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

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

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.

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

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.

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.

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.

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.

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

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.

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 maintainence of endometrial integrity and to the disease biology associated with altered levels of α6 integrin expression in the endometrium.

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

Development and validation of receptor occupancy pharmacodynamic assays used in the clinical development of the monoclonal antibody vedolizumab.

PubMed

Wyant, Tim; Estevam, Jose; Yang, Lili; Rosario, Maria

2016-03-01

Vedolizumab is a monoclonal antibody approved for use in ulcerative colitis and Crohn's disease. By specifically binding to α4 β7 integrin, vedolizumab prevents trafficking of lymphocytes to the gut, thereby interfering with disease pathology. During the clinical development program, the pharmacodynamic effect of vedolizumab was evaluated by 2 flow cytometry receptor occupancy assays: act-1 (ACT-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Here we describe the development and validation of these assays. The ACT-1 assay is a receptor occupancy free-site assay that uses a monoclonal antibody with the same binding epitope as vedolizumab to detect free (unbound) sites on α4 β7 integrin. The MAdCAM-1 assay used a soluble version of the natural ligand for α4 β7 integrin to detect free sites. The assays were validated using a fit-for-purpose approach throughout the clinical development of vedolizumab. Both the ACT-1 assay and the MAdCAM-1 assay demonstrated acceptable reproducibility and repeatability. The assays were sufficiently stable to allow for clinical use. During clinical testing the assays demonstrated that vedolizumab was able to saturate peripheral cells at all doses tested. Two pharmacodynamic receptor occupancy assays were developed and validated to assess the effect of vedolizumab on peripheral blood cells. The results of these assays demonstrated the practical use of flow cytometry to examine pharmacodynamic response in clinical trials. © 2015 International Clinical Cytometry Society.

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.

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

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.

An Rgd Sequence in the P2y2 Receptor Interacts with αVβ3 Integrins and Is Required for Go-Mediated Signal Transduction

PubMed Central

Erb, Laurie; Liu, Jun; Ockerhausen, Jonathan; Kong, Qiongman; Garrad, Richard C.; Griffin, Korey; Neal, Chris; Krugh, Brent; Santiago-Pérez, Laura I.; González, Fernando A.; Gresham, Hattie D.; Turner, John T.; Weisman, Gary A.

2001-01-01

The P2Y2 nucleotide receptor (P2Y2R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein–coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y2R to K562 erythroleukemia cells was inhibited by antibodies against αVβ3/β5 integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y2Rs indicated that αV integrins colocalized 10-fold better with the wild-type P2Y2R than with a mutant P2Y2R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y2R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal–regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca2+. Furthermore, an anti-αV integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y2R. Pertussis toxin, an inhibitor of Gi/o proteins, partially inhibited Ca2+ mobilization mediated by the wild-type P2Y2R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y2R-mediated activation of Go, but not Gq. Since CD47 has been shown to associate directly with Gi/o family proteins, these results suggest that interactions between P2Y2Rs, integrins, and CD47 may be important for coupling the P2Y2R to Go. PMID:11331301

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. PMID:26895964

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 antibodies and the laminin peptide HGD-6 activate the alpha 3 beta 1 integrin, which results in a downstream signaling cascade stimulating phagocytosis.

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

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

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.

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.

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

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 integrin-β1 via interaction with β1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-β1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR. PMID:23922889

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.

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.

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

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.

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.

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.

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 copies are found. PMID:22022374

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

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.

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

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 studies demonstrate a role for BM beta3-integrin in VEGF-induced mobilization of bone marrow-derived cells to the peripheral circulation and for the functionality of those vessels in which BM-derived cells become incorporated.

Characterization of a conformationally sensitive murine monoclonal antibody directed to the metal ion-dependent adhesion site face of integrin CD11b.

PubMed

Li, Rui; Haruta, Ikuko; Rieu, Philippe; Sugimori, Takashi; Xiong, Jian-Ping; Arnaout, M Amin

2002-02-01

Integrin binding to physiologic ligands requires divalent cations and an inside-out-driven switch of the integrin to a high-affinity state. Divalent cations at the metal ion-dependent adhesion site (MIDAS) face of the alpha subunit-derived A domain provide a direct bridge between ligands and the integrin, and it has been proposed that activation dependency is caused by reorientation of the surrounding residues relative to the metal ion, forming an optimal binding interface. To gain more insight into the functional significance of the protein movements on the MIDAS face, we raised and characterized a murine mAb 107 directed against the MIDAS face of the A domain from integrin CD11b. We find that mAb 107 behaves as a ligand mimic. It binds in a divalent-cation-dependent manner to solvent-exposed residues on the MIDAS face of CD11b, blocks interaction of 11bA or the holoreceptor with ligands, and inhibits spreading and phagocytosis by human neutrophils. However, in contrast to physiologic ligands, mAb 107 preferentially binds to the inactive low-affinity form of the integrin, suggesting that its antagonistic effects are exerted in part by stabilizing the receptor in the low-affinity state. These data support a functional relevance of the protein movements on the MIDAS face and suggest that stabilizing the A domain in the low-affinity state may have therapeutic benefit.

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

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

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

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.

A Single Disulfide Bond Disruption in the β3 Integrin Subunit Promotes Thiol/Disulfide Exchange, a Molecular Dynamics Study

PubMed Central

Levin, Lihie; Zelzion, Ehud; Nachliel, Esther; Gutman, Menachem; Tsfadia, Yossi; Einav, Yulia

2013-01-01

The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The β3 subunit of the platelet αIIbβ3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the β3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys567–Cys581 bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the αIIbβ3 integrin, which are essential for the native activation process. PMID:23527123

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 expression patterns on corneal tissue and corneal cells. Among the integrins identified, competition binding and infection experiments and biochemical assays pointed out αVβ1 and α3β1 to be of importance for HAdV-37 infection of corneal tissue. In the absence of a good animal model for EKC-causing HAdVs, we also developed an in vitro system with multilayer HCE cells and confirmed the relevance of the suggested integrins during HAdV-37 infection. PMID:27974569

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.

Matrix-regulated integrin αvβ5 maintains α5β1-dependent desmoplastic traits prognostic of neoplastic recurrence

PubMed Central

Franco-Barraza, Janusz; Francescone, Ralph; Luong, Tiffany; Shah, Neelima; Madhani, Raj; Cukierman, Gil; Dulaimi, Essel; Devarajan, Karthik; Egleston, Brian L; Nicolas, Emmanuelle; Katherine Alpaugh, R; Malik, Ruchi; Uzzo, Robert G; Hoffman, John P; Golemis, Erica A; Cukierman, Edna

2017-01-01

Desmoplasia, a fibrotic mass including cancer-associated fibroblasts (CAFs) and self-sustaining extracellular matrix (D-ECM), is a puzzling feature of pancreatic ductal adenocarcinoma (PDACs). Conflicting studies have identified tumor-restricting and tumor-promoting roles of PDAC-associated desmoplasia, suggesting that individual CAF/D-ECM protein constituents have distinguishable tumorigenic and tumor-repressive functions. Using 3D culture of normal pancreatic versus PDAC-associated human fibroblasts, we identified a CAF/D-ECM phenotype that correlates with improved patient outcomes, and that includes CAFs enriched in plasma membrane-localized, active α5β1-integrin. Mechanistically, we established that TGFβ is required for D-ECM production but dispensable for D-ECM-induced naïve fibroblast-to-CAF activation, which depends on αvβ5-integrin redistribution of pFAK-independent active α5β1-integrin to assorted endosomes. Importantly, the development of a simultaneous multi-channel immunofluorescence approach and new algorithms for computational batch-analysis and their application to a human PDAC panel, indicated that stromal localization and levels of active SMAD2/3 and α5β1-integrin distinguish patient-protective from patient-detrimental desmoplasia and foretell tumor recurrences, suggesting a useful new prognostic tool. DOI: http://dx.doi.org/10.7554/eLife.20600.001 PMID:28139197

Age-specific function of α5β1 integrin in microglial migration during early colonization of the developing mouse cortex.

PubMed

Smolders, Sophie Marie-Thérèse; Swinnen, Nina; Kessels, Sofie; Arnauts, Kaline; Smolders, Silke; Le Bras, Barbara; Rigo, Jean-Michel; Legendre, Pascal; Brône, Bert

2017-07-01

Microglia, the immune cells of the central nervous system, take part in brain development and homeostasis. They derive from primitive myeloid progenitors that originate in the yolk sac and colonize the brain mainly through intensive migration. During development, microglial migration speed declines which suggests that their interaction with the microenvironment changes. However, the matrix-cell interactions allowing dispersion within the parenchyma are unknown. Therefore, we aimed to better characterize the migration behavior and to assess the role of matrix-integrin interactions during microglial migration in the embryonic brain ex vivo. We focused on microglia-fibronectin interactions mediated through the fibronectin receptor α5β1 integrin because in vitro work indirectly suggested a role for this ligand-receptor pair. Using 2-photon time-lapse microscopy on acute ex vivo embryonic brain slices, we found that migration occurs in a saltatory pattern and is developmentally regulated. Most importantly, there is an age-specific function of the α5β1 integrin during microglial cortex colonization. At embryonic day (E) 13.5, α5β1 facilitates migration while from E15.5, it inhibits migration. These results indicate a developmentally regulated function of α5β1 integrin in microglial migration during colonization of the embryonic brain. © 2017 Wiley Periodicals, Inc.

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.

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.

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

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

The group B streptococcal alpha C protein binds alpha1beta1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells.

PubMed

Bolduc, Gilles R; Madoff, Lawrence C

2007-12-01

Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis and meningitis among neonates and a cause of morbidity among pregnant women and immunocompromised adults. GBS epithelial cell invasion is associated with expression of alpha C protein (ACP). Loss of ACP expression results in a decrease in GBS internalization and translocation across human cervical epithelial cells (ME180). Soluble ACP and its 170 amino acid N-terminal region (NtACP), but not the repeat protein RR', bind to ME180 cells and reduce internalization of wild-type GBS to levels obtained with an ACP-deficient isogenic mutant. In the current study, ACP colocalized with alpha(1)beta(1)-integrin, resulting in integrin clustering as determined by laser scanning confocal microscopy. NtACP contains two structural domains, D1 and D2. D1 is structurally similar to fibronectin's integrin-binding region (FnIII10). D1's (KT)D146 motif is structurally similar to the FnIII10 (RG)D1495 integrin-binding motif, suggesting that ACP binds alpha(1)beta(1)-integrin via the D1 domain. The (KT)D146A mutation within soluble NtACP reduced its ability to bind alpha(1)beta(1)-integrin and inhibit GBS internalization within ME180 cells. Thus ACP binding to human epithelial cell integrins appears to contribute to GBS internalization within epithelial cells.

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.

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

Inhibitory effects of OK-432 (Picibanil) on cellular proliferation and adhesive capacity of breast carcinoma cells.

PubMed

Horii, Yoshio; Iino, Yuichi; Maemura, Michio; Horiguchi, Jun; Morishita, Yasuo

2005-02-01

We investigated the potent inhibitory effects of OK-432 (Picibanil) on both cellular adhesion and cell proliferation of estrogen-dependent (MCF-7) or estrogen-independent (MDA-MB-231) breast carcinoma cells. Cellular proliferation of both MCF-7 and MDA-MB-231 cells was markedly inhibited in a dose-dependent manner, when the carcinoma cells were exposed to OK-432. Cell attachment assay demonstrated that incubation with OK-432 for 24 h reduced integrin-mediated cellular adhesion of both cell types. However, fluorescence activated cell sorter (FACS) analysis revealed that incubation with OK-432 for 24 h did not decrease the cell surface expressions of any integrins. These results suggest that the binding avidity of integrins is reduced by OK-432 without alteration of the integrin expression. We conclude that OK-432 inhibits integrin-mediated cellular adhesion as well as cell proliferation of breast carcinoma cells regardless of estrogen-dependence, and that these actions of OK-432 contribute to prevention or inhibition of breast carcinoma invasion and metastasis.

A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System.

PubMed

Qin, Yan; Garrison, Brian S; Ma, Wenjiang; Wang, Rui; Jiang, Aiping; Li, Jing; Mistry, Meeta; Bronson, Roderick T; Santoro, Daria; Franco, Charlotte; Robinton, Daisy A; Stevens, Beth; Rossi, Derrick J; Lu, Chafen; Springer, Timothy A

2018-06-12

Extracellular proTGF-β is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-β is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVβ8-dependent TGF-β activation. Lrrc33 -/- mice lack CNS vascular abnormalities associated with deficiency in TGF-β-activating integrins but have microglia with a reactive phenotype and after 2 months develop ascending paraparesis with loss of myelinated axons and death by 5 months. Whole bone marrow transplantation results in selective repopulation of Lrrc33 -/- brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33 -/- microglia in the same brain suggest that there is little spreading of TGF-β activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-β provide localized and selective activation of TGF-β. Copyright © 2018 Elsevier Inc. All rights reserved.

Streptococcal modulation of cellular invasion via TGF-beta1 signaling.

PubMed

Wang, Beinan; Li, Shaoying; Southern, Peter J; Cleary, Patrick P

2006-02-14

Group A Streptococcus (GAS) and other bacterial pathogens are known to interact with integrins as an initial step in a complex pathway of bacterial ingestion by host cells. Efficient GAS invasion depends on the interaction of bound fibronectin (Fn) with integrins and activation of integrin signaling. TGF-beta1 regulates expression of integrins, Fn, and other extracellular matrix proteins, and positively controls the integrin signaling pathway. Therefore, we postulated that TGF-beta1 levels could influence streptococcal invasion of mammalian cells. Pretreatment of HEp-2 cells with TGF-beta1 increased their capacity to ingest GAS when the bacteria expressed fibronectin-binding proteins (M1 or PrtF1). Western blots revealed significant induction of alpha5 integrin and Fn expression by HEp-2 cells in response to TGF-beta1. Increased ingestion of streptococci by these cells was blocked by a specific inhibitor of the TGF-beta1 receptor I and antibodies directed against alpha5 integrin and Fn, indicating that increased invasion depends on TGF-beta1 up-regulation of both the alpha5 integrin and Fn. The capacity of TGF-beta1 to up-regulate integrin expression and intracellular invasion by GAS was reproduced in primary human tonsil fibroblasts, which could be a source of TGF-beta1 in chronically infected tonsils. The relationship between TGF-beta1 and GAS invasion was strengthened by the observation that TGF-beta1 production was stimulated in GAS-infected primary human tonsil fibroblasts. These findings suggest a mechanism by which GAS induce a cascade of changes in mammalian tissue leading to elevated expression of the alpha5beta1 receptor, enhanced invasion, and increased opportunity for survival and persistence in their human host.

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.

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.

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.

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.

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

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

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.

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.

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

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.

Molecular mechanisms of the angiogenic effects of low-energy shock wave therapy: roles of mechanotransduction.

PubMed

Hatanaka, Kazuaki; Ito, Kenta; Shindo, Tomohiko; Kagaya, Yuta; Ogata, Tsuyoshi; Eguchi, Kumiko; Kurosawa, Ryo; Shimokawa, Hiroaki

2016-09-01

We have previously demonstrated that low-energy extracorporeal cardiac shock wave (SW) therapy improves myocardial ischemia through enhanced myocardial angiogenesis in a porcine model of chronic myocardial ischemia and in patients with refractory angina pectoris. However, the detailed molecular mechanisms for the SW-induced angiogenesis remain unclear. In this study, we thus examined the effects of SW irradiation on intracellular signaling pathways in vitro. Cultured human umbilical vein endothelial cells (HUVECs) were treated with 800 shots of low-energy SW (1 Hz at an energy level of 0.03 mJ/mm(2)). The SW therapy significantly upregulated mRNA expression and protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). The SW therapy also enhanced phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and Akt. Furthermore, the SW therapy enhanced phosphorylation of caveolin-1 and the expression of HUTS-4 that represents β1-integrin activity. These results suggest that caveolin-1 and β1-integrin are involved in the SW-induced activation of angiogenic signaling pathways. To further examine the signaling pathways involved in the SW-induced angiogenesis, HUVECs were transfected with siRNA of either β1-integrin or caveolin-1. Knockdown of either caveolin-1 or β1-integrin suppressed the SW-induced phosphorylation of Erk1/2 and Akt and upregulation of VEGF and eNOS. Knockdown of either caveolin-1 or β1-integrin also suppressed SW-induced enhancement of HUVEC migration in scratch assay. These results suggest that activation of mechanosensors on cell membranes, such as caveolin-1 and β1-integrin, and subsequent phosphorylation of Erk and Akt may play pivotal roles in the SW-induced angiogenesis. Copyright © 2016 the American Physiological Society.

Protopine inhibits heterotypic cell adhesion in MDA-MB-231 cells through down-regulation of multi-adhesive factors.

PubMed

He, Kai; Gao, Jian-Li

2014-01-01

A Chinese herb Corydalis yanhusuo W.T. Wang that showed anticancer and anti-angiogenesis effects in our previous studies was presented for further studies. In the present study, we studied the anticancer proliferation and adhesion effects of five alkaloids which were isolated from Corydalis yanhusuo. MTT dose response curves, cell migration assay, cell invasion assay, as well as three types of cell adhesive assay were performed on MDA-MB-231 human breast cancer cells. The mechanism of the compounds on inhibiting heterotypic cell adhesion were further explored by determining the expression of epidermal growth factor receptor (EGFR), Intercellular adhesion molecule 1 (ICAM-1), αv-integrin, β1-integrin and β5-integrin by western blotting assay. In five tested alkaloids, only protopine exhibited anti-adhesive and anti-invasion effects in MDA-MB-231 cells, which contributed to the anti-metastasis effect of Corydalis yanhusuo. The results showed that after treatment with protopine for 90 min, the expression of EGFR, ICAM-1, αv-integrin, β1-integrin and β5-integrin were remarkably reduced. The present results suggest that protopine seems to inhibit the heterotypic cell adhesion between MDA-MB-231 cells, and human umbilical vein endothelial cells by changing the expression of adhesive factors.

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

T cell costimulation via the integrin VLA-4 inhibits the actin-dependent centralization of signaling microclusters containing the adaptor SLP-76.

PubMed

Nguyen, Ken; Sylvain, Nicholas R; Bunnell, Stephen C

2008-06-01

Antigen-dependent T cell activation drives the formation of signaling microclusters containing the adaptor SLP-76. Costimulatory integrins regulate SLP-76 phosphorylation and could influence SLP-76 microclusters in the integrin-rich periphery of the immune synapse. We report that costimulation by the integrin VLA-4 (alpha4beta1) required SLP-76 domains implicated in microcluster assembly. Pro-adhesive ligands enlarged the contact and increased the number of SLP-76 microclusters regardless of their costimulatory potential. Costimulatory VLA-4 ligands also prevented the centralization of SLP-76, promoted microcluster persistence, prolonged lateral interactions between SLP-76 and its upstream kinase, ZAP-70, and retained SLP-76 in tyrosine-phosphorylated peripheral structures. SLP-76 centralization was driven by dynamic actin polymerization and was correlated with inward actin flows. VLA-4 ligation retarded these flows, even in the absence of SLP-76. These data suggest a widely applicable model of costimulation, in which integrins promote sustained signaling by attenuating cytoskeletal movements that drive the centralization and inactivation of SLP-76 microclusters.

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.

Cyclic RGD peptidomimetics containing 4- and 5-amino-cyclopropane pipecolic acid (CPA) templates as dual αVβ3 and α5β1 integrin ligands.

PubMed

Sernissi, Lorenzo; Trabocchi, Andrea; Scarpi, Dina; Bianchini, Francesca; Occhiato, Ernesto G

2016-02-15

4-Amino- and 5-amino-cyclopropane pipecolic acids (CPAs) with cis relative stereochemistry between the carboxylic and amino groups were used as templates to prepare cyclic peptidomimetics containing the RGD sequence as possible integrin binders. The peptidomimetic c(RGD8) built on the 5-amino-CPA displayed an inhibition activity (IC50=2.4nM) toward the αvβ3 integrin receptor (expressed in M21 human melanoma cell line) comparable to that of the most potent antagonists reported so far and it was ten times more active than the corresponding antagonist c(RGD7) derived from the isomeric 4-amino-CPA. Both compounds were also nanomolar ligands of the α5β1 integrin (expressed in human erythroleukemia cell line K562). These results suggest that the CPA-derived templates are suitable for the preparation of dual αvβ3 and α5β1 ligands to suppress integrin-mediated events as well as for targeted drug delivery in cancer therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

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

Receptor-based differences in human aortic smooth muscle cell membrane stiffness

NASA Technical Reports Server (NTRS)

Huang, H.; Kamm, R. D.; So, P. T.; Lee, R. T.

2001-01-01

Cells respond to mechanical stimuli with diverse molecular responses. The nature of the sensory mechanism involved in mechanotransduction is not known, but integrins may play an important role. The integrins are linked to both the cytoskeleton and extracellular matrix, suggesting that probing cells via integrins should yield different mechanical properties than probing cells via non-cytoskeleton-associated receptors. To test the hypothesis that the mechanical properties of a cell are dependent on the receptor on which the stress is applied, human aortic smooth muscle cells were plated, and magnetic beads, targeted either to the integrins via fibronectin or to the transferrin receptor by use of an IgG antibody, were attached to the cell surface. The resistance of the cell to deformation ("stiffness") was estimated by oscillating the magnetic beads at 1 Hz by use of single-pole magnetic tweezers at 2 different magnitudes. The ratio of bead displacements at different magnitudes was used to explore the mechanical properties of the cells. Cells stressed via the integrins required approximately 10-fold more force to obtain the same bead displacements as the cells stressed via the transferrin receptors. Cells stressed via integrins showed stiffening behavior as the force was increased, whereas this stiffening was significantly less for cells stressed via the transferrin receptor (P<0.001). Mechanical characteristics of vascular smooth muscle cells depend on the receptor by which the stress is applied, with integrin-based linkages demonstrating cell-stiffening behavior.

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.

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.

Calreticulin overexpression correlates with integrin-α5 and transforming growth factor-β1 expression in the atria of patients with rheumatic valvular disease and atrial fibrillation.

PubMed

Zhao, Fei; Zhang, Shijiang; Shao, Yongfeng; Wu, Yanhu; Qin, Jianwei; Chen, Yijiang; Chen, Liang; Gu, Haitao; Wang, Xiaowei; Huang, Chenjun; Zhang, Wei

2013-10-03

The aim of this study was to determine whether altered calreticulin expression and distribution contribute to the pathogenesis of atrial fibrillation (AF) associated with valvular heart disease (VHD). AF affects electrophysiological and structural changes that exacerbate AF. Atrial remodeling reportedly underlies AF generation, but the precise mechanism of atrial remodeling in AF remains unclear. Right and left atrial specimens were obtained from 68 patients undergoing valve replacement surgery. The patients were divided into sinus rhythm (SR; n=25), paroxysmal AF (PaAF; n=11), and persistent AF (PeAF; AF lasting >6 months; n=32) groups. Calreticulin, integrin-α5, and transforming growth factor-β1 (TGF-β1) mRNA and protein expression were measured. We also performed immunoprecipitation for calreticulin with either calcineurin B or integrin-α5. Calreticulin, integrin-α5, and TGF-β1 mRNA and protein expression were increased in the AF groups, especially in the left atrium in patients with mitral valve disease. Calreticulin interacted with both calcineurin B and integrin-α5. Integrin-α5 expression correlated with TGF-β1 expression, while calreticulin expression correlated with integrin-α5 and TGF-β1 expression. Despite similar cardiac function classifications, calreticulin expression was greater in the PeAF group than in the SR group. Calreticulin, integrin-α5, and TGF-β1 expression was increased in atrial tissue in patients with AF and was related to AF type, suggesting that calreticulin is involved in the pathogenesis of AF in VHD patients. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Coordinate regulation of estrogen-mediated fibronectin matrix assembly and epidermal growth factor receptor transactivation by the G protein-coupled receptor, GPR30.

PubMed

Quinn, Jeffrey A; Graeber, C Thomas; Frackelton, A Raymond; Kim, Minsoo; Schwarzbauer, Jean E; Filardo, Edward J

2009-07-01

Estrogen promotes changes in cytoskeletal architecture not easily attributed to the biological action of estrogen receptors, ERalpha and ERbeta. The Gs protein-coupled transmembrane receptor, GPR30, is linked to specific estrogen binding and rapid estrogen-mediated release of heparin-bound epidermal growth factor. Using marker rescue and dominant interfering mutant strategies, we show that estrogen action via GPR30 promotes fibronectin (FN) matrix assembly by human breast cancer cells. Stimulation with 17beta-estradiol or the ER antagonist, ICI 182, 780, results in the recruitment of FN-engaged integrin alpha5beta1 conformers to fibrillar adhesions and the synthesis of FN fibrils. Concurrent with this cellular response, GPR30 promotes the formation of Src-dependent, Shc-integrin alpha5beta1 complexes. Function-blocking antibodies directed against integrin alpha5beta1 or soluble Arg-Gly-Asp peptide fragments derived from FN specifically inhibited GPR30-mediated epidermal growth factor receptor transactivation. Estrogen-mediated FN matrix assembly and epidermal growth factor receptor transactivation were similarly disrupted in integrin beta1-deficient GE11 cells, whereas reintroduction of integrin beta1 into GE11 cells restored these responses. Mutant Shc (317Y/F) blocked GPR30-induced FN matrix assembly and tyrosyl phosphorylation of erbB1. Interestingly, relative to recombinant wild-type Shc, 317Y/F Shc was more readily retained in GPR30-induced integrin alpha5beta1 complexes, yet this mutant did not prevent endogenous Shc-integrin alpha5beta1 complex formation. Our results suggest that GPR30 coordinates estrogen-mediated FN matrix assembly and growth factor release in human breast cancer cells via a Shc-dependent signaling mechanism that activates integrin alpha5beta1.

Regional localization of the human integrin {beta}{sub 6} gene (ITGB6) to chromosome 2q24-q31

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

Fernandez-Ruiz, E.; Sanchez-Madrid, F.

The heterodimer {alpha}{sub v}{beta}{sub 6} acts as a fibronectin receptor for human carcinoma cells. The authors report here the regional localization of the {beta}{sub 6} gene to 2q24-q31 by fluorescence in situ hybridization coupled with GTG-banding. This gene is located close to the region to which genes coding for the {alpha} subunits of the integrins VLA-4 and vitronectin receptor (ITGA4 and ITGAV, respectively) have been previously mapped (2q31-q32). These data suggest a proximal position of the integrin {beta}{sub 6} locus (ITGB6) on this integrin gene cluster. Futhermore, double-labeling in situ hybridization experiments performed with {alpha}{sub 4} and {alpha}{sub v} probesmore » indicated a telomeric position of ITGAV with respect to ITGA4. 22 refs., 2 figs.« less

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.

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 oxidase pathway that can be inhibited by extracellular, but not intracellular SOD. PMID:11238444

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.

Salt bridge interactions within the β2 integrin α7 helix mediate force-induced binding and shear resistance ability.

PubMed

Zhang, Xiao; Li, Linda; Li, Ning; Shu, Xinyu; Zhou, Lüwen; Lü, Shouqin; Chen, Shenbao; Mao, Debin; Long, Mian

2018-01-01

The functional performance of the αI domain α 7 helix in β 2 integrin activation depends on the allostery of the α 7 helix, which axially slides down; therefore, it is critical to elucidate what factors regulate the allostery. In this study, we determined that there were two conservative salt bridge interaction pairs that constrain both the upper and bottom ends of the α 7 helix. Molecular dynamics (MD) simulations for three β 2 integrin members, lymphocyte function-associated antigen-1 (LFA-1; α L β 2 ), macrophage-1 antigen (Mac-1; α M β 2 ) and α x β 2 , indicated that the magnitude of the salt bridge interaction is related to the stability of the αI domain and the strength of the corresponding force-induced allostery. The disruption of the salt bridge interaction, especially with double mutations in both salt bridges, significantly reduced the force-induced allostery time for all three members. The effects of salt bridge interactions of the αI domain α 7 helix on β 2 integrin conformational stability and allostery were experimentally validated using Mac-1 constructs. The results demonstrated that salt bridge mutations did not alter the conformational state of Mac-1, but they did increase the force-induced ligand binding and shear resistance ability, which was consistent with MD simulations. This study offers new insight into the importance of salt bridge interaction constraints of the αI domain α 7 helix and external force for β 2 integrin function. © 2017 Federation of European Biochemical Societies.

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 disease. © 2014 by the American Association for the Study of Liver Diseases.

Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases.

PubMed

Melchior, Aurélie; Denys, Agnès; Deligny, Audrey; Mazurier, Joël; Allain, Fabrice

2008-02-01

Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.

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.

Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation

PubMed Central

García, Andrés J.; Vega, María D.; Boettiger, David

1999-01-01

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound α5 and β1 integrin subunits but not αv or β3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of α5β1 integrin bound to Fn, and differentiation was inhibited by anti-α5, but not anti-αv, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications. PMID:10069818

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

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.

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.

Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes.

PubMed

Capmany, G; Mart, M; Santaló, J; Bolton, V N

1998-10-01

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.

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

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.

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

Mapping Heart Development in Flies: Src42A Acts Non-Autonomously to Promote Heart Tube Formation in Drosophila

PubMed Central

Vanderploeg, Jessica; Jacobs, J. Roger

2017-01-01

Congenital heart defects, clinically identified in both small and large animals, are multifactorial and complex. Although heritable factors are known to have a role in cardiovascular disease, the full genetic aetiology remains unclear. Model organism research has proven valuable in providing a deeper understanding of the essential factors in heart development. For example, mouse knock-out studies reveal a role for the Integrin adhesion receptor in cardiac tissue. Recent research in Drosophila melanogaster (the fruit fly), a powerful experimental model, has demonstrated that the link between the extracellular matrix and the cell, mediated by Integrins, is required for multiple aspects of cardiogenesis. Here we test the hypothesis that Integrins signal to the heart cells through Src42A kinase. Using the powerful genetics and cell biology analysis possible in Drosophila, we demonstrate that Src42A acts in early events of heart tube development. Careful examination of mutant heart tissue and genetic interaction data suggests that Src42A’s role is independent of Integrin and the Integrin-related Focal Adhesion Kinase. Rather, Src42A acts non-autonomously by promoting programmed cell death of the amnioserosa, a transient tissue that neighbors the developing heart. PMID:29056682

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 matrix through the regulation of integrin expression to promote a decrease in fibrosis. Furthermore, this work demonstrates that this naturally occurring and commercially available supplement could be used to improve wound healing by impacting integrin expression, leading to a lower extracellular matrix requirement to achieve healing. Impact statement Scar formation during wound healing can be problematic for patients but there are limited therapies available to treat or prevent excess fibrosis at wound sites. This work examines the impact of quercetin, a flavonoid that decreases fibrosis, on wound healing, and relates quercetin's effects to changes in integrin expression on the surface of fibroblast cells. To our knowledge, this is the first report that quercetin alters integrin expression or that this impact may be part of the mechanism by which quercetin prevents fibrosis. This work demonstrates that quercetin can be used to modulate integrin expression and that this effect may in turn reduce fibrosis during wound healing. Furthermore, this paper identifies the modulation of integrin expression as a possible therapeutic target in preventing scars. This information could be used to improve therapeutics to aid in the cosmetic and functional results following wound healing.

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

Suppression of tumour growth by orally administered osteopontin is accompanied by alterations in tumour blood vessels.

PubMed

Rittling, S R; Wejse, P L; Yagiz, K; Warot, G A; Hui, T

2014-03-04

The integrin-binding protein osteopontin is strongly associated with tumour development, yet is an abundant dietary component as a constituent of human and bovine milk. Therefore, we tested the effect of orally administered osteopontin (o-OPN) on the development of subcutaneous tumours in mice. Bovine milk osteopontin was administered in drinking water to tumour-bearing immune-competent mice. Tumour growth, proliferation, necrosis, apoptosis and blood vessel size and number were measured. Expression of the α₉ integrin was determined. o-OPN suppressed tumour growth, increased the extent of necrosis, and induced formation of abnormally large blood vessels. Anti-OPN reactivity detected in the plasma of OPN-null mice fed OPN suggested that tumour-blocking peptides were absorbed during digestion, but the o-OPN effect was likely distinct from that of an RGD peptide. Expression of the α₉ integrin was detected on both tumour cells and blood vessels. Potential active peptides from the α₉ binding site of OPN were identified by mass spectrometry following in vitro digestion, and injection of these peptides suppressed tumour growth. These results suggest that peptides derived from o-OPN are absorbed and interfere with tumour growth and normal vessel development. o-OPN-derived peptides that target the α₉ integrin are likely involved.

Distinctive and selective route of PI3K/PKCα-PKCδ/RhoA-Rac1 signaling in osteoclastic cell migration.

PubMed

Kim, Jin-Man; Kim, Mi Yeong; Lee, Kyunghee; Jeong, Daewon

2016-12-05

Cell migration during specialized stages of osteoclast precursors, mononuclear preosteoclasts, and multinucleated mature osteoclasts remain uncertain. M-CSF- and osteopontin-induced osteoclastic cell migration was inhibited by function-blocking monoclonal antibodies specific to the integrin αv and β3 subunits, suggesting that integrin αvβ3 mediates migratory signaling induced by M-CSF and osteopontin. M-CSF and osteopontin stimulation was shown to regulate two branched signaling processes, PI3K/PKCα/RhoA axis and PI3K/PKCδ/Rac1 axis. Interestingly, inactivation of RhoA or Rac1 blocked preosteoclast and mature osteoclast migration but not osteoclast precursor migration in a transwell-based cell migration assay. Moreover, the inhibitory effect on preosteoclast and mature osteoclast migration induced by Rac1 inactivation was more effective than that by RhoA inactivation. Collectively, our findings suggest that osteoclast precursor migration depends on PI3K/PKCα-PKCδ signaling mediated via integrin αvβ3 bypassing RhoA and Rac1, whereas preosteoclast and mature osteoclast migration relies on PI3K/PKCα-PKCδ/RhoA-Rac1 axis signaling mediated via integrin αvβ3 with increased dependency on PKCδ/Rac1 signaling route as differentiation progresses. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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 metastases as compared to the primary tumor. For the first time, I demonstrate that tumor-associated integrin beta3 is elevated on bone metastases across all breast cancer subtypes, supporting the translational potential of targeting integrin beta3 in breast cancer patients with bone metastases. Integrin beta3 was weakly expressed on tumor cells in vitro and on tumor cells in the primary mammary fat pad (MFP). Additional analysis demonstrated that integrin beta3 on circulating tumor cells is dispensable for strong expression of integrin beta3 on subsequent bone metastases, suggested that integrin beta3 may be induced within the bone microenvironment. I identified transforming growth factor beta (TGF-beta) to be a potent inducer of integrin beta3 in vitro, and further demonstrate canonical TGF-beta signaling through the SMAD2 and SMAD3 (SMAD2/3) pathway is responsible for breast cancer upregulation of integrin beta3 induction on bone metastases, both in vitro and in vivo. Utilizing this information, I sought to evaluate the targeting potential of nanotherapy coated with a targeting ligand specific for integrin alphavbeta3. Nanotherapy has the potential to increase therapeutic efficacy and reduce toxicity versus traditional chemotherapies by enhancing drug delivery to specific targets of interest. I explored the localization potential of two nanoparticles with significantly different sizes: polysorbate (tween) 80 micelle nanoparticles (MPs, 12.5 nm) or perfluorocarbon (PFC) nanoparticles ( 250 nm). The smaller integrin alphavbeta3- targeted micelle nanoparticle (alphavbeta3-MP) could more effectively penetrate breast cancer bone metastases than larger integrin alphavbeta3-targeted PFC nanoparticles (alphavbeta3-PFCs). With these observations, I evaluated whether alphavbeta3-MP-mediated drug delivery could more effectively attenuate bone metastatic tumor burden and bone destruction than free drug delivery. Using the chemotherapeutic agent docetaxel (DTX), a potent microtubule inhibitor that is a first-line therapy for metastatic breast cancer, I observe that DTX is only weakly tumor- suppressive in our mouse model of breast cancer metastases. However, treating mice bearing breast cancer metastases with alphavbeta3-MP-delivery of a docetaxel-prodrug (DTX-PD) significantly reduced bone tumor burden and bone destruction, and with less hepatotoxicity. I observed a significant decrease in bone-residing tumor cell proliferation in mice treated with alphavbeta3-MP- delivery of DTX-PD, without overt osteoclast killing or inhibition of osteoclast formation. Together, these results provide support that nanotherapy-mediated attenuation of bone metastases and bone destruction occurs through enhanced drug efficacy against breast cancer cells within the bone. In this Dissertation, Chapter 1 will provide an overview of breast cancer, bone metastases, integrins, and the therapeutic potential of nanotherapy. In Chapter 2, my work on the expression and physiologic regulation of integrin beta3 on breast cancer during metastases will be explored. In Chapter 3, the role of the cytokine TGF-beta in regulating tumoral expression of integrin beta3 will be discussed. And in Chapter 4, I discuss the use of integrin alphavbeta3-targeted nanotherapy directed against breast cancer metastases. Collectively, I provide evidence that chemotherapeutic efficacy against breast cancer cells within bone can be enhanced by exploiting the expression of tumoral integrin beta3 at that metastatic site.

Identification of Cell Adhesive Sequences in the N-terminal Region of the Laminin α2 Chain*

PubMed Central

Hozumi, Kentaro; Ishikawa, Masaya; Hayashi, Takemitsu; Yamada, Yuji; Katagiri, Fumihiko; Kikkawa, Yamato; Nomizu, Motoyoshi

2012-01-01

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117–128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions. PMID:22654118

Integrin αvβ3-targeted dynamic contrast-enhanced magnetic resonance imaging using a gadolinium-loaded polyethylene gycol-dendrimer-cyclic RGD conjugate to evaluate tumor angiogenesis and to assess early antiangiogenic treatment response in a mouse xenograft tumor model.

PubMed

Chen, Wei-Tsung; Shih, Tiffany Ting Fang; Chen, Ran-Chou; Tu, Shin-Yang; Hsieh, Wen-Yuen; Yang, Pang-Chyr

2012-01-01

The purpose of this study was to validate an integrin αvβ3-targeted magnetic resonance contrast agent, PEG-G3-(Gd-DTPA)6-(cRGD-DTPA)2, for its ability to detect tumor angiogenesis and assess early response to antiangiogenic therapy using dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI). Integrin αvβ3-positive U87 cells and control groups were incubated with fluorescein-labeled cRGD-conjugated dendrimer, and the cellular attachment of the dendrimer was observed. DCE MRI was performed on mice bearing KB xenograft tumors using either PEG-G3-(Gd-DTPA)6-(cRGD-DTPA)2 or PEG-G3-(Gd-DTPA)6-(cRAD-DTPA)2. DCE MRI was also performed 2 hours after anti-integrin αvβ3 monoclonal antibody treatment and after bevacizumab treatment on days 3 and 6t. Using DCE MRI, the 30-minute contrast washout percentage was significantly lower in the cRGD-conjugate injection groups. The enhancement patterns were different between the two contrast injection groups. In the antiangiogenic therapy groups, a rapid increase in 30-minute contrast washout percentage was observed in both the LM609 and bevacizumab treatment groups, and this occurred before there was an observable decrease in tumor size. The integrin αvβ3 targeting ability of PEG-G3-(Gd-DTPA)6-(cRGD-DTPA)2 in vitro and in vivo was demonstrated. The 30-minute contrast washout percentage is a useful parameter for examining tumor angiogenesis and for the early assessment of antiangiogenic treatment response.

Inhibition of phosphatidylcholine-specific phospholipase C prevents bone marrow stromal cell senescence in vitro.

PubMed

Sun, Chunhui; Wang, Nan; Huang, Jie; Xin, Jie; Peng, Fen; Ren, Yinshi; Zhang, Shangli; Miao, Junying

2009-10-01

Bone marrow stromal cells (BMSCs) can proliferate in vitro and can be transplanted for treating many kinds of diseases. However, BMSCs become senescent with long-term culture, which inhibits their application. To understand the mechanism underlying the senescence, we investigated the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) and levels of integrin beta4, caveolin-1 and ROS with BMSC senescence. The activity of PC-PLC and levels of integrin beta4, caveolin-1 and ROS increased greatly during cell senescence. Selective inhibition of increased PC-PLC activity with D609 significantly decreased the number of senescence-associated beta galactosidase positive cells in BMSCs. Furthermore, D609 restored proliferation of BMSCs and their differentiation into adipocytes. Moreover, D609 suppressed the elevated levels of integrin beta4, caveolin-1 and ROS. The data suggest that PC-PLC is involved in senescence of BMSCs, and its function is associated with integrin beta4, caveolin-1 and ROS. (c) 2009 Wiley-Liss, Inc.

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

Laminin-111 peptide C16 regulates invadopodia activity of malignant cells through β1 integrin, Src and ERK 1/2.

PubMed

Siqueira, Adriane S; Pinto, Monique P; Cruz, Mário C; Smuczek, Basilio; Cruz, Karen S P; Barbuto, José Alexandre M; Hoshino, Daisuke; Weaver, Alissa M; Freitas, Vanessa M; Jaeger, Ruy G

2016-07-26

Laminin peptides influence tumor behavior. In this study, we addressed whether laminin peptide C16 (KAFDITYVRLKF, γ1 chain) would increase invadopodia activity of cells from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). We found that C16 stimulates invadopodia activity over time in both cell lines. Rhodamine-conjugated C16 decorates the edge of cells, suggesting a possible binding to membrane receptors. Flow cytometry showed that C16 increases activated β1 integrin, and β1 integrin miRNA-mediated depletion diminishes C16-induced invadopodia activity in both cell lines. C16 stimulates Src and ERK 1/2 phosphorylation, and ERK 1/2 inhibition decreases peptide-induced invadopodia activity. C16 also increases cortactin phosphorylation in both cells lines. Based on our findings, we propose that C16 regulates invadopodia activity over time of squamous carcinoma and fibrosarcoma cells, probably through β1 integrin, Src and ERK 1/2 signaling pathways.

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

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.

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.

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.

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 Trial Registry; Date of registration: 4 September 2013; Date of enrollment of the first study participant: 15 October 2013.

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.

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.

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.

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm.

PubMed

Coux, Gabriela; Cabada, Marcelo O

2006-04-28

Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.

Expression and putative function of fibronectin and its receptor (integrin alpha(5)beta(1)) in male and female gametes during bovine fertilization in vitro.

PubMed

Thys, Mirjan; Nauwynck, Hans; Maes, Dominiek; Hoogewijs, Maarten; Vercauteren, Dries; Rijsselaere, Tom; Favoreel, Herman; Van Soom, Ann

2009-09-01

Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm-egg interaction in human. Recently, it has been demonstrated that Fn--when present during bovine IVF--strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (alpha(5)beta(1)) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin alpha(5) on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin alpha(5) at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm-oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these results, the inhibitory effect of exogenously supplemented Fn seemed to be exerted on the male gamete by binding to the exposed integrin alpha(5)beta(1) receptor after acrosome reaction. The presence of endogenous Fn underneath the ZP together with integrin alpha(5) expression on oolemma and acrosome-reacted (AR) sperm cell surface suggests a 'velcro' interaction between the endogenous Fn ligand and corresponding receptors on both (AR) sperm cell and oolemma, initiating sperm-egg binding.

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

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.

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

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

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

Natalizumab in the treatment of Crohn’s disease

PubMed Central

Guagnozzi, Danila; Caprilli, Renzo

2008-01-01

The pathogenesis of Crohn’s disease (CD) is multifactorial and the activation of specific pathways of immunological system is important. In particular, the adhesion molecules (integrins) mediate the selective binding between the leukocytes and the endothelial cells regulating the migration of leukocytes into the normal and inflamed intestine. Selective adhesion molecule inhibitors interfere with the migration of leukocytes to the sites of inflammation by targeting adhesion molecules (α4-integrin or α4β7-integrin). Natalizumab is a humanized IgG4 anti-α4-integrin monoclonal antibody that inhibits both α4β7-integrin/mucosal addressin-cell adhesion molecule-1 (MadCAM-1) interaction and α4β1/vascular-cell adhesion molecule-1 (VCAM-1) binding. Pooled data from the four studies, analyzed in a Cochrane review, suggest that natalizumab is effective for induction of clinical response and remission in patients with moderately to severely active CD. In particular, natalizumab may be beneficial for patients with active inflammation or chronically active disease despite the use of conventional therapies with high level of C-reactive protein values at baseline time. Nevertheless, many problems about the utilization of natalizumab in CD remain unsolved (such as the high placebo response, the final definition of dosage and timing schedule, the definition of outcomes and the development of adverse events). PMID:19707360

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

Glycosylation controls cooperative PECAM-VEGFR2-β3 integrin functions at the endothelial surface for tumor angiogenesis.

PubMed

Imamaki, Rie; Ogawa, Kazuko; Kizuka, Yasuhiko; Komi, Yusuke; Kojima, Soichi; Kotani, Norihiro; Honke, Koichi; Honda, Takashi; Taniguchi, Naoyuki; Kitazume, Shinobu

2018-05-02

Most of the angiogenesis inhibitors clinically used in cancer treatment target the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway. However, the current strategies for treating angiogenesis have limited efficacy. The issue of how to treat angiogenesis and endothelial dysfunction in cancer remains a matter of substantial debate. Here we demonstrate a glycosylation-dependent regulatory mechanism for tumor angiogenesis. St6gal1 -/- mice, lacking the α2,6-sialylation enzyme, were shown to exhibit impaired tumor angiogenesis through enhanced endothelial apoptosis. In a previous study, St6gal1 -/- endothelial cells exhibited a reduction in the cell surface residency of platelet endothelial cell adhesion molecule (PECAM). In this study, we found that cooperative functionality of PECAM-VEGFR2-integrin β3 was disturbed in St6gal1 -/- mice. First, cell surface PECAM-VEGFR2 complexes were lost, and both VEGFR2 internalization and the VEGFR-dependent signaling pathway were enhanced. Second, enhanced anoikis was observed, suggesting that the absence of α2,6-sialic acid leads to dysregulated integrin signaling. Notably, ectopic expression of PECAM increased cell surface integrin-β3, indicating that the reduction of cell surface integrin-β3 involves loss-of-endothelial PECAM. The results suggest that the cell surface stability of these glycoproteins is significantly reduced by the lack of α2,6-sialic acid, leading to abnormal signal transduction. The present findings highlight that α2,6-sialylation is critically involved in endothelial survival by controlling the cell surface stability and signal transduction of angiogenic molecules, and could be a novel target for anti-angiogenesis therapy.

Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the alpha6-integrin subunit.

PubMed

Kashimata, M; Gresik, E W

1997-02-01

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) regulate branching morphogenesis of fetal mouse submandibular gland (SMG) rudiments in vitro. The EGF system (EGF, TGF-alpha, and their shared receptor, EGFR) also regulates expression of integrins and their ligands in the extracellular matrix. We show here that inhibition of EGFR tyrosine-kinase activity by a tyrphostin retards in vitro development of SMGs. Using total RNA isolated from pooled SMGs taken from intact mouse fetuses, mRNA transcripts for EGF, TGF-alpha, and EGFR were detected by reverse transcription-polymerase chain reaction (RT-PCR), and age-dependent variations in the levels of these mRNA were quantitatively determined by nuclease protection assays. These findings suggest that the EGF system is operative in the in vivo development of this gland. alpha6-Integrin subunit was localized by immunofluorescence at the basal surface of epithelial cells. Branching morphogenesis of cultured SMG rudiments was inhibited by anti-alpha6 antibodies. Synthesis of alpha6-subunit in cultured SMGs, detected by metabolic labeling and immunoprecipitation, was increased by EGF and drastically reduced by tyrphostin. RT-PCR revealed that mRNAs for alpha6- and beta1- and beta4-integrin subunits are expressed at all ages between embryonic day 13 and postnatal day 7. These findings suggest that 1) the EGF system is a physiologic regulator of development of fetal mouse SMG, and 2) one mechanism by which it acts may be by regulating expression of integrins, which in turn control interaction of epithelial cells with the extracellular matrix.

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.

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.

Wound Healing Is Defective in Mice Lacking Tetraspanin CD151

PubMed Central

Cowin, Allison J.; Adams, Damian; Geary, Sean M.; Wright, Mark D.; Jones, Jonathan C.R.; Ashman, Leonie K.

2010-01-01

The tetraspanin CD151 forms complexes in epithelial cell membranes with laminin-binding integrins α6 β4, α3 β1, and α6 β1, and modifies integrin-mediated cell migration in vitro. We demonstrate in this study that CD151 expression is upregulated in a distinct temporal and spatial pattern during wound healing, particularly in the migrating epidermal tongue at the wound edge, suggesting a role for CD151 in keratinocyte migration. We show that healing is significantly impaired in CD151-null mice, with wounds gaping wider at 7 days post-injury. The rate of re-epithelialization of the CD151-null wounds is adversely affected, with significantly less wound area being covered by migrating epidermal cells. Our studies reveal that although laminin levels are similar in wild-type and CD151-null wounds, the organization of the laminin in the basement membrane is impaired. Furthermore, upregulation of α6 and β4 integrin expression is adversely affected in CD151-null mice wounds. In contrast, we find no significant effect of CD151 gene knockout on α3 and β1 integrin expression in wound repair. We suggest that mice lacking the CD151 gene are defective in wound healing, primarily owing to impairment of the re-epithelialization process. This may be due to defective basement membrane formation and epithelial cell adhesion and migration. PMID:16410781

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

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.

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

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.

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

Connection between integrins and cell activation in rat adrenal glomerulosa cells: a role for Arg-Gly-Asp peptide in the activation of the p42/p44(mapk) pathway and intracellular calcium.

PubMed

Campbell, Shirley; Otis, Melissa; Côté, Mylène; Gallo-Payet, Nicole; Payet, Marcel Daniel

2003-04-01

Integrins are responsible for adhesion and activation of several intracellular cascades. The present study was aimed at determining whether the interaction between fibronectin and integrins could generate pathways involved in physiological functions of rat adrenal glomerulosa cells. Immunofluorescence studies and adhesion assays showed that fibronectin was the best matrix in promoting the formation of focal adhesion. Binding of glomerulosa cells to fibronectin, but not to collagen I or poly-L-lysine, involved the integrin-binding sequence Arg-Gly-Asp (RGD). Activation of glomerulosa cells with Arg-Gly-Asp-Ser (RGDS) induced an increase in [Ca(2+)](i), whereas fibronectin triggered a release of Ca(2+) from InsP(3)-sensitive Ca(2+) stores. Aldosterone secretion induced by ACTH, angiotensin II, and RGDS and proliferation were improved on fibronectin, compared with poly-L-lysine. The RGDS peptide induced a transient increase in the activity of the p42/p44(mapk), independent of phosphatidylinositol-3 kinase and protein kinase C. Integrins alpha(5) and alpha(V) as well as their fibronectin receptor partners beta(1) and beta(3), were identified. These results suggest that in rat adrenal glomerulosa cells, binding of the alpha(5)beta(1), alpha(v)beta(1), or alpha(v)beta(3) integrins to fibronectin is involved in the generation of two important signaling events, increase in intracellular calcium, and activation of the p42/p44(mapk) cascade, leading to cell proliferation and aldosterone secretion.

Upregulation of Fibronectin and the α5β1 and αvβ3 Integrins on Blood Vessels within the Cerebral Ischemic Penumbra

PubMed Central

Li, Longxuan; Liu, Fudong; Welser-Alves, Jennifer V.; McCullough, Louise D.; Milner, Richard

2012-01-01

Following focal cerebral ischemia, blood vessels in the ischemic border, or penumbra, launch an angiogenic response. In light of the critical role for fibronectin in angiogenesis, and the observation that fibronectin and its integrin receptors are strongly upregulated on angiogenic vessels in the hypoxic CNS, the aim of this study was to establish whether angiogenic vessels in the ischemic CNS also show this response. Focal cerebral ischemia was established in C57/Bl6 mice by middle cerebral artery occlusion (MCA:O), and brain tissue analyzed seven days following re-perfusion, a time at which angiogenesis is ongoing. Within the ischemic core, immunofluorescent (IF) studies demonstrated vascular expression of MECA-32, a marker of leaky cerebral vessels, and vascular breakdown, defined by loss of staining for the endothelial marker, CD31, and the vascular adhesion molecules, laminin, dystroglycan and α6 integrin. Within the ischemic penumbra, dual-IF with CD31 and Ki67 revealed the presence of proliferating endothelial cells, indicating ongoing angiogenesis. Significantly, vessels in the ischemic penumbra showed strong upregulation of fibronectin and the fibronectin receptors, α5β1 and αvβ3 integrins. Taken together with our recent finding that the α5β1 integrin plays an important role in promoting cerebral angiogenesis in response to hypoxia, these results suggest that stimulation of the fibronectin-α5β1 integrin signalling pathway may provide a novel approach to amplifying the intrinsic angiogenic response to cerebral ischemia. PMID:22056225

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.

CD147-targeting siRNA inhibits cell-matrix adhesion of human malignant melanoma cells by phosphorylating focal adhesion kinase.

PubMed

Nishibaba, Rie; Higashi, Yuko; Su, Juan; Furukawa, Tatsuhiko; Kawai, Kazuhiro; Kanekura, Takuro

2012-01-01

CD147/basigin, highly expressed on the surface of malignant tumor cells including malignant melanoma (MM) cells, plays a critical role in the invasiveness and metastasis of MM. Metastasis is an orchestrated process comprised of multiple steps including adhesion and invasion. Integrin, a major adhesion molecule, co-localizes with CD147/basigin on the cell surface. Using the human MM cell line A375 that highly expresses CD147/basigin, we investigated whether CD147/basigin is involved in adhesion in association with integrin. CD147/basigin was knocked-down using siRNA targeting CD147 to elucidate the role of CD147/basigin. Cell adhesion was evaluated by adhesion assay on matrix-coated plates. The localization of integrin was inspected under a confocal microscope and the expression and phosphorylation of focal adhesion kinase (FAK), a downstream kinase of integrin, were examined by western blot analysis. Silencing of CD147/basigin in A375 cells by siRNA induced the phosphorylation of FAK at Y397. Integrin identified on the surface of parental cells was distributed in a speckled fashion in the cytoplasm of CD147 knockdown cells, resulting in morphological changes from a round to a polygonal shape with pseudopodial protrusions. Silencing of CD147/basigin in A375 cells clearly weakened their adhesiveness to collagen I and IV. Our results suggest that CD147/basigin regulates the adhesion of MM cells to extracellular matrices and of integrin β1 signaling via the phosphorylation of FAK. © 2011 Japanese Dermatological Association.

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.

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.

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.

Integrin-mediated human glioblastoma cells adhesion, migration and invasion by native and recombinant phospholipases of Scorpio maurus venom glands.

PubMed

Krayem, Najeh; Abdelkefi-Koubaa, Zaineb; Gargouri, Youssef; Luis, José

2018-05-01

Integrins are a large family of cell surface receptors mediating the interaction of cells with their microenvironment and they play an important role in glioma biology. In the present work, we reported the anti-tumor effect of Sm-PLGV a phospholipase A 2 from Tunisian scorpion venom glands-as well as its recombinant forms expressed in Escherichia coli-through interference with integrin receptor function in malignant glioma cells U87. These phospholipases inhibited in a dose dependent manner the adhesion, migration and invasion onto fibrinogen and fibronectin without any cytotoxicity. We showed that Sm-PLGV and its recombinant constructs blocked U87 migration by reducing their velocity and directional persistence. The inhibitory effect was related to a blockage of the integrins αvβ3 and α5β1 function. Inactivation of the enzymatic activity of Sm-PLGV by chemical modification with p-bromophenacyl bromide did not affect its anti-tumor properties, suggesting the presence of 'pharmacological sites' distinct from the catalytic site in scorpion venom phospholipases A 2 . Copyright © 2018 Elsevier Inc. All rights reserved.

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 = 0.004), overall response rate (p = 0.003), and expression of ECAD (p = 0.015) were the independent good prognostic factors for OS. LD (p = 0.024), overall response rate (p < 0.001), and less than two metastasis (p = 0.003) were prognostic factors for longer PFS. These results suggest that ECAD expression may be useful as a prognostic indicator in patients with SCLC. © 2011 The Authors. APMIS © 2011 APMIS.

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

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

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.

Evidence for Integrin - Venus Kinase Receptor 1 Alliance in the Ovary of Schistosoma mansoni Females Controlling Cell Survival.

PubMed

Gelmedin, Verena; Morel, Marion; Hahnel, Steffen; Cailliau, Katia; Dissous, Colette; Grevelding, Christoph G

2017-01-01

In metazoan integrin signaling is an important process of mediating extracellular and intracellular communication processes. This can be achieved by cooperation of integrins with growth factor receptors (GFRs). Schistosoma mansoni is a helminth parasite inducing schistosomiasis, an infectious disease of worldwide significance for humans and animals. First studies on schistosome integrins revealed their role in reproductive processes, being involved in spermatogenesis and oogenesis. With respect to the roles of eggs for maintaining the parasite´s life cycle and for inducing the pathology of schistosomiasis, elucidating reproductive processes is of high importance. Here we studied the interaction of the integrin receptor Smβ-Int1 with the venus kinase receptor SmVKR1 in S. mansoni. To this end we cloned and characterized SmILK, SmPINCH, and SmNck2, three putative bridging molecules for their role in mediating Smβ-Int1/SmVKR1 cooperation. Phylogenetic analyses showed that these molecules form clusters that are specific for parasitic platyhelminths as it was shown for integrins before. Transcripts of all genes colocalized in the ovary. In Xenopus oocytes germinal vesicle breakdown (GVBD) was only induced if all members were simultaneously expressed. Coimmunoprecipitation results suggest that a Smβ-Int1-SmILK-SmPINCH-SmNck2-SmVKR1 complex can be formed leading to the phosphorylation and activation of SmVKR1. These results indicate that SmVKR1 can be activated in a ligand-independent manner by receptor-complex interaction. RNAi and inhibitor studies to knock-down SmILK as a representative complex member concurrently revealed effects on the extracellular matrix surrounding the ovary and oocyte localization within the ovary, oocyte survival, and egg production. By TUNEL assays, confocal laser scanning microscopy (CLSM), Caspase-3 assay, and transcript profiling of the pro-apoptotic BCL-2 family members BAK/BAX we obtained first evidence for roles of this signaling complex in mediating cell death in immature and primary oocytes. These results suggest that the Smβ-Int1/SmVKR1 signaling complex is important for differentiation and survival in oocytes of paired schistosomes.

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 specifically enhance the cytotoxicity against BEL-7402 compared with DOX-loaded PEG-CS-SA and doxorubicin hydrochlorate. This study suggests that RGD-modified PEG-CS-SA micelles are promising drug carriers for integrin-overexpressing tumor active targeting therapy.

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.

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

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.

Ligand-induced Epitope Masking: DISSOCIATION OF INTEGRIN α5β1-FIBRONECTIN COMPLEXES ONLY BY MONOCLONAL ANTIBODIES WITH AN ALLOSTERIC MODE OF ACTION.

PubMed

Mould, A Paul; Askari, Janet A; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A; Humphries, Martin J

2016-09-30

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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

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

Dwarfism in mice lacking collagen-binding integrins α2β1 and α11β1 is caused by severely diminished IGF-1 levels.

PubMed

Blumbach, Katrin; Niehoff, Anja; Belgardt, Bengt F; Ehlen, Harald W A; Schmitz, Markus; Hallinger, Ralf; Schulz, Jan-Niklas; Brüning, Jens C; Krieg, Thomas; Schubert, Markus; Gullberg, Donald; Eckes, Beate

2012-02-24

Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either α2β1 or α11β1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis.

Dwarfism in Mice Lacking Collagen-binding Integrins α2β1 and α11β1 Is Caused by Severely Diminished IGF-1 Levels*

PubMed Central

Blumbach, Katrin; Niehoff, Anja; Belgardt, Bengt F.; Ehlen, Harald W. A.; Schmitz, Markus; Hallinger, Ralf; Schulz, Jan-Niklas; Brüning, Jens C.; Krieg, Thomas; Schubert, Markus; Gullberg, Donald; Eckes, Beate

2012-01-01

Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either α2β1 or α11β1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis. PMID:22210772

Estradiol, tamoxifen and ICI 182,780 alter alpha3 and beta1 integrin expression and laminin-1 adhesion in oral squamous cell carcinoma cell cultures.

PubMed

Nelson, Katja; Helmstaedter, Victor; Moreau, Cynthia; Lage, Hermann

2008-01-01

Adhesion molecules such as integrins and extracellular matrix proteins like laminins have been identified to play an important role in cell proliferation, migration and invasion by regulating cell-extracellular matrix interaction in various cancers including oral squamous cell carcinoma (OSCC). In this study, the effect of estradiol (E2), and the E2 antagonists tamoxifen (TAM) and ICI 182,780 (ICI) on the expression of integrins and adhesion to laminin-1 in different OSCC in vitro models was analyzed. TAM and ICI inhibited growth in all OSCC cell lines. Dependent on estrogen receptor (ER) status E2 displayed a significant influence on growth after long-term administration. ICI reduced laminin-1 adhesion in all cell lines. beta1 Integrin transcription is reduced with TAM and E2 and alpha3 cell surface expression with TAM. This study shows that OSCC is estrogen and SERM sensitive and that these compounds can modulate cell-matrix interaction in part by modulating integrin expression and translation. The investigation also confirms that growth is significantly influenced by these adjuvant therapeutics. These data suggest that a greater understanding of basic biology and mechanisms of the ER and its ligands in oral squamous cells is needed to elucidate the use of specific pharmacological agents as therapeutics of anti-tumorigenic pathways.

JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets

PubMed Central

Naik, Meghna U.; Stalker, Timothy J.; Brass, Lawrence F.

2012-01-01

Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin αIIbβ3. Once platelet activation has occurred, integrin αIIbβ3 stabilizes thrombus formation by providing agonist-independent “outside-in” signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A–deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation. PMID:22271446

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

Nuclear Localization of the ERK MAP Kinase Mediated by Drosophila αPS2βPS Integrin and Importin-7

PubMed Central

James, Brian P.; Bunch, Thomas A.; Krishnamoorthy, Srinivasan; Perkins, Lizabeth A.

2007-01-01

The control of gene expression by the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK) requires its translocation into the nucleus. In Drosophila S2 cells nuclear accumulation of diphospho-ERK (dpERK) is greatly reduced by interfering double-stranded RNA against Drosophila importin-7 (DIM-7) or by the expression of integrin mutants, either during active cell spreading or after stimulation by insulin. In both cases, total ERK phosphorylation (on Westerns) is not significantly affected, and ERK accumulates in a perinuclear ring. Tyrosine phosphorylation of DIM-7 is reduced in cells expressing integrin mutants, indicating a mechanistic link between these components. DIM-7 and integrins localize to the same actin-containing peripheral regions in spreading cells, but DIM-7 is not concentrated in paxillin-positive focal contacts or stable focal adhesions. The Corkscrew (SHP-2) tyrosine phosphatase binds DIM-7, and Corkscrew is required for the cortical localization of DIM-7. These data suggest a model in which ERK phosphorylation must be spatially coupled to integrin-mediated DIM-7 activation to make a complex that can be imported efficiently. Moreover, dpERK nuclear import can be restored in DIM-7–deficient cells by Xenopus Importin-7, demonstrating that ERK import is an evolutionarily conserved function of this protein. PMID:17699602

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.

Development of Transgenic Cloned Pig Models of Skin Inflammation by DNA Transposon-Directed Ectopic Expression of Human β1 and α2 Integrin

PubMed Central

Staunstrup, Nicklas Heine; Madsen, Johannes; Primo, Maria Nascimento; Li, Juan; Liu, Ying; Kragh, Peter M.; Li, Rong; Schmidt, Mette; Purup, Stig; Dagnæs-Hansen, Frederik; Svensson, Lars; Petersen, Thomas K.; Callesen, Henrik; Bolund, Lars; Mikkelsen, Jacob Giehm

2012-01-01

Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage-induced inflammation and should be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis. PMID:22590584

Ligand-induced Epitope Masking

PubMed Central

Mould, A. Paul; Askari, Janet A.; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A.; Humphries, Martin J.

2016-01-01

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. PMID:27484800

Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors

PubMed Central

Goodman, Simon L.; Grote, Hans Juergen; Wilm, Claudia

2012-01-01

Summary The relationship between integrin expression and function in pathologies is often contentious as comparisons between human pathological expression and expression in cell lines is difficult. In addition, the expression of even integrins αvβ6 and αvβ8 in tumor cell lines is not comprehensively documented. Here, we describe rabbit monoclonal antibodies (RabMabs) against the extracellular domains of αv integrins that react with both native integrins and formalin fixed, paraffin embedded (FFPE) human tissues. These RabMabs, against αvβ3 (EM22703), αvβ5 (EM09902), αvβ6 (EM05201), αvβ8 (EM13309), and pan-αv (EM01309), recognize individual integrin chains in Western blots and in flow cytometry. EM22703 detected a ligand-induced binding site (LIBS), reporting an epitope enhanced by the binding of an RGD-peptide to αvβ3. αvβ8 was rarely expressed in human tumor specimens, and weakly expressed in non-small-cell lung carcinoma (NSCLC). However, ovarian carcinoma cell lines expressed αvβ8, as did some melanoma cells, whereas U87MG glioma lacked αvβ8 expression. We observed an unexpected strong expression of αvβ6 in tumor samples of invasive ductal breast adenoma, colorectal carcinoma (CRC), and NSCLC. αvβ3 was strongly expressed in some invasive NSCLC cohorts. Interestingly, PC3 prostate cell and human prostate tumors did not express αvβ3. The RabMabs stained plasma membranes in FFPE-immunohistochemistry (IHC) samples of tumor cell lines from lung, ovary, colon, prostate, squamous cell carcinoma of head and neck (SCCHN), breast, and pancreas carcinomas. The RabMabs are unique tools for probing αv integrin biology, and suggest that especially αvβ6 and αvβ8 biologies still have much to reveal. PMID:23213423

Force loading explains spatial sensing of ligands by cells

NASA Astrophysics Data System (ADS)

Oria, Roger; Wiegand, Tina; Escribano, Jorge; Elosegui-Artola, Alberto; Uriarte, Juan Jose; Moreno-Pulido, Cristian; Platzman, Ilia; Delcanale, Pietro; Albertazzi, Lorenzo; Navajas, Daniel; Trepat, Xavier; García-Aznar, José Manuel; Cavalcanti-Adam, Elisabetta Ada; Roca-Cusachs, Pere

2017-12-01

Cells can sense the density and distribution of extracellular matrix (ECM) molecules by means of individual integrin proteins and larger, integrin-containing adhesion complexes within the cell membrane. This spatial sensing drives cellular activity in a variety of normal and pathological contexts. Previous studies of cells on rigid glass surfaces have shown that spatial sensing of ECM ligands takes place at the nanometre scale, with integrin clustering and subsequent formation of focal adhesions impaired when single integrin-ligand bonds are separated by more than a few tens of nanometres. It has thus been suggested that a crosslinking ‘adaptor’ protein of this size might connect integrins to the actin cytoskeleton, acting as a molecular ruler that senses ligand spacing directly. Here, we develop gels whose rigidity and nanometre-scale distribution of ECM ligands can be controlled and altered. We find that increasing the spacing between ligands promotes the growth of focal adhesions on low-rigidity substrates, but leads to adhesion collapse on more-rigid substrates. Furthermore, disordering the ligand distribution drastically increases adhesion growth, but reduces the rigidity threshold for adhesion collapse. The growth and collapse of focal adhesions are mirrored by, respectively, the nuclear or cytosolic localization of the transcriptional regulator protein YAP. We explain these findings not through direct sensing of ligand spacing, but by using an expanded computational molecular-clutch model, in which individual integrin-ECM bonds—the molecular clutches—respond to force loading by recruiting extra integrins, up to a maximum value. This generates more clutches, redistributing the overall force among them, and reducing the force loading per clutch. At high rigidity and high ligand spacing, maximum recruitment is reached, preventing further force redistribution and leading to adhesion collapse. Measurements of cellular traction forces and actin flow speeds support our model. Our results provide a general framework for how cells sense spatial and physical information at the nanoscale, precisely tuning the range of conditions at which they form adhesions and activate transcriptional regulation.

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 chondryocyte- lineage cells regulate incisor eruption and perinatal bone formation in both intramembranously and endochondrally formed bones in young, rapidly growing mice. In contrast, the Osteocalcin-specific β1 integrin deletion had only minor effects on skeletal phenotype. PMID:25183373

Functional analysis of alpha5beta1 integrin and lipid rafts in invasion of epithelial cells by Porphyromonas gingivalis using fluorescent beads coated with bacterial membrane vesicles.

PubMed

Tsuda, Kayoko; Furuta, Nobumichi; Inaba, Hiroaki; Kawai, Shinji; Hanada, Kentaro; Yoshimori, Tamotsu; Amano, Atsuo

2008-01-01

Porphyromonas gingivalis, a periodontal pathogen, was previously suggested to exploit alpha5beta1 integrin and lipid rafts to invade host cells. However, it is unknown if the functional roles of these host components are distinct from one another during bacterial invasion. In the present study, we analyzed the mechanisms underlying P. gingivalis invasion, using fluorescent beads coated with bacterial membrane vesicles (MV beads). Cholesterol depletion reagents including methyl-beta-cyclodextrin (MbetaCD) drastically inhibited the entry of MV beads into epithelial cells, while they were less effective on bead adhesion to the cells. Bead entry was also abolished in CHO cells deficient in sphingolipids, components of lipid rafts, whereas adhesion was negligibly influenced. Following MbetaCD treatment, downstream events leading to actin polymerization were abolished; however, alpha5beta1 integrin was recruited to beads attached to the cell surface. Dominant-negative Rho GTPase Rac1 abolished cellular engulfment of the beads, whereas dominant-negative Cdc42 did not. Following cellular interaction with the beads, Rac1 was found to be translocated to the lipid rafts fraction, which was inhibited by MbetaCD. These results suggest that alpha5beta1 integrin, independent of lipid rafts, promotes P. gingivalis adhesion to epithelial cells, while the subsequent uptake process requires lipid raft components for actin organization, with Rho GTPase Rac1.

A dual-labeled knottin peptide for PET and near-infrared fluorescence imaging of integrin expression in living subjects

PubMed Central

Kimura, Richard H.; Miao, Zheng; Cheng, Zhen; Gambhir, Sanjiv S.; Cochran, Jennifer R.

2010-01-01

Previously, we used directed evolution to engineer mutants of the Ecballium elaterium trypsin inhibitor (EETI-II) knottin that bind to αvβ3 and αvβ5 integrin receptors with low nanomolar affinity, and showed that Cy5.5- or 64Cu-DOTA-labeled knottin peptides could be used to image integrin expression in mouse tumor models using near-infrared fluorescence (NIRF) imaging or positron emission tomography (PET). Here, we report the development of a dual-labeled knottin peptide conjugated to both NIRF and PET imaging agents for multimodality imaging in living subjects. We created an orthogonally-protected peptide-based linker for stoichiometric coupling of 64Cu-DOTA and Cy5.5 onto the knottin N-terminus, and confirmed that conjugation did not affect binding to αvβ3 and αvβ5 integrins. NIRF and PET imaging studies in tumor xenograft models showed that Cy5.5 conjugation significantly increased kidney uptake and retention compared to the knottin peptide labeled with 64Cu-DOTA alone. In the tumor, the dual-labeled 64Cu-DOTA/Cy5.5 knottin probe showed decreased wash-out leading to significantly better retention (p < 0.05) compared to the 64Cu-DOTA-labeled knottin probe. Tumor uptake was significantly reduced (p < 0.05) when the dual-labeled probe was co-injected with an excess of unlabeled competitor and when tested in a tumor model with lower levels of integrin expression. Finally, plots of tumor-to-background tissue ratios for Cy5.5 versus 64Cu uptake were well correlated over several time points post injection, demonstrating pharmacokinetic cross validation of imaging labels. This dual-modality NIRF/PET imaging agent is promising for further development in clinical applications where high sensitivity and high-resolution are desired, such as detection of tumors located deep within the body and image-guided surgical resection. PMID:20131753

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.

Cell Attachment to the Extracellular Matrix Induces Proteasomal Degradation of p21CIP1 via Cdc42/Rac1 Signaling

PubMed Central

Bao, Wenjie; Thullberg, Minna; Zhang, Hongquan; Onischenko, Anatoli; Strömblad, Staffan

2002-01-01

The cyclin-dependent kinase 2 (Cdk2) inhibitors p21CIP1 and p27KIP1 are negatively regulated by anchorage during cell proliferation, but it is unclear how integrin signaling may affect these Cdk2 inhibitors. Here, we demonstrate that integrin ligation led to rapid reduction of p21CIP1 and p27KIP1 protein levels in three distinct cell types upon attachment to various extracellular matrix (ECM) proteins, including fibronectin (FN), or to immobilized agonistic anti-integrin monoclonal antibodies. Cell attachment to FN did not rapidly influence p21CIP1 mRNA levels, while the protein stability of p21CIP1 was decreased. Importantly, the down-regulation of p21CIP1 and p27KIP1 was completely blocked by three distinct proteasome inhibitors, demonstrating that integrin ligation induced proteasomal degradation of these Cdk2 inhibitors. Interestingly, ECM-induced proteasomal proteolysis of a ubiquitination-deficient p21CIP1 mutant (p21K6R) also occurred, showing that the proteasomal degradation of p21CIP1 was ubiquitin independent. Concomitant with our finding that the small GTPases Cdc42 and Rac1 were activated by attachment to FN, constitutively active (ca) Cdc42 and ca Rac1 promoted down-regulation of p21CIP1. However, dominant negative (dn) Cdc42 and dn Rac1 mutants blocked the anchorage-induced degradation of p21CIP1, suggesting that an integrin-induced Cdc42/Rac1 signaling pathway activates proteasomal degradation of p21CIP1. Our results indicate that integrin-regulated proteasomal proteolysis might contribute to anchorage-dependent cell cycle control. PMID:12052868

Expression of alpha V integrin is modulated by Epstein-Barr virus nuclear antigen 3C and the metastasis suppressor Nm23-H1 through interaction with the GATA-1 and Sp1 transcription factors

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

Choudhuri, Tathagata; Verma, Subhash C.; Lan, Ke

Epstein-Barr virus (EBV) is a lymphotrophic herpesvirus infecting most of the world's population. It is associated with a number of human lymphoid and epithelial tumors and lymphoproliferative diseases in immunocompromised patients. A subset of latent EBV antigens is required for immortalization of primary B-lymphocytes. The metastatic suppressor Nm23-H1 which is downregulated in human invasive breast carcinoma reduces the migration and metastatic activity of breast carcinoma cells when expressed from a heterologous promoter. Interestingly, the EBV nuclear antigen 3C (EBNA3C) reverses these activities of Nm23-H1. The alpha V integrins recognize a variety of ligands for signaling and are involved in cellmore » migration and proliferation and also serve as major receptors for extracellular-matrix-mediated cell adhesion and migration. The goal of this study was to determine if Nm23-H1 and EBNA3C can modulate alpha V integrin expression and downstream activities. The results of our studies indicate that Nm23-H1 downregulates alpha V intregrin expression in a dose responsive manner. In contrast, EBNA3C can upregulate alpha V integrin expression. Furthermore, the study showed that the association of the Sp1 and GATA transcription factors with Nm23-H1 is required for modulation of the alpha V integrin activity. Thus, these results suggest a direct correlation between the alpha V integrin expression and the interaction of Nm23-H1 with EBNA3C.« less

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.

Effect of P to A Mutation of the N-Terminal Residue Adjacent to the Rgd Motif on Rhodostomin: Importance of Dynamics in Integrin Recognition

PubMed Central

Chen, Yi-Chun; Chang, Yao-Tsung; Chang, Yung-Sheng; Huang, Chun-Hao; Chuang, Woei-Jer

2012-01-01

Rhodostomin (Rho) is an RGD protein that specifically inhibits integrins. We found that Rho mutants with the P48A mutation 4.4–11.5 times more actively inhibited integrin α5β1. Structural analysis showed that they have a similar 3D conformation for the RGD loop. Docking analysis also showed no difference between their interactions with integrin α5β1. However, the backbone dynamics of RGD residues were different. The values of the R2 relaxation parameter for Rho residues R49 and D51 were 39% and 54% higher than those of the P48A mutant, which caused differences in S2, Rex, and τe. The S2 values of the P48A mutant residues R49, G50, and D51 were 29%, 14%, and 28% lower than those of Rho. The Rex values of Rho residues R49 and D51 were 0.91 s−1 and 1.42 s−1; however, no Rex was found for those of the P48A mutant. The τe values of Rho residues R49 and D51 were 9.5 and 5.1 times lower than those of P48A mutant. Mutational study showed that integrin α5β1 prefers its ligands to contain (G/A)RGD but not PRGD sequences for binding. These results demonstrate that the N-terminal proline residue adjacent to the RGD motif affect its function and dynamics, which suggests that the dynamic properties of the RGD motif may be important in Rho's interaction with integrin α5β1. PMID:22238583

Expression of FLNa in human melanoma cells regulates the function of integrin α1β1 and phosphorylation and localisation of PKB/AKT/ERK1/2 kinases.

PubMed

Krebs, Kristi; Ruusmann, Anu; Simonlatser, Grethel; Velling, Teet

2015-12-01

FLNa is a ubiquitous cytoskeletal protein that links transmembrane receptors, including integrins, to F-actin and functions as a signalling intermediate. We investigated FLNa's role in the function of integrin-type collagen receptors, EGF-EGFR signalling and regulation of PKB/Akt and ERK1/2. Using FLNa-deficient M2 human melanoma cells, and same cells expressing EGFP-FLNa (M2F) or its Ig-like repeats 1-8+24, 8-15+24 and 16-24, we found that in M2F and M2 8-15+24 cells, EGF induced the increased phosphorylation of PKB/Akt and ERK1/2. In M2F cells EGF induced the localisation of these kinases to cell nucleus and lamellipodia, respectively, and the ERK1/2 phosphorylation-dependent co-immunoprecipitation of FLNa with ERK1/2. Only M2F and M2 8-15+24 cells adhered to and spread on type I collagen whereas on fibronectin all cells behaved similarly. α1β1 and α2β1 were the integrin-type collagen receptors expressed on these cells with primarily α1β1 localising to focal contacts and affecting cell adhesion and migration in a manner dependent on FLNa or its Ig-like repeats 8-15. Our results suggest a role for FLNa repeats 8-15 in the α1-subunit-dependent regulation of integrin α1β1 function, EGF-EGFR signalling to PKB/Akt and ERK1/2, identify ERK1/2 in EGF-induced FLNa-associated protein complexes, and show that the function of different integrins is subjected to differential regulation by FLNa. Copyright © 2015. Published by Elsevier GmbH.

Extracellular Membrane-proximal Domain of HAb18G/CD147 Binds to Metal Ion-dependent Adhesion Site (MIDAS) Motif of Integrin β1 to Modulate Malignant Properties of Hepatoma Cells*

PubMed Central

Li, Yong; Wu, Jiao; Song, Fei; Tang, Juan; Wang, Shi-Jie; Yu, Xiao-Ling; Chen, Zhi-Nan; Jiang, Jian-Li

2012-01-01

Several lines of evidence suggest that HAb18G/CD147 interacts with the integrin variants α3β1 and α6β1. However, the mechanism of the interaction remains largely unknown. In this study, mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, was used to study the CD147-integrin β1 subunit interaction. CD147 in human hepatocellular carcinoma (HCC) cells was interfered with by small hairpin RNA. Nude mouse xenograft model and metastatic model of HCC were used to detect the role of CD147 in carcinogenesis and metastasis. We found that the extracellular membrane-proximal domain of HAb18G/CD147 (I-type domain) binds at the metal ion-dependent adhesion site in the βA domain of the integrin β1 subunit, and Asp179 in the I-type domain of HAb18G/CD147 plays an important role in the interaction. The levels of the proteins that act downstream of integrin, including focal adhesion kinase (FAK) and phospho-FAK, were decreased, and the cytoskeletal structures of HCC cells were rearranged bearing the HAb18G/CD147 deletion. Simultaneously, the migration and invasion capacities, secretion of matrix metalloproteinases, colony formation rate in vitro, and tumor growth and metastatic potential in vivo were decreased. These results indicate that the interaction of HAb18G/CD147 extracellular I-type domain with the integrin β1 metal ion-dependent adhesion site motif activates the downstream FAK signaling pathway, subsequently enhancing the malignant properties of HCC cells. PMID:22130661

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.

IL-2 Enhances Gut Homing Potential of Human Naive Regulatory T Cells Early in Life.

PubMed

Hsu, Peter S; Lai, Catherine L; Hu, Mingjing; Santner-Nanan, Brigitte; Dahlstrom, Jane E; Lee, Cheng Hiang; Ajmal, Ayesha; Bullman, Amanda; Arbuckle, Susan; Al Saedi, Ahmed; Gacis, Lou; Nambiar, Reta; Williams, Andrew; Wong, Melanie; Campbell, Dianne E; Nanan, Ralph

2018-06-15

Recent evidence suggests early environmental factors are important for gut immune tolerance. Although the role of regulatory T (Treg) cells for gut immune homeostasis is well established, the development and tissue homing characteristics of Treg cells in children have not been studied in detail. In this article, we studied the development and homing characteristics of human peripheral blood Treg cell subsets and potential mechanisms inducing homing molecule expression in healthy children. We found contrasting patterns of circulating Treg cell gut and skin tropism, with abundant β7 integrin + Treg cells at birth and increasing cutaneous lymphocyte Ag (CLA + ) Treg cells later in life. β7 integrin + Treg cells were predominantly naive, suggesting acquisition of Treg cell gut tropism early in development. In vitro, IL-7 enhanced gut homing but reduced skin homing molecule expression in conventional T cells, whereas IL-2 induced a similar effect only in Treg cells. This effect was more pronounced in cord compared with adult blood. Our results suggest that early in life, naive Treg cells may be driven for gut tropism by their increased sensitivity to IL-2-induced β7 integrin upregulation, implicating a potential role of IL-2 in gut immune tolerance during this critical period of development. Copyright © 2018 by The American Association of Immunologists, Inc.

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

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.

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

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 activities of the alpha 6 beta 4 integrin. PMID:1500432

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

Integrin αMβ2 is differently expressed by subsets of human osteoclast precursors and mediates adhesion of classical monocytes to bone

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

Sprangers, Sara, E-mail: s.l.sprangers@acta.nl; Schoenmaker, Ton, E-mail: t.schoenmaker@acta.nl; Department of Periodontology, Academic Centre for Dentistry Amsterdam

Bone-degrading osteoclasts are formed through fusion of their monocytic precursors. In the population of human peripheral blood monocytes, three distinct subsets have been identified: classical, intermediate and non-classical monocytes. We have previously shown that when the monocyte subsets are cultured on bone, significantly more osteoclasts are formed from classical monocytes than from intermediate or non-classical monocytes. Considering that this difference does not exist when monocyte subsets are cultured on plastic, we hypothesized that classical monocytes adhere better to the bone surface compared to intermediate and non-classical monocytes. To investigate this, the different monocyte subsets were isolated from human peripheral bloodmore » and cultured on slices of human bone in the presence of the cytokine M-CSF. We found that classical monocytes adhere better to bone due to a higher expression of the integrin αMβ2 and that their ability to attach to bone is significantly decreased when the integrin is blocked. This suggests that integrin αMβ2 mediates attachment of osteoclast precursors to bone and thereby enables the formation of osteoclasts.« less

Cytohesin 1 regulates homing and engraftment of human hematopoietic stem and progenitor cells.

PubMed

Rak, Justyna; Foster, Katie; Potrzebowska, Katarzyna; Talkhoncheh, Mehrnaz Safaee; Miharada, Natsumi; Komorowska, Karolina; Torngren, Therese; Kvist, Anders; Borg, Åke; Svensson, Lena; Bonnet, Dominique; Larsson, Jonas

2017-02-23

Adhesion is a key component of hematopoietic stem cell regulation mediating homing and retention to the niche in the bone marrow. Here, using an RNA interference screen, we identify cytohesin 1 (CYTH1) as a critical mediator of adhesive properties in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs). Knockdown of CYTH1 disrupted adhesion of HSPCs to primary human mesenchymal stroma cells. Attachment to fibronectin and ICAM1, 2 integrin ligands, was severely impaired, and CYTH1-deficient cells showed a reduced integrin β1 activation response, suggesting that CYTH1 mediates integrin-dependent functions. Transplantation of CYTH1-knockdown cells to immunodeficient mice resulted in significantly lower long-term engraftment levels, associated with a reduced capacity of the transplanted cells to home to the bone marrow. Intravital microscopy showed that CYTH1 deficiency profoundly affects HSPC mobility and localization within the marrow space and thereby impairs proper lodgment into the niche. Thus, CYTH1 is a novel major regulator of adhesion and engraftment in human HSPCs through mechanisms that, at least in part, involve the activation of integrins. © 2017 by The American Society of Hematology.

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

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

Kawamoto, Eiji; Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507; Okamoto, Takayuki, E-mail: okamotot@doc.medic.mie-u.ac.jp

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

Herpes simplex virus type 2 glycoprotein H interacts with integrin αvβ3 to facilitate viral entry and calcium signaling in human genital tract epithelial cells.

PubMed

Cheshenko, Natalia; Trepanier, Janie B; González, Pablo A; Eugenin, Eliseo A; Jacobs, William R; Herold, Betsy C

2014-09-01

Herpes simplex virus (HSV) entry requires multiple interactions at the cell surface and activation of a complex calcium signaling cascade. Previous studies demonstrated that integrins participate in this process, but their precise role has not been determined. These studies were designed to test the hypothesis that integrin αvβ3 signaling promotes the release of intracellular calcium (Ca2+) stores and contributes to viral entry and cell-to-cell spread. Transfection of cells with small interfering RNA (siRNA) targeting integrin αvβ3, but not other integrin subunits, or treatment with cilengitide, an Arg-Gly-Asp (RGD) mimetic, impaired HSV-induced Ca2+ release, viral entry, plaque formation, and cell-to-cell spread of HSV-1 and HSV-2 in human cervical and primary genital tract epithelial cells. Coimmunoprecipitation studies and proximity ligation assays indicated that integrin αvβ3 interacts with glycoprotein H (gH). An HSV-2 gH-null virus was engineered to further assess the role of gH in the virus-induced signaling cascade. The gH-2-null virus bound to cells and activated Akt to induce a small Ca2+ response at the plasma membrane, but it failed to trigger the release of cytoplasmic Ca2+ stores and was impaired for entry and cell-to-cell spread. Silencing of integrin αvβ3 and deletion of gH prevented phosphorylation of focal adhesion kinase (FAK) and the transport of viral capsids to the nuclear pore. Together, these findings demonstrate that integrin signaling is activated downstream of virus-induced Akt signaling and facilitates viral entry through interactions with gH by activating the release of intracellular Ca2+ and FAK phosphorylation. These findings suggest a new target for HSV treatment and suppression. Herpes simplex viruses are the leading cause of genital disease worldwide, the most common infection associated with neonatal encephalitis, and a major cofactor for HIV acquisition and transmission. There is no effective vaccine. These epidemiological findings underscore the urgency to develop novel HSV treatment or prevention strategies. This study addresses this gap by further defining the signaling pathways the virus usurps to enter human genital tract epithelial cells. Specifically, the study defines the role played by integrins and by the viral envelope glycoprotein H in entry and cell-to-cell spread. This knowledge will facilitate the identification of new targets for the development of treatment and prevention. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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-down cells and αvβ3low cells was not altered. In summary, these results suggest that the αvβ3 integrin enhances cancer cell invasion through increased cellular stiffness and enhanced cytoskeletal remodeling dynamics, which enables the cells to generate and transmit contractile forces to overcome the steric hindrance of 3D ECMs.

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

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.

Molecular Mechanotransduction: how forces trigger cytoskeletal dynamics

NASA Astrophysics Data System (ADS)

Ehrlicher, Allen

2012-02-01

Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders, such as cardiac failure and pulmonary injury. Despite detailed knowledge of the cytoskeleton's structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein, filamin A (FLNa) as a central mechanotransduction element of the cytoskeleton by using Fluorescence Loss After photoConversion (FLAC), a novel high-speed alternative to FRAP. We reconstituted a minimal system consisting of actin filaments, FLNa and two FLNa-binding partners: the cytoplasmic tail of ß-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with ß integrin tails connecting to the actin cytoskeleton by binding directly to filamin. FilGAP is a FLNa-binding GTPase-activating protein specific for Rac, which in vivo regulates cell spreading and bleb formation. We demonstrate that both externally-imposed bulk shear and myosin II driven forces differentially regulate the binding of integrin and FilGAP to FLNa. Consistent with structural predictions, strain increases ß-integrin binding to FLNa, whereas it causes FilGAP to dissociate from FLNa, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify the first molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signaling molecules. Moreover, GAP activity has been shown to switch cell movement from mesenchymal to amoeboid motility, suggesting that mechanical forces directly impact the invasiveness of cancer.

Expression of laminin-5 and integrins in actinic cheilitis and superficially invasive squamous cell carcinomas of the lip.

PubMed

Peixoto da-Silva, Janaína; Lourenço, Silvia; Nico, Marcello; Silva, Filomena H; Martins, Marília Trierveiler; Costa-Neves, Adriana

2012-10-15

The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5γ2 chain as well as α3, β1 subunits of α3β1 heterodimer and β4 subunit of integrin α6β4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of β1, β4 and α3 integrins, and SISCCs lacked β1, β4 and α3 integrins in the invasive front. AC cases were negative for the Ln-5γ2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5γ2 chain expression in the invasive front of the tumor. Integrin β1, β4 and α3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5γ2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype. Copyright © 2012 Elsevier GmbH. All rights reserved.

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

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

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.

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.

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.

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

Scaffold composition affects cytoskeleton organization, cell-matrix interaction and the cellular fate of human mesenchymal stem cells upon chondrogenic differentiation.

PubMed

Li, Yuk Yin; Choy, Tze Hang; Ho, Fu Chak; Chan, Pui Barbara

2015-06-01

The stem cell niche, or microenvironment, consists of soluble, matrix, cell and mechanical factors that together determine the cellular fates and/or differentiation patterns of stem cells. Collagen and glycosaminoglycans (GAGs) are important scaffolding materials that can mimic the natural matrix niche. Here, we hypothesize that imposing changes in the scaffold composition or, more specifically, incorporating GAGs into the collagen meshwork, will affect the morphology, cytoskeletal organization and integrin expression profiles, and hence the fate of human mesenchymal stem cells (MSCs) upon the induction of differentiation. Using chondrogenesis as an example, we microencapsulated MSCs in three scaffold systems that had varying matrix compositions: collagen alone (C), aminated collagen (AC) and aminated collagen with GAGs (ACG). We then induced the MSCs to differentiate toward a chondrogenic lineage, after which, we characterized the cell viability and morphology, as well as the level of cytoskeletal organization and the integrin expression profile. We also studied the fate of the MSCs by evaluating the major chondrogenic markers at both the gene and protein level. In C, MSC chondrogenesis was successfully induced and MSCs that spread in the scaffolds had a clear actin cytoskeleton; they expressed integrin α2β1, α5 and αv; promoted sox9 nuclear localization transcription activation; and upregulated the expression of chondrogenic matrix markers. In AC, MSC chondrogenesis was completely inhibited but the scaffold still supported cell survival. The MSCs did not spread and they had no actin cytoskeleton; did not express integrin α2 or αv; they failed to differentiate into chondrogenic lineage cells even on chemical induction; and there was little colocalization or functional interaction between integrin α5 and fibronectin. In ACG, although the MSCs did not express integrin α2, they did express integrin αv and there was strong co-localization and hence functional binding between αv and fibronectin. In addition, vimentin was the dominant cytoskeletal protein in these cells, and the chondrogenic marker genes were expressed but at a much lower level than in the MSCs encapsulated in C alone. This work suggests the importance of controlling the matrix composition as a strategy to manipulate cell-matrix interactions (through changes in the integrin expression profile and cytoskeleton organization), and hence stem cell fates. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-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.

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.

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.

The role of the micro-pattern and nano-topography of hydroxyapatite bioceramics on stimulating osteogenic differentiation of mesenchymal stem cells.

PubMed

Zhao, Cancan; Wang, Xiaoya; Gao, Long; Jing, Linguo; Zhou, Quan; Chang, Jiang

2018-06-01

The micro/nano hybrid structure is considered to be a biomaterial characteristic to stimulate osteogenesis by mimicking the three-dimensional structure of the bone matrix. However, the mechanism of the hybrid structure induced osteogenic differentiation of stem cells is still unknown. For elucidating the mechanisms, one of the challenge is to directly fabricate micro/nano hybrid structure on bioceramics because of its brittleness. In this study, hydroxyapatite (HA) bioceramics with the micro/nano hybrid structure were firstly fabricated via a hydrothermal treatment and template method, and the effect of the different surface structures on the expression of integrins, BMP2 signaling pathways and cell-cell communication was investigated. Interestingly, the results suggested that the osteogenic differentiation induced by micro/nano structures was modulated first through activating integrins and then further activating BMP2 signaling pathway and cell-cell communication, while activated BMP2 could in turn activate integrins and Cx43-related cell-cell communication. Furthermore, differences in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cell-cell communication were observed, in which nanorod and micropattern structures activated different integrin subunits, BMP downstream receptors and Cx43. This finding may explain the synergistic effect of the micro/nano hybrid structure on the activation of osteogenic differentiation of BMSCs. Based on our study, we concluded that the different activation mechanisms of micro- and nano-structures led to the synergistic stimulatory effect on integrin activation and osteogenesis, in which not only the direct contact of cells on micro/nano structure played an important role, but also other surface characteristics such as protein adsorption might contribute to the bioactive effect. The micro/nano hybrid structure has been found to have synergistic bioactivity on osteogenesis. However, it is still a challenge to fabricate the hybrid structure directly on the bioceramics, and the role of micro- and nano-structure, in particular the mechanism of the micro/nano-hybrid structure induced stem cell differentiation is still unknown. In this study, we firstly fabricated hydroxyapatite bioceramics with the micro/nano hybrid structure, and then investigated the effect of different surface structure on expression of integrins, BMP2 signaling pathways and cell-cell communication. Interestingly, we found that the osteogenic differentiation induced by structure was modulated first through activating integrins and then further activating BMP2 signaling pathway and cell-cell communication, and activated BMP2 could in turn activate some integrin subunits and Cx43-related cell-cell communication. Furthermore, differences in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cell-cell communication were observed, in which nanorod and micropattern structures activated different integrin subunits, BMP downstream receptors and Cx43. This finding may explain the synergistic effect of the micro/nano hybrid structure on the activation of osteogenic differentiation of BMSCs. Based on our study, we concluded that the different activation mechanisms of micro- and nano-structures led to the synergistic stimulatory effect on integrin activation and osteogenesis, in which not only the direct contact of cells on micro/nano structure played an important role, but also other surface characteristics such as protein adsorption might contribute to the bioactive effect. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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.

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

PubMed

Chigaev, Alexandre; Smagley, Yelena; Sklar, Larry A

2011-05-17

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

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 tumor development and progression through PI3K activation via the galectin-3- N -glycan complex. Mol Cancer Res; 16(6); 1024-34. ©2018 AACR . ©2018 American Association for Cancer Research.

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.

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.

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.

Novel Mechanistic Link between Focal Adhesion Remodeling and Glucose-stimulated Insulin Secretion*

PubMed Central

Rondas, Dieter; Tomas, Alejandra; Soto-Ribeiro, Martinho; Wehrle-Haller, Bernhard; Halban, Philippe A.

2012-01-01

Actin cytoskeleton remodeling is well known to be positively involved in glucose-stimulated pancreatic β cell insulin secretion. We have observed glucose-stimulated focal adhesion remodeling at the β cell surface and have shown this to be crucial for glucose-stimulated insulin secretion. However, the mechanistic link between such remodeling and the insulin secretory machinery remained unknown and was the major aim of this study. MIN6B1 cells, a previously validated model of primary β cell function, were used for all experiments. Total internal reflection fluorescence microscopy revealed the glucose-responsive co-localization of focal adhesion kinase (FAK) and paxillin with integrin β1 at the basal cell surface after short term stimulation. In addition, blockade of the interaction between β1 integrins and the extracellular matrix with an anti-β1 integrin antibody (Ha2/5) inhibited short term glucose-induced phosphorylation of FAK (Tyr-397), paxillin (Tyr-118), and ERK1/2 (Thr-202/Tyr-204). Pharmacological inhibition of FAK activity blocked glucose-induced actin cytoskeleton remodeling and glucose-induced disruption of the F-actin/SNAP-25 association at the plasma membrane as well as the distribution of insulin granules to regions in close proximity to the plasma membrane. Furthermore, FAK inhibition also completely blocked short term glucose-induced activation of the Akt/AS160 signaling pathway. In conclusion, these results indicate 1) that glucose-induced activation of FAK, paxillin, and ERK1/2 is mediated by β1 integrin intracellular signaling, 2) a mechanism whereby FAK mediates glucose-induced actin cytoskeleton remodeling, hence allowing docking and fusion of insulin granules to the plasma membrane, and 3) a possible functional role for the Akt/AS160 signaling pathway in the FAK-mediated regulation of glucose-stimulated insulin secretion. PMID:22139838

Gd-EDDA/HYNIC-RGD as an MR molecular probe imaging integrin alphanubeta3 receptor-expressed tumor-MR molecular imaging of angiogenesis.

PubMed

Huo, Tianlong; Du, Xiangke; Zhang, Sen; Liu, Xia; Li, Xubing

2010-02-01

The aim of this study is to develop a novel MR probe containing arginine-glycine-aspartic acid (RGD) motif for imaging integrin alphanubeta3 receptor-expressed tumor. Commercially available HYNIC-RGD conjugated with co-ligand EDDA was labeled with Gd(3+), and the mixture was isolated and purified by solid phase extract (SPE) to get the entire probe Gd-EDDA/HYNIC-RGD. Human hepatocellular carcinoma (HHCC) cell line BEL-7402 was cultured and the cells harvested and suspended in serum-free Dulbecco's modified Eagle medium (DMEM) were subcutaneously inoculated into athymic nude mice for tumor growth. In vitro cell binding assay to integrin alphanubeta3 receptor and cell viability experiments were conducted. The in vivo imaging of the three arms of xenografts were performed by MR scan with a dedicated animal coil at time points of 0, 30, 60, 90min and 24-h post-intravenous injection (p.i.). Three arms of nude mice then were sacrificed for histological examination to confirm the imaging results. Gd-EDDA/HYNIC-RGD was successfully isolated by SPE and validity was verified on signal enhancement through in vitro and in vivo experiments. The nude mice model bearing HHCC was well established. There was approx. 30% signal enhancement on T1WI FSE images at 90min post-intravenous injection of the Gd-EDDA/HYNIC-RGD compared with baseline, and the signal to time curve is straightforward over time in the span of 0-90min p.i., while the control arms do not show this tendency. Gd-EDDA/HYNIC-RGD has the potential to serve as an MR probe detecting integrin alphanubeta3 receptor-expressed tumor. Copyright (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Acquisition of epithelial-mesenchymal transition phenotype in the tamoxifen-resistant breast cancer cell: a new role for G protein-coupled estrogen receptor in mediating tamoxifen resistance through cancer-associated fibroblast-derived fibronectin and β1-integrin signaling pathway in tumor cells.

PubMed

Yuan, Jie; Liu, Manran; Yang, Li; Tu, Gang; Zhu, Qing; Chen, Maoshan; Cheng, Hong; Luo, Haojun; Fu, Weijie; Li, Zhenhua; Yang, Guanglun

2015-05-21

Acquired tamoxifen resistance remains the major obstacle to breast cancer endocrine therapy. β1-integrin was identified as one of the target genes of G protein-coupled estrogen receptor (GPER), a novel estrogen receptor recognized as an initiator of tamoxifen resistance. Here, we investigated the role of β1-integrin in GPER-mediated tamoxifen resistance in breast cancer. The expression of β1-integrin and biomarkers of epithelial-mesenchymal transition were evaluated immunohistochemically in 53 specimens of metastases and paired primary tumors. The function of β1-integrin was investigated in tamoxifen-resistant (MCF-7R) subclones, derived from parental MCF-7 cells, and MCF-7R β1-integrin-silenced subclones in MTT and Transwell assays. Involved signaling pathways were identified using specific inhibitors and Western blotting analysis. GPER, β1-integrin and mesenchymal biomarkers (vimentin and fibronectin) expression in metastases increased compared to the corresponding primary tumors; a close expression pattern of β1-integrin and GPER were in metastases. Increased β1-integrin expression was also confirmed in MCF-7R cells compared with MCF-7 cells. This upregulation of β1-integrin was induced by agonists of GPER and blocked by both antagonist and knockdown of it in MCF-7R cells. Moreover, the epidermal growth factor receptor/extracellular regulated protein kinase (EGFR/ERK) signaling pathway was involved in this transcriptional regulation since specific inhibitors of these kinases also reduced the GPER-induced upregulation of β1-integrin. Interestingly, silencing of β1-integrin partially rescued the sensitivity of MCF-7R cells to tamoxifen and the α5β1-integrin subunit is probably responsible for this phenomenon. Importantly, the cell migration and epithelial-mesenchymal transition induced by cancer-associated fibroblasts, or the product of cancer-associated fibroblasts, fibronectin, were reduced by knockdown of β1-integrin in MCF-7R cells. In addition, the downstream kinases of β1-integrin including focal adhesion kinase, Src and AKT were activated in MCF-7R cells and may be involved in the interaction between cancer cells and cancer-associated fibroblasts. GPER/EGFR/ERK signaling upregulates β1-integrin expression and activates downstream kinases, which contributes to cancer-associated fibroblast-induced cell migration and epithelial-mesenchymal transition, in MCF-7R cells. GPER probably contributes to tamoxifen resistance via interaction with the tumor microenvironment in a β1-integrin-dependent pattern. Thus, β1-integrin may be a potential target to improve anti-hormone therapy responses in breast cancer patients.

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

In vitro cell studies of technetium-99m labeled RGD-HYNIC peptide, a comparison of tricine and EDDA as co-ligands.

PubMed

Su, Zi-Fen; He, Jiang; Rusckowski, Mary; Hnatowich, Donald J

2003-02-01

The level of alpha(V)beta(3) integrins on endothelial cells is elevated in angiogenesis. The high binding specificity to alpha(V)beta(3) integrins of peptides containing Arg-Gly-Asp (RGD) residues suggests that the radiolabeled RGD peptides may be useful as tumor specific imaging agents. In this research, cyclised peptides containing Arg-Gly-Asp (RGD) and Arg-Gly-Glu (RGE, as control) residues were conjugated with HYNIC and labeled with (99m)Tc. The goal was to evaluate the influence of co-ligand, either tricine or ethylenediamine-N,N'-diacetic acid (EDDA) on protein and integrin binding and on cellular uptake in culture. The n-octanol/water partition coefficient, binding to bovine serum albumin (BSA) and human umbilical vein endothelial (HUVE) cells, and cell lysate distributions of the radiolabeled peptides were evaluated. The co-ligands had a significant effect on the labeling efficiency of the HYNIC conjugates and on certain properties of the (99m)Tc complexes. The labeling efficiency with tricine was 10 fold higher and BSA binding was over 8 fold greater compared to EDDA. Both RGD labels showed higher (6 to 28 fold) binding to HUVE cells than that of the RGE labels, indicating binding specificity. After cell-lysis, only a small percentage of the total RGD label that accumulated in the cells was found bound to cellular proteins (9% of RGD/tricine and 5% of RGD/EDDA), implying that over 90% of the radiolabeled peptides were internalized for both radiolabeled RGDs. The number of the RGD molecules bound to proteins was estimated to be approximately three per cell, suggesting that only a small number of alpha(V)beta(3) integrin proteins are expressed on the cells. Apart from the differences in radiolabeling, the only important effect of substituting EDDA for tricine as co-ligand on the HYNIC-peptides was the lower degree of serum protein binding. In spite of the lower serum protein binding potential, in vivo tumor accumulation of the RGD/EDDA may not be improved compared to RGD/tricine since quantitation of the cell binding results suggests that the number of alpha(V)beta(3) integrin proteins per cell might be limited.

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.

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

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.

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.

Caveolin 3-mediated integrin β1 signaling is required for the proliferation of folliculostellate cells in rat anterior pituitary gland under the influence of extracellular matrix.

PubMed

Horiguchi, Kotaro; Fujiwara, Ken; Ilmiawati, Cimi; Kikuchi, Motoshi; Tsukada, Takehiro; Kouki, Tom; Yashiro, Takashi

2011-07-01

Folliculostellate (FS) cells in the anterior pituitary gland are believed to have multifunctional properties. Using transgenic rats that express green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary gland (S100b-GFP rats), we recently revealed that FS cells in primary culture exhibited marked proliferation in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. In a process referred to as matricrine action, FS cells receive ECM as a signal through their receptors, which results in morphological and functional changes. In this study, we investigated matricrine signaling in FS cells and observed that the proliferation of FS cells is mediated by integrin β1, which is involved in various signaling pathways for cell migration and proliferation in response to ECM. Then, we analyzed downstream events of the integrin β1 signaling pathway in the proliferation of FS cells and identified caveolin 3 as a potential candidate molecule. Caveolin 3 is a membrane protein that binds cholesterol and a number of signaling molecules that interact with integrin β1. Using specific small interfering RNA of caveolin 3, the proliferation of FS cells was inhibited. Furthermore, caveolin 3 drove activation of the mitogen-activated protein kinase (MAPK) signaling cascades, which resulted in upregulation of cyclin D1 in FS cells. These findings suggest that matricrine signaling in the proliferation of FS cells was transduced by a caveolin 3-mediated integrin β1 signaling pathway and subsequent activation of the MAPK pathway. © 2011 Society for Endocrinology

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.

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.

Activation states of blood eosinophils in asthma

PubMed Central

Johansson, Mats W.

2014-01-01

Asthma is characterized by airway inflammation rich in eosinophils. Airway eosinophilia is associated with exacerbations and has been suggested to play a role in airway remodeling. Recruitment of eosinophils from the circulation requires that blood eosinophils become activated, leading to their arrest on the endothelium and extravasation. Circulating eosinophils can be envisioned as potentially being in different activation states, including non-activated, pre-activated or “primed”, or fully activated. In addition, the circulation can potentially be deficient of pre-activated or activated eosinophils, because such cells have marginated on activated endothelium or extravasated into the tissue. A number of eosinophil-surface proteins, including CD69, L-selectin, intercellular adhesion molecule-1 (ICAM-1, CD54), CD44, P-selectin glycoprotein ligand-1 (PSGL-1, CD162), cytokine receptors, Fc receptors, integrins including αM integrin (CD11b), and activated conformations of Fc receptors and integrins have been proposed to report cell activation. Variation in eosinophil activation states may be associated with asthma activity. Eosinophil-surface proteins proposed to be activation markers, with a particular focus on integrins, and evidence for associations between activation states of blood eosinophils and features of asthma are reviewed here. Partial activation of β1 and β2 integrins on blood eosinophils, reported by monoclonal antibodies (mAb) N29 and KIM-127, is associated with impaired pulmonary function and airway eosinophilia, respectively, in non-severe asthma. The association with lung function does not occur in severe asthma, presumably due to greater eosinophil extravasation, specifically of activated or pre-activated cells, in severe disease. PMID:24552191

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

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.

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.

SNARE-mediated trafficking of {alpha}{sub 5}{beta}{sub 1} integrin is required for spreading in CHO cells

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

Skalski, Michael; Coppolino, Marc G.

2005-10-07

In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cellmore » spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of {alpha}{sub 5}{beta}{sub 1} integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading.« less

Strategies that target leukocyte traffic in inflammatory bowel diseases: recent developments.

PubMed

Rivera-Nieves, Jesús

2015-11-01

We review the most recent developments regarding the targeting of molecules involved in the traffic of leukocytes for the treatment of inflammatory bowel diseases (IBD). We discuss the most important findings of one published phase II trial that targeted the β7 integrin (etrolizumab), two phase II trials that targeted the α4β7 integrin ligand: mucosal addressin cell adhesion molecule 1 (MAdCAM-1, PF-00547659), a phase II trial targeting the chemokine IP-10 (CXCL10) in Crohn's, and a phase II trial that targeted the sphingosine-1-phosphate receptor-1: ozanimod in patients with ulcerative colitis. Targeting molecules involved in leukocyte traffic has recently become an effective and well tolerated strategy for the treatment of IBD. Novel approaches now not only target the integrins on the lymphocyte surface, but also its endothelial ligand: MAdCAM-1. As with vedolizumab, antibodies against MAdCAM-1 appear most effective in ulcerative colitis rather than in Crohn's. Targeting chemokines or their receptors does not appear to have the same efficacy as those that target the most stable integrin: immunoglobulin superfamily interactions between the lymphocyte and endothelium. Preliminary results also suggest that the sphingosine-1-phosphate pathway might also be targeted therapeutically in IBD, no longer with parenterally administered antibodies but with orally administered small molecules.

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

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.

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.

CD98 at the crossroads of adaptive immunity and cancer

PubMed Central

Cantor, Joseph M.; Ginsberg, Mark H.

2012-01-01

Adaptive immunity, a vertebrate specialization, adds memory and exquisite specificity to the basic innate immune responses present in invertebrates while conserving metabolic resources. In adaptive immunity, antigenic challenge requires extremely rapid proliferation of rare antigen-specific lymphocytes to produce large, clonally expanded effector populations that neutralize pathogens. Rapid proliferation and resulting clonal expansion are dependent on CD98, a protein whose well-conserved orthologs appear restricted to vertebrates. Thus, CD98 supports lymphocyte clonal expansion to enable protective adaptive immunity, an advantage that could account for the presence of CD98 in vertebrates. CD98 supports lymphocyte clonal expansion by amplifying integrin signals that enable proliferation and prevent apoptosis. These integrin-dependent signals can also provoke cancer development and invasion, anchorage-independence and the rapid proliferation of tumor cells. CD98 is highly expressed in many cancers and contributes to formation of tumors in experimental models. Strikingly, vertebrates, which possess highly conserved CD98 proteins, CD98-binding integrins and adaptive immunity, also display propensity towards invasive and metastatic tumors. In this Commentary, we review the roles of CD98 in lymphocyte biology and cancer. We suggest that the CD98 amplification of integrin signaling in adaptive immunity provides survival benefits to vertebrates, which, in turn, bear the price of increased susceptibility to cancer. PMID:22499670

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

PubMed

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

2017-06-01

Cholangiocarcinoma (CCA) is a slow-growing but highly metastatic cancer. Its metastatic potential largely explains its high mortality rate. A recognized risk factor for CCA development is infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis. We previously reported that the excretory-secretory products (ESPs) of C. sinensis promoted the three-dimensional aggregation and invasion of CCA cells. In the present study, a quantitative real-time PCR array of extracellular matrix (ECM) and adhesion molecules was used to examine the regulatory mechanism of ESP-mediated CCA cell migration and invasion. In particular, the expression levels of integrin α isoforms and β4 were upregulated in response to ESPs. Increased expression of integrin β4 was probably correlated with activation of focal adhesion kinase (FAK) and the steroid receptor coactivator (Src) family kinase and the subsequent activation of two downstream focal adhesion molecules, paxillin and vinculin. Moreover, inhibition of FAK/Src activation reduced paxillin and vinculin phosphorylation and attenuated ESP-induced CCA cell migration and invasion. These findings suggest that the integrin β4-FAK/Src signaling axis may play a crucial role in clonorchiasis-associated CCA metastasis during tumor progression. Copyright © 2017 Elsevier B.V. All rights reserved.

NRP1 knockdown promotes the migration and invasion of human neuroblastoma-derived SK‑N‑AS cells via the activation of β1 integrin expression.

PubMed

Ishizuka, Yoshiaki; Koshinaga, Tsugumichi; Hirano, Takayuki; Nagasaki-Maeoka, Eri; Watanabe, Yosuke; Hoshi, Reina; Yoshizawa, Shinsuke; Sugito, Kiminobu; Kawashima, Hiroyuki; Uekusa, Shota; Fukuda, Noboru; Soma, Masayoshi; Fujiwara, Kyoko

2018-07-01

Neuropilin 1 (NRP1) is a transmembrane glycoprotein, which regulates many aspects of cellular function by functioning as co-receptor of various ligands. Recent studies have suggested that NRP1 promotes tumorigenesis, not only by activating the growth of tumor vessels, but also by activating the growth or migration of tumor cells themselves. The present study was performed to elucidate the roles of NRP1 in the development and/or progression of neuroblastoma (NB). In contrast to previous observations in various types of cancer, the analysis of public datasets indicated that lower levels of NRP1 expression were significantly associated with a shorter survival period of patients with NB. Consistent with this finding, wound-healing assay and Matrigel invasion assay revealed that NB cells in which NRP1 was knocked down exhibited increased migratory and invasive abilities. Further analyses indicated that β1 integrin expression was markedly increased in NB cells in which NRP1 was knocked down, and NB cells in which β1 integrin was knocked down exhibited decreased migratory and invasive abilities. The results presented herein indicate that NRP1 exerts tumor suppressive effects in NB, at least in part by regulating the expression of β1 integrin.

PI3Kδ promotes CD4(+) T-cell interactions with antigen-presenting cells by increasing LFA-1 binding to ICAM-1.

PubMed

Garçon, Fabien; Okkenhaug, Klaus

2016-05-01

Activation of T lymphocytes by peptide/major histocompatibility complex on antigen-presenting cells (APCs) involves dynamic contacts between the two cells, during which T cells undergo marked morphological changes. These interactions are facilitated by integrins. Activation of the T cells increases the binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) expressed by T cells to intercellular adhesion molecule (ICAM)-1 and ICAM-2 expressed by APCs. The signalling pathways that control integrin affinities are incompletely defined. The phosphoinositide 3-kinases (PI3Ks) generate second-messenger signalling molecules that control cell growth, proliferation, differentiation and trafficking. Here we show that in T cells, PI3Kδ attenuates the activation of Rac1, but sustains the activation of Rap1. Consequently, PI3Kδ increases LFA-1-dependent adhesion to form stable conjugates with APCs. Increased Rap1 activity and LFA-1 adhesion were only in part mediated by the downstream kinase Akt, suggesting the involvement of additional phosphatidylinositol(3,4,5)P3-binding proteins. These results establish a link between PI3K activity, cytoskeletal changes and integrin binding and help explain the impaired T-cell-dependent immune responses in PI3Kδ-deficient mice.

Granulocyte-macrophage and macrophage colony-stimulating factors differentially regulate alpha v integrin expression on cultured human macrophages.

PubMed

De Nichilo, M O; Burns, G F

1993-03-15

The colony-stimulating factors (CSFs) greatly influence mature macrophage function in vitro: macrophage (M)-CSF induces maturation of monocytes and enhances differentiated cell function; granulocyte-macrophage (GM)-CSF stimulates a variety of antimicrobial functions. In vivo M-CSF is thought to promote differentiation, and GM-CSF is thought to potentiate the inflammatory response. One mechanism by which these differential effects may be achieved is through the receptor-mediated interaction of macrophages with their extracellular matrix. Here we show that M-CSF induces specifically the expression of the alpha v beta 5 integrin receptor, whereas GM-CSF rapidly induces mRNA and surface expression of the alpha v beta 3 integrin. The M-CSF-treated cells acquire a flattened epitheloid phenotype, and on vitronectin the alpha v beta 5 is located in adhesion plaques. These cells do not bind collagen or laminin. In contrast, cells treated with GM-CSF adopt an elongated phenotype on a number of substrates, including collagen and laminin, and express alpha v beta 3 at the leading edge of cells on vitronectin. These results suggest that a primary means by which the CSFs exert their individual effects on mature cells may be through regulating integrin expression.

NAD+ Biosynthesis Ameliorates a Zebrafish Model of Muscular Dystrophy

PubMed Central

Goody, Michelle F.; Kelly, Meghan W.; Reynolds, Christine J.; Khalil, Andre; Crawford, Bryan D.; Henry, Clarissa A.

2012-01-01

Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex– or integrin alpha7–deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction with integrin alpha6 to reduce muscle degeneration. Taken together, these results define a novel cell adhesion pathway that may have future therapeutic relevance for a broad spectrum of muscular dystrophies. PMID:23109907

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 antiangiogenic therapy.

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

Recombinant Nonstructural 3 Protein, rNS3, of Hepatitis C Virus Along With Recombinant GP96 Induce IL-12, TNFα and α5integrin Expression in Antigen Presenting Cells

PubMed Central

Hajizadeh, Mohammad Reza; Mokarram, Pooneh; Kamali sarvestani, Eskandar; Bolhassani, Azam; Mostafavi Pour, Zohreh

2013-01-01

Background Hepatitis C virus (HCV) infection is the main cause of chronic liver disease and to date there has been no vaccine development to prevent this infection. Among non-structural HCV proteins, NS3 protein is an excellent goal for a therapeutic vaccine, due to its large size and less variation in conserved regions. The immunogenic properties of heat shock proteins (HSPs) for instance GP96 have prompted investigations into their function as strong adjuvant to improve innate and adaptive immunity. Objectives The aim of this study was to examine additive effects of recombinant GP96 (rGP96) fragments accompanied by rNS3 on expression levels of α5integrin and pro-inflammatory cytokines, IL-12 and TNFα, in Antigen Presenting Cells (APCs). Materials and Methods Recombinant viral proteins (rNS3 and rRGD-NS3), N-terminal and C-terminal fragments of GP96 were produced and purified from E. coli in order to treat the cells; mouse spleen Dendritic Cells (DCs) and THP-1 macrophages. Results Our results showed that rNT-GP96 alone significantly increases the expression level of IL-12, TNFα and α5integrin in THP-1 macrophages and DCs, while IL-12 and TNFα expression levels were unaffected by either rNS3 or rRGD-NS3. Interestingly, the co-addition of these recombinant proteins with rNT-GP96 increased IL-12, TNFα and α5integrin expression. Pearson Correlation showed a direct association between α5integrin with IL-12 and TNF-α expression. Conclusions we have highlighted the role of rNS3 plus rNT-GP96 mediated by α5integrin in producing IL-12 and TNFα. It can be suggested that rNT-GP96 could enhance immunity characteristic of rNS3 protein via production of pro-inflammatory cytokines. PMID:24032046

Expression of mucosal addressin cell adhesion molecule 1 on vessel endothelium of gastric mucosa in patients with nodular gastritis

PubMed Central

Ohara, Hiroshi; Isomoto, Hajime; Wen, Chun-Yang; Ejima, Chieko; Murata, Masahiro; Miyazaki, Masanobu; Takeshima, Fuminao; Mizuta, Yohei; Murata, Ikuo; Koji, Takehiko; Nagura, Hiroshi; Kohno, Shigeru

2003-01-01

AIM: The interaction of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) with integrin α4β7 mediates lymphocyte recruitment into mucosa-associated lymphoid tissue (MALT). Nodular gastritis is characterized by a unique military pattern on endoscopy representing increased numbers of lymphoid follicles with germinal center, strongly associated with H pylori infection. The purpose of this study was to address the implication of the MAdCAM-1/integrin β7 pathway in NG. METHODS: We studied 17 patients with NG and H pylori infection and 19 H pylori-positive and 14 H pylori-negative controls. A biopsy sample was taken from the antrum and snap-frozen for immunohistochemical analysis of MAdCAM-1 and integrin β7. In simultaneous viewing of serial sections, the percentage of MAdCAM-1-positive to von Willebrand factor-positive vessels was calculated. We also performed immunostaining with anti-CD20, CD4, CD8 and CD68 antibodies to determine the lymphocyte subsets co-expressing integrin β7. RESULTS: Vascular endothelial MAdCAM-1 expression was more enhanced in gastric mucosa with than without H pylori infection. Of note, the percentages of MAdCAM-1-positive vessels were significantly higher in the lamina propria of NG patients than in H pylori-positive controls. Strong expression of MAdCAM-1 was identified adjacent to lymphoid follicles and dense lymphoid aggregates. Integrin β7-expressing mononuclear cells, mainly composed of CD20 and CD4 lymphocytes, were associated with vessels lined with MAdCAM-1-expressing endothelium. CONCLUSION: Our results suggest that the MAdCAM-1/ integrin α4β7 homing system may participate in gastric inflammation in response to H pylori-infection and contributes to MALT formation, typically leading to the development of NG. PMID:14669317

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

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

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.

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

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.

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

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.

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

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

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

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.

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

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

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

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

NASA Technical Reports Server (NTRS)

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

2003-01-01

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

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.

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.

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

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

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

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

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

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

Choi, Yoon Pyo; Kim, Baek Gil; Department of Pathology, Yonsei University College of Medicine, Seoul

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

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

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.

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.

Leukocyte Anti-Trafficking Strategies: Current Status and Future Directions.

PubMed

Sands, Bruce E

2017-01-01

In inflammatory bowel diseases (IBD), a pivotal step in the initiation and perpetuation of mucosal inflammation entails the recruitment of inflammatory leukocytes to the gut. Understanding the carefully coordinated series of molecular events that culminate in the recruitment of leukocytes to the gut has resulted in novel interventions with new capabilities in treating both Crohn's disease and ulcerative colitis. Key Messages: Natalizumab, an anti-α4 integrin antibody, was the first agent to demonstrate the efficacy of this approach for the induction and maintenance of response and remission in Crohn's disease. Widespread adoption was mitigated by the previously unknown risk of progressive multifocal leukoencephalopathy (PML) with this approach. Current approaches employ a more selective inhibition of adhesion molecules targeting the gut to avoid broad suppression of surveillance for JC virus, the causal pathogen of PML. Subsequently, vedolizumab, a humanized anti-α4β7 integrin antibody, has demonstrated efficacy in patients with IBD and has an excellent safety profile. To date, there have been no cases of PML in patients treated with vedolizumab, suggesting that this more selective agent does not have the same risk for PML as natalizumab. Other agents target β7 integrin (etrolizumab) and mucosal addressin cellular adhesion molecule-1, the endothelial ligand of α4β7 integrin. Efforts to inhibit the chemokine receptor CCR9 using the agent CCX282-B in Crohn's disease were not successful. An orally administered anti-α4 integrin compound showed some promise in a phase 2 trial but raises concern for PML. Finally, the S1P1 receptor agonist ozanimod showed promise in early trials in ulcerative colitis. In summary, anti-trafficking agents have the potential to provide safe and effective therapy for IBD, and are a burgeoning class of novel agents. © 2017 S. Karger AG, Basel.

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.

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.

Monocyte-endothelial adhesion in chronic rheumatoid arthritis. In situ detection of selectin and integrin-dependent interactions.

PubMed Central

Grober, J S; Bowen, B L; Ebling, H; Athey, B; Thompson, C B; Fox, D A; Stoolman, L M

1993-01-01

Blood monocytes are the principal reservoir for tissue macrophages in rheumatoid synovitis. Receptor-mediated adhesive interactions between circulating cells and the synovial venules initiate recruitment. These interactions have been studied primarily in cultured endothelial cells. Thus the functional activities of specific adhesion receptors, such as the endothelial selectins and the leukocytic integrins, have not been evaluated directly in diseased tissues. We therefore examined monocyte-microvascular interactions in rheumatoid synovitis by modifying the Stamper-Woodruff frozen section binding assay initially developed to study lymphocyte homing. Specific binding of monocytes to venules lined by low or high endothelium occurred at concentrations as low as 5 x 10(5) cells/ml. mAbs specific for P-selectin (CD62, GMP-140/PADGEM) blocked adhesion by > 90% in all synovitis specimens examined. In contrast, P-selectin-mediated adhesion to the microvasculature was either lower or absent in frozen sections of normal foreskin and placenta. mAbs specific for E-selectin (ELAM-1) blocked 20-50% of monocyte attachment in several RA synovial specimens but had no effect in others. mAbs specific for LFA-1, Mo1/Mac 1, the integrin beta 2-chain, and L-selectin individually inhibited 30-40% of adhesion. An mAb specific for the integrin beta 1-chain inhibited the attachment of elutriated monocytes up to 20%. We conclude that P-selectin associated with the synovial microvasculature initiates shear-resistant adhesion of monocytes in the Stamper-Woodruff assay and stabilizes bonds formed by other selectins and the integrins. Thus the frozen section binding assay permits direct evaluation of leukocyte-microvascular adhesive interactions in inflamed tissues and suggests a prominent role for P-selectin in monocyte recruitment in vivo. Images PMID:7685772

Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein.

PubMed

Kumar, Devender; Ristow, Laura C; Shi, Meiqing; Mukherjee, Priyanka; Caine, Jennifer A; Lee, Woo-Yong; Kubes, Paul; Coburn, Jenifer; Chaconas, George

2015-12-01

Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration.

Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein

PubMed Central

Kumar, Devender; Ristow, Laura C.; Shi, Meiqing; Mukherjee, Priyanka; Caine, Jennifer A.; Lee, Woo-Yong; Kubes, Paul; Coburn, Jenifer; Chaconas, George

2015-01-01

Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d -/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration. PMID:26684456

Modulation of focal adhesion constituents and their down-stream events by EGF: On the cross-talk of integrins and growth factor receptors.

PubMed

Eberwein, Philipp; Laird, Dougal; Schulz, Simon; Reinhard, Thomas; Steinberg, Thorsten; Tomakidi, Pascal

2015-10-01

Within the concept of integrin growth factor receptor (GFR) cross-talk, little is known about the effects of GFRs on focal adhesions (FAs). Therefore, we tested the hypothesis whether EGF can modulate constituents of FAs and subsequent down-stream events. To this end, EGF-treated keratinocytes were subjected to combined fluorescence imaging and western blotting, to quantify expression and/or activation of molecules, involved in integrin GFR cross-talk, and receptor proximal and distal signaling events. Generally, EGF response revealed an amplified redistribution or activation of molecules under study, which will be explained in detail from the plasma membrane to the cell interior. In addition to significant activation of EGF receptor (EGFR) at tyrosine Tyr845, a remarkable redistribution was detectable for the focal adhesion constituents, integrin ß1 and ß3, and zyxin. Increased activation also applied to focal adhesion kinase (FAK) by phosphorylation at Tyr397, Tyr576, and Src at Tyr418, while total FAK remained unchanged. Risen activity was seen as well for the analyzed distal down-stream events, p190RhoGAP and MAP kinases p42/44. Intriguingly, Src-specific inhibitor Herbimycin A abrogated the entire EGF response except FAK Tyr397 phosphorylation, independent of EGF presence. Mechanistically, our results show that EGF modulates adhesion in a dual fashion, by firstly redistributing focal adhesion constituents to adhesion sites, but also by amplifying levels of activated RhoA antagonist p190RhoGAP, important for cell motility. Further, the findings suggest that the observed EGF response underlies an EGFR integrin cross-talk under recruitment of receptor proximal FAK and Src, and MAP kinase and p190RhoGAP as receptor distal events. Copyright © 2015 Elsevier B.V. All rights reserved.

ADAP interactions with talin and kindlin promote platelet integrin αIIbβ3 activation and stable fibrinogen binding

PubMed Central

Kang, Jian; Kahner, Bryan; Ye, Feng; Ginsberg, Mark H.; Shattil, Sanford J.

2014-01-01

ADAP is a hematopoietic-restricted adapter protein that promotes integrin activation and is a carrier for other adapter proteins, Src kinase–associated phosphoprotein 1 (SKAP1) and SKAP2. In T lymphocytes, SKAP1 is the ADAP-associated molecule that activates integrins through direct linkages with Rap1 effectors (regulator of cell adhesion and polarization enriched in lymphoid tissues; Rap1-interacting adapter molecule). ADAP also promotes integrin αIIbβ3 activation in platelets, which lack SKAP1, suggesting an ADAP integrin–regulatory pathway different from those in lymphocytes. Here we characterized a novel association between ADAP and 2 essential integrin-β cytoplasmic tail-binding proteins involved in αIIbβ3 activation, talin and kindlin-3. Glutathione S-transferase pull-downs identified distinct regions in ADAP necessary for association with kindlin or talin. ADAP was physically proximal to talin and kindlin-3 in human platelets, as assessed biochemically, and by immunofluorescence microscopy and proximity ligation. Relative to wild-type mouse platelets, ADAP-deficient platelets exhibited reduced co-localization of talin with αIIbβ3, and reduced irreversible fibrinogen binding in response to a protease activated receptor 4 (PAR4) thrombin receptor agonist. When ADAP was heterologously expressed in Chinese hamster ovary cells co-expressing αIIbβ3, talin, PAR1, and kindlin-3, it associated with an αIIbβ3/talin complex and enabled kindlin-3 to promote agonist-dependent ligand binding to αIIbβ3. Thus, ADAP uniquely promotes activation of and irreversible fibrinogen binding to platelet αIIbβ3 through interactions with talin and kindlin-3. PMID:24523237

Osteoblast-secreted WISP-1 promotes adherence of prostate cancer cells to bone via the VCAM-1/integrin α4β1 system.

PubMed

Chang, An-Chen; Chen, Po-Chun; Lin, Yu-Feng; Su, Chen-Ming; Liu, Ju-Fang; Lin, Tien-Huang; Chuang, Show-Mei; Tang, Chih-Hsin

2018-07-10

Bone metastasis is a frequent occurrence in prostate cancer (PCa) that is associated with severe complications such as fracture, bone pain and hypercalcemia. The cross-talk between metastatic cancer cells and bone is critical to the development and progression of bone metastases. In our previous data, we have described how the involvement of the Wnt-induced secreted protein-1/vascular cell adhesion molecule-1 (WISP-1/VCAM-1) system in this tumor-bone interaction contributes to human PCa cell motility. In this study, we found that WISP-1 regulates bone mineralization by inducing bone morphogenetic protein-2 (BMP2), BMP4 and osteopontin (OPN) expression in osteoblasts. We also found that WISP-1 inhibited RANKL-dependent osteoclastogenesis. Moreover, osteoblast-derived WISP-1 enhanced VCAM-1 expression in PCa cells and subsequently promoted the adherence of cancer cells to osteoblasts. Furthermore, endothelin-1 (ET-1) expression in PCa cells was regulated by osteoblast-derived WISP-1, which promoted integrin α4β1 expression in osteoblasts via the MAPK pathway. Pretreatment of PCa cells with VCAM-1 antibody or osteoblasts with integrin α4β1 antibody attenuated the adherence of PCa cells to osteoblasts, suggesting that integrin α4β1 serves as a ligand that captures VCAM-1 + metastatic tumor cells adhering to osteoblasts. Our findings reveal that osteoblast-derived WISP-1 plays a key role in regulating the adhesion of PCa cells to osteoblasts via the VCAM-1/integrin α4β1 system. Osteoblast-derived WISP-1 is a promising target for the prevention and inhibition of PCa-bone interaction. Copyright © 2018 Elsevier B.V. All rights reserved.

Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A

PubMed Central

Austin, Pamela; Heller, Markus; Williams, David E.; McIntosh, Lawrence P.; Vogl, A. Wayne; Foster, Leonard J.; Andersen, Raymond J.; Roberge, Michel; Roskelley, Calvin D.

2010-01-01

Background Neopetrosiamide A (NeoA) is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown. Methodology/Principal Findings We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of β1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: β1 integrin and numerous α integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD) proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that β1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane. Conclusions/Significance NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface. PMID:20520768

Identification of Human Cathelicidin Peptide LL-37 as a Ligand for Macrophage Integrin αMβ2 (Mac-1, CD11b/CD18) that Promotes Phagocytosis by Opsonizing Bacteria

PubMed Central

Lishko, Valeryi K.; Moreno, Benjamin; Podolnikova, Nataly P.; Ugarova, Tatiana P.

2016-01-01

LL-37, a cationic antimicrobial peptide, has numerous immune-modulating effects. However, the identity of a receptor(s) mediating the responses in immune cells remains uncertain. We have recently demonstrated that LL-37 interacts with the αMI-domain of integrin αMβ2 (Mac-1), a major receptor on the surface of myeloid cells, and induces a migratory response in Mac-1-expressing monocyte/macrophages as well as activation of Mac-1 on neutrophils. Here, we show that LL-37 and its C-terminal derivative supported strong adhesion of various Mac-1-expressing cells, including HEK293 cells stably transfected with Mac-1, human U937 monocytic cells and murine IC-21 macrophages. The cell adhesion to LL-37 was partially inhibited by specific Mac-1 antagonists, including mAb against the αM integrin subunit and neutrophil inhibitory factor, and completely blocked when anti-Mac-1 antibodies were combined with heparin, suggesting that cell surface heparan sulfate proteoglycans act cooperatively with integrin Mac-1. Coating both Gram-negative and Gram-positive bacteria with LL-37 significantly potentiated their phagocytosis by macrophages, and this process was blocked by a combination of anti-Mac-1 mAb and heparin. Furthermore, phagocytosis by wild-type murine peritoneal macrophages of LL-37-coated latex beads, a model of foreign surfaces, was several fold higher than that of untreated beads. By contrast, LL-37 failed to augment phagocytosis of beads by Mac-1-deficient macrophages. These results identify LL-37 as a novel ligand for integrin Mac-1 and demonstrate that the interaction between Mac-1 on macrophages and bacteria-bound LL-37 promotes phagocytosis. PMID:27990411

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 demonstrated differences in osteoblast rather than osteoclast parameters. Together, these results suggest that integrin signals generated during growth enhance the acquisition of a skeletal mass, structure, and strength to withstand the mechanical loads generated by weight-bearing.

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

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.

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.

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.

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.

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

Cell motion predicts human epidermal stemness

PubMed Central

Toki, Fujio; Tate, Sota; Imai, Matome; Matsushita, Natsuki; Shiraishi, Ken; Sayama, Koji; Toki, Hiroshi; Higashiyama, Shigeki

2015-01-01

Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that α6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation. PMID:25897083

Loss of SLP-76 expression within myeloid cells confers resistance to neutrophil-mediated tissue damage while maintaining effective bacterial killing.

PubMed

Clemens, Regina A; Lenox, Laurie E; Kambayashi, Taku; Bezman, Natalie; Maltzman, Jonathan S; Nichols, Kim E; Koretzky, Gary A

2007-04-01

The Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is an adaptor molecule critical for immunoreceptor and integrin signaling in multiple hemopoietic lineages. We showed previously that SLP-76 is required for neutrophil function in vitro, including integrin-induced adhesion and production of reactive oxygen intermediates, and to a lesser extent, FcgammaR-induced calcium flux and reactive oxygen intermediate production. It has been difficult to determine whether SLP-76 regulates neutrophil responses in vivo, because Slp-76(-/-) mice exhibit marked defects in thymocyte and vascular development, as well as platelet and mast cell function. To circumvent these issues, we generated mice with targeted loss of SLP-76 expression within myeloid cells. Neutrophils obtained from these animals failed to respond to integrin activation in vitro, similar to Slp-76(-/-) cells. Despite these abnormalities, SLP-76-deficient neutrophils migrated normally in vivo in response to Staphylococcus aureus infection and efficiently cleared micro-organisms. Interestingly, SLP-76-deficient neutrophils did not induce a robust inflammatory response in the localized Shwartzman reaction. Collectively, these data suggest that disruption of integrin signaling via loss of SLP-76 expression differentially impairs neutrophil functions in vivo, with preservation of migration and killing of S. aureus but reduction in LPS-induced tissue damage and vascular injury.

Strategies that Target Leukocyte Traffic in IBD: Recent Developments

PubMed Central

Rivera-Nieves, Jesús

2015-01-01

Purpose of review We review the most recent developments regarding the targeting of molecules involved in the traffic of leukocytes for the treatment of IBD. Recent Findings We discuss the most important findings of one published phase II trial that targeted the β7 integrin (Etrolizumab), two phase II trials that targeted the α4β7 integrin ligand: Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1, PF-00547659), a phase II targeting the chemokine IP-10 (CXCL10) in Crohn’s and a phase II trial that targeted the sphingosine-1-phosphate receptor-1 (S1P1): ozanimod in patients with ulcerative colitis (UC). Summary Targeting molecules involved in leukocyte traffic has recently become an effective and safe strategy for the treatment of IBD. Novel approaches now not only target the integrins on the lymphocyte surface, but also its endothelial ligand: MAdCAM-1. As with vedolizumab, antibodies against MAdCAM-1 appear most effective in ulcerative colitis rather than in Crohn’s. Targeting chemokines or their receptors does not appear to have the same efficacy as those that target the most stable integrin:immunoglobulin superfamily interactions between the lymphocyte and endothelium. Preliminary results also suggest that the sphingosine-1-phosphate pathway might also be targeted therapeutically in IBD, no longer with parenterally administered antibodies but with orally administered small molecules. PMID:26398681

Mouse ES cells have a potential to differentiate into odontoblast-like cells using hanging drop method.

PubMed

Kawai, R; Ozeki, N; Yamaguchi, H; Tanaka, T; Nakata, K; Mogi, M; Nakamura, H

2014-05-01

We examined whether mouse embryonic stem (ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVβ3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The angiogenic factor CCN1 promotes adhesion and migration of circulating CD34+ progenitor cells: potential role in angiogenesis and endothelial regeneration.

PubMed

Grote, Karsten; Salguero, Gustavo; Ballmaier, Matthias; Dangers, Marc; Drexler, Helmut; Schieffer, Bernhard

2007-08-01

Tissue regeneration involves the formation of new blood vessels regulated by angiogenic factors. We reported recently that the expression of the angiogenic factor CCN1 is up-regulated under various pathophysiologic conditions within the cardiovascular system. Because CD34+ progenitor cells participate in cardiovascular tissue regeneration, we investigated whether CCN1-detected for the first time in human plasma-promotes the recruitment of CD34+ progenitor cells to endothelial cells, thereby enhancing endothelial proliferation and neovascularization. In this study, we demonstrated that CCN1 and supernatants from CCN1-stimulated human CD34+ progenitor cells promoted proliferation of endothelial cells and angiogenesis in vitro and in vivo. In addition, CCN1 induced migration and transendothelial migration of CD34+ cells and the release of multiple growth factors, chemokines, and matrix metalloproteinase-9 (MMP-9) from these cells. Moreover, the CCN1-specific integrins alpha(M)beta(2) and alpha(V)beta(3) are expressed on CD34+ cells and CCN1 stimulated integrin-dependent signaling. Furthermore, integrin antagonists (RGD-peptides) suppressed both binding of CCN1 to CD34+ cells and CCN1-induced adhesion of CD34+ cells to endothelial cells. These data suggest that CCN1 promotes integrin-dependent recruitment of CD34+ progenitor cells to endothelial cells, which may contribute to paracrine effects on angiogenesis and tissue regeneration.

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

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

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

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.

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

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.

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

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

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

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.

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.

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

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.

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

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

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

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.

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

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

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

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

Arf6 controls platelet spreading and clot retraction via integrin αIIbβ3 trafficking

PubMed Central

Huang, Yunjie; Joshi, Smita; Xiang, Binggang; Kanaho, Yasunori; Li, Zhenyu; Bouchard, Beth A.; Moncman, Carole L.

2016-01-01

Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate–ribosylation factor 6 (Arf6) is a small guanosine triphosphate–binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbβ3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)–labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbβ3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbβ3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function. PMID:26738539

Arf6 controls platelet spreading and clot retraction via integrin αIIbβ3 trafficking.

PubMed

Huang, Yunjie; Joshi, Smita; Xiang, Binggang; Kanaho, Yasunori; Li, Zhenyu; Bouchard, Beth A; Moncman, Carole L; Whiteheart, Sidney W

2016-03-17

Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate-ribosylation factor 6 (Arf6) is a small guanosine triphosphate-binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbβ3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)-labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbβ3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbβ3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function. © 2016 by The American Society of Hematology.

Surface potential-governed cellular osteogenic differentiation on ferroelectric polyvinylidene fluoride trifluoroethylene films.

PubMed

Tang, Bolin; Zhang, Bo; Zhuang, Junjun; Wang, Qi; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

2018-07-01

Surface potential of biomaterials can dramatically influence cellular osteogenic differentiation. In this work, a wide range of surface potential on ferroelectric polyvinylidene fluoride trifluoroethylene (P(VDF-TrFE)) films was designed to get insight into the interfacial interaction of cell-charged surface. The P(VDF-TrFE) films poled by contact electric poling at various electric fields obtained well stabilized surface potential, with wide range from -3 to 915 mV. The osteogenic differentiation level of cells cultured on the films was strongly dependent on surface potential and reached the optimum at 391 mV in this system. Binding specificity assay indicated that surface potential could effectively govern the binding state of the adsorbed fibronectin (FN) with integrin. Molecular dynamic (MD) simulation further revealed that surface potential brought a significant difference in the relative distance between RGD and synergy PHSRN sites of adsorbed FN, resulting in a distinct integrin-FN binding state. These results suggest that the full binding of integrin α5β1 with both RGD and PHSRN sites of FN possesses a strong ability to activate osteogenic signaling pathway. This work sheds light on the underlying mechanism of osteogenic differentiation behavior on charged material surfaces, and also provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. The ferroelectric P(VDF-TrFE) films with steady and a wide range of surface potential were designed to understand underlying mechanism of cell-charged surface interaction. The results showed that the charged surface well favored upregulation of osteogenic differentiation of MC3T3-E1 cells, and more importantly, a highest level occurred on the film with a moderate surface potential. Experiments and molecular dynamics simulation demonstrated that the surface potential could govern fibronectin conformation and then the integrin-fibronectin binding. We propose that a full binding state of integrin α5β1 with fibronectin induces effective activation of integrin-mediated FAK/ERK signaling pathway to upregulate cellular osteogenic differentiation. This work provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

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.

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.

A novel device to stretch multiple tissue samples with variable patterns: application for mRNA regulation in tissue-engineered constructs.

PubMed

Imsirovic, Jasmin; Derricks, Kelsey; Buczek-Thomas, Jo Ann; Rich, Celeste B; Nugent, Matthew A; Suki, Béla

2013-01-01

A broad range of cells are subjected to irregular time varying mechanical stimuli within the body, particularly in the respiratory and circulatory systems. Mechanical stretch is an important factor in determining cell function; however, the effects of variable stretch remain unexplored. In order to investigate the effects of variable stretch, we designed, built and tested a uniaxial stretching device that can stretch three-dimensional tissue constructs while varying the strain amplitude from cycle to cycle. The device is the first to apply variable stretching signals to cells in tissues or three dimensional tissue constructs. Following device validation, we applied 20% uniaxial strain to Gelfoam samples seeded with neonatal rat lung fibroblasts with different levels of variability (0%, 25%, 50% and 75%). RT-PCR was then performed to measure the effects of variable stretch on key molecules involved in cell-matrix interactions including: collagen 1α, lysyl oxidase, α-actin, β1 integrin, β3 integrin, syndecan-4, and vascular endothelial growth factor-A. Adding variability to the stretching signal upregulated, downregulated or had no effect on mRNA production depending on the molecule and the amount of variability. In particular, syndecan-4 showed a statistically significant peak at 25% variability, suggesting that an optimal variability of strain may exist for production of this molecule. We conclude that cycle-by-cycle variability in strain influences the expression of molecules related to cell-matrix interactions and hence may be used to selectively tune the composition of tissue constructs.

Differential expression of extracellular matrix constituents and cell adhesion molecules between malignant pleural mesothelioma and mesothelial hyperplasia.

PubMed

Alì, Greta; Borrelli, Nicla; Riccardo, Giannini; Proietti, Agnese; Pelliccioni, Serena; Niccoli, Cristina; Boldrini, Laura; Lucchi, Marco; Mussi, Alfredo; Fontanini, Gabriella

2013-11-01

Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm associated with asbestos exposure. Currently, the molecular mechanisms that induce MPM development are still unknown. The purpose of this study was to identify new molecular biomarkers for mesothelial carcinogenesis. We analyzed a panel of 84 genes involved in extracellular matrix remodeling and cell adhesion by polymerase chain reaction (PCR) array in 15 samples of epithelioid mesothelioma and 10 samples of reactive mesothelial hyperplasia (MH; 3 of 25 samples were inadequate for mRNA analysis). To validate the differentially expressed genes identified by PCR array, we analyzed 27 more samples by immunohistochemistry, in addition to the 25 samples already studied. Twenty-five genes were differentially expressed in MPM and MH by PCR array. Of these we studied matrix metalloproteinase 7 (MMP7), MMP14, CD44, and integrin, alpha3 expression by immunohistochemistry in 26 epithelioid MPM and 26 MH samples from the entire series of 52 cases. We observed higher MMP14 and integrin, alpha3 expression in MPM samples compared with MH samples (p = 0.000002 and p = 0.000002, respectively). Conversely, CD44 expression was low in most (57.7%) mesothelioma samples but only in 11.5% of the MH samples (p = 0.0013). As regards MMP7, we did not observe differential expression between MH and MPM samples. We have extensively studied genes involved in cell adhesion and extracellular matrix remodeling in MPM and MH samples, gaining new insight into the pathophysiology of mesothelioma. Moreover, our data suggest that these factors could be potential biomarkers for MPM.

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

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

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

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

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.

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.

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

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

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.

Identification and characterization of integrin-binding sialoprotein (IBSP) genes in reptile and amphibian.

PubMed

Shintani, Seikou; Kamakura, Naofumi; Kobata, Mitsuhiko; Toyosawa, Satoru; Onishi, Tomoyuki; Sato, Akie; Kawasaki, Kazuhiko; Weiss, Kenneth M; Ooshima, Takashi

2008-11-15

Integrin-binding sialoprotein (IBSP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family; and the whole SIBLING family is further included in a larger secretory calcium-binding phosphoprotein (SCPP) family. SIBLING proteins are known to construct a part of the non-collagenous extracellular matrices of calcified tissues, and considered to have arisen by duplication and subsequent divergent evolution of a single ancient gene. To understand the alterations of SIBLING molecules associated with the evolution of calcified tissues in vertebrates, we initiated a search for lower vertebrate orthologs of SIBLING genes. In the present study, an IBSP ortholog from a reptile (caiman) and two distinct orthologs from an amphibian (African clawed toad) were identified and characterized. As expected, the toad IBSP genes were transcribed only in calcified tissue (jaw and tibia), as also seen in mammals. The caiman, toad, avian, and mammalian IBSPs share several unique features specific for IBSP and apparently have similar properties. Furthermore, analysis of the sequences suggested that the IBSP molecule might have gradually intensified its functions related to calcification during its evolutionary process through tetrapods.

Separation of integrin-dependent adhesion from morphological changes based on differential PLC specificities.

PubMed

Wooten, D K; Teague, T K; McIntyre, B W

1999-01-01

In normal lymphocytes an inside-out signal up-regulating integrin adhesion is followed by a ligand-mediated outside-in cell spreading signal. Protein kinase C (PKC) inhibition blocks lymphocyte adherence to and spreading on fibronectin. In contrast, putative PLC inhibitors yield distinct differences with respect to adhesion and morphology. The phosphatidylinositol-specific phospholipase C (PLC) inhibitor neomycin blocked spreading of CD3/CD28-activated T cells on fibronectin by disrupting adhesion. Furthermore, when an additional inside-out signal for fibronectin adhesion is unnecessary such as with HPB-ALL T leukemic or phorbol-myristate-acetate-treated normal T cells, neomycin treatment does not alter adhesion or morphology. However, the phosphatidylcholine-specific PLC inhibitor D609 abrogates cell spreading without affecting adhesion to fibronectin in these cells as well as the CD3/CD28-activated T cells. These results strongly suggest that inside-out signaling for the integrin alpha4beta1 in lymphocytes proceeds through phosphatidylinositol-specific PLC and PKC, whereas the outside-in signal utilizes phosphatidylcholine-specific PLC and PKC.

Mechanism of mast cell adhesion to human tenocytes in vitro.

PubMed

Behzad, Hayedeh; Tsai, Shu-Huei; Nassab, Paulina; Mousavizadeh, Rouhollah; McCormack, Robert G; Scott, Alex

2015-01-01

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell-fibroblast co-culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5β1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte-conditioned media; the tenocyte-derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5β1 integrin and tenocyte fibronectin. Additionally, tenocyte-derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5β1 integrin mediates binding of mast cells to human tenocyte and to tenocyte-derived ECM proteins, in particular fibronectin. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Is there a role for vedolizumab in the treatment of ulcerative colitis and Crohn’s disease?

PubMed Central

Gilroy, Leah; Allen, Patrick B

2014-01-01

Inflammatory bowel disease (IBD) is an important cause of morbidity and mortality for millions of patients worldwide. Current treatment options include corticosteroids, 5-aminosalicylates, immunosuppressants, and TNFα antagonists. However, these are frequently ineffective in achieving sustained response and remission over time. At present, gastroenterologists lack safe and effective treatments if patients fail anti-TNF therapy. Vedolizumab is a promising new agent for IBD patients refractory to anti-TNF therapy. Vedolizumab is an integrin antagonist which is thought to act by reducing inflammation by selectively inhibiting leukocyte migration in the gut. Emerging evidence from clinical trials suggests a potential role for vedolizumab in both ulcerative colitis (UC) and Crohn’s disease (CD), particularly in patients who have previously failed biological therapy. The safety profile of vedolizumab appears reasonable, possibly because it has a “gut-selective” mode of action, with no reported cases of progressive multifocal leukoencephalopathy, a condition which has been linked to another integrin antagonist, natalizumab. This review discusses the available evidence for integrin antagonists and their potential role in the management of IBD. PMID:24899819

Hyperpolarisation of cultured human chondrocytes following cyclical pressure-induced strain: evidence of a role for alpha 5 beta 1 integrin as a chondrocyte mechanoreceptor.

PubMed

Wright, M O; Nishida, K; Bavington, C; Godolphin, J L; Dunne, E; Walmsley, S; Jobanputra, P; Nuki, G; Salter, D M

1997-09-01

Mechanical stimuli influence chondrocyte metabolism, inducing changes in intracellular cyclic adenosine monophosphate and proteoglycan production. We have previously demonstrated that primary monolayer cultures of human chondrocytes have an electrophysiological response after intermittent pressure-induced strain characterised by a membrane hyperpolarisation of approximately 40%. The mechanisms responsible for these changes are not fully understood but potentially involve signalling molecules such as integrins that link extracellular matrix with cytoplasmic components. The results reported in this paper demonstrate that the transduction pathways involved in the hyperpolarisation response of human articular chondrocytes in vitro after cyclical pressure-induced strain involve alpha 5 beta 1 integrin. We have demonstrated, using pharmacological inhibitors of a variety of intracellular signalling pathways, that the actin cytoskeleton, the phospholipase C calmodulin pathway, and both tyrosine protein kinase and protein kinase C activities are important in the transduction of the electrophysiological response. These results suggest that alpha 5 beta 1 is an important chondrocyte mechanoreceptor and a potential regulator of chondrocyte function.

Structural basis of the interaction between integrin α6β4 and plectin at the hemidesmosomes

PubMed Central

de Pereda, José M; Lillo, M Pilar; Sonnenberg, Arnoud

2009-01-01

The interaction between the integrin α6β4 and plectin is essential for the assembly and stability of hemidesmosomes, which are junctional adhesion complexes that anchor epithelial cells to the basement membrane. We describe the crystal structure at 2.75 Å resolution of the primary α6β4–plectin complex, formed by the first pair of fibronectin type III domains and the N-terminal region of the connecting segment of β4 and the actin-binding domain of plectin. Two missense mutations in β4 (R1225H and R1281W) linked to nonlethal forms of epidermolysis bullosa prevent essential intermolecular contacts. We also present two structures at 1.75 and 2.05 Å resolution of the β4 moiety in the absence of plectin, which reveal a major rearrangement of the connecting segment of β4 on binding to plectin. This conformational switch is correlated with the way α6β4 promotes stable adhesion or cell migration and suggests an allosteric control of the integrin. PMID:19242489

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 in young boys (age <2 years). The expression of the beta1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of beta1D integrin was significantly reduced in the alpha7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of alpha7B in laminin alpha2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in alpha7 deficient mice, implies that the alpha7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

An assay for evoked locomotor behavior in Drosophila reveals a role for integrins in ethanol sensitivity and rapid ethanol tolerance.

PubMed

Bhandari, Poonam; Kendler, Kenneth S; Bettinger, Jill C; Davies, Andrew G; Grotewiel, Mike

2009-10-01

Ethanol induces similar behavioral responses in mammals and the fruit fly, Drosophila melanogaster. By coupling assays for ethanol-related behavior to the genetic tools available in flies, a number of genes have been identified that influence physiological responses to ethanol. To enhance the utility of the Drosophila model for investigating genes involved in ethanol-related behavior, we explored the value of an assay that measures the sedative effects of ethanol on negative geotaxis, an evoked locomotor response. We established eRING (ethanol Rapid Iterative Negative Geotaxis) as an assay for quantitating the sedative effects of ethanol on negative geotaxis (i.e., startle-induced climbing). We validated the assay by assessing acute sensitivity to ethanol and rapid ethanol tolerance in several different control strains and in flies with mutations known to disrupt these behaviors. We also used eRING in a candidate screen to identify mutants with altered ethanol-related behaviors. Negative geotaxis measured in eRING assays was dose-dependently impaired by ethanol exposure. Flies developed tolerance to the intoxicating effects of ethanol when tested during a second exposure. Ethanol sensitivity and rapid ethanol tolerance varied across 4 control strains, but internal ethanol concentrations were indistinguishable in the 4 strains during a first and second challenge with ethanol. Ethanol sensitivity and rapid ethanol tolerance, respectively, were altered in flies with mutations in amnesiac and hangover, genes known to influence these traits. Additionally, mutations in the beta integrin gene myospheroid and the alpha integrin gene scab increased the initial sensitivity to ethanol and enhanced the development of rapid ethanol tolerance without altering internal ethanol concentrations. The eRING assay is suitable for investigating genetic mechanisms that influence ethanol sensitivity and rapid ethanol tolerance. Ethanol sensitivity and rapid ethanol tolerance depend on the function of alpha and beta integrins in flies.

[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.

Evaluation of 99mTc-3PRGD2 integrin receptor imaging in hepatocellular carcinoma tumour-bearing mice: comparison with 18F-FDG metabolic imaging.

PubMed

Zheng, Jieling; Miao, Weibing; Huang, Chao; Lin, Haoxue

2017-07-01

Our study was designed to explore the utility of 99m Tc-HYNIC-PEG 4 -E[PEG 4 -c(RGDfK)] 2 ( 99m Tc-3PRGD 2 ) for the detection of hepatocellular carcinoma (HCC) and specifically to compare the diagnostic performance of 99m Tc-3PRGD 2 integrin receptor imaging and 2-18-fluoro-2-deoxy-D-glucose ( 18 F-FDG) metabolic imaging in a nude mouse model. 99m Tc-3PRGD 2 was synthesized using a HYNIC-3PRGD 2 lyophilized kit with 99m TcO 4 labelling. The nude mouse animal model was established by subcutaneously injecting 5 × 10 7 /ml HepG2 cells into the shoulder flank of each mouse. Biodistribution studies were performed at 0.5, 1, 2 and 4 h after intravenous administration of 0.37 MBq of 99m Tc-3PRGD 2 . Immunohistochemistry was performed to evaluate the expression level of integrin αvβ3 in the HCC tissues. Dynamic imaging was performed using list-mode after the administration of 55.5 MBq of 99m Tc-3PRGD 2 , to reconstruct the multiphase images and acquire the best initial scan time. At 8, 12, 16, 20 and 24 days after inoculation with HepG2 cells, 55.5 MBq of 99m Tc-3PRGD 2 and 37 MBq of 18 F-FDG were injected successively into the nude mouse model, subsequently, simultaneous SPECT/PET imaging was performed to calculate the tumour volume and tumour uptake of 99m Tc-3PRGD 2 and 18 F-FDG. The biodistribution study first validated that the tumour uptake of 99m Tc-3PRGD 2 at the different time points was higher than that of all the other organs tested in the experiment, except for the kidney. Integrin αvβ3 expressed highly in early stage HCC and declined for further necrosis of the tumour tissue. Subcutaneous tumours were visualized clearly with excellent contrast under 99m Tc-3PRGD 2 SPECT/CT imaging, and the multiphase imaging comparison showed the tumours were prominent at 0.5 h, suggesting that the best initial scan time is 0.5 h post-injection. The comparison of the imaging results of the two methods showed that 99m Tc-3PRGD 2 integrin receptor imaging was more sensitive than 18 F-FDG metabolic imaging for the detection of early stage HCC, meanwhile the tumour uptake of 99m Tc-3PRGD 2 was consistently higher than that of 18 F-FDG. However, as tumour necrosis further increased in HCC tissues, the uptake of 18 F-FDG was higher than that of 99m Tc-3PRGD 2 . Our study demonstrated that 99m Tc-3PRGD 2 is a valuable tumour molecular probe for the detection of early stage HCC compared with 18 F-FDG, meriting further investigation of 99m Tc-3PRGD 2 as a novel SPECT tracer for tumour imaging.

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

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.

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.

Na+/H+ exchange activity during phagocytosis in human neutrophils: role of Fcgamma receptors and tyrosine kinases

PubMed Central

1996-01-01

In neutrophils, binding and phagocytosis facilitate subsequent intracellular killing of microorganisms. Activity of Na+/H+ exchangers (NHEs) participates in these events, especially in regulation of intracellular pH (pHi) by compensating for the H+ load generated by the respiratory burst. Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutrophils. The purpose of this study was to identify which NHE(s) are expressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis. Exposure of cells to the phagocytic stimulus opsonized zymosan (OpZ) induced a transient cytosolic acidification followed by a prolonged alkalinization. The latter was inhibited in Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H+ exchange. Reverse transcriptase PCR and cDNA sequencing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed. Immunoblotting of purified plasma membranes with isoform- specific antibodies confirmed the presence of NHE-1 protein in neutrophils. Since phagocytosis involves Fcgamma (FcgammaR) and complement receptors such as CR3 (a beta2 integrin) which are linked to pathways involving alterations in intracellular [Ca2+]i and tyrosine phosphorylation, we studied these pathways in relation to activation of NHE-1. Cross-linking of surface bound antibodies (mAb) directed against FcgammaRs (FcgammaRII > FcgammaRIII) but not beta2 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-beta2 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE- 1 activation involved cooperation between integrins and FcgammaRs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcgammaR cross-linking suggesting that tyrosine phosphorylation was involved in NHE-I activation. An increase in [Ca2+]i was not required for NHE-1 activation because neither removal of extracellular Ca2+ nor buffering of changes in [Ca2+]i inhibited alkalinization after OpZ or Fc-gammaR cross-linking. In summary, Fc-gammaRs and beta2 integrins cooperate in activation of NHE- 1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation. PMID:8601583

A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

PubMed

Hocking, D C; Smith, R K; McKeown-Longo, P J

1996-04-01

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

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.

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.

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.

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

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

PubMed Central

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

2013-01-01

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

Crystal structure of isoflurane bound to integrin LFA-1 supports a unified mechanism of volatile anesthetic action in the immune and central nervous systems

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

Zhang, Hongmin; Astrof, Nathan S.; Liu, Jin-Huan

2009-09-15

Volatile anesthetics (VAs), such as isoflurane, induce a general anesthetic state by binding to specific targets (i.e., ion channels) in the central nervous system (CNS). Simultaneously, VAs modulate immune functions, possibly via direct interaction with alternative targets on leukocytes. One such target, the integrin lymphocyte function-associated antigen-1 (LFA-1), has been shown previously to be inhibited by isoflurane. A better understanding of the mechanism by which isoflurane alters protein function requires the detailed information about the drug-protein interaction at an atomic level. Here, we describe the crystal structure of the LFA-1 ligand-binding domain (I domain) in complex with isoflurane at 1.6more » {angstrom}. We discovered that isoflurane binds to an allosteric cavity previously implicated as critical for the transition of LFA-1 from the low- to the high-affinity state. The isoflurane binding site in the I domain involves an array of amphiphilic interactions, thereby resembling a 'common anesthetic binding motif' previously predicted for authentic VA binding sites. These results suggest that the allosteric modulation of protein function by isoflurane, as demonstrated for the integrin LFA-1, might represent a unified mechanism shared by the interactions of volatile anesthetics with targets in the CNS. Crystal structure of isoflurane bound to integrin LFA-1 supports a unified mechanism of volatile anesthetic action in the immune and central nervous systems.« less

Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition.

PubMed

Civra, Andrea; Giuffrida, Maria Gabriella; Donalisio, Manuela; Napolitano, Lorenzo; Takada, Yoshikazu; Coulson, Barbara S; Conti, Amedeo; Lembo, David

2015-05-08

Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine, and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2β1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2β1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a minilibrary of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20-amino acid peptide containing both DGE and RGD motifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2β1 by means of the DGE motif and inhibits rotavirus attachment to the cell surface. These findings suggest the potential anti-rotavirus activity of equine lactadherin and support the feasibility of developing an anti-rotavirus peptide that acts by hindering virus-receptor binding. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Detection of ultrastructural changes in genetically altered and exercised skeletal muscle using PS-OCT

NASA Astrophysics Data System (ADS)

Pasquesi, James J.; Schlachter, Simon C.; Boppart, Marni D.; Chaney, Eric; Kaufman, Stephen J.; Boppart, Stephen A.

2006-02-01

Birefringence of skeletal muscle has been associated with the ultrastructure of individual sarcomeres, specifically the arrangement of A-bands corresponding to the thick myosin filaments. Murine skeletal muscle (gastrocnemius) was imaged with a fiber-based PS-OCT imaging system to determine the level of birefringence present in the tissue under various conditions. In addition to muscle controls from wild-type mice, muscle from abnormal mice included: genetically-modified (mdx) mice which model human muscular dystrophy, transgenic mice exhibiting an overexpression of integrin (α7β1), and transgenic integrin (α7β1)knockout mice. Comparisons were also made between rested and exercised muscles to determine the effects of exercise on muscle birefringence for each of these normal and abnormal conditions. The PS-OCT images revealed that the presence of birefringence was similar in the rested muscle with dystrophy-like features (i.e., lacking the structural protein dystrophin - mdx) and in the integrin (α7β1)knockout muscle when compared to the normal (wild-type) control. However, exercising these abnormal muscle tissues drastically reduced the presence of birefringence detected by the PS-OCT system. The muscle exhibiting an overexpression of integrin (α7β1) remained heavily birefringent before and after exercise, similar to the normal (wild-type) muscle. These results suggest that there is a distinct relationship between the degree of birefringence detected using PS-OCT and the sarcomeric ultrastructure present within skeletal muscle.

Fibronectin on extracellular vesicles from microvascular endothelial cells is involved in the vesicle uptake into oligodendrocyte precursor cells

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

Osawa, Sho; Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511; Kurachi, Masashi

We previously reported transplantation of brain microvascular endothelial cells (MVECs) into cerebral white matter infarction model improved the animal's behavioral outcome by increasing the number of oligodendrocyte precursor cells (OPCs). We also revealed extracellular vesicles (EVs) derived from MVECs promoted survival and proliferation of OPCs in vitro. In this study, we investigated the mechanism how EVs derived from MVECs contribute to OPC survival and proliferation. Protein mass spectrometry and enzyme-linked immunosorbent assay revealed fibronectin was abundant on the surface of EVs from MVECs. As fibronectin has been reported to promote OPC survival and proliferation via integrin signaling pathway, we blocked themore » binding between fibronectin and integrins using RGD sequence mimics. Blocking the binding, however, did not attenuate the survival and proliferation promoting effect of EVs on OPCs. Flow cytometric and imaging analyses revealed fibronectin on EVs mediates their internalization into OPCs by its binding to heparan sulfate proteoglycan on OPCs. OPC survival and proliferation promoted by EVs were attenuated by blocking the internalization of EVs into OPCs. These lines of evidence suggest that fibronectin on EVs mediates their internalization into OPCs, and the cargo of EVs promotes survival and proliferation of OPCs, independent of integrin signaling pathway. - Highlights: • Fibronectin exists on the surface of extracellular vesicles from endothelial cells. • Integrin signaling is not involved in effects of extracellular vesicles on OPCs. • Fibronectin on the surface of extracellular vesicles mediates their uptake into OPCs.« less

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.

Mechanisms of action of nonpeptide hormones on resveratrol-induced antiproliferation of cancer cells.

PubMed

Lin, Hung-Yun; Hsieh, Meng-Ti; Cheng, Guei-Yun; Lai, Hsuan-Yu; Chin, Yu-Tang; Shih, Ya-Jung; Nana, André Wendindondé; Lin, Shin-Ying; Yang, Yu-Chen S H; Tang, Heng-Yuan; Chiang, I-Jen; Wang, Kuan

2017-09-01

Nonpeptide hormones, such as thyroid hormone, dihydrotestosterone, and estrogen, have been shown to stimulate cancer proliferation via different mechanisms. Aside from their cytosolic or membrane-bound receptors, there are receptors on integrin α v β 3 for nonpeptide hormones. Interaction between hormones and integrin α v β 3 can induce signal transduction and eventually stimulate cancer cell proliferation. Resveratrol induces inducible COX-2-dependent antiproliferation via integrin α v β 3 . Resveratrol and hormone-induced signals are both transduced by activated extracellular-regulated kinases 1 and 2 (ERK1/2); however, hormones promote cell proliferation, while resveratrol induces antiproliferation in cancer cells. Hormones inhibit resveratrol-stimulated phosphorylation of p53 on Ser15, resveratrol-induced nuclear COX-2 accumulation, and formation of p53-COX-2 nuclear complexes. Subsequently, hormones impair resveratrol-induced COX-2-/p53-dependent gene expression. The inhibitory effects of hormones on resveratrol action can be blocked by different antagonists of specific nonpeptide hormone receptors but not integrin α v β 3 blockers. Results suggest that nonpeptide hormones inhibit resveratrol-induced antiproliferation in cancer cells downstream of the interaction between ligand and receptor and ERK1/2 activation to interfere with nuclear COX-2 accumulation. Thus, the surface receptor sites for resveratrol and nonpeptide hormones are distinct and can induce discrete ERK1/2-dependent downstream antiproliferation biological activities. It also indicates the complex pathways by which antiproliferation is induced by resveratrol in various physiological hormonal environments. . © 2017 New York Academy of Sciences.

Reduced immunohistochemical expression of adhesion molecules in vitiligo skin biopsies.

PubMed

Reichert Faria, Adriane; Jung, Juliana Elizabeth; Silva de Castro, Caio César; de Noronha, Lucia

2017-03-01

Because defects in adhesion impairment seem to be involved in the etiopathogenesis of vitiligo, this study aimed to compare the immunohistochemical expression of several adhesion molecules in the epidermis of vitiligo and non lesional vitiligo skin. Sixty-six specimens of lesional and non lesional skin from 33 volunteers with vitiligo were evaluated by immunohistochemistry using anti-beta-catenin, anti-E-cadherin, anti-laminin, anti-beta1 integrin, anti-collagen IV, anti-ICAM-1 and anti-VCAM-1 antibodies. Biopsies of vitiligo skin demonstrated a significant reduction in the expression of laminin and integrin. The average value of the immunohistochemically positive reaction area of the vitiligo specimens was 3053.2μm 2 , compared with the observed value of 3431.8μm 2 in non vitiligo skin (p=0.003) for laminin. The immuno-positive area was 7174.6μm 2 (vitiligo) and 8966.7μm 2 (non lesional skin) for integrin (p=0.042). A reduction in ICAM-1 and VCAM-1 expression in the basal layer of the epidermis in vitiligo samples was also observed (p=0.001 and p<0.001, respectively). However, no significant differences were observed with respect to the expression of beta-catenin, E-cadherin, and collagen IV between vitiligo and non lesional skin. Our results suggest that an impairment in adhesion exists in vitiligo skin, which is supported by the diminished immunohistochemical expression of laminin, beta1 integrin, ICAM-1 and VCAM-1. Copyright © 2017 Elsevier GmbH. All rights reserved.

Luteogenic Hormones Act through a Vascular Endothelial Growth Factor-Dependent Mechanism to Up-Regulate α5β1 and αvβ3 Integrins, Promoting the Migration and Survival of Human Luteinized Granulosa Cells

PubMed Central

Rolaki, Alexandra; Coukos, George; Loutradis, Dimitris; DeLisser, Horace M.; Coutifaris, Christos; Makrigiannakis, Antonis

2007-01-01

The formation of the corpus luteum (CL) is critical for the establishment of a successful pregnancy. After ovulation, the CL develops from the remnants of the ovulated ovarian follicle. This process, which involves varying cell-matrix interactions, is poorly characterized. To understand the role and potential regulation of cell-matrix interactions in the formation of the CL, we investigated the expression and activity of the matrix protein fibronectin (FN) and several of its integrin receptors on luteinized granulosa cells (GCs). In situ, FN and several FN-binding integrins were detected around luteinizing GCs during the early luteal phase, although expression declined in the late luteal phase. In vitro, GCs released FN, and stimulation of these cells with human chorionic gonadotropin increased the surface expression of FN, α5β1, and αvβ3. Up-regulation of these proteins on GCs was reproduced by stimulation with vascular endothelial growth factor (VEGF) and was inhibited by anti-VEGF antibody. Lastly, expression of α5β1 and αvβ3 mediated adhesion to FN, facilitated migration, and prevented apoptosis. These data suggest that in vivo luteogenic hormones, in part through a VEGF-dependent mechanism, stimulate selected integrin-matrix adhesive interactions that promote the motility and survival of GCs and thus contribute to the formation and preservation of the CL. PMID:17456762

Polymerized laminin-332 matrix supports rapid and tight adhesion of keratinocytes, suppressing cell migration.

PubMed

Kariya, Yoshinobu; Sato, Hiroki; Katou, Naoko; Kariya, Yukiko; Miyazaki, Kaoru

2012-01-01

Laminin-332 (α3ß3γ2) (Lm332) supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or γ1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the α3 and α6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK) almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin α3ß1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin α6ß4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin α6ß4 and α3ß1, whereas unassembled soluble Lm332 supports cell migration.

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

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

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

Cocrystal structure of the ICAP1 PTB domain in complex with a KRIT1 peptide

PubMed Central

Liu, Weizhi; Boggon, Titus J.

2013-01-01

Integrin cytoplasmic domain-associated protein-1 (ICAP1) is a suppressor of integrin activation and directly binds to the cytoplasmic tail of β1 integrins; its binding suppresses integrin activation by competition with talin. Krev/Rap1 interaction trapped-1 (KRIT1) releases ICAP1 suppression of integrin activation by sequestering ICAP1 away from integrin cytoplasmic tails. Here, the cocrystal structure of the PTB domain of ICAP1 in complex with a 29-­amino-acid fragment (residues 170–198) of KRIT1 is presented to 1.7 Å resolution [the resolution at which 〈I/σ(I)〉 = 2.9 was 1.83 Å]. In previous studies, the structure of ICAP1 with integrin β1 was determined to 3.0 Å resolution and that of ICAP1 with the N-terminal portion of KRIT1 (residues 1–­198) was determined to 2.54 Å resolution; therefore, this study provides the highest resolution structure yet of ICAP1 and allows further detailed analysis of the interaction of ICAP1 with its minimal binding region in KRIT1. PMID:23695561

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.

Age Increases Monocyte Adhesion on Collagen

NASA Astrophysics Data System (ADS)

Khalaji, Samira; Zondler, Lisa; Kleinjan, Fenneke; Nolte, Ulla; Mulaw, Medhanie A.; Danzer, Karin M.; Weishaupt, Jochen H.; Gottschalk, Kay-E.

2017-05-01

Adhesion of monocytes to micro-injuries on arterial walls is an important early step in the occurrence and development of degenerative atherosclerotic lesions. At these injuries, collagen is exposed to the blood stream. We are interested whether age influences monocyte adhesion to collagen under flow, and hence influences the susceptibility to arteriosclerotic lesions. Therefore, we studied adhesion and rolling of human peripheral blood monocytes from old and young individuals on collagen type I coated surface under shear flow. We find that firm adhesion of monocytes to collagen type I is elevated in old individuals. Pre-stimulation by lipopolysaccharide increases the firm adhesion of monocytes homogeneously in older individuals, but heterogeneously in young individuals. Blocking integrin αx showed that adhesion of monocytes to collagen type I is specific to the main collagen binding integrin αxβ2. Surprisingly, we find no significant age-dependent difference in gene expression of integrin αx or integrin β2. However, if all integrins are activated from the outside, no differences exist between the age groups. Altered integrin activation therefore causes the increased adhesion. Our results show that the basal increase in integrin activation in monocytes from old individuals increases monocyte adhesion to collagen and therefore the risk for arteriosclerotic plaques.

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.

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

Structure-Guided Design of a High-Affinity Platelet Integrin αIIbβ3 Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS | Center for Cancer Research

Cancer.gov

A Better Fit. An improved anticoagulant drug called RUC-2 (ball and stick structure) fits snugly into its binding pocket on integrin (blue), a protein found on the surface of platelets. RUC-2 binds both subunits of integrin, inhibiting the excessive blood coagulation that can lead to strokes and heart attacks. Unlike similar drugs that alter integrin's structure when they bind

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

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.

Regulation of COX-2–mediated signaling by α3 type IV noncollagenous domain in tumor angiogenesis

PubMed Central

Boosani, Chandra Shekhar; Mannam, Arjuna P.; Cosgrove, Dominic; Silva, Rita; Hodivala-Dilke, Kairbaan M.; Keshamouni, Venkateshwar G.

2007-01-01

Human α3 chain, a noncollagenous domain of type IV collagen [α3(IV)NC1], inhibits angiogenesis and tumor growth. These biologic functions are partly attributed to the binding of α3(IV)NC1 to αVβ3 and α3β1 integrins. α3(IV)NC1 binds αVβ3 integrin, leading to translation inhibition by inhibiting focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathways. In the present study, we evaluated the role of α3β1 and αVβ3 integrins in tube formation and regulation of cyclooxygenase-2 (COX-2) on α3(IV)NC1 stimulation. We found that although both integrins were required for the inhibition of tube formation by α3(IV)NC1 in endothelial cells, only α3β1 integrin was sufficient to regulate COX-2 in hypoxic endothelial cells. We show that binding of α3(IV)NC1 to α3β1 integrin leads to inhibition of COX-2–mediated pro-angiogenic factors, vascular endothelial growth factor, and basic fibroblast growth factor by regulating IκBα/NFκB axis, and is independent of αVβ3 integrin. Furthermore, β3 integrin–null endothelial cells, when treated with α3(IV)NC1, inhibited hypoxia-mediated COX-2 expression, whereas COX-2 inhibition was not observed in α3 integrin–null endothelial cells, indicating that regulation of COX-2 by α3(IV)NC1 is mediated by integrin α3β1. Our in vitro and in vivo findings demonstrate that α3β1 integrin is critical for α3(IV)NC1-mediated inhibition of COX-2–dependent angiogenic signaling and inhibition of tumor progression. PMID:17426256

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

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.

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

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.

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

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.

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

PubMed

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

2016-07-01

The definitive role of ganglioside GM2 in mediating tumor-induced growth and progression is still unknown. Here we report a novel role of ganglioside GM2 in mediating tumor cell migration and uncovered its mechanism. Data shows differential expression levels of GM2-synthase as well as GM2 in different human cancer cells. siRNA mediated knockdown of GM2-synthase in CCF52, A549 and SK-RC-26B cells resulted in significant inhibition of tumor cell migration as well as invasion in vitro without affecting cellular proliferation. Over-expression of GM2-synthase in low-GM2 expressing SK-RC-45 cells resulted in a consequent increase in migration thus confirming the potential role GM2 and its downstream partners play in tumor cell migration and motility. Further, treatment of SK-RC-45 cells with exogenous GM2 resulted in a dramatic increase in migratory and invasive capacity with no change in proliferative capacity, thereby confirming the role of GM2 in tumorigenesis specifically by mediating tumor migration and invasion. Gene expression profiling of GM2-synthase silenced cells revealed altered expression of several genes involved in cell migration primarily those controlling the integrin mediated signaling. GM2-synthase knockdown resulted in decreased phosphorylation of FAK, Src as well as Erk, while over-expression and/or exogenous GM2 treatment caused increased FAK and Erk phosphorylation respectively. Again, GM2 mediated invasion and Erk phosphorylation is blocked in integrin knockdown SK-RC-45 cells, thus confirming that GM2 mediated migration and phosphorylation of Erk is integrin dependent. Finally, confocal microscopy suggested co-localization while co-immunoprecipitation and surface plasmon resonance (SPR) confirmed direct interaction of membrane bound ganglioside, GM2 with the integrin receptor. Copyright © 2016 Elsevier B.V. All rights reserved.

MR angiogenesis imaging with Robo4- vs. αVβ3-targeted nanoparticles in a B16/F10 mouse melanoma model

PubMed Central

Boles, Kent S.; Schmieder, Anne H.; Koch, Alexander W.; Carano, Richard A. D.; Wu, Yan; Caruthers, Shelton D.; Tong, Raymond K.; Stawicki, Scott; Hu, Grace; Scott, Michael J.; Zhang, Huiying; Reynolds, Benton A.; Wickline, Samuel A.; Lanza, Gregory M.

2010-01-01

The primary objective of this study was to utilize MR molecular imaging to compare the 3-dimensional spatial distribution of Robo4 and αVβ3-integrin as biosignatures of angiogenesis, in a rapidly growing, syngeneic tumor. B16-F10 melanoma-bearing mice were imaged with magnetic resonance (MR; 3.0 T) 11 d postimplantation before and after intravenous administration of either Robo4- or αVβ3-targeted paramagnetic nanoparticles. The percentage of MR signal-enhanced voxels throughout the tumor volume was low and increased in animals receiving αVβ3- and Robo4-targeted nanoparticles. Neovascular signal enhancement was predominantly associated with the tumor periphery (i.e., outer 50% of volume). Microscopic examination of tumors coexposed to the Robo4- and αVβ3-targeted nanoparticles corroborated the MR angiogenesis mapping results and further revealed that Robo4 expression generally colocalized with αVβ3-integrin. Robo4- and αVβ3-targeted nanoparticles were compared to irrelevant or nontargeted control groups in all modalities. These results suggest that αVβ3-integrin and Robo4 are useful biomarkers for noninvasive MR molecular imaging in syngeneic mouse tumors, but αVβ3-integrin expression was more detectable by MR at 3.0 T than Robo4. Noninvasive, neovascular assessments of the MR signal of Robo4, particularly combined with αVβ3-integrin expression, may help define tumor character prior to and following cancer therapy.—Boles, K. S., Schmieder, A. H., Koch, A. W., Carano, R. A. D., Wu, Y., Caruthers, S. D., Tong, R. K., Stawicki, S., Hu, G., Scott, M. J., Zhang, H., Reynolds, B. A., Wickline, S. A., and Lanza, G. M. MR angiogenesis imaging with Robo4- vs. αVβ3-targeted nanoparticles in a B16/F10 mouse melanoma model. PMID:20585027

Coexpression of α6β4 Integrin and Guanine Nucleotide Exchange Factor Net1 Identifies Node-Positive Breast Cancer Patients at High Risk for Distant Metastasis

PubMed Central

Gilcrease, Michael Z.; Kilpatrick, Shannan K.; Woodward, Wendy A.; Zhou, Xiao; Nicolas, Marlo M.; Corley, Lynda J.; Fuller, Gregory N.; Tucker, Susan L.; Diaz, Leslie K.; Buchholz, Thomas A.; Frost, Jeffrey A.

2009-01-01

Preclinical data indicate that α6β4 integrin signaling through Ras homolog gene family, member A, plays an important role in tumor cell motility. The objective of this study was to determine whether the combined expression of α6β4 integrin and neuroepithelioma transforming gene 1 (Net1), a guanine nucleotide exchange factor specific for Ras homolog gene family member A, is associated with adverse clinical outcome in breast cancer patients. Immunohistochemical expression of each protein was evaluated in a tumor tissue microarray prepared from the primary tumors of 94 node-positive patients with invasive breast carcinoma treated with total mastectomy and doxorubicin-based chemotherapy without radiation with a median follow-up of 12.5 years. Associations between staining results and multiple clinicopathologic variables were investigated. Although there was no significant association between α6β4 integrin or Net1 expression and clinical outcome when each marker was considered individually, coexpression of α6β4 and Net1 was associated with decreased distant metastasis–free survival (P = 0.030). In the subset of patients with hormone receptor–positive tumors, coexpression of α6β4 and Net1 was associated with a decrease in distant metastasis–free and overall survival (P < 0.001 and P = 0.006, respectively). Although an association between human epidermal growth factor receptor 2 expression and coexpression of α6β4 and Net1 (P = 0.008) was observed, coexpression of α6β4 and Net1 (hazard ratio, 1.63; P = 0.02) and lymphovascular invasion (hazard ratio, 2.35; P = 0.02) were the only factors independently associated with the development of distant metastasis in multivariate analysis. These findings suggest that coexpression of α6β4 integrin and Net1 could be a useful biomarker for aggressive disease in node-positive breast cancer patients. PMID:19124484

Association of high microvessel αvβ3 and low PTEN with poor outcome in stage 3 neuroblastoma: rationale for using first in class dual PI3K/BRD4 inhibitor, SF1126

PubMed Central

Singh, Alok R.; Joshi, Shweta; Vega, Francisco M.; Guo, Pinzheng; Xu, Jingying; Groshen, Susan; Ye, Wei; Millard, Melissa; Campan, Mihaela; Morales, Guillermo; Garlich, Joseph R.; Laird, Peter W.; Seeger, Robert C.; Shimada, Hiroyuki

2017-01-01

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Our previous studies showed that the angiogenic integrin αvβ3 was increased in high-risk metastatic (stage 4) NB compared with localized neuroblastomas. Herein, we show that integrin αvβ3 was expressed on 68% of microvessels in MYCN-amplified stage 3 neuroblastomas, but only on 34% (means) in MYCN-non-amplified tumors (p < 0.001; n = 54). PTEN, a tumor suppressor involved in αvβ3 signaling, was expressed in neuroblastomas either diffusely, focally or not at all (immunohistochemistry). Integrin αvβ3 was expressed on 60% of tumor microvessels when PTEN was negative or focal, as compared to 32% of microvessels in tumors with diffuse PTEN expression (p < 0.001). In a MYCN transgenic mouse model, loss of one allele of PTEN promoted tumor growth, illustrating the potential role of PTEN in neuroblastoma pathogenesis. Interestingly, we report the novel dual PI-3K/BRD4 activity of SF1126 (originally developed as an RGD-conjugated pan PI3K inhibitor). SF1126 inhibits BRD4 bromodomain binding to acetylated lysine residues with histone H3 as well as PI3K activity in the MYCN amplified neuroblastoma cell line IMR-32. Moreover, SF1126 suppressed MYCN expression and MYCN associated transcriptional activity in IMR-32 and CHLA136, resulting in overall decrease in neuroblastoma cell viability. Finally, treatment of neuroblastoma tumors with SF1126 inhibited neuroblastoma growth in vivo. These data suggest integrin αvβ3, MYCN/BRD4 and PTEN/PI3K/AKT signaling as biomarkers and hence therapeutic targets in neuroblastoma and support testing of the RGD integrin αvβ3-targeted PI-3K/BRD4 inhibitor, SF1126 as a therapeutic strategy in this specific subgroup of high risk neuroblastoma. PMID:28881723

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

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.

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

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

Mechanotransduction across the cell surface and through the cytoskeleton

NASA Technical Reports Server (NTRS)

Wang, N.; Butler, J. P.; Ingber, D. E.

1993-01-01

Mechanical stresses were applied directly to cell surface receptors with a magnetic twisting device. The extracellular matrix receptor, integrin beta 1, induced focal adhesion formation and supported a force-dependent stiffening response, whereas nonadhesion receptors did not. The cytoskeletal stiffness (ratio of stress to strain) increased in direct proportion to the applied stress and required intact microtubules and intermediate filaments as well as microfilaments. Tensegrity models that incorporate mechanically interdependent struts and strings that reorient globally in response to a localized stress mimicked this response. These results suggest that integrins act as mechanoreceptors and transmit mechanical signals to the cytoskeleton. Mechanotransduction, in turn, may be mediated simultaneously at multiple locations inside the cell through force-induced rearrangements within a tensionally integrated cytoskeleton.

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.

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.

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.

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

Permeability Changes of Integrin-Containing Multivesicular Structures Triggered by Picornavirus Entry

PubMed Central

Soonsawad, Pan; Weerachatyanukul, Wattana; Rintanen, Nina; Espinoza, Juan; McNerney, Gregory; Marjomäki, Varpu; Cheng, R. Holland

2014-01-01

Cellular uptake of clustered α2β1-integrin induces the formation of membrane compartments that subsequently mature into a multivesicular body (MVB). Enhanced internalization mediated by clustered integrins was observed upon infection by the picornavirus echovirus 1 (EVI). We elucidated the structural features of virus-induced MVBs (vMVBs) in comparison to antibody-induced control MVBs (mock infection) by means of high-pressure cryo fixation of cells followed by immuno electron tomography during early entry of the virus. Three-dimensional tomograms revealed a marked increase in the size and complexity of these vMVBs and the intraluminal vesicles (ILVs) at 2 and 3.5 hours post infection (p.i.), in contrast to the control MVBs without virus. Breakages in the membranes of vMVBs were detected from tomograms after 2 and especially after 3.5 h suggesting that these breakages could facilitate the genome release to the cytoplasm. The in situ neutral-red labeling of viral genome showed that virus uncoating starts as early as 30 min p.i., while an increase of permeability was detected in the vMVBs between 1 and 3 hours p.i., based on a confocal microscopy assay. Altogether, the data show marked morphological changes in size and permeability of the endosomes in the infectious entry pathway of this non-enveloped enterovirus and suggest that the formed breakages facilitate the transfer of the genome to the cytoplasm for replication. PMID:25299706

Functional Interaction of CD154 Protein with α5β1 Integrin Is Totally Independent from Its Binding to αIIbβ3 Integrin and CD40 Molecules*

PubMed Central

El Fakhry, Youssef; Alturaihi, Haydar; Yacoub, Daniel; Liu, Lihui; Guo, Wenyan; Leveillé, Claire; Jung, Daniel; Khzam, Lara Bou; Merhi, Yahye; Wilkins, John A.; Li, Hongmin; Mourad, Walid

2012-01-01

In addition to its classical CD40 receptor, CD154 also binds to αIIbβ3, α5β1, and αMβ2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbβ3, and α5β1 receptors. We found that the binding affinity of CD154 for αIIbβ3 is ∼4-fold higher than for α5β1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbβ3 and show that CD154 residues involved in its binding to CD40 or αIIbβ3 are distinct from those implicated in its interaction to α5β1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5β1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5β1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors. PMID:22461623

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 properties of eosinophils in BAL. Dampening of β(2) -integrin function of eosinophils in transit to inflamed airway may contribute to the decrease in lung inflammation caused by anti-IL-5. © 2012 Blackwell Publishing Ltd.

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 minimal effects on properties of eosinophils in BAL. Dampening of β2-integrin function of eosinophils in transit to inflamed airway may contribute to the decrease in lung inflammation caused by anti-IL-5. PMID:23414537

Spatial distribution of osteopontin, CD44v6 and podoplanin in the lining epithelium of odontogenic keratocyst, and their biological relevance.

PubMed

Kechik, Khamisah Awang; Siar, Chong Huat

2018-02-01

The odontogenic keratocyst (OKC) remains the most challenging jaw cyst to treat because of its locally-aggressive behaviour and high recurrence potential. Emerging evidence suggests that osteopontin, its receptors CD44v6 and integrin α v , and podoplanin, have a role in the local invasiveness of this cyst. However the spatial distribution characteristics of these pro-invasive markers in the lining epithelium of OKC, and their association with the clinicopathologic parameters of OKC are largely unexplored. This study sought to address these issues in comparison with dentigerous cysts (DCs) and radicular cysts (RCs) and to evaluate their biological relevance. A sample consisting of 20 OKC cases, 10 DCs and 10 RCs was subjected to immunohistochemical staining for osteopontin, CD44v6 and integrin α v , and podoplanin, and semiquantitative analysis was performed. All factors (except integrin α v ) were detected heterogeneously in the constitutive layers of the lining epithelium in all three cyst types. Key observations were significant upregulation of CD44v6 and podoplanin in OKC compared to DCs and RCs, suggesting that these protein molecules may play crucial roles in promoting local invasiveness in OKC (P<0.05). Osteopontin underexpression and distribution patterns were indistinctive among all three cysts indicating its limited role as pro-invasive factor. Clinical parameters showed no significant correlations with all protein factors investigated. Present findings suggest that an osteopontin low CD44v6 high and podoplanin high immunoprofile most probably represent epithelial signatures of OKC and are markers of local invasiveness in this cyst. Copyright © 2017 Elsevier Inc. All rights reserved.

HAMLET binding to α-actinin facilitates tumor cell detachment.

PubMed

Trulsson, Maria; Yu, Hao; Gisselsson, Lennart; Chao, Yinxia; Urbano, Alexander; Aits, Sonja; Mossberg, Ann-Kristin; Svanborg, Catharina

2011-03-08

Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in β1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed.

HAMLET Binding to α-Actinin Facilitates Tumor Cell Detachment

PubMed Central

Trulsson, Maria; Yu, Hao; Gisselsson, Lennart; Chao, Yinxia; Urbano, Alexander; Aits, Sonja; Mossberg, Ann-Kristin; Svanborg, Catharina

2011-01-01

Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in β1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed. PMID:21408150

Structural and Functional Characterization of the Kindlin-1 Pleckstrin Homology Domain*

PubMed Central

Yates, Luke A.; Lumb, Craig N.; Brahme, Nina N.; Zalyte, Ruta; Bird, Louise E.; De Colibus, Luigi; Owens, Raymond J.; Calderwood, David A.; Sansom, Mark S. P.; Gilbert, Robert J. C.

2012-01-01

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin β-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P3; KD ∼100 μm) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P3 as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P2 on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P3 is affected by the presence of PO4 ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species. PMID:23132860

HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes.

PubMed

Domínguez-Catzín, Victoria; Reveles-Espinoza, Alicia-María; Sánchez-Ramos, Janet; Cruz-Cadena, Raúl; Lemus-Hernández, Diana; Garrido, Efraín

2017-04-03

Cervical cancer is the fourth cause of death worldwide by cancer in women and is a disease associated to persistent infection with human papillomavirus (HPV), particularly from two high-risk types HPV16 and 18. The virus initiates its replicative cycle infecting cells located in the basal layer of the epithelium, where a small population of epithelial stem cells is located performing important functions of renewal and maintenance of the tissue. Viral E2 gene is one of the first expressed after infection and plays relevant roles in the replicative cycle of the virus, modifying fundamental processes in the infected cells. Thus, the aim of the present study was to demonstrate the presence of hierarchic subpopulations in HaCaT cell line and evaluate the effect of HPV16-E2 expression, on their biological processes. HaCaT-HPV16-E2 cells were generated by transduction of HaCaT cell line with a lentiviral vector. The α6-integrin-CD71 expression profile was established by immunostaining and flow cytometric analysis. After sorting, cell subpopulations were analyzed in biological assays for self-renewal, clonogenicity and expression of stemness factors (RT-qPCR). We identified in HaCaT cell line three different subpopulations that correspond to early differentiated cells (α6-integrin dim ), transitory amplifying cells (α6-integrin bri /CD71 bri ) and progenitor cells (α6-integrin bri /CD71 dim ). The last subpopulation showed stem cell characteristics, such as self-renewal ability, clonogenicity and expression of the well-known stem cell factors SOX2, OCT4 and NANOG, suggesting they are stem-like cells. Interestingly, the expression of HPV16-E2 in HaCaT cells changed its α6-integrin-CD71 immunophenotype modifying the relative abundance of the cell subpopulations, reducing significantly the percentage of α6-integrin bri /CD71 dim cells. Moreover, the expression of the stem cell markers was also modified, increasing the expression of SOX2 and NANOG, but decreasing notably the expression of OCT4. Our data demonstrated the presence of a small subpopulation with epithelial "progenitor cells" characteristics in the HaCaT cell line, and that HPV16-E2 expression on these cells induces early differentiation.

Therapeutic Targeting of Eosinophil Adhesion and Accumulation in Allergic Conjunctivitis

PubMed Central

Baiula, Monica; Bedini, Andrea; Carbonari, Gioia; Dattoli, Samantha Deianira; Spampinato, Santi

2012-01-01

Considerable evidence indicates that eosinophils are important effectors of ocular allergy. Increased worldwide prevalence of allergic eye pathologies has stimulated the identification of novel drug targets, including eosinophils and adhesion molecules. Accumulation of eosinophils in the eye is a key event in the onset and maintenance of allergic inflammation and is mediated by different adhesion molecules. Antihistamines with multiple mechanisms of action can be effective during the early and late phases of allergic conjunctivitis by blocking the interaction between β1 integrins and vascular cell adhesion molecule (VCAM)-1. Small molecule antagonists that target key elements in the process of eosinophil recruitment have been identified and reinforce the validity of α4β1 integrin as a therapeutic target. Glucocorticoids are among the most effective drugs for ocular allergy, but their use is limited by adverse effects. Novel dissociated glucocorticoids can prevent eosinophil accumulation and induce apoptosis of eosinophils, making them promising candidates for ophthalmic drugs. This article reviews recent understanding of the role of adhesion molecules in eosinophil recruitment in the inflamed conjunctiva along with effective treatments for allergic conjunctivitis. PMID:23271999

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

PubMed

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

2015-11-27

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

Synthesis and evaluation of two NIR fluorescent cyclic RGD penta-peptides for targeting integrins

NASA Astrophysics Data System (ADS)

Ye, Yunpeng; Bloch, Sharon; Xu, Baogang; Achilefu, Samuel

2006-02-01

Interest in novel RGD peptides has been increasingly growing as the interactions between RGD peptides and integrins are the basis for a variety of cellular functions and medical applications such as modulation of cell adhesion, invasion, tumor angiogenesis, and metastasis. In particular, we have been interested in novel NIR fluorescent RGD peptides as potential optical contrast agents for in vivo tumor optical imaging. Therefore, two cyclic RGD penta-peptides conjugated with a NIR fluorescent carbocyanine (Cypate), i.e. lactam-based cyclo[RGDfK(Cypate)] (1) and disulfide-containing Cypate-cyclo(CRGDC)-NH II (2), were designed and synthesized. The competitive binding assay between the purified α vβ 3 integrin and the peptide ligands using 125I-echistatin as a tracer showed that 1 had a higher receptor binding affinity (IC 50~10 -7 M) than 2 (IC 50~10 -6 M). Furthermore, the internalization of 1 in A549 cells in vitro was less than 2, as revealed by fluorescence microscopy. These results suggest that both the lactam- and disulfide-based cyclic RGD penta-peptides should be further studied structurally and functionally to elucidate the advantages of each class of compounds.

Lysophosphatidic Acid Initiates Epithelial to Mesenchymal Transition and Induces β-Catenin-mediated Transcription in Epithelial Ovarian Carcinoma*

PubMed Central

Burkhalter, Rebecca J.; Westfall, Suzanne D.; Liu, Yueying; Stack, M. Sharon

2015-01-01

During tumor progression, epithelial ovarian cancer (EOC) cells undergo epithelial-to-mesenchymal transition (EMT), which influences metastatic success. Mutation-dependent activation of Wnt/β-catenin signaling has been implicated in gain of mesenchymal phenotype and loss of differentiation in several solid tumors; however, similar mutations are rare in most EOC histotypes. Nevertheless, evidence for activated Wnt/β-catenin signaling in EOC has been reported, and immunohistochemical analysis of human EOC tumors demonstrates nuclear staining in all histotypes. This study addresses the hypothesis that the bioactive lipid lysophosphatidic acid (LPA), prevalent in the EOC microenvironment, functions to regulate EMT in EOC. Our results demonstrate that LPA induces loss of junctional β-catenin, stimulates clustering of β1 integrins, and enhances the conformationally active population of surface β1 integrins. Furthermore, LPA treatment initiates nuclear translocation of β-catenin and transcriptional activation of Wnt/β-catenin target genes resulting in gain of mesenchymal marker expression. Together these data suggest that LPA initiates EMT in ovarian tumors through β1-integrin-dependent activation of Wnt/β-catenin signaling, providing a novel mechanism for mutation-independent activation of this pathway in EOC progression. PMID:26175151

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

PubMed

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

2012-11-01

The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail-bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear. We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β, and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co-expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell-matrix interactions regulating BMP signalling at this site of caudal morphogenesis. Laminin532 could interact with α6-containing integrin to direct differentiation of the specialised VER cells from surface ectoderm. Copyright © 2012 Wiley Periodicals, Inc.

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

PubMed Central

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

2013-01-01

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

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

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

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.

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.

Non-Invasive Detection of Lung Inflammation by Near-Infrared Fluorescence Imaging Using Bimodal Liposomes.

PubMed

Desu, Hari R; Wood, George C; Thoma, Laura A

2016-01-01

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome results in respiratory obstruction and severe lung inflammation. Critical characteristics of ALI are alveolar edema, infiltration of leukocytes (neutrophils and monocytes), release of pro-inflammatory cytokines and chemokines into broncho-alveolar lavage fluid, and activation of integrin receptors. The purpose of the study was to demonstrate non-invasive detection of lung inflammation using integrin receptor targeted fluorescence liposomes. An inflammation similar to that observed in ALI was elicited in rodents by intra-tracheal instillation of interleukin-1beta (IL-1beta). Cyclic arginine glycine-(D)-aspartic acid-peptide (cRGD-peptide) grafted fluorescence liposomes were administered to ALI induced male Sprague-Dawley rats for targeting lung integrin receptors. Near-infrared fluorescence imaging (NIRFI) was applied for visualization and quantitation of lung inflammation. NIRFI signals were correlated with inflammatory cellular and biochemical markers of lungs. A positive correlation was observed between NIRF signals and lung inflammation markers. Compared to control group, an intense NIRF signal was observed in ALI induced rats in the window 6-24 h post-IL-1beta instillation. Interaction of integrin receptors with targeted liposomes was assumed to contribute to intense NIRF signal. RT-PCR studies showed an elevated lung expression of alphavbeta5 integrin receptors, 12 h post-IL-1beta instillation. In vitro studies demonstrated integrin receptor specificity of targeted liposomes. These targeted liposomes showed binding to alphavbeta5 integrin receptors expressed on alveolar cells. Non-invasive detection of lung inflammation was demonstrated using a combination of integrin receptor targeting and NIRFI.

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.

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

Emergence and subsequent functional specialization of kindlins during evolution of cell adhesiveness

PubMed Central

Meller, Julia; Rogozin, Igor B.; Poliakov, Eugenia; Meller, Nahum; Bedanov-Pack, Mark; Plow, Edward F.; Qin, Jun; Podrez, Eugene A.; Byzova, Tatiana V.

2015-01-01

Kindlins are integrin-interacting proteins essential for integrin-mediated cell adhesiveness. In this study, we focused on the evolutionary origin and functional specialization of kindlins as a part of the evolutionary adaptation of cell adhesive machinery. Database searches revealed that many members of the integrin machinery (including talin and integrins) existed before kindlin emergence in evolution. Among the analyzed species, all metazoan lineages—but none of the premetazoans—had at least one kindlin-encoding gene, whereas talin was present in several premetazoan lineages. Kindlin appears to originate from a duplication of the sequence encoding the N-terminal fragment of talin (the talin head domain) with a subsequent insertion of the PH domain of separate origin. Sequence analysis identified a member of the actin filament–associated protein 1 (AFAP1) superfamily as the most likely origin of the kindlin PH domain. The functional divergence between kindlin paralogues was assessed using the sequence swap (chimera) approach. Comparison of kindlin 2 (K2)/kindlin 3 (K3) chimeras revealed that the F2 subdomain, in particular its C-terminal part, is crucial for the differential functional properties of K2 and K3. The presence of this segment enables K2 but not K3 to localize to focal adhesions. Sequence analysis of the C-terminal part of the F2 subdomain of K3 suggests that insertion of a variable glycine-rich sequence in vertebrates contributed to the loss of constitutive K3 targeting to focal adhesions. Thus emergence and subsequent functional specialization of kindlins allowed multicellular organisms to develop additional tissue-specific adaptations of cell adhesiveness. PMID:25540429

Enhanced homing permeability and retention of bone marrow stromal cells (BMSC) by non-invasive pulsed focused ultrasound

PubMed Central

Ziadloo, Ali; Burks, Scott R.; Gold, Eric M.; Lewis, Bobbi K.; Chaudhry, Aneeka; Merino, Maria J.; Frenkel, Victor; Frank, Joseph A.

2012-01-01

Bone marrow stromal cells (BMSC) have shown significant promise in the treatment of disease, but their therapeutic efficacy is often limited by inefficient homing of systemically-administered cells, which results in low numbers of cells accumulating at sites of pathology. BMSC home to areas of inflammation where local expression of integrins and chemokine gradients are present. We demonstrated that non-destructive pulsed focused ultrasound (pFUS) exposures that emphasize the mechanical effects of ultrasound-tissue interactions induced local and transient elevations of chemoattractants (i.e., cytokines, integrins, and growth factors) in the murine kidney. pFUS-induced upregulation of cytokines occurred through approximately 1 day post-treatment and returned to contralateral kidney levels by day 3. This window of significant increases in cytokine expression was accompanied by local increases of other trophic factors and integrins that have been shown to promote BMSC homing. When BMSC were administered intravenously following pFUS treatment to a single kidney, enhanced homing, permeability, and retention of BMSC was observed in the treated kidney versus the contralateral kidney. Histological analysis revealed up to 8 times more BMSC in the peritubular regions of the treated kidneys on days 1 and 3 post-treatment. Furthermore, cytokine levels in pFUS-treated kidneys following BMSC administration were found to be similar to controls, suggesting modulation of cytokine levels by BMSC. pFUS could potentially improve cell-based therapies as a noninvasive modality to target BMSC homing by establishing local chemoattractant gradients and increasing expression of integrins to enhance tropism of BMSC toward treated tissues. PMID:22593018

Alpha lipoic acid selectively inhibits proliferation and adhesion to fibronectin of v-H-ras-transformed 3Y1 cells.

PubMed

Yamasaki, Masao; Iwase, Masahiro; Kawano, Kazuo; Sakakibara, Yoichi; Suiko, Masahito; Nishiyama, Kazuo

2012-05-01

Here, we focused on the effects of racemic α-lipoic acid on proliferation and adhesion properties of 3Y1 rat fibroblasts and the v-H-ras-transformed derivative, HR-3Y1-2 cells. Racemic α-lipoic acid inhibited proliferation of HR-3Y1-2 but not 3Y1 cells at 0.3 and 1.0 mM. R-(+)-α-lipoic acid also inhibited proliferation of HR-3Y1-2 cells equivalent to that of racemic α-lipoic acid. In addition, racemic α-lipoic acid decreased intracellular reactive oxygen species levels in HR-3Y1 cells but not 3Y1 cells. Next, we evaluated the effects of racemic α-lipoic acid on cell adhesion to fibronectin. The results indicated that racemic α-lipoic acid decreased adhesive ability of HR-3Y1-2 cells to fibronectin-coated plates. As blocking antibody experiment revealed that β1-integrin plays a key role in cell adhesion in this experimental system, the effects of racemic α-lipoic acid on the expression of β1-integrin were examined. The results indicated that racemic α-lipoic acid selectively downregulated the expression of cell surface β1-integrin expression in HR-3Y1-2 cells. Intriguingly, exogenous hydrogen peroxide upregulated cell surface β1-integrin expression in 3Y1 cells. Taken together, these data suggest that reduction of intracellular reactive oxygen species levels by α-lipoic acid could be an effective means of ameliorating abnormal growth and adhesive properties in v-H-ras transformed cells.

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

PubMed

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

2015-01-01

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

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.

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

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.

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.

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.

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

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

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.

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

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

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.

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.

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.

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

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.

Insulin-like Growth Factor (IGF) Signaling Requires αvβ3-IGF1-IGF Type 1 Receptor (IGF1R) Ternary Complex Formation in Anchorage Independence, and the Complex Formation Does Not Require IGF1R and Src Activation

PubMed Central

Fujita, Masaaki; Takada, Yoko K.; Takada, Yoshikazu

2013-01-01

Integrin αvβ3 plays a role in insulin-like growth factor 1 (IGF1) signaling (integrin-IGF1 receptor (IGF1R) cross-talk) in non-transformed cells in anchorage-dependent conditions. We reported previously that IGF1 directly binds to αvβ3 and induces αvβ3-IGF1-IGF1R ternary complex formation in these conditions. The integrin-binding defective IGF1 mutant (R36E/R37E) is defective in inducing ternary complex formation and IGF signaling, whereas it still binds to IGF1R. We studied if IGF1 can induce signaling in anchorage-independent conditions in transformed Chinese hamster ovary cells that express αvβ3 (β3-CHO) cells. Here we describe that IGF1 signals were more clearly detectable in anchorage-independent conditions (polyHEMA-coated plates) than in anchorage-dependent conditions. This suggests that IGF signaling is masked by signals from cell-matrix interaction in anchorage-dependent conditions. IGF signaling required αvβ3 expression, and R36E/R37E was defective in inducing signals in polyHEMA-coated plates. These results suggest that αvβ3-IGF1 interaction, not αvβ3-extracellular matrix interaction, is essential for IGF signaling. Inhibitors of IGF1R, Src, AKT, and ERK1/2 did not suppress αvβ3-IGF-IGF1R ternary complex formation, suggesting that activation of these kinases are not required for ternary complex formation. Also, mutations of the β3 cytoplasmic tail (Y747F and Y759F) that block β3 tyrosine phosphorylation did not affect IGF1R phosphorylation or AKT activation. We propose a model in which IGF1 binding to IGF1R induces recruitment of integrin αvβ3 to the IGF-IGF1R complex and then β3 and IGF1R are phosphorylated. It is likely that αvβ3 should be together with the IGF1-IGF1R complex for triggering IGF signaling. PMID:23243309

Synergistic action of cisplatin and echistatin in MDA-MB-231 breast cancer cells.

PubMed

Czarnomysy, Robert; Surażyński, Arkadiusz; Popławska, Bożena; Rysiak, Edyta; Pawłowska, Natalia; Czajkowska, Anna; Bielawski, Krzysztof; Bielawska, Anna

2017-03-01

The aim of our study was to determine whether the use of cisplatin in the presence echistatin in MDA-MB-231 breast cancer cells leads to a reduction of toxic effects associated with the use of cisplatin. The expression of β 1 -integrin and insulin-like growth factor 1 receptor (IGF-IR), signaling pathway protein expression: protein kinase B (AKT), mitogen-activated protein kinases (ERK1/ERK2), nuclear factor kappa B (NFκB), and caspase-3 and -9 activity was measured after 24 h of incubation with tested compounds to explain detailed molecular mechanism of induction of apoptosis. The viability of MDA-MB-231 breast cancer cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Annexin V-FITC/propidium iodide staining assay was performed to detect the induction of apoptosis. Inhibition DNA biosynthesis was determined by [ 3 H]thymidine incorporation into DNA. The expression of of β 1 -integrin, IGF-IR, AKT, ERK1/ERK2, NFκB, caspase-3 and -9 was evaluated using Western blot. The results suggest that treatment of MDA-MB-231 breast cancer cells for 24 h cisplatin plus echistatin severely inhibits cell growth and activates apoptosis by upregulation of caspase-3 and -9 expressions. The effect was stronger than treatment cisplatin and echistatin alone. In this study, we have found that cisplatin plus echistatin treatment decreases collagen biosynthesis in MDA-MB-231 breast cancer cells stronger than the individual compounds. The inhibition was found to be dependent on the β 1 -integrin and IGF receptor activation. A significant reduction of ERK1/ERK2, AKT expression in cancer cells after cisplatin plus echistatin treatment was also found. The cancer cells treated by echistatin, cisplatin, and in particular the combination of both compounds drastically increased expression of NFκB transcription factor. Our results suggest that combined therapy cisplatin plus echistatin is a possible way to improve selectiveness of cisplatin. This mechanism probably is due to downregulation of expression of β 1 -integrin and IGF-IR receptors, and the signaling pathway proteins induced by these receptors. Our results suggest that therapy cisplatin plus echistatin is a possible way to improve selectiveness of cisplatin.

Evaluation of 111In-Labeled Cyclic RGD Peptides: Effects of Peptide and Linker Multiplicity on Their Tumor Uptake, Excretion Kinetics and Metabolic Stability

PubMed Central

Shi, Jiyun; Zhou, Yang; Chakraborty, Sudipta; Kim, Young-Seung; Jia, Bing; Wang, Fan; Liu, Shuang

2011-01-01

Purpose: The purpose of this study was to demonstrate the valence of cyclic RGD peptides, P-RGD (PEG4-c(RGDfK): PEG4 = 15-amino-4,710,13-tetraoxapentadecanoic acid), P-RGD2 (PEG4-E[c(RGDfK)]2, 2P-RGD4 (E{PEG4-E[c(RGDfK)]2}2, 2P4G-RGD4 (E{PEG4-E[G3-c(RGDfK)]2}2: G3 = Gly-Gly-Gly) and 6P-RGD4 (E{PEG4-E[PEG4-c(RGDfK)]2}2) in binding to integrin αvβ3, and to assess the impact of peptide and linker multiplicity on biodistribution properties, excretion kinetics and metabolic stability of their corresponding 111In radiotracers. Methods: Five new RGD peptide conjugates (DOTA-P-RGD (DOTA =1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid), DOTA-P-RGD2, DOTA-2P-RGD4, DOTA-2P4G-RGD4, DOTA-6P-RGD4), and their 111In complexes were prepared. The integrin αvβ3 binding affinity of cyclic RGD conjugates were determined by a competitive displacement assay against 125I-c(RGDyK) bound to U87MG human glioma cells. Biodistribution, planar imaging and metabolism studies were performed in athymic nude mice bearing U87MG human glioma xenografts. Results: The integrin αvβ3 binding affinity of RGD conjugates follows the order of: DOTA-6P-RGD4 (IC50 = 0.3 ± 0.1 nM) ~ DOTA-2P4G-RGD4 (IC50 = 0.2 ± 0.1 nM) ~ DOTA-2P-RGD4 (IC50 = 0.5 ± 0.1 nM) > DOTA-3P-RGD2 (DOTA-PEG4-E[PEG4-c(RGDfK)]2: IC50 = 1.5 ± 0.2 nM) > DOTA-P-RGD2 (IC50 = 5.0 ± 1.0 nM) >> DOTA-P-RGD (IC50 = 44.3 ± 3.5 nM) ~ c(RGDfK) (IC50 = 49.9 ± 5.5 nM) >> DOTA-6P-RGK4 (IC50 = 437 ± 35 nM). The fact that DOTA-6P-RGK4 had much lower integrin αvβ3 binding affinity than DOTA-6P-RGD4 suggests that the binding of DOTA-6P-RGD4 to integrin αvβ3 is RGD-specific. This conclusion is consistent with the lower tumor uptake for 111In(DOTA-6P-RGK4) than that for 111In(DOTA-6P-RGD4). It was also found that the G3 and PEG4 linkers between RGD motifs have a significant impact on the integrin αvβ3-targeting capability, biodistribution characteristics, excretion kinetics and metabolic stability of 111In-labeled cyclic RGD peptides. Conclusion: On the basis of their integrin αvβ3 binding affinity and tumor uptake of their corresponding 111In radiotracers, it was conclude that 2P-RGD4, 2P4G-RGD4 and 6P-RGD4 are most likely bivalent in binding to integrin αvβ3, and extra RGD motifs might contribute to the long tumor retention times of 111In(DOTA-2P-RGD4), 111In(DOTA-2P4G-RGD4) and 111In(DOTA-6P-RGD4) than that of 111In(DOTA-3P-RGD3) at 72 h p.i. Among the 111In-labeled cyclic RGD tetramers evaluated in the glioma model, 111In(DOTA-2P4G-RGD4) has very high tumor uptake with the best tumor/kidney and tumor/liver ratios, suggesting that 90Y(DOTA-2P4G-RGD4) and 177Lu(DOTA-2P4G-RGD4) might have the potential for targeted radiotherapy of integrin αvβ3-positive tumors. PMID:21850213

αvß3-Integrin-Targeted Magnetic Resonance Imaging for the Assessment of Early Antiangiogenic Therapy Effects in Orthotopic Breast Cancer Xenografts.

PubMed

Kazmierczak, Philipp Maximilian; Schneider, Moritz; Habereder, Thomas; Hirner-Eppeneder, Heidrun; Eschbach, Ralf S; Moser, Matthias; Reiser, Maximilian F; Lauber, Kirsten; Nikolaou, Konstantin; Cyran, Clemens C

2016-11-01

The aim of this study was to investigate magnetic resonance imaging (MRI) with αvß3-integrin-targeted ultrasmall superparamagnetic iron oxide nanoparticles (RGD-USPIO) for the in vivo monitoring of early antiangiogenic therapy effects in experimental breast cancer. Orthotopic human breast cancer (MDA-MB-231) xenograft-bearing severe combined immunodeficiency mice were imaged before and after a 1-week therapy with the vascular endothelial growth factor receptor-antibody bevacizumab or placebo (n = 10 per group, daily intraperitoneal injections of bevacizumab or a volume-equivalent placebo solution, respectively) on a clinical 3 T scanner (Magnetom Skyra; Siemens Healthcare, Erlangen, Germany) before and 60 minutes after the intravenous injection of RGD-USPIO (P04000; Guerbet, Villepinte, France). R2 relaxometry employing a T2-weighted spin-echo sequence with 4 echo times (echo time, 20/40/60/80 milliseconds; repetition time, 3800 milliseconds; matrix, 128 × 128; field of view, 50 × 50; slice thickness, 1.2 mm; time to acquisition, 25 minutes) was used as semiquantitative measure to determine RGD-USPIO endothelial binding. In addition, the T2-weighted images were used to perform volumetric tumor response assessments. Imaging results were validated by ex vivo multiparametric immunohistochemistry with regard to αvß3-integrin expression, microvascular density (CD31), proliferation (Ki-67), and apoptosis (TUNEL). RGD-USPIO endothelial binding was significantly reduced after vascular endothelial growth factor inhibition, compared with the control group in which an increased endothelial binding was detected ([INCREMENT]R2Therapy = -0.80 ± 0.78 s; [INCREMENT]R2Control = +0.27 ± 0.59 s; P = 0.002). Correspondingly, immunohistochemistry revealed a significantly lower αvß3-integrin expression (91 ± 30 vs 357 ± 72; P < 0.001), microvascular density (CD31, 109 ± 46 vs 440 ± 208; P < 0.001), tumor cell proliferation (Ki-67, 4040 ± 1373 vs 6530 ± 1217; P < 0.001), as well as significantly higher apoptosis (TUNEL, 11186 ± 4387 vs 4017 ± 1191; P = 0.004) in the therapy compared with the control group. Contrary to the changes in αvß3-integrin expression detected by RGD-USPIO MRI, morphology-based tumor response assessments did not show a significant intergroup difference in tumor volume development over the course of the experiment (ΔVolTherapy +71 ± 40 μL vs ΔVolControl +125 ± 81 μL; P > 0.05). RGD-USPIO MRI allows for the noninvasive assessment of αvß3-integrin expression in the investigated breast cancer model. RGD-USPIO MRI may be applicable for the in vivo monitoring of early antiangiogenic therapy effects in experimental breast cancer, generating possible complementary molecular imaging biomarkers to morphology-based tumor response assessments.

Dissecting the role of matrix metalloproteinases (MMP) and integrin alpha(v)beta3 in angiogenesis in vitro: absence of hemopexin C domain bioactivity, but membrane-Type 1-MMP and alpha(v)beta3 are critical.

PubMed

Nisato, Riccardo E; Hosseini, Ghamartaj; Sirrenberg, Christian; Butler, Georgina S; Crabbe, Thomas; Docherty, Andrew J P; Wiesner, Matthias; Murphy, Gillian; Overall, Christopher M; Goodman, Simon L; Pepper, Michael S

2005-10-15

Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin alpha(v)beta3-mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti-membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin alpha(v)beta3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin alpha(v)beta(3). Moreover, no specific binding of pro-MMP-2 to integrin alpha(v)beta3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin alpha(v)beta3 , and this in a PEX-independent manner. Likewise, integrin alpha(v)beta3 -expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and alpha(v)beta3 - dependent but MMP-2 independent and does not support a role for PEX in alpha(v)beta3 integrin binding or in modulating angiogenesis in this system.

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

Insulin promotes cell migration by regulating PSA-NCAM

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

Monzo, Hector J.; Coppieters, Natacha; Department of Anatomy and Medical Imaging, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland

Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cellmore » migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration. - Highlights: • Insulin modulates PSA-NCAM turnover through upregulation of p-FAK. • P-FAK modulates αv-integrin/PSA-NCAM clustering. • αv-integrin acts as a carrier for PSA-NCAM endocytosis. • Cell migration is promoted by cell surface PSA. • Insulin promotes PSA-dependent migration in vitro.« less

α6β1- and αV-integrins are required for long-term self-renewal of murine embryonic stem cells in the absence of LIF.

PubMed

Cattavarayane, Sandhanakrishnan; Palovuori, Riitta; Tanjore Ramanathan, Jayendrakishore; Manninen, Aki

2015-02-27

The growth properties and self-renewal capacity of embryonic stem (ES) cells are regulated by their immediate microenvironment such as the extracellular matrix (ECM). Integrins, a central family of cellular ECM receptors, have been implicated in these processes but their specific role in ES cell self-renewal remains unclear. Here we have studied the effects of different ECM substrates and integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) using short-term assays as well as long-term cultures. Removal of LIF from ES cell culture medium induced morphological differentiation of ES cells into polarized epistem cell-like cells. These cells maintained epithelial morphology and expression of key stemness markers for at least 10 passages in the absence of LIF when cultured on laminin, fibronectin or collagen IV substrates. The specific functional roles of α6-, αV- and β1-integrin subunits were dissected using stable lentivirus-mediated RNAi methodology. β1-integrins were required for ES cell survival in long-term cultures and for the maintenance of stem cell marker expression. Inhibition of α6-integrin expression compromised self-renewal on collagen while αV-integrins were required for robust ES cell adhesion on laminin. Analysis of the stemness marker expression revealed subtle differences between α6- and αV-depleted ES cells but the expression of both was required for optimal self-renewal in long-term ES cell cultures. In the absence of LIF, long-term ES cell cultures adapt an epistem cell-like epithelial phenotype and retain the expression of multiple stem cell markers. Long-term maintenance of such self-renewing cultures depends on the expression of β1-, α6- and αV-integrins.

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

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.

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

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.

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

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.

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

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

The interaction of protein-tyrosine phosphatase α (PTPα) and RACK1 protein enables insulin-like growth factor 1 (IGF-1)-stimulated Abl-dependent and -independent tyrosine phosphorylation of PTPα.

PubMed

Khanna, Ranvikram S; Le, Hoa T; Wang, Jing; Fung, Thomas C H; Pallen, Catherine J

2015-04-10

Protein tyrosine phosphatase α (PTPα) promotes integrin-stimulated cell migration in part through the role of Src-phosphorylated PTPα-Tyr(P)-789 in recruiting and localizing p130Cas to focal adhesions. The growth factor IGF-1 also stimulates PTPα-Tyr-789 phosphorylation to positively regulate cell movement. This is in contrast to integrin-induced PTPα phosphorylation, that induced by IGF-1 can occur in cells lacking Src family kinases (SFKs), indicating that an unknown kinase distinct from SFKs can target PTPα. We show that this IGF-1-stimulated tyrosine kinase is Abl. We found that PTPα binds to the scaffold protein RACK1 and that RACK1 coordinates the IGF-1 receptor, PTPα, and Abl in a complex to enable IGF-1-stimulated and Abl-dependent PTPα-Tyr-789 phosphorylation. In cells expressing SFKs, IGF-1-stimulated phosphorylation of PTPα is mediated by RACK1 but is Abl-independent. Furthermore, expressing the SFKs Src and Fyn in SFK-deficient cells switches IGF-1-induced PTPα phosphorylation to occur in an Abl-independent manner, suggesting that SFK activity dominantly regulates IGF-1/IGF-1 receptor signaling to PTPα. RACK1 is a molecular scaffold that integrates growth factor and integrin signaling, and our identification of PTPα as a RACK1 binding protein suggests that RACK1 may coordinate PTPα-Tyr-789 phosphorylation in these signaling networks to promote cell migration. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Type I Collagen and Collagen Mimetics as Angiogenesis Promoting Superpolymers

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

Twardowski, T.; Fertala, A.; Orgel, J.P.R.O.

Angiogenesis, the development of blood vessels from the pre-existing vasculature, is a key component of embryogenesis and tissue regeneration. Angiogenesis also drives pathologies such as tumor growth and metastasis, and hemangioma development in newborns. On the other hand, promotion of angiogenesis is needed in tissues with vascular insufficiencies, and in bioengineering, to endow tissue substitutes with appropriate microvasculatures. Therefore, much research has focused on defining mechanisms of angiogenesis, and identifying pro- and anti-angiogenic molecules. Type I collagen, the most abundant protein in humans, potently stimulates angiogenesis in vitro and in vivo. Crucial to its angiogenic activity appears to be ligationmore » and possibly clustering of endothelial cell (EC) surface {alpha}1{beta}1/{alpha}2{beta}1 integrin receptors by the GFPGER502-507 sequence of the collagen fibril. However, additional aspects of collagen structure and function that may modulate its angiogenic properties are discussed. Moreover, type I collagen and fibrin, another angiogenic polymer, share several structural features. These observations suggest strategies for creating 'angiogenic superpolymers', including: modifying type I collagen to influence its biological half-life, immunogenicity, and integrin binding capacity; genetically engineering fibrillar collagens to include additional integrin binding sites or angiogenic determinants, and remove unnecessary or deleterious sequences without compromising fibril integrity; and exploring the suitability of poly(ortho ester), PEG-lysine copolymer, tubulin, and cholesteric cuticle as collagen mimetics, and suggesting means of modifying them to display ideal angiogenic properties. The collagenous and collagen mimetic angiogenic superpolymers described here may someday prove useful for many applications in tissue engineering and human medicine.« less

αvβ6 Integrin Regulates Renal Fibrosis and Inflammation in Alport Mouse

PubMed Central

Hahm, Kyungmin; Lukashev, Matvey E.; Luo, Yi; Yang, William J.; Dolinski, Brian M.; Weinreb, Paul H.; Simon, Kenneth J.; Chun Wang, Li; Leone, Diane R.; Lobb, Roy R.; McCrann, Donald J.; Allaire, Normand E.; Horan, Gerald S.; Fogo, Agnes; Kalluri, Raghu; Shield, Charles F.; Sheppard, Dean; Gardner, Humphrey A.; Violette, Shelia M.

2007-01-01

The transforming growth factor (TGF)-β-inducible integrin αvβ6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-β. Herein, we show that αvβ6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture’s syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of αvβ6 in renal disease, we studied the effects of function-blocking αvβ6 monoclonal antibodies (mAbs) and genetic ablation of the β6 subunit on kidney fibrosis in Col4A3−/− mice, a mouse model of Alport syndrome. Expression of αvβ6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with αvβ6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in β6-deficient Alport mice. Transcript profiling of kidney tissues showed that αvβ6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-β RII treatment, suggesting shared regulatory functions of αvβ6 and TGF-β. These findings demonstrate that αvβ6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target. PMID:17200187

TNFα pathway blockade ameliorates toxic effects of FSGS plasma on podocyte cytoskeleton and β3 integrin activation.

PubMed

Bitzan, Martin; Babayeva, Sima; Vasudevan, Anil; Goodyer, Paul; Torban, Elena

2012-12-01

In the absence of mutant genes encoding components of the podocyte slit diaphragm, about 30-50 % of children with primary glucocorticoid-resistant focal segmental glomerulosclerosis (FSGS) develop recurrent proteinuria and slowly progressive FSGS lesions following renal transplantation. Recurrence of FSGS in the allograft strongly suggests a circulating factor that disturbs normal podocyte biology. To date, the nature of the circulating factor is unclear, and there is no cure for the recurrent form of FSGS (R-FSGS). Cultured differentiated human podocytes were exposed to the plasmapheresis effluent or blood plasma samples from pediatric patients with recurrent or primary FSGS; in some cases, podocytes were pre-incubated with specific antibodies to block the tumor necrosis factor-alpha (TNFα) signaling pathway. Integrity of focal adhesion complexes and actin cytoskeleton were investigated by immunofluorescent microscopy. Plasmapheresis effluent from an R-FSGS child or fresh plasma from two children with primary FSGS rapidly disturbed the cytoskeleton of normal human podocytes in vitro. Plasma from a child with R-FSGS also activated β3 integrin and dispersed focal adhesion complexes. The effects were reversed by pre-incubation with antibodies against TNFα or either of the two TNFα receptors. When our patient with R-FSGS became resistant to plasmapheresis, we initiated treatment with twice weekly etanercept injections and then infliximab. Within 3 weeks of regular anti-TNFα therapy, the patient achieved sustained partial remission of proteinuria, allowing us to wean her off plasmapheresis completely. We suggest that in some FSGS patients, disruption of the podocyte cytoskeleton and β3 integrin-mediated podocyte attachment are driven by the TNFα pathway.

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.

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.

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.

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.

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.

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

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.

PD-1 blocks lytic granule polarization with concomitant impairment of integrin outside-in signaling in the natural killer cell immunological synapse.

PubMed

Huang, Yu; Chen, Zhiying; Jang, Joon Hee; Baig, Mirza S; Bertolet, Grant; Schroeder, Casey; Huang, Shengjian; Hu, Qian; Zhao, Yong; Lewis, Dorothy E; Qin, Lidong; Zhu, Michael Xi; Liu, Dongfang

2018-04-18

The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. We sought to determine the effect of PD-1 signaling on NK cells. PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling blocked lytic granule polarization in NK cells, which was accompanied by failure of integrin-linked kinase, a key molecule in the integrin outside-in signaling pathway, to accumulate in the immunological synapse after NK-target cell conjugation. Our results suggest that NK cell cytotoxicity is inhibited by PD-1 engagement, which blocks lytic granule polarization to the NK cell immunological synapse with concomitant impairment of integrin outside-in signaling. This study provides novel mechanistic insights into how PD-1 inhibition disrupts NK cell function. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Priming Endothelial Cells With a Melanoma-Derived Extracellular Matrix Triggers the Activation of αvβ3/VEGFR2 Axis.

PubMed

Helal-Neto, Edward; Brandão-Costa, Renata M; Saldanha-Gama, Roberta; Ribeiro-Pereira, Cristiane; Midlej, Victor; Benchimol, Marlene; Morandi, Verônica; Barja-Fidalgo, Christina

2016-11-01

The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvβ3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Orphan nuclear receptor TR3/Nur77 improves wound healing by upregulating the expression of integrin β4.

PubMed

Niu, Gengming; Ye, Taiyang; Qin, Liuliang; Bourbon, Pierre M; Chang, Cheng; Zhao, Shengqiang; Li, Yan; Zhou, Lei; Cui, Pengfei; Rabinovitz, Issac; Mercurio, Arthur M; Zhao, Dezheng; Zeng, Huiyan

2015-01-01

Tissue repair/wound healing, in which angiogenesis plays an important role, is a critical step in many diseases including chronic wound, myocardial infarction, stroke, cancer, and inflammation. Recently, we were the first to report that orphan nuclear receptor TR3/Nur77 is a critical mediator of angiogenesis and its associated microvessel permeability. Tumor growth and angiogenesis induced by VEGF-A, histamine, and serotonin are almost completely inhibited in Nur77 knockout mice. However, it is not known whether TR3/Nur77 plays any roles in wound healing. In these studies, skin wound-healing assay was performed in 3 types of genetically modified mice having various Nur77 activities. We found that ectopic induction of Nur77 in endothelial cells of mice is sufficient to improve skin wound healing. Although skin wound healing in Nur77 knockout mice is comparable to the wild-type control mice, the process is significantly delayed in the EC-Nur77-DN mice, in which a dominant negative Nur77 mutant is inducibly and specifically expressed in mouse endothelial cells. By a loss-of-function assay, we elucidate a novel feed-forward signaling pathway, integrin β4 → PI3K → Akt → FAK, by which TR3 mediates HUVEC migration. Furthermore, TR3/Nur77 regulates the expression of integrin β4 by targeting its promoter activity. In conclusion, expression of TR3/Nur77 improves wound healing by targeting integrin β4. TR3/Nur77 is a potential candidate for proangiogenic therapy. The results further suggest that TR3/Nur77 is required for pathologic angiogenesis but not for developmental/physiologic angiogenesis and that Nur77 and its family members play a redundant role in normal skin wound healing. © FASEB.

Micro-positron emission tomography/contrast-enhanced computed tomography imaging of orthotopic pancreatic tumor-bearing mice using the αvβ₃ integrin tracer ⁶⁴Cu-labeled cyclam-RAFT-c(-RGDfK-)₄.

PubMed

Aung, Winn; Jin, Zhao-Hui; Furukawa, Takako; Claron, Michael; Boturyn, Didier; Sogawa, Chizuru; Tsuji, Atsushi B; Wakizaka, Hidekatsu; Fukumura, Toshimitsu; Fujibayashi, Yasuhisa; Dumy, Pascal; Saga, Tsuneo

2013-09-01

The purpose of this study was to develop a clinically relevant orthotopic xenotransplantation model of pancreatic cancer and to perform a preclinical evaluation of a new positron emission tomography (PET) imaging probe, ⁶⁴Cu-labeled cyclam-RAFT-c(-RGDfK-)₄ peptide (⁶⁴Cu-RAFT-RGD), using this model. Varying degrees of αvβ₃ integrin expression in several human pancreatic cancer cell lines were examined by flow cytometry and Western blotting. The cell line BxPC-3, which is stably transfected with a red fluorescence protein (RFP), was used for surgical orthotopic implantation. Orthotopic xenograft was established in the pancreas of recipient nude mice. An in vivo probe biodistribution and receptor blocking study, preclinical PET imaging coregistered with contrast-enhanced computed tomography (CECT) comparing ⁶⁴Cu-RAFT-RGD and ¹⁸F-fluoro-2-deoxy-d-glucose (¹⁸F-FDG) accumulation in tumor, postimaging autoradiography, and histologic and immunohistochemical examinations were done. Biodistribution evaluation with a blocking study confirmed that efficient binding of probe to tumor is highly αvβ₃ integrin specific. ⁶⁴Cu-RAFT-RGD PET combined with CECT provided for precise and easy detection of cancer lesions. Autoradiography, histologic, and immunohistochemical examinations confirmed the accumulation of ⁶⁴Cu-RAFT-RGD in tumor versus nontumor tissues. In comparative PET studies, ⁶⁴Cu-RAFT-RGD accumulation provided better tumor contrast to background than ¹⁸F-FDG. Our results suggest that ⁶⁴Cu-RAFT-RGD PET imaging is potentially applicable for the diagnosis of αvβ₃ integrin-expressing pancreatic tumors.

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

PubMed

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

2016-04-20

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

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

PubMed Central

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

2016-01-01

Background: Glaucoma is a progressive optic neuropathy characterized by degeneration of neurons due to loss of retinal ganglion cells (RGCs). High intraocular pressure (HIOP), the main risk factor, causes the optic nerve damage. However, the precise mechanism of HIOP-induced RGC death is not yet completely understood. This study was conducted to determine apoptosis of RGC-5 cells induced by elevated hydrostatic pressures, explore whether laminin is associated with apoptosis under pressure, whether laminin can protect RGCs from apoptosis and affirm the mechanism that regulates the process of RGCs survival. Methods: RGC-5 cells were exposed to 0, 20, 40, and 60 mmHg in a pressurized incubator for 6, 12, and 24 h, respectively. The effect of elevated hydrostatic pressure on RGC-5 cells was measured by Annexin V-fluorescein isothiocyanate/propidium iodide staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Western blotting of cleaved caspase-3 protein. Location and expression of laminin were detected by immunofluorescence. The expression of β1-integrin, phosphorylation of focal adhesion kinase (FAK) and protein kinase B (PKB, or AKT) were investigated with real-time polymerase chain reaction and Western blotting analysis. Results: Elevated hydrostatic pressure induced apoptosis in cultured RGC-5 cells. Pressure with 40 mmHg for 24 h induced a maximum apoptosis. Laminin was declined in RGC-5 cells after exposing to 40 mmHg for 24 h. After pretreating with laminin, RGC-5 cells survived from elevated pressure. Furthermore, β1-integrin and phosphorylation of FAK and AKT were increased compared to 40 mmHg group. Conclusions: The data show apoptosis tendency of RGC-5 cells with elevated hydrostatic pressure. Laminin can protect RGC-5 cells against high pressure via β1-integrin/FAK/AKT signaling pathway. These results suggest that the decreased laminin of RGC-5 cells might be responsible for apoptosis induced by elevated hydrostatic pressure, and laminin or activating β1-integrin/FAK/AKT pathway might be potential treatments to prevent RGC loss in glaucomatous optic neuropathy. PMID:27064044

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. The effect on cultured endothelial cells and the rapid decrease after delivery suggests the presence of a circulating serum factor which increases endothelial cell [Ca2+]i and enhances adhesion molecule expression.

Ofloxacin induces apoptosis via β1 integrin-EGFR-Rac1-Nox2 pathway in microencapsulated chondrocytes

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

Sheng, Zhi-Guo; Huang, Wei; Liu, Yu-Xiang

2013-02-15

Quinolones (QNs)-induced arthropathy is an important toxic side-effect in immature animals leading to the restriction of their therapeutic use in pediatrics. Ofloxacin, a typical QN, was found to induce the chondrocytes apoptosis in the early phase (12–48 h) of arthropathy in our previous study. However, the exact mechanism(s) is unclear. Microencapsulated juvenile rabbit joint chondrocytes, a three-dimensional culture system, is utilized to perform the present study. Ofloxacin, at a therapeutically relevant concentration (10 μg/ml), disturbs the interaction between β1 integrin and activated intracellular signaling proteins at 12 h, which is inhibited when supplementing Mg{sup 2+}. Intracellular reactive oxygen species (ROS)more » significantly increases in a time-dependent manner after exposure to ofloxacin for 12–48 h. Furthermore, ofloxacin markedly enhances the level of activated Rac1 and epidermal growth factor receptor (EGFR) phosphorylation, and its inhibition in turn reduces the ROS production, apoptosis and Rac1 activation. Silencing Nox2, Rac1 or supplementing Mg{sup 2+} inhibits ROS accumulation, apoptosis occurrence and EGFR phosphorylation induced by ofloxacin. However, depletion of Nox2, Rac1 and inhibition of EGFR do not affect ofloxacin-mediated loss of interaction between β1 integrin and activated intracellular signaling proteins. In addition, ofloxacin also induces Vav2 phosphorylation, which is markedly suppressed after inactivating EGFR or supplementing Mg{sup 2+}. These results suggest that ofloxacin causes Nox2-mediated intracellular ROS production by disrupting the β1 integrin function and then activating the EGFR-Vav2-Rac1 pathway, finally resulting in apoptosis within 12–48 h exposure. The present study provides a novel insight regarding the potential role of Nox-driven ROS in QNs-induced arthropathy. - Highlights: ► Ofloxacin induces Nox2-driven ROS in encapsulated chondrocyte at 12–48 h. ► Ofloxacin stimulates ROS production via the β1 integrin-EGFR-Vav2-Rac1 pathway. ► Ofloxacin induces ROS-dependent apoptosis in encapsulated chondrocyte at 12–48 h.« less

Significance of Epidermal Growth Factor Receptor in the Radiation Resistance of Glioblastoma Tumors

NASA Astrophysics Data System (ADS)

Petrás, Miklós; Lajtos, Tamás; Pintye, Éva; Feuerstein, Burt G.; Szöllősi, János; Vereb, György

2008-12-01

In the United States, a dramatically increased incidence and mortality of brain tumors have been observed over the past decades. Of the ˜44 thousand new cases of primary malignant and benign brain tumors diagnosed per year, high grade astrocytomas or multiform glioblastomas show particularly bad prognosis in spite of therapeutic developments. Current management of multiform glioblastoma includes the most extensive surgical resection possible, followed by adjuvant radio- and chemotherapy. However, treatment is frequently hampered by decreased radiosensitivity of the tumor. Recent studies revealed that subpopulations of glioblastoma cells show amplified checkpoint activation of the cell cycle upon ionizing radiation, which induces overactivation of DNA repair processes and leads to maintained proliferation rate as well as clinically observed radioresistance and recurrence of the tumor over time. In addition, overexpression of some transmembrane receptors has also been implicated in radioresistance. However, the role of the overexpressed proteins can only be interpreted reliably if their multi-faceted molecular interactions are properly characterized. Thus, based on recent evidence for the functional crosstalk between certain cell adhesion molecules and receptor tyrosine kinases, we have examined the molecular interactions of the receptor tyrosine kinase EGFR and the cell adhesion molecule β1-integrin using flow cytometric and microscopic fluorescence resosnance energy transfer (FRET) measurements on two cellular model systems showing similar expression patterns to low and high grade astrocytomas. On the one hand, U251 glioblastoma clones established by introducing varying amounts of extra chromosome 7 into the cells, and on the other hand stable, high and low EGFR expressing transfenctant U251 NCI sublines were investigated. The results revealed that increased EGFR and β1-integrin expression levels correlate with stronger EGFR—β1-integrin heteroassociation, while concurrently the EGFR homoassociation is decreased, suggesting that β1-integrins may dynamically modulate the homoassociation state of EGFR receptors. This functional relationship may play an important role in decreasing radiosensitivity and tumor progression, especially since the EGFR—β1-integrin molecular interaction appears to promote radioresistance via the Akt pathway.

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 integrin β3 recapitulates the sex-dependent neurochemical and behavioral attributes of ASD. Using state-of-the-art techniques, we show that presynaptic 5-HT function is altered in these mice, and that the localization of 5-HT transporters to specific compartments within the synapse, disrupted by the integrin β3 Pro33 mutation, is critical for appropriate reuptake of 5-HT. Our studies provide fundamental insight into the genetic network regulating 5-HT neurotransmission in the CNS that is also associated with ASD risk. Copyright © 2017 the authors 0270-6474/17/3711272-14$15.00/0.

Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells

NASA Technical Reports Server (NTRS)

D'Alonzo, Richard C.; Kowalski, Aaron J.; Denhardt, David T.; Nickols, G. Allen; Partridge, Nicola C.

2002-01-01

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

Active elastic dimers: cells moving on rigid tracks.

PubMed

Lopez, J H; Das, Moumita; Schwarz, J M

2014-09-01

Experiments suggest that the migration of some cells in the three-dimensional extracellular matrix bears strong resemblance to one-dimensional cell migration. Motivated by this observation, we construct and study a minimal one-dimensional model cell made of two beads and an active spring moving along a rigid track. The active spring models the stress fibers with their myosin-driven contractility and α-actinin-driven extendability, while the friction coefficients of the two beads describe the catch and slip-bond behaviors of the integrins in focal adhesions. In the absence of active noise, net motion arises from an interplay between active contractility (and passive extendability) of the stress fibers and an asymmetry between the front and back of the cell due to catch-bond behavior of integrins at the front of the cell and slip-bond behavior of integrins at the back. We obtain reasonable cell speeds with independently estimated parameters. We also study the effects of hysteresis in the active spring, due to catch-bond behavior and the dynamics of cross linking, and the addition of active noise on the motion of the cell. Our model highlights the role of α-actinin in three-dimensional cell motility and does not require Arp2/3 actin filament nucleation for net motion.

Differential Dynamics of Platelet Contact and Spreading

PubMed Central

Lee, Dooyoung; Fong, Karen P.; King, Michael R.; Brass, Lawrence F.; Hammer, Daniel A.

2012-01-01

Platelet spreading is critical for hemostatic plug formation and thrombosis. However, the detailed dynamics of platelet spreading as a function of receptor-ligand adhesive interactions has not been thoroughly investigated. Using reflection interference contrast microscopy, we found that both adhesive interactions and PAR4 activation affect the dynamics of platelet membrane contact formation during spreading. The initial growth of close contact area during spreading was controlled by the combination of different immobilized ligands or PAR4 activation on fibrinogen, whereas the growth of the total area of spreading was independent of adhesion type and PAR4 signaling. We found that filopodia extend to their maximal length and then contract over time; and that filopodial protrusion and expansion were affected by PAR4 signaling. Upon PAR4 activation, the integrin αIIbβ3 mediated close contact to fibrinogen substrata and led to the formation of ringlike patterns in the platelet contact zone. A systematic study of platelet spreading of GPVI-, α2-, or β3-deficient platelets on collagen or fibrinogen suggests the integrin α2 is indispensable for spreading on collagen. The platelet collagen receptors GPVI and α2 regulate integrin αIIbβ3-mediated platelet spreading on fibrinogen. This work elucidates quantitatively how receptor-ligand adhesion and biochemical signals synergistically control platelet spreading. PMID:22325269

Adhesion to the extracellular matrix is positively regulated by retinoic acid in HepG2 cells.

PubMed

Massimi, Mara; Devirgiliis, Laura Conti

2007-02-01

In this work, we aimed to investigate the possible modulation of cell-matrix interactions by retinoic acid (RA), in view of the well-known role of the extracellular matrix (ECM) and integrins in hepatocyte differentiation and proliferation. For this purpose, we analysed the adhesion ability of HepG2 cells on different substrates in the presence and absence of RA evaluating both the expression and cellular localisation of major proteins involved in focal contacts, using Western blot and confocal microscopy. A positive and substrate-dependent effect of RA on cell-matrix adhesion was observed after long-term culture. The increased adhesiveness in the treated cells was accompanied by an enhanced expression of beta1 and alpha3 integrin subunits, together with a redistribution of beta1 receptors clustered at the basal surface. In contrast, the levels of focal adhesion kinase (FAK), paxillin and alpha-actinin were unchanged, as was the phosphorylation state of FAK. Nonetheless, a stronger association between beta1 integrin and intracytoplasmatic proteins of focal contacts was observed in coimmunoprecipitation experiments after RA treatment, suggesting improved connection with the actin cytoskeleton. These results are consistent with previously described antiproliferative and differentiative effects of RA on transformed hepatocytes, and confirm the hypothesis of a direct influence of RA on specific adhesion molecules.

Altered expression of extracellular matrix molecules and their receptors in chronic pancreatitis and pancreatic adenocarcinoma in comparison with normal pancreas.

PubMed

Shimoyama, S; Gansauge, F; Gansauge, S; Oohara, T; Beger, H G

1995-12-01

The aim of this study was to elucidate the expression and distribution patterns of both integrins and extracellular matrix (ECM) molecules in chronic pancreatitis (CP) and pancreatic adenocarcinoma (PC) compared with normal pancreas (NP). Expression of nine alpha-subunits (alpha 2-alpha 6, alpha V, alpha L, alpha M, and alpha X), four beta-subunits (beta 1, beta 3-beta 5), and four ECM molecules (type IV collagen, laminin, fibronectin, and vitronectin) was investigated immunohistochemically. In CP, all integrins except alpha V showed nearly the same staining patterns compared with NP. Some acinar cells in CP expressed alpha V. Whereas alpha 2, alpha 3, and alpha 6 expression was stronger and diffuse, no alpha 5 expression was seen in PC. Basement membrane (BM) showed continuous staining in CP, whereas it showed discontinuous/absent staining in PC with antitype IV collagen, laminin, and vitronectin antibodies. Some carcinoma cells showed reverse correlation between alpha 2, alpha 3, and alpha 6 expression and type IV collagen and laminin expression. Fibronectin showed diffuse stromal expression in CP and PC. Some acinar cells or duct cells in CP carcinoma cells in PC showed intracellular VN expression. These results suggest that these integrins and ECM molecules are involved in inflammatory and malignant processes in pancreas.

Equine herpesvirus 1 entry via endocytosis is facilitated by alphaV integrins and an RSD motif in glycoprotein D.

PubMed

Van de Walle, Gerlinde R; Peters, Sarah T; VanderVen, Brian C; O'Callaghan, Dennis J; Osterrieder, Nikolaus

2008-12-01

Equine herpesvirus 1 (EHV-1) is a member of the Alphaherpesvirinae, and its broad tissue tropism suggests that EHV-1 may use multiple receptors to initiate virus entry. EHV-1 entry was thought to occur exclusively through fusion at the plasma membrane, but recently entry via the endocytic/phagocytic pathway was reported for Chinese hamster ovary cells (CHO-K1 cells). Here we show that cellular integrins, and more specifically those recognizing RGD motifs such as alphaVbeta5, are important during the early steps of EHV-1 entry via endocytosis in CHO-K1 cells. Moreover, mutational analysis revealed that an RSD motif in the EHV-1 envelope glycoprotein D (gD) is critical for entry via endocytosis. In addition, we show that EHV-1 enters peripheral blood mononuclear cells predominantly via the endocytic pathway, whereas in equine endothelial cells entry occurs mainly via fusion at the plasma membrane. Taken together, the data in this study provide evidence that EHV-1 entry via endocytosis is triggered by the interaction between cellular integrins and the RSD motif present in gD and, moreover, that EHV-1 uses different cellular entry pathways to infect important target cell populations of its natural host.

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

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

Accurate evaluation of the biological performance of biomaterials requires the correct assessment of their native-like cell ligation properties. However, cell attachment studies often overlook the details of the substrate-cell binding mechanisms, be they integrin-mediated or non-specific, and ignore the class- and species-specificities of the cell adhesion receptor involved. In this work we have used different collagen (Col) substrates (fibrillar collagens I, II and III and network-forming Col IV), containing different affinity cell-recognition motifs, to establish the influence of the receptor identity and species-specificity on collagen-cell interactive properties. Receptor expression was varied by using cells of different origin, or transfecting collagen-binding integrins into integrin-null cells. These include mouse C2C12 myoblasts transfected with human α1, α2, α10 or α11; human fibrosarcoma HT1080 cells which constitutively express only human α2β1, and rat glioma Rugli cells, with only rat α1β1. Using these lines, the nature of integrin binding sites was studied in order to delineate the bioactivity of different collagen substrates. Integrin ligation was studied on collagen coatings alongside synthetic (GFOGER/GLOGEN) and Toolkit (Col II-28/Col III-7) triple-helical peptides to evaluate (1) their affinity towards different integrins and (2) to confirm the activity of the inserted integrin in the transfected cells. Thin films of dermal and tendon Col I were used to evaluate the influence of the carbodiimide (EDC)-based treatment on the cellular response on Col of different origin. The results showed that the binding properties of transfected C2C12 cells to collagens depend on the identity of inserted integrin. Similar ligation characteristics were observed using α1+ and α10+ cells, but these were distinct from the similar binding features of α2+ and α11+ cells. Recombinant human and rat-α1 I domain binding to collagens and peptides correlated with 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.

Quantitative Protein Sulfenic Acid Analysis Identifies Platelet Releasate-Induced Activation of Integrin β2 on Monocytes via NADPH Oxidase.

PubMed

Li, Ru; Klockenbusch, Cordula; Lin, Liwen; Jiang, Honghui; Lin, Shujun; Kast, Juergen

2016-12-02

Physiological stimuli such as thrombin, or pathological stimuli such as lysophosphatidic acid (LPA), activate platelets. The activated platelets bind to monocytes through P-selectin-PSGL-1 interactions but also release the contents of their granules, commonly called "platelet releasate". It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS is considered to be a potential regulator of protein function. In a previous study, we used THP-1 monocytic cells exposed to LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that are influenced by reversible cysteine oxidation. To gain a better understanding of the redox regulation of monocytes in atherosclerosis, we have now altered the modified biotin switch to selectively quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite as reducing agent in the modified biotin switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites highlighted the relevant, previously missed biological process of monocyte transendothelial migration, which included integrin β 2 . Flow cytometry validated the activation of LFA-1 (α L β 2 ) and Mac-1 (α M β 2 ), two subfamilies of integrin β 2 complexes, on human primary monocytes following platelet releasate treatment. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Production of ROS and activation of LFA-1 in human primary monocytes were independent of P-selectin-PSGL-1 interaction. Our results proved the modified biotin switch assay to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.

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

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

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

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

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.

Inhibition of untransformed prostaglandin H(2) production and stretch-induced contraction of rabbit pulmonary arteries by indoxam, a selective secretory phospholipase A(2) inhibitor.

PubMed

Tanabe, Yoshiyuki; Saito, Maki; Morikawa, Yuki; Kamataki, Akihisa; Sawai, Takashi; Hirose, Masamichi; Nakayama, Koichi

2011-01-01

Involvement of secretory phospholipase A(2) (sPLA(2)) in the stretch-induced production of untransformed prostaglandin H(2) (PGH(2)) in the endothelium of rabbit pulmonary arteries was investigated. The stretch-induced contraction was significantly inhibited by indoxam, a selective inhibitor for sPLA(2), and NS-398, a selective inhibitor for cyclooxygenase-2 (COX-2). Indoxam inhibited the RGD-sensitive-integrin-independent production of untransformed PGH(2), but did not affect the RGD-sensitive-integrin-dependent production of thromboxane A(2) (TXA(2)). These results suggest that the stretch-induced contraction and untransformed PGH(2) production was mediated by sPLA(2)-COX-2 pathway, making it a new possible target for pharmacological intervention of pulmonary artery contractility.