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Integrin Activation (integrin + activation)

Distribution by Scientific Domains

Medical Sciences	93%

Selected Abstracts

Involvement of the ,3 E749ATSTFTN756 region in stabilizing integrin ,IIb,3 -ligand interaction

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2003
P. E. M. H. Litjens
Summary., Platelet integrin ,IIb,3 must be activated via intracellular mechanisms before it binds soluble ligands, and it is thought to be activated at its extracellular site by surface-bound ligands. Integrin activation is associated with rearrangement of the cytoskeleton and phosphorylation of proteins that become localized in focal contacts. In these processes, the cytoplasmic tail of the ,-subunit plays a central role. We introduced peptides homologous to the E749ATSTFTN756 domain (E,N peptide) and the T755NITYRGT762 domain (T,T peptide) of ,3 in streptolysin O-permeabilized platelets and analyzed the initial interaction with soluble fibronectin, fibrinogen and PAC-1 after stimulation with thrombin. E,N peptide left the initial binding of fibronectin intact but interfered with stable receptor occupancy. E,N peptide also inhibited fibrinogen binding, thereby reducing the formation of large aggregates. Strikingly, E,N peptide did not disturb the binding of PAC-1, which is known to reflect activation of the integrin. E,N peptide also inhibited tyrosine phosphorylation of focal adhesion kinase, a response known to be dependent on ,IIb,3. T,T peptide did not affect these processes. In a model for outside-in integrin activation, E,N peptide disrupted the binding of CHO cells expressing ,IIb,3 to surface-bound ligand. Again, T,T peptide had no effect. We conclude that the E749ATSTFTN756 region of the ,3 -tail stabilizes the binding of soluble and surface-bound ligand to integrin ,IIb,3 via a mechanism that involves the phosphorylation of FAK. [source]

SLIC-1/sorting nexin,20: A novel sorting nexin that directs subcellular distribution of PSGL-1

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2008
Ulrich
Abstract P-Selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like glycoprotein expressed on the surface of leukocytes that serves as the major ligand for the selectin family of adhesion molecules and functions in leukocyte tethering and rolling on activated endothelium and platelets. Previous studies have implicated the highly conserved cytoplasmic domain of PSGL-1 in regulating outside-in signaling of integrin activation. However, molecules that physically and functionally interact with this domain are not completely defined. Using a yeast two-hybrid screen with the cytoplasmic domain of PSGL-1 as bait, a novel protein designated selectin ligand interactor cytoplasmic-1 (SLIC-1) was isolated. Computer-based homology search revealed that SLIC-1 was the human orthologue for the previously identified mouse sorting nexin,20. Direct interaction between SLIC-1 and PSGL-1 was specific as indicated by co-immunoprecipitation and motif mapping. Colocalization experiments demonstrated that SLIC-1 contains a Phox homology domain that binds phosphoinositides and targets the PSGL-1/SLIC-1 complex to endosomes. Deficiency in the murine homologue of SLIC-1 did not modulate PSGL-1-dependent signaling nor alter neutrophil adhesion through PSGL-1. We conclude that SLIC-1 serves as a sorting molecule that cycles PSGL-1 into endosomes with no impact on leukocyte recruitment. [source]

Mechanically Strained Cells of the Osteoblast Lineage Organize Their Extracellular Matrix Through Unique Sites of ,V,3 -Integrin Expression

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2000
Magdalena Wozniak
Abstract Bone cells transduce mechanical signals into anabolic biochemical responses. However, the mechanisms of mechanotransduction are unknown. To address this issue, we performed studies in primary cells of the human osteoblast lineage grown on collagen/vitronectin-coated supports. We discovered that mechanical strain stimulated a redistribution of the ,v,3 -integrin to irregular plaque-like areas at the cell-extracellular matrix surface. Proteins involved in integrin-matrix interactions in focal adhesions, vinculin and talin, did not localize to the plaque-like areas of ,v,3 -expression, but signaling molecules such as focal adhesion kinase (FAK) did. Mechanical strain increased the number and size of the plaques defined by surface expression of ,v,3 -integrin. Osteopontin was secreted as a cross-linked macromolecular complex, likely through the action of tissue transglutaminase that also was found in the plaques of ,v,3 -integrin cell-matrix interaction. Mechanical strain increased mineralization of the extracellular matrix that developed in these plaques in ,v,3 -integrin-dependent manner. Because the plaque-like areas of cell-matrix interaction exhibit macromolecular assembly and mineralization, we conclude that they may represent subcellular domains of bone formation and that ,v,3 -integrin activation represents one mechanism by which mechanical strain stimulates bone formation. [source]

Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2007
M. G. TOMLINSON
Summary. Background:,Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcR,-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). Objective:,To determine why GPVI,FcR, signals poorly, or not at all, in response to collagen in hematopoietic cell lines, despite robust responses to the GPVI-reactive snake venom toxin convulxin. Methods and results:,Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay, a sensitive readout for sustained ITAM signaling, we demonstrate collagen-induced GPVI,FcR, signaling in hematopoietic cell lines. This is accompanied by relatively weak but sustained protein tyrosine phosphorylation, in contrast to the stronger but transient response to convulxin. Sustained signaling by collagen is also observed in platelets and is necessary for the maintenance of spreading on collagen. Finally, in cell lines, the inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), which is not expressed on platelets but is present on most hematopoietic cells, inhibits GPVI responses to collagen but not convulxin. Conclusion:,The inability of previous studies to readily detect GPVI collagen signaling in cell lines is probably because of the weak but sustained nature of the signal and the presence of the inhibitory collagen receptor LAIR-1. In platelets, we propose that GPVI,FcR, has evolved to transmit sustained signals in order to maintain spreading over several hours, as well as facilitating rapid activation through release of feedback agonists and integrin activation. The establishment of a cell line NFAT assay will facilitate the molecular dissection of GPVI signaling and the identification of GPVI antagonists in drug discovery. [source]

Platelet integrin ,IIb,3: activation mechanisms

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2007
Y.-Q. MA
Summary., Integrin ,IIb,3 plays a critical role in platelet aggregation, a central response in hemostasis and thrombosis. This function of ,IIb,3 depends upon a transition from a resting to an activated state such that it acquires the capacity to bind soluble ligands. Diverse platelet agonists alter the cytoplasmic domain of ,IIb,3 and initiate a conformational change that traverses the transmembrane region and ultimately triggers rearrangements in the extracellular domain to permit ligand binding. The membrane-proximal regions of ,IIb and ,3 cytoplasmic tails, together with the transmembrane segments of the subunits, contact each other to form a complex which restrains the integrin in the resting state. It is unclasping of this complex that induces integrin activation. This clasping/unclasping process is influenced by multiple cytoplasmic tail binding partners. Among them, talin appears to be a critical trigger of ,IIb,3 activation, but other binding partners, which function as activators or suppressors, are likely to act as co-regulators of integrin activation. [source]

A novel functional role for the highly conserved , -subunit KVGFFKR motif distinct from integrin ,IIb,3 activation processes

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2006
K. AYLWARD
Summary.,Background: The highly conserved integrin , -subunit membrane-proximal motif KVGFFKR plays a decisive role in modulating the activation of integrin ,IIb,3. Previously, we have shown that a platelet permeable palmityl (pal)-peptide with this seven amino acid sequence can directly activate ,IIb,3 leading to platelet aggregation. Objectives: To investigate further the role of the KVGFFKR motif in integrin ,IIb,3 function. Methods: We used two sequence-specific complementary model systems, palmityl pal-peptides in platelets, and mutant ,IIb,3 -expressing Chinese Hamster Ovary (CHO) cell lines. Results: In platelets we show that the two phenylalanine amino acids in pal-KVGFFKR (pal-FF) peptide are critical for stimulating platelet aggregation. Pal-FF peptide treatment of platelets also gives rise to a tyrosine phosphorylation signal despite the presence of inhibitors of fibrinogen binding. In CHO cells, a double alanine substitution, ,IIb(F992A, F993A),3, induces constitutive integrin activation but prevents actin stress fiber formation upon adhesion to fibrinogen, suggesting that ,IIb,3 -mediated cytoskeletal reorganization is also dependent on F992 and F993. This further highlights a critical role for the two phenylalanine residues in both of these ,IIb,3 -mediated processes. Conclusion: In addition to regulating integrin ,IIb,3 activation state, the KVGFFKR motif also influences cytoskeletal reorganization. This activity is critically determined by F992 and F993 within the seven amino acid sequence. [source]

Involvement of the ,3 E749ATSTFTN756 region in stabilizing integrin ,IIb,3 -ligand interaction

Chemokine Signaling to Lymphocyte Integrins Under Shear Flow

MICROCIRCULATION, Issue 1 2009
RONEN ALON
ABSTRACT The arrest of lymphocytes at target vascular sites depends on the rapid activation of their integrins by specialized endothelial chemokines. For over a decade, the mechanisms by which these chemokines trigger initial integrin-mediated adhesiveness and subsequent adhesion strengthening and crawling over endothelial surfaces have been dissected in vitro using flow chamber setups. These studies revealed that lymph node chemokines and subsets of inflammatory chemokines, collectively termed "arrest chemokines," can trigger the fastest measurable inside-out integrin activation events. Recent studies indicate that shear forces applied on lymphocytes are instrumental in these rapid activation processes. Different GTPases have been implicated in these activation processes. As these enzymes contribute to successive integrin activation and redistribution processes in both early and prolonged contacts there is a growing need to dissect in vitro and validate in vivo specific signaling routes involved in early and late integrin activation events controlling lymphocyte arrest and their subsequent crawling to sites of diapedesis. In this article, we present an overview of both early and recent shear-flow studies of integrin activation in lymphocytes and discuss future perspectives of integrin activation research in vitro and in vivo. [source]

The p110, Isoform of PI3K Differentially Regulates ,1 and ,2 Integrin-Mediated Monocyte Adhesion and Spreading and Modulates Diapedesis

MICROCIRCULATION, Issue 6 2006
ALEXANDER M. FERREIRA
ABSTRACT Objective: Leukocyte diapedesis is misregulated in inflammatory disease and depends on the binding of monocytic LFA-1 and VLA-4 to endothelial ICAM-1 and VCAM-1, respectively. The authors hypothesized that these different molecular interactions elicit specific signaling cascades within monocytes regulating specific steps in adhesion, motility, and diapedesis. Methods: The authors employed the PI3K p110, catalytic subunit specific inhibitor IC87114 (2 , M) and the broad-spectrum PI3K inhibitory agents LY294002 (50 , M) and wortmannin (100 nM), to examine the role of PI3K, in monocyte diapedesis through endothelial monolayers and its role in monocyte adhesion and spreading upon carpets of ICAM-1 or VCAM-1. They further explored the effects of PI3K, inhibition on the activation state of , 1 and , 2 integrins with immunocytochemistry and flow cytometry. Results: In human peripheral blood monocytes IC87114 was as effective as wortmannin and LY294002 at inhibiting diapedesis, however, in THP-1 cells LY294002 and wortmannin caused a 5-fold reduction in diapedesis, while IC87114 only decreased diapedesis 2-fold. PI3K, activity was specifically required for THP-1 cell adhesion and spreading on VCAM-1, but not on ICAM-1 protein substrates. Flow cytometric analysis demonstrated that PI3K, inhibition decreased the amount of conformationally active , 1-integrins, while having no effect on the prevalence of conformationally active , 2-integrins expressed on the cell surface. In addition, PI3K, inhibition resulted in a 4-fold decrease in the activation state of Rac-1 and Cdc42. Conclusions: These results demonstrate the specific necessity of PI3K, in regulating monocytic integrin activation and the general role of PI3K signaling during diapedesis, implicating PI3K as a target for therapeutic intervention. [source]

Possible Steps Involved in the Transition to Stationary Adhesion of Rolling Neutrophils: A Brief Review

MICROCIRCULATION, Issue 6 2000
C. WAYNE SMITHArticle first published online: 26 JAN 2010
ABSTRACT The transition from rolling to firm adhesion is a phenomenon frequently observed when neutrophils are interacting with activated endothelium in vitro or in vivo under physiologically relevant shear stress. The mechanisms leading to this activation are poorly understood, though selectin-dependent tethering and CD18-integrin-dependent adhesion are known to be involved. This transition may involve a sequence of interactions that trigger sufficient integrin activation to allow cell arrest under flow. Recent evidence is reviewed in support of the concept that integrin (Mac-1 and LFA-1) activation results from signaling that occurs through selectin binding, chemotactic factor stimulation, and, possibly, LFA-1 binding. [source]

Glucocorticoids increase ,5 integrin expression and adhesion of synovial fibroblasts but inhibit ERK signaling, migration, and cartilage invasion

ARTHRITIS & RHEUMATISM, Issue 12 2009
Torsten Lowin
Objective In rheumatoid arthritis (RA), integrins mediate cell adhesion, migration, and invasion, and their expression is regulated by cytokines and growth factors. The aim of this study was to investigate whether hormones such as cortisol or other steroids can influence integrin expression and function in the synovial cells of patients with RA. Methods We performed immunofluorescence and fluorescence-activated cell sorting analyses to quantify surface integrin levels. Adhesion and migration assays were performed to study the function of synovial fibroblasts (SFs). ERK activation was measured by cellular activation of a signaling enzyme-linked immunosorbent assay. Invasion of SFs into cartilage was determined in the SCID mouse coimplantation model of RA in vivo. Results In RA, expression of integrin subunits ,5, ,v, and ,1 was higher at the site of invasion compared with the sublining zone. Testosterone and 17,-estradiol had no influence on integrin levels, but cortisol up-regulated expression of the ,5 subunit in a time-dependent and dose-dependent manner. In addition, cortisol increased the adhesion of SFs to fibronectin and inhibited ERK signaling upon integrin activation or upon stimulation with tumor necrosis factor. Small interfering RNA or a neutralizing antibody to ,5 integrin increased SF migration, indicating that up-regulated ,5 integrin is responsible for an immobile phenotype. In addition, in the SCID mouse model, SF invasion into cartilage was attenuated by glucocorticoid treatment in vivo. Conclusion Glucocorticoids increase integrin expression and the adhesion of cells to fibronectin, inhibit ERK signaling, and down-regulate the invasiveness of SFs in vivo. This study demonstrates that an important antiinflammatory aspect of glucocorticoids is regulating the expression and function of ,5 integrin. [source]

Targeted mast cell silencing protects against joint destruction and angiogenesis in experimental arthritis in mice

ARTHRITIS & RHEUMATISM, Issue 6 2007
Manfred Kneilling
Objective Induction of arthritis with autoantibodies against glucose-6-phosphate isomerase (GPI) is entirely independent of T cells and B cells but is strictly dependent on the presence of mast cells. Here, we used this disease model to analyze whether exclusive intraarticular mast cell reconstitution is sufficient for disease induction and whether targeted mast cell silencing can prevent neoangiogenesis and joint destruction, 2 hallmarks of rheumatoid arthritis. Methods Ankle swelling and clinical index scores were determined after injection of either K/BxN mouse,derived serum or control serum in wild-type Kit+/Kit+ mice, congenic mast cell,deficient KitW/KitW - v mice, or mast cell,deficient KitW/KitW - v mice reconstituted with mast cells, either by intraperitoneal or selective intraarticular injection. Angiogenesis was quantified in vivo by measuring activated ,v,3 integrin using 18F,galacto-RGD and positron emission tomography. In addition, staining of joint tissue with hematoxylin and eosin, Giemsa, ,3, and ,-actin was performed. The effect of mast cell stabilization by treatment with cromolyn or salbutamol was investigated in C57BL/6 or BALB/c mice. Results Comparing wild-type mice, mast cell,deficient KitW/KitW - v mice, and mast cell,reconstituted KitW/KitW - v mice, we first showed that intraarticular and intraperitoneal mast cell engraftment fully restores susceptibility to antibody-induced arthritis, angiogenesis, and ,v,3 integrin activation. Importantly, selective mast cell silencing with either salbutamol or cromolyn prevented ,v,3 integrin activation, angiogenesis, and joint destruction. Conclusion Mast cell engraftment fully restores susceptibility to ,v,3 integrin activation, angiogenesis, and joint destruction in GPI antibody,induced arthritis. Importantly, selective mast cell stabilization prevents ,v,3 integrin activation, angiogenesis, and joint destruction. [source]

R-Ras promotes tumor growth of cervical epithelial cells

CANCER, Issue 3 2003
Héctor Rincón-Arano B.S.
Abstract BACKGROUND R-Ras is 55% identical to H-Ras. However, these two oncogenes seem to have different tumor-transforming potential. R-Ras induced cell transformation in fibroblasts but not in other cell types. R-Ras also reportedly induces a more invasive phenotype in breast epithelial cells through integrin activation. The authors studied the mechanisms whereby R-Ras induces a malignant phenotype. METHODS Dominant negative (R-Ras43N) and constitutively active (R-Ras87L) mutants of R-Ras were stably transfected into human cervical epithelium C33A cells. Transfected cells were analyzed for adhesion, cell spreading, migration, and growth in culture and in nude mice. The activity of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-K) also was determined by Western blot analysis and by in vitro kinase assays. RESULTS R-Ras87L-transfected cells, but not R-Ras43 N-transfected cells, had a higher growth rate in nude mice and in culture compared with control cells. None of the transfected C33A cells showed an increase in cell adhesion to fibronectin or collagen I, nor did they show an increment of ,1 integrin affinity. However, cells that expressed R-Ras87L, but not cells that expressed R-Ras 43N, presented a marked increase in cell spreading and migration through collagen-coated membranes. Increases in cell proliferation, spreading, and migration induced by R-Ras87L were inhibited by the PI 3-K inhibitor LY294002. In addition, PI 3-K activity, but not ERK activity, was increased only in cells that expressed R-Ras87L. CONCLUSIONS These data suggest that the oncogene R-Ras promotes tumor growth of cervical epithelial cells and increases their migration potential over collagen through a pathway that involves PI 3-K. Cancer 2003;97:575,85. © 2003 American Cancer Society. DOI 10.1002/cncr.11093 [source]

Immune synapses formed with measles virus-infected dendritic cells are unstable and fail to sustain T cell activation

CELLULAR MICROBIOLOGY, Issue 8 2007
Yoanna Shishkova
Summary Interaction with dendritic cells (DCs) is considered as central to immunosuppression induced by viruses, including measles virus (MV). Commonly, viral infection of DCs abrogates their ability to promote T cell expansion, yet underlying mechanisms at a cellular level are undefined. We found that MV-infected DCs only subtly differed from LPS-matured with regard to integrin activation, acquisition of a migratory phenotype and motility. Similarly, the organization of MV-DC/T cell interfaces was consistent with that of functional immune synapses with regard to CD3 clustering and MHC class II surface recruitment. The majority of MV-DC/T cell conjugates was, however, unstable and only promoted abortive T cell activation. Thus, MV-infected DCs retain activities required for initiating, but not sustaining T cell conjugation and activation. This is partially rescued if surface expression of the MV glycoproteins on DCs is abolished by infection with a recombinant MV encoding VSV G protein instead, indicating that these contribute directly to synapse destabilization and thereby act as effectors of T cell inhibition. [source]