose Metronomic Anti-Angiogenic Chemotherapy Efficacy

    DTIC Science & Technology

    2008-08-01

    AD_________________ Award Number: W81XWH-04-1-0697 TITLE: Alpha -v Integrin Targeted PET Imaging of...SUBTITLE 5a. CONTRACT NUMBER Alpha -v Integrin Targeted PET Imaging of Breast Cancer Angiogenesis and Low- Dose Metronomic Anti-Angiogenic...Evaluation of biodistribution and anti-tumor effect of a dimeric RGD peptide-paclitaxel conjugate in mice with breast cancer” was published in Eur J Nucl

  9. Biomimetic design of platelet adhesion inhibitors to block integrin α2β1-collagen interactions: I. Construction of an affinity binding model.

    PubMed

    Zhang, Lin; Sun, Yan

    2014-04-29

    Platelet adhesion on a collagen surface through integrin α2β1 has been proven to be significant for the formation of arterial thrombus. However, the molecular determinants mediating the integrin-collagen complex remain unclear. In the present study, the dynamics of integrin-collagen binding and molecular interactions were investigated using molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) analysis. Hydrophobic interaction is identified as the major driving force for the formation of the integrin-collagen complex. On the basis of the MD simulation and MM-PBSA results, an affinity binding model (ABM) of integrin for collagen is constructed; it is composed of five residues, including Y157, N154, S155, R288, and L220. The ABM has been proven to capture the major binding motif contributing 84.8% of the total binding free energy. On the basis of the ABM, we expect to establish a biomimetic design strategy of platelet adhesion inhibitors, which would be beneficial for the development of potent peptide-based drugs for thrombotic diseases.

  10. α1β1 Integrin/Rac1-Dependent Mesangial Invasion of Glomerular Capillaries in Alport Syndrome

    PubMed Central

    Zallocchi, Marisa; Johnson, Brianna M.; Meehan, Daniel T.; Delimont, Duane; Cosgrove, Dominic

    2014-01-01

    Alport syndrome, hereditary glomerulonephritis with hearing loss, results from mutations in type IV collagen COL4A3, COL4A4, or COL4A5 genes. The mechanism for delayed glomerular disease onset is unknown. Comparative analysis of Alport mice and CD151 knockout mice revealed progressive accumulation of laminin 211 in the glomerular basement membrane. We show mesangial processes invading the capillary loops of both models as well as in human Alport glomeruli, as the likely source of this laminin. l-NAME salt–induced hypertension accelerated mesangial cell process invasion. Cultured mesangial cells showed reduced migratory potential when treated with either integrin-linked kinase inhibitor or Rac1 inhibitor, or by deletion of integrin α1. Treatment of Alport mice with Rac1 inhibitor or deletion of integrin α1 reduced mesangial cell process invasion of the glomerular capillary tuft. Laminin α2–deficient Alport mice show reduced mesangial process invasion, and cultured laminin α2–null cells showed reduced migratory potential, indicating a functional role for mesangial laminins in progression of Alport glomerular pathogenesis. Collectively, these findings predict a role for biomechanical insult in the induction of integrin α1β1–dependent Rac1-mediated mesangial cell process invasion of the glomerular capillary tuft as an initiation mechanism of Alport glomerular pathology. PMID:23911822

  11. Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone.

    PubMed

    El-Amin, Saadiq F; Attawia, Mohamed; Lu, Helen H; Shah, Asist K; Chang, Richard; Hickok, Noreen J; Tuan, Rocky S; Laurencin, Cato T

    2002-01-01

    The use of biodegradable polymers in the field of orthopaedic surgery has gained increased popularity, as surgical pins and screws, and as potential biological scaffolds for repairing cartilage and bone defects. One such group of polymers that has gained considerable attention are the polyesters, poly(lactide-co-glycolide) (PLAGA) and polylactic acid (PLA), because of their minimal tissue inflammatory response, favorable biocompatibility and degradation characteristics. The objective of this study was to evaluate human osteoblastic cell adherence and growth on PLAGA and PLA scaffolds by examining integrin receptor (alpha2, alpha3, alpha4, alpha5, alpha6 and beta1) expression. Primary human osteoblastic cells isolated from trabecular bone adhered efficiently to both PLAGA and PLA, with the rate of adherence on PLAGA comparable to that of control tissue culture polystyrene (TCPS), and significantly higher than on PLA polymers at 3, 6 and 12 h. Human osteoblastic phenotypic expression, alkaline phosphatase (ALP) activity was positive on both degradable matrices, whereas osteocalcin levels were significantly higher on cells grown on PLAGA than on PLA composites. Interestingly, the integrin subunits, alpha2, alpha3, alpha4, alpha5, alpha6 and beta1 were all expressed at higher levels by osteoblasts cultured on PLAGA than those on PLA as analyzed by westerns blots and by flow cytometry. Among the integrins, alpha2, beta5 and beta1 showed the greatest difference in levels between the two surfaces. Thus, both PLA and PLAGA support osteoblastic adhesion and its accompanying engagement of integrin receptor and expression of osteocalcin and ALP. However PLAGA consistently appeared to be a better substrate for osteoblastic cells based on these parameters. This study is one of the first to investigate the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable polymers PLAGA and PLA, and to examine the expression of their key

  12. In situ analysis of integrin and growth factor receptor signaling pathways in human glioblastomas suggests overlapping relationships with focal adhesion kinase activation.

    PubMed

    Riemenschneider, Markus J; Mueller, Wolf; Betensky, Rebecca A; Mohapatra, Gayatry; Louis, David N

    2005-11-01

    Deregulated integrin signaling is common in cancers, including glioblastoma. Integrin binding and growth factor receptor signaling activate focal adhesion kinase (FAK) and subsequently up-regulate extracellular regulated kinases (ERK-1/2), leading to cell-cycle progression and cell migration. Most studies of this pathway have used in vitro systems or tumor lysate-based approaches. We examined these pathways primarily in situ using a panel of 30 glioblastomas and gene expression arrays, immunohistochemistry, and fluorescence in situ hybridization, emphasizing the histological distribution of molecular changes. Within individual tumors, increased expression of FAK, p-FAK, paxillin, ERK-1/2, and p-ERK-1/2 occurred in regions of elevated EGFR and/or PDGFRA expression. Moreover, FAK activation levels correlated with EGFR and PDGFRA expression, and p-FAK and EGFR expression co-localized at the single-cell level. In addition, integrin expression was enriched in EGFR/PDGFRA-overexpressing areas but was more regionally confined than FAK, p-FAK, and paxillin. Integrins beta8 and alpha5beta1 were most commonly expressed, often in a perinecrotic or perivascular pattern. Taken together, our data suggest that growth factor receptor overexpression facilitates alterations in the integrin signaling pathway. Thus, FAK may act in glioblastoma as a downstream target of growth factor signaling, with integrins enhancing the impact of such signaling in the tumor microenvironment.

  13. Integrin-specific mechanoresponses to compression and extension probed by cylindrical flat-ended AFM tips in lung cells.

    PubMed

    Acerbi, Irene; Luque, Tomás; Giménez, Alícia; Puig, Marta; Reguart, Noemi; Farré, Ramon; Navajas, Daniel; Alcaraz, Jordi

    2012-01-01

    Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ~1 µm(2) cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca(2+) signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

  14. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rossier, Olivier; Giannone, Grégory; CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux

    Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements andmore » interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.« less

  15. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking.

    PubMed

    Rossier, Olivier; Giannone, Grégory

    2016-04-10

    Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells. Copyright © 2015. Published by Elsevier Inc.

  16. The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL-C3G-mediated activation of Rap1.

    PubMed

    Nolz, Jeffrey C; Nacusi, Lucas P; Segovis, Colin M; Medeiros, Ricardo B; Mitchell, Jason S; Shimizu, Yoji; Billadeau, Daniel D

    2008-09-22

    WAVE2 regulates T cell receptor (TCR)-stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL-C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL-C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.

  17. The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL–C3G-mediated activation of Rap1

    PubMed Central

    Nolz, Jeffrey C.; Nacusi, Lucas P.; Segovis, Colin M.; Medeiros, Ricardo B.; Mitchell, Jason S.; Shimizu, Yoji; Billadeau, Daniel D.

    2008-01-01

    WAVE2 regulates T cell receptor (TCR)–stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL–C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL–C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation. PMID:18809728

  18. Beta1 integrin inhibits apoptosis induced by cyclic stretch in annulus fibrosus cells via ERK1/2 MAPK pathway.

    PubMed

    Zhang, Kai; Ding, Wei; Sun, Wei; Sun, Xiao-jiang; Xie, You-zhuan; Zhao, Chang-qing; Zhao, Jie

    2016-01-01

    Low back pain is associated with intervertebral disc degeneration (IVDD) due to cellular loss through apoptosis. Mechanical factors play an important role in maintaining the survival of the annulus fibrosus (AF) cells and the deposition of extracellular matrix. However, the mechanisms that excessive mechanical forces lead to AF cell apoptosis are not clear. The present study was to look for how AF cells sense mechanical changes. In vivo experiments, the involvement of mechanoreceptors in apoptosis was examined by RT-PCR and/or immunoblotting in the lumbar spine of rats subjected to unbalanced dynamic and static forces. In vitro experiments, we investigated apoptotic signaling pathways in untransfected and transfected AF cells with the lentivirus vector for rat β1 integrin overexpression after cyclic stretch. Apoptosis in AF cells was assessed using flow cytometry, Hoechst 33258 nuclear staining. Western blotting was used to analyze expression of β1 integrin and caspase-3 and ERK1/2 MAPK signaling molecules. In the rat IVDD model, unbalanced dynamic and static forces induced apoptosis of disc cells, which corresponded to decreased expression of β1 integrin. Cyclic stretch-induced apoptosis in rat AF cells correlated with the activation of caspase-3 and with decreased levels of β1 integrin and the phosphorylation levels of ERK1/2 activation level. However, the overexpression of β1 integrin in AF cells ameliorated cyclic stretch-induced apoptosis and decreased caspase-3 activation. Furthermore, ERK1/2-specific inhibitor promotes apoptosis in vector β1-infected AF cells. These results suggest that the disruption of β1 integrin signaling may underlie disc cell apoptosis induced by mechanical stress. Further work is necessary to fully elucidate the pathophysiological mechanisms that underlie IVDD caused by unbalanced dynamic and static forces.

  19. RGD peptide-mediated chitosan-based polymeric micelles targeting delivery for integrin-overexpressing tumor cells.

    PubMed

    Cai, Li-Li; Liu, Ping; Li, Xi; Huang, Xuan; Ye, Yi-Qing; Chen, Feng-Ying; Yuan, Hong; Hu, Fu-Qiang; Du, Yong-Zhong

    2011-01-01

    Solid tumors need new blood vessels to feed and nourish them as well as to allow tumor cells to escape into the circulation and lodge in other organs, which is termed "angiogenesis." Some tumor cells within solid tumors can overexpress integrins α(v)β(3) and α(v)β(5), which can specifically recognize the peptide motif Arg-Gly-Asp (RGD). Thus, the targeting of RGD-modified micelles to tumor vasculature is a promising strategy for tumor-targeting treatment. RGD peptide (GSSSGRGDSPA) was coupled to poly(ethylene glycol)-modified stearic acid-grafted chitosan (PEG-CS-SA) micelles via chemical reaction in the presence of N,N'-Disuccinimidyl carbonate. The critical micelle concentration of the polymeric micelles was determined by measuring the fluorescence intensity of pyrene as a fluorescent probe. The micelle size, size distribution, and zeta potential were measured by light scattering and electrophoretic mobility. Doxorubicin (DOX) was chosen as a model anticancer drug to investigate the drug entrapment efficiency, in vitro drug-release profile, and in vitro antitumor activities of drug-loaded RGD-PEG-CS-SA micelles in cells that overexpress integrins (α(ν)β(3) and α(ν)β(5)) and integrin-deficient cells. Using DOX as a model drug, the drug encapsulation efficiency could reach 90%, and the in vitro drug-release profiles suggested that the micelles could be used as a controlled-release carrier for the hydrophobic drug. Qualitative and quantitative analysis of cellular uptake indicated that RGD-modified micelles could significantly increase the DOX concentration in integrin-overexpressing human hepatocellular carcinoma cell line (BEL-7402), but not in human epithelial carcinoma cell line (Hela). The competitive cellular-uptake test showed that the cellular uptake of RGD-modified micelles in BEL-7402 cells was significantly inhibited in the presence of excess free RGD peptides. In vitro cytotoxicity tests demonstrated DOX-loaded RGD-modified micelles could

  20. The depletion of MARVELD1 leads to murine placenta accreta via integrin β4-dependent trophoblast cell invasion.

    PubMed

    Chen, Yue; Zhang, Hui; Han, Fang; Yue, Lei; Qiao, Chunxiao; Zhang, Yao; Dou, Peng; Liu, Weizhe; Li, Yu

    2018-03-01

    The placenta is a remarkable organ, it serves as the interface between the mother and the fetus. Proper invasion of trophoblast cells is required for a successful pregnancy. Previous studies have found that the adhesion molecule integrin β4 plays important roles during trophoblast cell invasion. Here, we found that the overall birth rate of the MARVELD1 knockout mouse is much lower than that of the wild-type mouse (p < 0.001). In E18.5 MARVELD1 knockout mice, we observed an over-invasion of trophoblast cells, and indeed, the pregnant mice had a partial placenta accreta phenotype. The HTR8/SVneo cell line was used as an in vitro model to elucidate the underlying mechanisms of MARVELD1-mediated trophoblast invasion. We detected a diminished expression of integrin β4 upon the downregulation of MARVELD1 and enhanced migrate and invasive abilities of trophoblast cells both in vivo and in vitro. The integrin β4 rescue assay also supported the results. In conclusion, this study found that MARVELD1 mediated the invasion of trophoblast cells via regulating the expression of integrin β4 during placenta development. © 2017 Wiley Periodicals, Inc.

  1. Vascular Induction of a Disintegrin and Metalloprotease 17 by Angiotensin II Through Hypoxia Inducible Factor 1α

    PubMed Central

    Obama, Takashi; Takayanagi, Takehiko; Kobayashi, Tomonori; Bourne, Allison M.; Elliott, Katherine J.; Charbonneau, Martine; Dubois, Claire M.

    2015-01-01

    BACKGROUND A disintegrin and metalloprotease 17 (ADAM17) is a membrane-spanning metalloprotease overexpressed in various cardiovascular diseases such as hypertension and atherosclerosis. However, little is known regarding the regulation of ADAM17 expression in the cardiovascular system. Here, we test our hypothesis that angiotensin II induces ADAM17 expression in the vasculature. METHODS Cultured vascular smooth muscle cells were stimulated with 100nM angiotensin II. Mice were infused with 1 μg/kg/minute angiotensin II for 2 weeks. ADAM17 expression was evaluated by a promoter–reporter construct, quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry. RESULTS In vascular smooth muscle cells, angiotensin II increased ADAM17 protein expression, mRNA, and promoter activity. We determined that the angiotensin II response involves hypoxia inducible factor 1α and a hypoxia responsive element. In angiotensin II–infused mice, marked induction of ADAM17 and hypoxia inducible factor 1α was seen in vasculatures in heart and kidney, as well as in aortae, by immunohistochemistry. CONCLUSIONS Angiotensin II induces ADAM17 expression in the vasculatures through a hypoxia inducible factor 1α–dependent transcriptional upregulation, potentially contributing to end-organ damage in the cardiovascular system. PMID:24871629

  2. Imaging Metastasis Using an Integrin-Targeting Chain-Shaped Nanoparticle

    PubMed Central

    Peiris, Pubudu M.; Toy, Randall; Doolittle, Elizabeth; Pansky, Jenna; Abramowski, Aaron; Tam, Morgan; Vicente, Peter; Tran, Emily; Hayden, Elliott; Camann, Andrew; Mayer, Aaron; Erokwu, Bernadette O.; Berman, Zachary; Wilson, David; Baskaran, Harihara; Flask, Chris A.; Keri, Ruth A.; Karathanasis, Efstathios

    2012-01-01

    While the enhanced permeability and retention effect may promote the preferential accumulation of nanoparticles into well-vascularized primary tumors, it is ineffective in the case of metastases hidden within a large population of normal cells. Due to their small size, high dispersion to organs, and low vascularization, metastatic tumors are less accessible to targeted nanoparticles. To tackle these challenges, we designed a nanoparticle for vascular targeting based on an αvβ3 integrin-targeted nanochain particle composed of four iron oxide nanospheres chemically linked in a linear assembly. The chain-shaped nanoparticles enabled enhanced ‘sensing’ of the tumor-associated remodeling of the vascular bed offering increased likelihood of specific recognition of metastatic tumors. Compared to spherical nanoparticles, the chain-shaped nanoparticles resulted in superior targeting of αvβ3 integrin due to geometrically enhanced multivalent docking. We performed multimodal in vivo imaging (Fluorescence Molecular Tomography and Magnetic Resonance Imaging) in a non-invasive and quantitative manner, which showed that the nanoparticles targeted metastases in the liver and lungs with high specificity in a highly aggressive breast tumor model in mice. PMID:23005348

  3. Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure

    NASA Technical Reports Server (NTRS)

    Maniotis, A. J.; Chen, C. S.; Ingber, D. E.

    1997-01-01

    We report here that living cells and nuclei are hard-wired such that a mechanical tug on cell surface receptors can immediately change the organization of molecular assemblies in the cytoplasm and nucleus. When integrins were pulled by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoskeleton and nucleus. Actin microfilaments mediated force transfer to the nucleus at low strain; however, tearing of the actin gel resulted with greater distortion. In contrast, intermediate filaments effectively mediated force transfer to the nucleus under both conditions. These filament systems also acted as molecular guy wires to mechanically stiffen the nucleus and anchor it in place, whereas microtubules acted to hold open the intermediate filament lattice and to stabilize the nucleus against lateral compression. Molecular connections between integrins, cytoskeletal filaments, and nuclear scaffolds may therefore provide a discrete path for mechanical signal transfer through cells as well as a mechanism for producing integrated changes in cell and nuclear structure in response to changes in extracellular matrix adhesivity or mechanics.

  4. Connective tissue growth factor and integrin αvβ6: a new pair of regulators critical for ductular reaction and biliary fibrosis in mice.

    PubMed

    Pi, Liya; Robinson, Paulette M; Jorgensen, Marda; Oh, Seh-Hoon; Brown, Alicia R; Weinreb, Paul H; Trinh, Thu Le; Yianni, Protopapadakis; Liu, Chen; Leask, Andrew; Violette, Shelia M; Scott, Edward W; Schultz, Gregory S; Petersen, Bryon E

    2015-02-01

    Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell-matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvβ6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvβ6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter-driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvβ6 during liver injury induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen-inducible Cre-loxP system down-regulated integrin αvβ6 in DDC-damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvβ6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis-related genes, smaller areas of alpha-smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvβ6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)-β1 activation in vitro. CTGF and integrin αvβ6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF-β1. CTGF and integrin αvβ6 are potential therapeutic targets to control DRs and fibrosis in related liver

  5. A near-infrared phthalocyanine dye-labeled agent for integrin αvβ6-targeted theranostics of pancreatic cancer.

    PubMed

    Gao, Duo; Gao, Liquan; Zhang, Chenran; Liu, Hao; Jia, Bing; Zhu, Zhaohui; Wang, Fan; Liu, Zhaofei

    2015-06-01

    Integrin αvβ6 is widely upregulated in variant malignant cancers but is undetectable in normal organs, making it a promising target for cancer diagnostic imaging and therapy. Using streptavidin-biotin chemistry, we synthesized an integrin αvβ6-targeted near-infrared phthalocyanine dye-labeled agent, termed Dye-SA-B-HK, and investigated whether it could be used for cancer imaging, optical imaging-guided surgery, and phototherapy in pancreatic cancer mouse models. Dye-SA-B-HK specifically bound to integrin αvβ6 in vitro and in vivo with high receptor binding affinity. Using small-animal optical imaging, we detected subcutaneous and orthotopic BxPC-3 human pancreatic cancer xenografts in vivo. Upon optical image-guidance, the orthotopically growing pancreatic cancer lesions could be successfully removed by surgery. Using light irradiation, Dye-SA-B-HK manifested remarkable antitumor effects both in vitro and in vivo. (18)F-FDG positron emission tomography (PET) imaging and ex vivo fluorescence staining validated the observed decrease in proliferation of treated tumors by Dye-DA-B-HK phototherapy. Tissue microarray results revealed overexpression of integrin αvβ6 in over 95% cases of human pancreatic cancer, indicating that theranostic application of Dye-DA-B-HK has clear translational potential. Overall, the results of this study demonstrated that integrin αvβ6-specific Dye-SA-B-HK is a promising theranostic agent for the management of pancreatic cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. A novel leukocyte adhesion deficiency caused by expressed but nonfunctional β2 integrins Mac-1 and LFA-1

    PubMed Central

    Hogg, Nancy; Stewart, Mairi P.; Scarth, Sarah L.; Newton, Rebecca; Shaw, Jacqueline M.; Law, S.K. Alex; Klein, Nigel

    1999-01-01

    In the leukocyte adhesion deficiency (LAD)-1 syndrome, there is diminished expression of β2(CD18) integrins. This is caused by lesions in the β2-subunit gene and gives rise to recurrent bacterial infections, impaired pus formation, and poor wound healing. We describe a patient with clinical features compatible with a moderately severe phenotype of LAD-1 but who expresses the β2 integrins lymphocyte function– associated molecule (LFA)-1 and Mac-1 at 40%–60% of normal levels. This level of expression should be adequate for normal integrin function, but both the patient's Mac-1 on neutrophils and LFA-1 on T cells failed to bind ligands such as fibrinogen and intercellular adhesion molecule (ICAM)-1, respectively, or to display a β2-integrin activation epitope after adhesion-inducing stimuli. Unexpectedly, divalent cation treatment induced the patient's T cells to bind to ICAM-2 and ICAM-3. Sequencing of the patient's two CD18 alleles revealed the mutations S138P and G273R. Both mutations are in the β2-subunit conserved domain, with S138P a putative divalent cation coordinating residue in the metal ion–dependent adhesion site (MIDAS) motif. After K562 cell transfection with α subunits, the mutated S138P β subunit was coexpressed but did not support function, whereas the G273R mutant was not expressed. In summary, the patient described here exhibits failure of the β2 integrins to function despite adequate levels of cell-surface expression. PMID:9884339

  7. Integrin receptor involvement in actin cable formation in an in vitro model of events associated with wound contraction.

    PubMed

    Stephens, P; Genever, P G; Wood, E J; Raxworthy, M J

    1997-01-01

    Actin cables have been reported to act in vivo as contractile 'purse strings' capable of closing embryonic wounds through generation of circumferential tension. Furthermore, their involvement in wounds within in vitro model systems suggests that actin cable contraction may be an important mechanism involved in the process of wound closure. The aim of this study therefore, was to investigate the appearance of actin cables in a contracting fibroblast populated collagen lattice, an in vitro model of events associated with wound contraction. Utilising this in vitro model, the time-course of actin cable production was investigated and the involvement of integrin receptors analysed using immunofluorescent labelling techniques. Over a period of hours distinct cellular cable-like structures developed at the edges of collagen lattices coinciding with the onset of contraction. Cellular organisation within the cable was evident as was polymerisation of actin microfilaments into elongated stress fibres forming a continuous cell-cell 'actin cable' around the circumference of the lattice. Immunolocalisation demonstrated that integrin receptor subunits beta 1 and alpha 2 but not alpha 5 were involved in apparent intimate cell-cell contact between juxtaposed fibroblasts within this actin cable. This study demonstrates the involvement of integrin receptors in actin cable formation within collagen lattice systems undergoing reorganisation. Such integrin involvement may enable participating cells to respond to the tensional status of their surrounding environment and via cell-cell communication, to permit a co-ordinated contraction of the cable. It is concluded that integrin receptor involvement in active actin cable contraction may be involved in the process of wound contraction.

  8. SEL1L Regulates Adhesion, Proliferation and Secretion of Insulin by Affecting Integrin Signaling

    PubMed Central

    Diaferia, Giuseppe R.; Cirulli, Vincenzo; Biunno, Ida

    2013-01-01

    SEL1L, a component of the endoplasmic reticulum associated degradation (ERAD) pathway, has been reported to regulate the (i) differentiation of the pancreatic endocrine and exocrine tissue during the second transition of mouse embryonic development, (ii) neural stem cell self-renewal and lineage commitment and (iii) cell cycle progression through regulation of genes related to cell-matrix interaction. Here we show that in the pancreas the expression of SEL1L is developmentally regulated, such that it is readily detected in developing islet cells and in nascent acinar clusters adjacent to basement membranes, and becomes progressively restricted to the islets of Langherans in post-natal life. This peculiar expression pattern and the presence of two inverse RGD motifs in the fibronectin type II domain of SEL1L protein indicate a possible interaction with cell adhesion molecules to regulate islets architecture. Co-immunoprecipitation studies revealed SEL1L and ß1-integrin interaction and, down-modulation of SEL1L in pancreatic ß-cells, negatively influences both cell adhesion on selected matrix components and cell proliferation likely due to altered ERK signaling. Furthermore, the absence of SEL1L protein strongly inhibits glucose-stimulated insulin secretion in isolated mouse pancreatic islets unveiling an important role of SEL1L in insulin trafficking. This phenotype can be rescued by the ectopic expression of the ß1-integrin subunit confirming the close interaction of these two proteins in regulating the cross-talk between extracellular matrix and insulin signalling to create a favourable micro-environment for ß-cell development and function. PMID:24324549

  9. The translational blocking of α5 and α6 integrin subunits affects migration and invasion, and increases sensitivity to carboplatin of SKOV-3 ovarian cancer cell line

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Villegas-Pineda, Julio César, E-mail: jcvillegas@cinvestav.mx; Toledo-Leyva, Alfredo, E-mail: toledo_leyva@hotmail.com; Osorio-Trujillo, Juan Carlos, E-mail: clostrujillo2@yahoo.com.mx

    Epithelial ovarian cancer is the most lethal gynecologic malignancy. Integrins, overexpressed in cancer, are involved in various processes that favor the development of the disease. This study focused on determining the degree of involvement of α5, α6 and β3 integrin subunits in the establishment/development of epithelial ovarian cancer (EOC), such as proliferation, migration, invasion, and response to carboplatin. The translation of the α5, α6 and β3 integrins was blocked using morpholines, generating morphant cells for these proteins, which were corroborated by immunofluorescence assays. WST-1 proliferation assay showed that silencing of α5, α6, and β3 integrins does not affect the survivalmore » of morphants. Wound healing and transwell chamber assays showed that blocking α5 and α6 integrins decrease, in lesser and greater level respectively, the migratory and the invasive capacity of SKOV-3 cells. Finally, blocking α5 and α6 integrins partially sensitized the cells response to carboplatin, while blocking integrin β3 generated resistance to this drug. Statistical analyses were performed with the GraphPad Prism 5.0 software employing one way and two-way ANOVA tests; data are shown as average±SD. Results suggest that α5 and α6 integrins could become good candidates for chemotherapy targets in EOC.« less

  10. 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

  11. INTEGRIN-MEDIATED CELL ATTACHMENT SHOWS TIME-DEPENDENT UPREGULATION OF GAP JUNCTION COMMUNICATION.

    EPA Science Inventory


    Integrin-mediated Cell Attachment Shows Time-Dependent Upregulation of Gap Junction
    Communication

    Rachel Grindstaff and Carl Blackman, National Health & Environmental Effects Research
    Laboratory, Office of Research and Development, US EPA, Research Triang...

  12. β1 integrin signaling promotes neuronal migration along vascular scaffolds in the post-stroke brain.

    PubMed

    Fujioka, Teppei; Kaneko, Naoko; Ajioka, Itsuki; Nakaguchi, Kanako; Omata, Taichi; Ohba, Honoka; Fässler, Reinhard; García-Verdugo, José Manuel; Sekiguchi, Kiyotoshi; Matsukawa, Noriyuki; Sawamoto, Kazunobu

    2017-02-01

    Cerebral ischemic stroke is a main cause of chronic disability. However, there is currently no effective treatment to promote recovery from stroke-induced neurological symptoms. Recent studies suggest that after stroke, immature neurons, referred to as neuroblasts, generated in a neurogenic niche, the ventricular-subventricular zone, migrate toward the injured area, where they differentiate into mature neurons. Interventions that increase the number of neuroblasts distributed at and around the lesion facilitate neuronal repair in rodent models for ischemic stroke, suggesting that promoting neuroblast migration in the post-stroke brain could improve efficient neuronal regeneration. To move toward the lesion, neuroblasts form chain-like aggregates and migrate along blood vessels, which are thought to increase their migration efficiency. However, the molecular mechanisms regulating these migration processes are largely unknown. Here we studied the role of β1-class integrins, transmembrane receptors for extracellular matrix proteins, in these migrating neuroblasts. We found that the neuroblast chain formation and blood vessel-guided migration critically depend on β1 integrin signaling. β1 integrin facilitated the adhesion of neuroblasts to laminin and the efficient translocation of their soma during migration. Moreover, artificial laminin-containing scaffolds promoted neuroblast chain formation and migration toward the injured area. These data suggest that laminin signaling via β1 integrin supports vasculature-guided neuronal migration to efficiently supply neuroblasts to injured areas. This study also highlights the importance of vascular scaffolds for cell migration in development and regeneration. Copyright © 2017 3-V Biosciences. Published by Elsevier B.V. All rights reserved.

  13. SU9516 Increases α7β1 Integrin and Ameliorates Disease Progression in the mdx Mouse Model of Duchenne Muscular Dystrophy.

    PubMed

    Sarathy, Apurva; Wuebbles, Ryan D; Fontelonga, Tatiana M; Tarchione, Ashley R; Mathews Griner, Lesley A; Heredia, Dante J; Nunes, Andreia M; Duan, Suzann; Brewer, Paul D; Van Ry, Tyler; Hennig, Grant W; Gould, Thomas W; Dulcey, Andrés E; Wang, Amy; Xu, Xin; Chen, Catherine Z; Hu, Xin; Zheng, Wei; Southall, Noel; Ferrer, Marc; Marugan, Juan; Burkin, Dean J

    2017-06-07

    Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene, resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex (DGC), which links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction-induced muscle damage. The α7β1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, that promoted increased α7β1 integrin expression. Here we show that SU9516 leads to increased α7B integrin in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7β1 integrin in skeletal muscle, ameliorated pathology, and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and Ste20-related proline alanine rich kinase (SPAK)/OSR1 signaling pathways. This study identifies a first in-class α7 integrin-enhancing small-molecule compound with potential for the treatment of DMD. Copyright © 2017 The American Society of Gene and Cell Therapy. All rights reserved.

  14. [Distribution diversity of integrins and calcium channels on major human and mouse host cells of Leptospira species].

    PubMed

    Li, Cheng-xue; Zhao, Xin; Qian, Jing; Yan, Jie

    2012-07-01

    To determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species. The expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay. β1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets. There is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

  15. Loss-of-function FERMT1 mutations in kindler syndrome implicate a role for fermitin family homolog-1 in integrin activation.

    PubMed

    Lai-Cheong, Joey E; Parsons, Maddy; Tanaka, Akio; Ussar, Siegfried; South, Andrew P; Gomathy, Sethuraman; Mee, John B; Barbaroux, Jean-Baptiste; Techanukul, Tanasit; Almaani, Noor; Clements, Suzanne E; Hart, Ian R; McGrath, John A

    2009-10-01

    Kindler syndrome is an autosomal recessive disorder characterized by skin atrophy and blistering. It results from loss-of-function mutations in the FERMT1 gene encoding the focal adhesion protein, fermitin family homolog-1. How and why deficiency of fermitin family homolog-1 results in skin atrophy and blistering are unclear. In this study, we investigated the epidermal basement membrane and keratinocyte biology abnormalities in Kindler syndrome. We identified altered distribution of several basement membrane proteins, including types IV, VII, and XVII collagens and laminin-332 in Kindler syndrome skin. In addition, reduced immunolabeling intensity of epidermal cell markers such as beta1 and alpha6 integrins and cytokeratin 15 was noted. At the cellular level, there was loss of beta4 integrin immunolocalization and random distribution of laminin-332 in Kindler syndrome keratinocytes. Of note, active beta1 integrin was reduced but overexpression of fermitin family homolog-1 restored integrin activation and partially rescued the Kindler syndrome cellular phenotype. This study provides evidence that fermitin family homolog-1 is implicated in integrin activation and demonstrates that lack of this protein leads to pathological changes beyond focal adhesions, with disruption of several hemidesmosomal components and reduced expression of keratinocyte stem cell markers. These findings collectively provide novel data on the role of fermitin family homolog-1 in skin and further insight into the pathophysiology of Kindler syndrome.

  16. The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen

    PubMed Central

    Malcor, Jean-Daniel; Bax, Daniel; Hamaia, Samir W.; Davidenko, Natalia; Best, Serena M.; Cameron, Ruth E.; Farndale, Richard W.; Bihan, Dominique

    2016-01-01

    Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2β1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2β1 and α1β1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons. PMID:26854392

  17. ATP release due to Thy-1–integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation

    PubMed Central

    Henríquez, Mauricio; Herrera-Molina, Rodrigo; Valdivia, Alejandra; Alvarez, Alvaro; Kong, Milene; Muñoz, Nicolás; Eisner, Verónica; Jaimovich, Enrique; Schneider, Pascal; Quest, Andrew F. G.; Leyton, Lisette

    2011-01-01

    Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion. PMID:21502139

  18. A cyclic-RGD-BioShuttle functionalized with TMZ by DARinv “Click Chemistry” targeted to αvβ3 integrin for therapy

    PubMed Central

    Braun, Klaus; Wiessler, Manfred; Pipkorn, Rüdiger; Ehemann, Volker; Bäuerle, Tobias; Fleischhacker, Heinz; Müller, Gabriele; Lorenz, Peter; Waldeck, Waldemar

    2010-01-01

    Clinical experiences often document, that a successful tumor control requires high doses of drug applications. It is widely believed that unavoidable adverse reactions could be minimized by using gene-therapeutic strategies protecting the tumor-surrounding healthy tissue as well as the bone-marrow. One new approach in this direction is the use of “Targeted Therapies” realizing a selective drug targeting to gain effectual amounts at the target site, even with drastically reduced application doses. MCF-7 breast cancer cells expressing the αvβ3 [alpha(v)beta(3)] integrin receptor are considered as appropriate candidates for such a targeted therapy. The modularly composed BioShuttle carrier consisting of different units designed to facilitate the passage across the cell membranes and for subcellular addressing of diagnostic and/or therapeutic molecules could be considered as an eligible delivery platform. Here we used the cyclic RGD-BioShuttle as a carrier for temozolomide (TMZ) at the αvβ3 integrin receptor realizing local TMZ concentrations sufficient for cell killing. The IC50 values are 12 µMol/L in the case of cRGD-BioShuttle-TMZ and 100 µMol/L for underivatized TMZ, which confirms the advantage of TMZ reformulation to realize local concentrations sufficient for cell killing. Our paper focuses on the design, synthesis and application of the cRGD-BioShuttle conjugate composed of the cyclic RGD, a αvβ3 integrin-ligand, ligated to the cytotoxic drug TMZ. The ligation was carried out by the Diels Alder Reaction with inverse electron demand (DARinv). PMID:20922134

  19. β5 Integrin Up-Regulation in Brain-Derived Neurotrophic Factor Promotes Cell Motility in Human Chondrosarcoma

    PubMed Central

    Li, Te-Mao; Fong, Yi-Chin; Liu, Shan-Chi; Chen, Po-Chun; Tang, Chih-Hsin

    2013-01-01

    Chondrosarcoma is a primary malignant bone cancer, with a potent capacity to invade locally and cause distant metastasis; it has a poor prognosis and shows a predilection for metastasis to the lungs. Brain derived neurotrophic factor (BDNF) is a small-molecule protein from the neurotrophin family of growth factors that is associated with the disease status and outcomes of cancers. However, the effect of BDNF on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that human chondrosarcoma tissues showed significant expression of BDNF, which was higher than that in normal cartilage and primary chondrocytes. We also found that BDNF increased the migration and expression of β5 integrin in human chondrosarcoma cells. In addition, knockdown of BDNF expression markedly inhibited migratory activity. BDNF-mediated migration and β5 integrin up-regulation were attenuated by antibody, inhibitor, or siRNA against the TrkB receptor. Pretreatment of chondrosarcoma cells with PI3K, Akt, and NF-κB inhibitors or mutants also abolished BDNF-promoted migration and integrin expression. The PI3K, Akt, and NF-κB signaling pathway was activated after BDNF treatment. Taken together, our results indicate that BDNF enhances the migration of chondrosarcoma by increasing β5 integrin expression through a signal transduction pathway that involves the TrkB receptor, PI3K, Akt, and NF-κB. BDNF thus represents a promising new target for treating chondrosarcoma metastasis. PMID:23874483

  20. α1β1 integrin/Rac1-dependent mesangial invasion of glomerular capillaries in Alport syndrome.

    PubMed

    Zallocchi, Marisa; Johnson, Brianna M; Meehan, Daniel T; Delimont, Duane; Cosgrove, Dominic

    2013-10-01

    Alport syndrome, hereditary glomerulonephritis with hearing loss, results from mutations in type IV collagen COL4A3, COL4A4, or COL4A5 genes. The mechanism for delayed glomerular disease onset is unknown. Comparative analysis of Alport mice and CD151 knockout mice revealed progressive accumulation of laminin 211 in the glomerular basement membrane. We show mesangial processes invading the capillary loops of both models as well as in human Alport glomeruli, as the likely source of this laminin. L-NAME salt-induced hypertension accelerated mesangial cell process invasion. Cultured mesangial cells showed reduced migratory potential when treated with either integrin-linked kinase inhibitor or Rac1 inhibitor, or by deletion of integrin α1. Treatment of Alport mice with Rac1 inhibitor or deletion of integrin α1 reduced mesangial cell process invasion of the glomerular capillary tuft. Laminin α2-deficient Alport mice show reduced mesangial process invasion, and cultured laminin α2-null cells showed reduced migratory potential, indicating a functional role for mesangial laminins in progression of Alport glomerular pathogenesis. Collectively, these findings predict a role for biomechanical insult in the induction of integrin α1β1-dependent Rac1-mediated mesangial cell process invasion of the glomerular capillary tuft as an initiation mechanism of Alport glomerular pathology. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  1. Developmental control of integrin expression regulates Th2 effector homing

    USDA-ARS?s Scientific Manuscript database

    Integrin CD18, a component of the LFA-1 complex that also includes CD11a, is essential for Th2, but not Th1, cell homing, but the explanation for this phenomenon remains obscure. In this study, we investigate the mechanism by which Th2 effector responses require the LFA-1 complex. CD11a-deficient T ...

  2. Mutations of Cystic Fibrosis Transmembrane Conductance Regulator Gene Cause a Monocyte-Selective Adhesion Deficiency.

    PubMed

    Sorio, Claudio; Montresor, Alessio; Bolomini-Vittori, Matteo; Caldrer, Sara; Rossi, Barbara; Dusi, Silvia; Angiari, Stefano; Johansson, Jan E; Vezzalini, Marzia; Leal, Teresinha; Calcaterra, Elisa; Assael, Baroukh M; Melotti, Paola; Laudanna, Carlo

    2016-05-15

    Cystic fibrosis (CF) is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Persistent lung inflammation, characterized by increasing polymorphonuclear leukocyte recruitment, is a major cause of the decline in respiratory function in patients with CF and is a leading cause of morbidity and mortality. CFTR is expressed in various cell types, including leukocytes, but its involvement in the regulation of leukocyte recruitment is unknown. We evaluated whether CF leukocytes might present with alterations in cell adhesion and migration, a key process governing innate and acquired immune responses. We used ex vivo adhesion and chemotaxis assays, flow cytometry, immunofluorescence, and GTPase activity assays in this study. We found that chemoattractant-induced activation of β1 and β2 integrins and of chemotaxis is defective in mononuclear cells isolated from patients with CF. In contrast, polymorphonuclear leukocyte adhesion and chemotaxis were normal. The functionality of β1 and β2 integrins was restored by treatment of CF monocytes with the CFTR-correcting drugs VRT325 and VX809. Moreover, treatment of healthy monocytes with the CFTR inhibitor CFTR(inh)-172 blocked integrin activation by chemoattractants. In a murine model of lung inflammation, we found that integrin-independent migration of CF monocytes into the lung parenchyma was normal, whereas, in contrast, integrin-dependent transmigration into the alveolar space was impaired. Finally, signal transduction analysis showed that, in CF monocytes, chemoattractant-triggered activation of RhoA and CDC42 Rho small GTPases (controlling integrin activation and chemotaxis, respectively) was strongly deficient. Altogether, these data highlight the critical regulatory role of CFTR in integrin activation by chemoattractants in monocytes and identify CF as a new, cell type-selective leukocyte adhesion deficiency disease, providing new insights into CF

  3. Simulated physiological stretch increases expression of extracellular matrix proteins in human bladder smooth muscle cells via integrin α4/αv-FAK-ERK1/2 signaling pathway.

    PubMed

    Chen, Shulian; Peng, Chuandu; Wei, Xin; Luo, Deyi; Lin, Yifei; Yang, Tongxin; Jin, Xi; Gong, Lina; Li, Hong; Wang, Kunjie

    2017-08-01

    To investigate the effect of simulated physiological stretch on the expression of extracellular matrix (ECM) proteins and the role of integrin α4/αv, focal adhesion kinase (FAK), extracellular regulated protein kinases 1/2 (ERK1/2) in the stretch-induced ECM protein expression of human bladder smooth muscle cells (HBSMCs). HBSMCs were seeded onto silicone membrane and subjected to simulated physiological stretch at the range of 5, 10, and 15% elongation. Expression of primary ECM proteins in HBSMCs was analyzed by real-time polymerase chain reaction and Western blot. Specificity of the FAK and ERK1/2 was determined by Western blot with FAK inhibitor and ERK1/2 inhibitor (PD98059). Specificity of integrin α4 and integrin αv was determined with small interfering ribonucleic acid (siRNA) transfection. The expression of collagen I (Col1), collagen III (Col3), and fibronectin (Fn) was increased significantly under the simulated physiological stretch of 10 and 15%. Integrin α4 and αv, FAK, ERK1/2 were activated by 10% simulated physiological stretch compared with the static condition. Pretreatment of ERK1/2 inhibitor, FAK inhibitor, integrin α4 siRNA, or integrin αv siRNA reduced the stretch-induced expression of ECM proteins. And FAK inhibitor decreased the stretch-induced ERK1/2 activity and ECM protein expression. Integrin α4 siRNA or integrin αv siRNA inhibited the stretch-induced activity of FAK. Simulated physiological stretch increases the expression of ECM proteins in HBSMCs, and integrin α4/αv-FAK-ERK1/2 signaling pathway partly modulates the mechano-transducing process.

  4. Canine chondrodysplasia caused by a truncating mutation in collagen-binding integrin alpha subunit 10.

    PubMed

    Kyöstilä, Kaisa; Lappalainen, Anu K; Lohi, Hannes

    2013-01-01

    The skeletal dysplasias are disorders of the bone and cartilage tissues. Similarly to humans, several dog breeds have been reported to suffer from different types of genetic skeletal disorders. We have studied the molecular genetic background of an autosomal recessive chondrodysplasia that affects the Norwegian Elkhound and Karelian Bear Dog breeds. The affected dogs suffer from disproportionate short stature dwarfism of varying severity. Through a genome-wide approach, we mapped the chondrodysplasia locus to a 2-Mb region on canine chromosome 17 in nine affected and nine healthy Elkhounds (praw = 7.42×10(-6), pgenome-wide = 0.013). The associated locus contained a promising candidate gene, cartilage specific integrin alpha 10 (ITGA10), and mutation screening of its 30 exons revealed a nonsense mutation in exon 16 (c.2083C>T; p.Arg695*) that segregated fully with the disease in both breeds (p = 2.5×10(-23)). A 24% mutation carrier frequency was indicated in NEs and an 8% frequency in KBDs. The ITGA10 gene product, integrin receptor α10-subunit combines into a collagen-binding α10β1 integrin receptor, which is expressed in cartilage chondrocytes and mediates chondrocyte-matrix interactions during endochondral ossification. As a consequence of the nonsense mutation, the α10-protein was not detected in the affected cartilage tissue. The canine phenotype highlights the importance of the α10β1 integrin in bone growth, and the large animal model could be utilized to further delineate its specific functions. Finally, this study revealed a candidate gene for human chondrodysplasias and enabled the development of a genetic test for breeding purposes to eradicate the disease from the two dog breeds.

  5. Focal Adhesion Kinase Regulates Fibroblast Migration via Integrin beta-1 and Plays a Central Role in Fibrosis

    PubMed Central

    Zhao, Xue-Ke; Cheng, Yiju; Liang Cheng, Ming; Yu, Lei; Mu, Mao; Li, Hong; Liu, Yang; Zhang, Baofang; Yao, Yumei; Guo, Hui; Wang, Rong; Zhang, Quan

    2016-01-01

    Lung fibrosis is a major medical problem for the aging population worldwide. Fibroblast migration plays an important role in fibrosis. Focal Adhesion Kinase (FAK) senses the extracellular stimuli and initiates signaling cascades that promote cell migration. This study first examined the dose and time responses of FAK activation in human lung fibroblasts treated with platelet derived growth factor BB (PDGF-BB). The data indicate that FAK is directly recruited by integrin β1 and the subsequent FAK activation is required for fibroblast migration on fibronectin. In addition, the study has identified that α5β1 and α4β1 are the major integrins for FAK-mediated fibroblast migration on fibronect. In contrast, integrins αvβ3, αvβ6, and αvβ8 play a minor but distinct role in fibroblast migration on fibronectin. FAK inhibitor significantly reduces PDGF-BB stimulated fibroblast migration. Importantly, FAK inhibitor protects bleomycin-induced lung fibrosis in mice. FAK inhibitor blocks FAK activation and significantly reduces signaling cascade of fibroblast migration in bleomycin-challenged mice. Furthermore, FAK inhibitor decreases lung fibrotic score, collagen accumulation, fibronectin production, and myofibroblast differentiation in in bleomycin-challenged mice. These data demonstrate that FAK mediates fibroblast migration mainly via integrin β1. Furthermore, the findings suggest that targeting FAK signaling is an effective therapeutic strategy against fibrosis. PMID:26763945

  6. The Role of Integrin Signaling in Breast Cancer.

    DTIC Science & Technology

    1997-10-01

    specificity. Cell. 80:199-211. 15 Unpublished Data Shaw, L.M. 16. Wary, K.K., F . Mainiero, S.J. Isakoff, E.E. Marcantonio , and F.G. Giancotti. 1996. The...receptors: achieving specificity through cooperation. 1995. Trends in Cell Biol. 5:419-423. 8. Friedrichs, K., P. Ruiz, F . Franke, I. Gille, H.-J.Terpe, and...793. 12. Falcioni, R., V. Turchi, P. Vitullo, G. Navarra, F . Ficari, F . Cavaliere, A. Sacchi, and R. Mariani-Constantini. 1994. Integrin 1P4 expression

  7. Advancement in integrin facilitated drug delivery.

    PubMed

    Arosio, Daniela; Casagrande, Cesare

    2016-02-01

    The research of integrin-targeted anticancer agents has recorded important advancements in ingenious design of delivery systems, based either on the prodrug approach, or on nanoparticle carriers, but for now, none of these has reached a clinical stage of development. Past work in this area has been extensively reviewed by us and others. Thus, the purpose and scope of the present review is to survey the advancement reported in the last 3years, with focus on innovative delivery systems that appear to afford openings for future developments. These systems exploit the labelling with conventional and novel integrin ligands for targeting the interface of cancer cells and of endothelial cells involved in cancer angiogenesis, with the proteins of the extracellular matrix, in the circulation, in tissues, and in tumour stroma, as the site of progression and metastatic evolution of the disease. Furthermore, these systems implement the expertise in the development of nanomedicines to the purpose of achieving preferential biodistribution and uptake in cancer tissues, internalisation in cancer cells, and release of the transported drugs at intracellular sites. The assessment of the value of controlling these factors, and their combination, for future developments requires support of biological testing in appropriate mechanistic models, but also imperatively demand confirmation in therapeutically relevant in vivo models for biodistribution, efficacy, and lack of off-target effects. Thus, among many studies, we have tried to point out the results supported by relevant in vivo studies, and we have emphasised in specific sections those addressing the medical needs of drug delivery to brain tumours, as well as the delivery of oligonucleotides modulating gene-dependent pathological mechanism. The latter could constitute the basis of a promising third branch in the therapeutic armamentarium against cancer, in addition to antibody-based agents and to cytotoxic agents. Copyright © 2015

  8. Coated platelets function in platelet-dependent fibrin formation via integrin αIIbβ3 and transglutaminase factor XIII

    PubMed Central

    Mattheij, Nadine J.A.; Swieringa, Frauke; Mastenbroek, Tom G.; Berny-Lang, Michelle A.; May, Frauke; Baaten, Constance C.F.M.J.; van der Meijden, Paola E.J.; Henskens, Yvonne M.C.; Beckers, Erik A.M.; Suylen, Dennis P.L.; Nolte, Marc W.; Hackeng, Tilman M.; McCarty, Owen J.T.; Heemskerk, Johan W.M.; Cosemans, Judith M.E.M.

    2016-01-01

    Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin αIIbβ3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin αIIbβ3. Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin αIIbβ3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in αIIbβ3 (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial αIIbβ3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin αIIbβ3. PMID:26721892

  9. Connective tissue growth factor inhibits gastric cancer peritoneal metastasis by blocking integrin α3β1-dependent adhesion.

    PubMed

    Chen, Chiung-Nien; Chang, Cheng-Chi; Lai, Hong-Shiee; Jeng, Yung-Ming; Chen, Chia-I; Chang, King-Jeng; Lee, Po-Huang; Lee, Hsinyu

    2015-07-01

    Connective tissue growth factor (CTGF) plays important roles in normal and pathological conditions. The aim of this study was to investigate the role of CTGF in peritoneal metastasis as well as the underlying mechanism in gastric cancer progression. CTGF expression levels for wild-type and stable overexpression clones were determined by Western blotting and quantitative polymerase chain reaction (Q-PCR). Univariate and multivariate analyses, immunohistochemistry, and survival probability analyses were performed on gastric cancer patients. The extracellular matrix components involved in CTGF-regulated adhesion were determined. Recombinant CTGF was added to cells or coinoculated with gastric cancer cells into mice to evaluate its therapeutic potential. CTGF overexpression and treatment with the recombinant protein significantly inhibited cell adhesion. In vivo peritoneal metastasis demonstrated that CTGF-stable transfectants markedly decreased the number and size of tumor nodules in the mesentery. Statistical analysis of gastric cancer patient data showed that patients expressing higher CTGF levels had earlier TNM staging and a higher survival probability after the surgery. Integrin α3β1 was the cell adhesion molecule mediating gastric cancer cell adhesion to laminin, and blocking of integrin α3β1 prevented gastric cancer cell adhesion to recombinant CTGF. Coimmunoprecipitation results indicated that CTGF binds to integrin α3. Coinoculation of recombinant CTGF and gastric cancer cell lines in mice showed effective inhibition of peritoneal dissemination. Our results suggested that gastric cancer peritoneal metastasis is mediated through integrin α3β1 binding to laminin, and CTGF effectively blocks the interaction by binding to integrin α3β1, thus demonstrating the therapeutic potential of recombinant CTGF in gastric cancer patients.

  10. The lutheran/basal cell adhesion molecule promotes tumor cell migration by modulating integrin-mediated cell attachment to laminin-511 protein.

    PubMed

    Kikkawa, Yamato; Ogawa, Takaho; Sudo, Ryo; Yamada, Yuji; Katagiri, Fumihiko; Hozumi, Kentaro; Nomizu, Motoyoshi; Miner, Jeffrey H

    2013-10-25

    Cell-matrix interactions are critical for tumor cell migration. Lutheran (Lu), also known as basal cell adhesion molecule (B-CAM), competes with integrins for binding to laminin α5, a subunit of LM-511, a major component of basement membranes. Here we show that the preferential binding of Lu/B-CAM to laminin α5 promotes tumor cell migration. The attachment of Lu/B-CAM transfectants to LM-511 was slightly weaker than that of control cells, and this was because Lu/B-CAM disturbed integrin binding to laminin α5. Lu/B-CAM induced a spindle cell shape with pseudopods and promoted cell migration on LM-511. In addition, blocking with an anti-Lu/B-CAM antibody led to a flat cell shape and inhibited migration on LM-511, similar to the effects of an activating integrin β1 antibody. We conclude that tumor cell migration on LM-511 requires that Lu/B-CAM competitively modulates cell attachment through integrins. We suggest that this competitive interaction is involved in a balance between static and migratory cell behaviors.

  11. HPW-RX40 restores anoikis sensitivity of human breast cancer cells by inhibiting integrin/FAK signaling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, I-Hua; Shih, Hsin-Chu; Hsieh, Pei-Wen

    Anoikis is defined as apoptosis, which is induced by inappropriate cell–matrix interactions. Cancer cells with anoikis resistance tend to undergo metastasis, and this phenomenon has been reported to be associated with integrin and FAK activity. HPW-RX40 is a derivative of 3,4-methylenedioxy-β-nitrostyrene, which is known to prevent platelet aggregation by inhibition of integrin. In the present study, we investigated the effect of HPW-RX40 on an anoikis-resistant human breast cancer cell line MDA-MB-231. HPW-RX40 inhibited cell aggregation and induced cell death in suspending MDA-MB-231 cells, but had only little effect on the monolayer growth of adherent cells. Analysis of caspase activation andmore » poly (ADP-ribose) polymerase (PARP) cleavage confirmed anoikis in HPW-RX40-treated suspending cancer cells. HPW-RX40 also affected the Bcl-2 family proteins in detached cancer cells. Furthermore, HPW-RX40 inhibited detachment-induced activation of FAK and the downstream phosphorylation of Src and paxillin, but did not affect this pathway in adherent cancer cells. We also found that the expression and activation of β1 integrin in MDA-MB-231 cells were reduced by HPW-RX40. The combination of HPW-RX40 with an EGFR inhibitor led to enhanced anoikis and inhibition of the FAK pathway in breast cancer cells. Taken together, our results suggest that HPW-RX40 restores the anoikis sensitivity in the metastatic breast cancer cells by inhibiting integrin and subsequent FAK activation, and reveal a potential strategy for prevention of tumor metastasis. - Highlights: • The β-nitrostyrene derivative, HPW-RX40, induces anoikis in human breast cancer cells. • HPW-RX40 inhibits the integrin/FAK signaling pathway. • The combination of HPW-RX40 with an EGFR inhibitor leads to enhanced anoikis. • HPW-RX40 may have a potential to prevent the spread of metastatic breast cancer.« less

  12. Integrin α5β1 Inhibition by CLT-28643 Reduces Postoperative Wound Healing in a Mouse Model of Glaucoma Filtration Surgery.

    PubMed

    Van Bergen, Tine; Zahn, Grit; Caldirola, Patrizia; Fsadni, Mario; Caram-Lelham, Ninus; Vandewalle, Evelien; Moons, Lieve; Stalmans, Ingeborg

    2016-11-01

    To evaluate the therapeutic potential of the small molecule integrin α5β1 inhibitor, CLT-28643, to improve the filtering surgery outcome in a mouse model. Different dose regimens and administration routes of the inhibitor were compared with mitomycin C (MMC), the gold standard in clin ical practice. The efficacy of CLT-28643 on surgical outcome was studied in a mouse model for filtering surgery (n = 40 eyes from 20 mice per group). Single and repeated subconjunctival (SCJ) injections (1 or 2 μg) and topical eye drops (10 μg) of the integrin inhibitor were compared with 2-minute administration of MMC 0.02%. Bleb size, survival, and signs of toxicity were examined until 28 days after surgery. Immunohistochemical analysis of angiogenesis, inflammation, collagen deposition, and integrin α5β1 expression were performed on postoperative days 3, 8, 14, and 28. A masked observer performed all the assessments. Immunostaining showed that integrin α5β1 was highly expressed in the bleb at early time-points after surgery and that CLT-28643 inhibited this upregulation. Efficacy was shown to be dose-dependent for the integrin inhibitor CLT-28643 for bleb area and survival, and the wound healing process. While 2-μg single injection of CLT-28643 improved bleb characteristics in a similar way as 10-μg administered by eye drops and MMC, repeated injections of 2 μg showed superior efficacy compared to MMC, with no corneal toxicity. Administration of the integrin α5β1 inhibitor CLT-28643 has therapeutic potential as an adjunct to glaucoma surgery, possibly with a superior efficacy and tolerability compared with MMC when used at the optimal dose.

  13. PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

    PubMed Central

    González Wusener, Ana E.; González, Ángela; Nakamura, Fumihiko; Arregui, Carlos O.

    2016-01-01

    ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration. PMID:26700725

  14. Alpha-V Integrin Targeted PET Imagining of Breast Cancer Angiogenesis and Lose-Dose Metronomic Anti-Angiogenic Chemotherapy Efficacy

    DTIC Science & Technology

    2005-08-01

    Chen X, Plasencia C, Hou Y, Neamati N. Synthesis and Biological Evaluation of Dimeric RGD Peptide-Paclitaxel Conjugate as Model for Integrin Targeted...Plasencia C, Hou Y, Neamati N. Synthesis and biological evaluation of dimeric RGD peptide-paclitaxel conjugate as a model for integrin-targeted drug...Targeted Imaging of Lung Cancer. Neoplasia 2005;7:271-279. 6. Chen X, Plasencia C, Hou Y, Neamati N. Synthesis and Biological Evaluation of Dimeric RGD

  15. Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair.

    PubMed

    Girault, Alban; Chebli, Jasmine; Privé, Anik; Trinh, Nguyen Thu Ngan; Maillé, Emilie; Grygorczyk, Ryszard; Brochiero, Emmanuelle

    2015-09-04

    Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K(+) channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before. Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair. Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.

  16. Targeting αvβ3 and αvβ5 integrins inhibits pulmonary metastasis in an intratibial xenograft osteosarcoma mouse model

    PubMed Central

    Gvozdenovic, Ana; Boro, Aleksandar; Meier, Daniela; Bode-Lesniewska, Beata; Born, Walter; Muff, Roman; Fuchs, Bruno

    2016-01-01

    Osteosarcoma is an aggressive bone cancer that has a high propensity for metastasis to the lungs. Patients with metastatic disease face a very poor prognosis. Therefore, novel therapeutics, efficiently suppressing the metastatic process, are urgently needed. Integrins play a pivotal role in tumor cell adhesion, motility and metastasis. Here, we evaluated αvβ3 and αvβ5 integrin inhibition with cilengitide as a novel metastasis-suppressive therapeutic approach in osteosarcoma. Immunohistochemical analysis of αvβ3 and αvβ5 integrins expression in a tissue microarray of tumor specimens collected from osteosarcoma patients revealed that αvβ5 integrin is mainly found on tumor cells, whereas αvβ3 is predominantly expressed by stromal cells. In vitro functional assays demonstrated that cilengitide dose-dependently inhibited de novo adhesion, provoked detachment and inhibited migration of osteosarcoma cell lines. Cilengitide induced a decline in cell viability, blocked the cell cycle in the G1 phase and caused anoikis by activation of the Hippo pathway. In a xenograft orthotopic mouse model cilengitide minimally affected intratibial primary tumor growth but, importantly, suppressed pulmonary metastasis. The data demonstrate that targeting αvβ3 and αvβ5 integrins in osteosarcoma should be considered as a novel therapeutic option for patients with metastatic disease. PMID:27409827

  17. CD44-mediated activation of α5β1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and Fibronectin-enriched matrices

    PubMed Central

    McFarlane, Suzanne; McFarlane, Cheryl; Montgomery, Nicola; Hill, Ashleigh; Waugh, David J.J.

    2015-01-01

    CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of β1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the β1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with β1-integrin and repressed HA-induced, CD44-mediated activation of β1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and β1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche. PMID:26447611

  18. Skeletal phenotype of growing transgenic mice that express a function-perturbing form of beta1 integrin in osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; Amblard, D.; Nishimura, Y.; Iwaniec, U. T.; Kim, J-B; Almeida, E. A. C.; Damsky, C. D.; Wronski, T. J.; van der Meulen, M. C. H.

    2005-01-01

    Skeletal modeling entails the deposition of large amounts of extracellular matrix (ECM) to form structures tailored to withstand increasing mechanical loads during rapid growth. Specific ECM molecules bind to integrin receptors on the cell surface, thereby triggering a cascade of signaling events that affect critical cell functions. To evaluate the role of integrins during skeletal growth, transgenic mice were engineered to express a function-perturbing fragment of beta1 integrin consisting of the transmembrane domain and cytoplasmic tail under the control of the osteocalcin promoter (TG mice). Thus, transgene expression was targeted to mature cells of the osteoblast lineage, and herein we show that cultured cells resembling osteocytes from 90-day-old TG mice display impaired adhesion to collagen I, a ligand for beta1 integrin. To determine the influence of beta1 integrin on bones that are responsible for providing structural support during periods of rapid growth, we examined the phenotype of the appendicular skeleton in TG mice compared to wild type (WT) mice. According to radiographs, bones from mice of both genotypes between 14 and 90 days of age appeared similar in gross structure and density, although proximal tibiae from 35-90 days old TG mice were less curved than those of WT mice (72-92% TG/WT). Although there were only mild and transient differences in absolute bone mass and strength, once normalized to body mass, the tibial dry mass (79.1% TG/WT females), ash mass (78.5% TG/WT females), and femoral strength in torsion (71.6% TG/WT females) were reduced in TG mice compared to WT mice at 90 days of age. Similar effects of genotype on bone mass and curvature were observed in 1-year-old retired breeders, indicating that these phenotypic differences between TG and WT mice were stable well into adulthood. Effects of genotype on histomorphometric indices of cancellous bone turnover were minimal and evident only transiently during growth, but when present they

  19. Integrin-linked kinase modulates longevity and thermotolerance in C. elegans through neuronal control of HSF-1

    PubMed Central

    Kumsta, Caroline; Ching, Tsui-Ting; Nishimura, Mayuko; Davis, Andrew E; Gelino, Sara; Catan, Hannah H; Yu, Xiaokun; Chu, Chu-Chiao; Ong, Binnan; Panowski, Siler H; Baird, Nathan; Bodmer, Rolf; Hsu, Ao-Lin; Hansen, Malene

    2014-01-01

    Integrin-signaling complexes play important roles in cytoskeletal organization and cell adhesion in many species. Components of the integrin-signaling complex have been linked to aging in both Caenorhabditis elegans and Drosophila melanogaster, but the mechanism underlying this function is unknown. Here, we investigated the role of integrin-linked kinase (ILK), a key component of the integrin-signaling complex, in lifespan determination. We report that genetic reduction of ILK in both C. elegans and Drosophila increased resistance to heat stress, and led to lifespan extension in C. elegans without majorly affecting cytoskeletal integrity. In C. elegans, longevity and thermotolerance induced by ILK depletion was mediated by heat-shock factor-1 (HSF-1), a major transcriptional regulator of the heat-shock response (HSR). Reduction in ILK levels increased hsf-1 transcription and activation, and led to enhanced expression of a subset of genes with roles in the HSR. Moreover, induction of HSR-related genes, longevity and thermotolerance caused by ILK reduction required the thermosensory neurons AFD and interneurons AIY, which are known to play a critical role in the canonical HSR. Notably, ILK was expressed in neighboring neurons, but not in AFD or AIY, implying that ILK reduction initiates cell nonautonomous signaling through thermosensory neurons to elicit a noncanonical HSR. Our results thus identify HSF-1 as a novel effector of the organismal response to reduced ILK levels and show that ILK inhibition regulates HSF-1 in a cell nonautonomous fashion to enhance stress resistance and lifespan in C. elegans. PMID:24314125

  20. Defining the Role of Integrin Alpha 11 in Wound Healing and Fibrosis

    DTIC Science & Technology

    2009-09-01

    Introduction Scleroderma is characterized by fibrosis, the replacement of healthy tissue with collagenous matrix. The collagen-binding integrins are...Itga11 and determine its role in the etiology of fibrosis and scleroderma . Body The heterozygous (Het) phenotype of one intact Itga11 allele and one