alphadbeta2 integrin is a ligand for vascular cell adhesion molecule-1.
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Animals↗
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Mesenchymal progenitor cells communicate via alpha and beta integrins with a three-dimensional collagen type I matrix.
BACKGROUND/AIMS: The aim of our study was to investigate interactions of mesenchymal progenitor cells (MPCs) with collagen matrices. METHODS: Human bone-marrow-derived MPCs were cultivated in collagen type I gels with and without inhibition of beta(1)-integrin by a specific antibody. Collagen gel contraction, cell morphology, expression of integrin subunits and several genes related to matrix synthesis and turnover as well as MPC differentiation were analyzed over 14 days. RESULTS: Human MPCs markedly contracted free-floating collagen gels. Contraction was nearly completely inhibited by blocking beta(1)-integrin. Cellular morphology was elongated in the absence and mostly round in the presence of the antibody. Expression of integrin alpha(1), alpha(2) and beta(1) subunits showed several changes partly dependent on beta(1)-integrin blocking. Expression of matrix metalloproteinase-1 was elevated irrespective of beta(1)-integrin blocking and tenascin-C was subsequently induced during gel contraction. Spontaneous induction of chondrogenic, osteogenic or adipogenic differentiation was observed neither in the presence nor in the absence of the beta(1)-integrin antibody. CONCLUSION: Our results indicate that the interaction of human MPCs with fibrillar collagen type I involves beta(1)- and alpha-integrin subunits and induces changes in gene expression related to extracellular matrix synthesis and turnover but not differentiation to the chondrogenic, osteogenic or adipogenic phenotype.
Antibodies, Monoclonal↗
Effect of oxidation on the platelet-activating properties of low-density lipoprotein.
OBJECTIVE: Because of the large variation in oxidizing procedures and susceptibility to oxidation of low-density lipoprotein (LDL) and the lack in quantification of LDL oxidation, the role of oxidation in LDL-platelet contact has remained elusive. This study aims to compare platelet activation by native LDL (nLDL) and oxidized LDL (oxLDL). METHODS AND RESULTS: After isolation, nLDL was dialyzed against FeSO4 to obtain LDL oxidized to well-defined extents varying between 0% and >60%. The oxLDL preparations were characterized with respect to their platelet-activating properties. An increase in LDL oxidation enhances platelet activation via 2 independent pathways, 1 signaling via p38(MAPK) phosphorylation and 1 via Ca2+ mobilization. Between 0% and 15% oxidation, the p38(MAPK) route enhances fibrinogen binding induced by thrombin receptor (PAR-1)-activating peptide (TRAP), and signaling via Ca2+ is absent. At >30% oxidation, p38(MAPK) signaling increases further and is accompanied by Ca2+ mobilization and platelet aggregation in the absence of a second agonist. Despite the increase in p38(MAPK) signaling, synergism with TRAP disappears and oxLDL becomes an inhibitor of fibrinogen binding. Inhibition is accompanied by binding of oxLDL to the scavenger receptor CD36, which is associated with the fibrinogen receptor, alpha(IIb)beta3. CONCLUSIONS: At >30% oxidation, LDL interferes with ligand binding to integrin alpha(IIb)beta3, thereby attenuating platelet functions.
Blood Platelets↗
Differential regulation of airway epithelial integrins by growth factors.
The pattern of integrin expression on human airway epithelium changes significantly in injury or inflammation. In particular, two integrins, the fibronectin receptor, alpha 5 beta 1 and the fibronectin/tenascin receptor alpha v beta 6, are expressed at low or undetectable levels in normal airways in vivo but are induced in response to airway epithelial injury. We investigated the effects of various growth factors known to be present in the airways on the expression of constitutively expressed and inducible airway epithelial integrins using flow cytometry. In primary cultures of human airway epithelial cells, transforming growth factor-beta 1 (TGF beta 1) dramatically increased expression of alpha v beta 6 and essentially did not affect the expression of any other integrin, including alpha 5 beta 1. In contrast, epidermal growth factor (EGF) upregulated surface levels of both alpha v beta 6 and alpha 5 beta 1. Together, TGF beta 1 and EGF had an additive effect on alpha v beta 6 and alpha 5 beta 1 expression while increasing levels of alpha 2 beta 1 and decreasing expression of alpha 3 beta 1- and alpha 6-containing integrins. In contrast, the transformed airway epithelial cell line, BEAS-2B, expressed a markedly different repertoire of integrins. Integrin expression on BEAS-2B cells was not affected by any of the growth factors tested in this study. These results demonstrate that, in primary cultures of human airway epithelial cells, the pattern of integrin expression can be dramatically altered by growth factors. The inducible integrins, alpha v beta 6, and alpha 5 beta 1 are most subject to regulation by growth factors and expression of each of these can be differentially regulated. The differential regulation of the two principal fibronectin receptors on airway epithelial cells suggests that they may mediate different cellular responses to fibronectin.
Antibodies, Monoclonal↗
Neutrophil transmigration across human airway epithelial monolayers: mechanisms and dependence on electrical resistance.
To examine neutrophil transepithelial migration in the basolateral-to-luminal direction, bronchial epithelial cells (16HBE) were grown at an air-medium interface on the lower face of permeable supports, and resistance across each membrane was recorded before measuring neutrophil transmigration over 2 h. Subconfluent monolayers (resistance < 250 Omega) permitted high spontaneous migration of neutrophils (7.4+/-1%), which was further enhanced (29.7+/-3%) in response to interleukin (IL)-8 (100 ng/ml). Confluent monolayers (250 to 700 Omega) showed low spontaneous migration (2+/- 0.5%) but responded markedly to IL-8 (12.4+/-1.3%). Left in culture, 16HBE resistances continued to increase and were associated with minimal spontaneous migration (< 0.5%) or responses to IL-8. Using cells in the 250 to 700 Omega range, neutrophil migration to IL-8 was dose-dependent and was enhanced when epithelial cells were incubated with a combination of tumor necrosis factor-alpha and interferon-gamma. Neutrophil migration was stimulus-specific and was reduced by preincubation of epithelial cells with a F(ab')(2) anti-intercellular adhesion molecule (ICAM)-1, or by preincubation of neutrophils with anti-CD18, anti-CD11a, anti-CD11b, or anti-CD11c, but not by anti-CD11d, indicating a role for beta(2)-integrin-ICAM-1 interaction in the migration process.
Antibodies↗
A mouse model for the study of recurrent corneal epithelial erosions: alpha9beta1 integrin implicated in progression of the disease.
PURPOSE: To describe an in vivo mouse model for the study of recurrent corneal erosion syndrome (RCES) in mice and to characterize the changes in alpha9 integrin expression during wound healing. METHODS: Corneal epithelial debridement wounds of two sizes (1.5 and 2.5 mm) were made on the ocular surface of BALB/c mice and were evaluated at various times after wounding. Corneas were processed either as whole mounts and stained with propidium iodide and an antibody against alpha9 integrin or for bromodeoxyuridine analyses of cell proliferation. A separate study involved analyses of corneal wound healing over time in individual mice with large and small debridement wounds. Mice were anesthetized once per week and their corneas stained with fluorescein to assess the quality of the corneal epithelium. After 6 weeks, mice were killed and eyes processed for study by immunofluorescence in either whole mounts or frozen sections. RESULTS: Whole mount confocal microscopy showed open wounds on the ocular surface of mice at 1 and 2 weeks after large wounds were created, but not after small wounds. In addition, alpha9 integrin was upregulated during healing, and changes were observed in alpha9 integrin localization at the limbus with large wounds but not with small wounds. Although only 1 of 16 corneas with small wounds had erosions at 1 and 2 weeks, 11 of 16 with large wounds had erosions. However, by 6 weeks, 13 of 16 eyes showed signs of erosion whether wounds were small or large. With large wounds, RCES corneas frequently showed numerous goblet cells adjacent to a limbus lacking alpha9 integrin. Corneas from mice with documented RCES showed both retention of alpha9 integrin and tenascin-C expression at the anterior stromal-epithelial interface as well as impaired relocalization of alpha3beta1 integrin to the basement membrane zone. CONCLUSIONS: These data show that spontaneous recurrent corneal erosions occur in a mouse model after manual creation of a single wound by debridement. Differences between the healing of small (1.5 mm) and large (2.5 mm) wounds were observed. Large wounds often resulted in the presence of goblet cells on the central cornea and a loss of alpha9 integrin at the limbus. Small wounds never showed differences in the localization of alpha9 integrin at the limbus, and no goblet cells were observed in the central cornea. More studies are needed to understand the causes of erosions in these mice.
Animals↗
Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis.
It has been shown that osteopontin (OPN) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). However, the molecular mechanism of OPN action is yet to be elucidated. Splenic monocytes obtained from arthritic mice exhibited a significant capacity for cell migration toward thrombin-cleaved OPN but not toward full-length OPN. Migratory monocytes expressed alpha9 and alpha4 integrins. Since cleavage of OPN by thrombin exposes the cryptic epitope recognized by alpha9 and alpha4 integrins, we investigated the role of the cryptic epitope SLAYGLR in a murine RA model by using a specific antibody (M5) reacting to SLAYGLR sequence. The M5 antibody could abrogate monocyte migration toward the thrombin-cleaved form of OPN. Importantly, M5 antibody could inhibit the proliferation of synovium, bone erosion, and inflammatory cell infiltration in arthritic joints. Thus, we demonstrated that a cryptic epitope, the SLAYGLR sequence of murine OPN, is critically involved in the pathogenesis of a murine model of RA.
Animals↗
Integrin expression during epithelial migration and restratification in the tenascin-C-deficient mouse cornea.
In the unwounded cornea, tenascin-C localizes to a short stretch of the basement membrane zone at the corneoscleral junction or limbus. To determine whether the function of the limbus is affected by the absence of tenascin-C, mice possessing a deletion of tenascin-C and strain-matched wild-type mice are used in corneal debridement wounding experiments. The expression of integrins (alpha3, alpha9, and beta4) in the tenascin-C knockout corneas is evaluated by producing polyclonal cytoplasmic domain antipeptide sera and performing immunofluorescence microscopy. In addition, we evaluate the localization of several other proteins involved in wound healing, including fibronectin, laminin beta1, nidogen/entactin, and VCAM-1, in both the tenascin knockout and wild-type mice. There are no differences in healing rate, scarring, or neovascularization after corneal debridement wounds. alpha9 integrin is expressed at the limbal border of unwounded tenascin-C knockout animals and is upregulated during migration only after the larger wounds. At 8 weeks after larger wounds, the localization of alpha9 again becomes restricted to the limbal border. Results show that tenascin-C is not required for development or maintenance of the corneal limbus or for normal re-epithelialization of corneal epithelial cells after debridement wounding.
Animals↗
Immunolocalization of tenascin-C, alpha9 integrin subunit, and alphavbeta6 integrin during wound healing in human oral mucosa.
Tenascin-C (TN-C) and its isoforms are multidomain extracellular matrix (ECM) proteins that are believed to be involved in the regulation of stromal-epithelial interactions. Some of the interactions between TN-C and cells are mediated by integrins. In this study we analyzed the expression of TN-C and its large molecular weight splice isoform (TN-C(L)) and the putative TN-C-binding alpha9 and alphavbeta6 integrins during human wound repair. In 3-day-old oral mucosal wounds, immunoreactivity for alpha9 integrin localized abundantly at the migrating basal wound epithelial cells. TN-C and TN-C(L) were localized in the matrix between and underneath alpha9-expressing epithelial cells. In parallel with gradual downregulation of alpha9 integrin immunoreactivity in 7-day and older wounds, the expression of alphavbeta6 integrin was temporarily induced. Integrin alphavbeta6 co-localized in the same area as TN-C and TN-C(L) immunoreactivity at the cell-cell contacts of the basal and suprabasal cell layers of the wound epithelium. During granulation tissue formation and reorganization from 7 to 28 days after wounding, TN-C and TN-C(L) were abundantly localized in the granulation tissue. The findings show that TN-C(L) is expressed under the migrating epithelial front and in the granulation tissue during matrix deposition in wound repair. Preferential localization of alpha9 integrin in migrating epithelial cells and of alphavbeta6 integrin in epithelium after wound closure suggests different functions for these integrins in wound repair.
Alternative Splicing↗
Upregulation of alpha 9 integrin and tenascin during epithelial regeneration after debridement in the cornea.
Stratified epithelia are exposed to abrasive forces and are required to respond rapidly to injury to minimize fluid loss and the risk for microbial infection. Healing involves a cell migratory phase to reestablish barrier function and cell proliferation to restratify the epithelium. Cell migration during re-epithelialization involves cell sliding, termed sheet movement, during which cells retain their cell-cell junctions while dynamically altering their shape and cell-substrate interactions to permit movement across the exposed wound bed. Proteins of the integrin family of receptor molecules modulate cell shape, cell migration, and signal transduction in many cell types. In epithelial cells, integrins of the beta 1 family have been implicated in regulating cell proliferation and differentiation, alpha 9 beta b1 is one of the newer members of the integrin beta family and has been recently shown to function as a tenascin receptor. Although little is known about its function in vivo, studies in developing mouse cornea and eyelid suggest that it may play a role in epithelial differentiation. Using a debridement wound model in the mouse cornea, we show in this study that (a) in response to small debridement wounds that close without cell proliferation, alpha 9 integrin protein and mRNA are not induced during migration but are induced during restratification, (b) larger debridement wounds that require cell proliferation to generate the cells necessary for sheet movement result in a dramatic induction of alpha 9 protein and its mRNA during both migration and restratification, and (c) tenascin, an alpha 9 beta 1 ligand, accumulates beneath epithelial cells during restratification but not during cell migration. Therefore, alpha 9 integrin protein production and tenascin accumulation are dynamically regulated in response to corneal epithelial injury.
Animals↗
Sustained alterations in biodistribution of stem/progenitor cells in Tie2Cre+ alpha4(f/f) mice are hematopoietic cell autonomous.
We have generated Tie2Cre+alpha4(f/f) mice with documented alpha4-integrin ablation in hematopoietic and endothelial cells. A prominent feature in this model is a sustained, significant increase in circulating progenitors at levels higher than the levels seen with Tie2Cre+VCAM-1(f/f) mice. To test whether phenotypic differences are due to contributions by ligands other than VCAM-1 in bone marrow, or to alpha4-deficient endothelial cells or pericytes, we carried out transplantation experiments using these mice as donors or as recipients. Changes in progenitor biodistribution after transplantation were seen only with alpha4-deficient donor cells, suggesting that these cells were necessary and sufficient to reproduce the phenotype with no discernible contribution by alpha4-deficient nonhematopoietic cells. Because several similarities are seen after transplantation between our results and those with CXCR4-/- donor cells, the data suggest that VLA4/VCAM-1 and CXCR4/CXCL12 pathways contribute to a nonredundant, ongoing signaling required for bone marrow retention of progenitor cells during homeostasis.
Animals↗
ICAM-1-dependent pathways regulate colonic eosinophilic inflammation.
Eosinophilic inflammation is a common feature of numerous eosinophil-associated gastrointestinal (EGID) diseases. Central to eosinophil migration into the gastrointestinal tract are the integrin-mediated interactions with adhesion molecules. Although the mechanisms regulating eosinophil homing into the small intestine have begun to be elucidated, the adhesion pathways responsible for eosinophil trafficking into the large intestine are unknown. We investigated the role of adhesion pathways in eosinophil recruitment into the large intestine during homeostasis and disease. First, using a hapten-induced colonic injury model, we demonstrate that in contrast to the small intestine, eosinophil recruitment into the colon is regulated by a beta7 -integrin addressin cell adhesion molecule-1-independent pathway. Characterization of integrin expression on colonic eosinophils by flow cytometry analysis revealed that colonic CC chemokine receptor 3+ eosinophils express the intercellular adhesion molecule-1 (ICAM-1) counter-receptor integrins alphaL, alphaM, and beta2. Using ICAM-1-deficient mice and anti-ICAM-1 neutralizing antibodies, we show that hapten-induced colonic eosinophilic inflammation is critically dependent on ICAM-1. These studies demonstrate that beta2 -integrin/ICAM-1-dependent pathways are integral to eosinophil recruitment into the colon during GI inflammation associated with colonic injury.
Animals↗
Drosophila PS2 integrin mediates RGD-dependent cell-matrix interactions.
Integrins are a family of transmembrane glycoproteins that mediate cell-matrix and cell-cell interactions. We have transfected cultured Drosophila cells with genes that express the Drosophila PS2 integrin. We demonstrate that this integrin is expressed on the surface of the cells and can mediate cell spreading on an undefined component of fetal calf serum or on the purified vertebrate matrix molecules vitronectin and fibronectin. Additionally, PS2 integrin can cause cell spreading on RGD peptide. The spreading on matrix components or RGD peptide can be inhibited by soluble RGD peptide and is dependent on divalent cations.
Animals↗
Analyses of PS integrin functions during Drosophila development.
The Drosophila position-specific (PS) antigens are homologues of the vertebrate integrins, a family of transmembrane proteins that function in cell-matrix and cell-cell adhesion. The common beta subunit of PS integrins (PS beta) is encoded by the lethal(l)myospheroid gene (mys) and is required during wing, eye and muscle development. By expressing PS beta protein at defined developmental periods, we have shown that PS integrins are required throughout pupation, but not earlier, for normal development of wings. In contrast, the key requirement for PS integrins in eye development occurs only in the late pupa. Furthermore, PS integrins are apparently not required for the differentiation of the ommatidial cells; only for their organization. These results are consistent with roles for PS integrins in the interactions between the wing epithelia during the two phases of pupal wing expansion and in maintaining the attachment of a fully formed fenestrated membrane to the basement membrane of the retina. We have also examined the functional significance of alternative splicing of the transcript of the mys gene using P element-mediated transformation to introduce transgenes producing only one of the two spliced forms of PS beta. We find that either form is sufficient to rescue postembryonic mys phenotypes in the wing, eye and muscle but that both of the two splice forms are necessary to rescue the mys embryonic defects. This result indicates a requirement for the alternative splicing of mys during embryogenesis. The location of the alternative exons suggests that the two forms of the PS beta integrin subunit may interact with alternative alpha subunits and/or ligands.
Animals↗
Functions of the cytoplasmic domain of the beta PS integrin subunit during Drosophila development.
Integrins constitute a family of membrane-spanning, heterodimeric proteins that mediate adhesive interactions between cells and surrounding extracellular matrices (or other cells) and participate in signal transduction. We are interested in assessing integrin functions in the context of developing Drosophila melanogaster. This report, using mutants of the beta PS subunit encoded by the myospheroid (mys) locus, analyzes the relationships between integrin protein structure and developmental functions in an intact organism. As a first step in this analysis, we demonstrated the ability of a fragment of wild-type mys genomic DNA, introduced into the germ line in a P-element vector P[mys+], to rescue phenotypes attributed to lack of (or defects in) the endogenous beta PS during several discrete morphogenetic events. We then produced in vitro a series of modifications of the wild-type P[mys+] transposon, which encode beta PS derivatives with mutations within the small and highly conserved cytoplasmic domain. In vivo analysis of these mutant transposons led to the following conclusions. (1) The cytoplasmic tail of beta PS is essential for all developmental functions of the protein that were assayed. (2) An intron at a conserved position in the DNA sequence encoding the cytoplasmic tail is thought to participate in important alternative splicing events in vertebrate beta integrin subunit genes, but is not required for the developmental functions of the mys gene assayed here. (3) Phosphorylation on two conserved tyrosines found in the C terminus of the beta PS cytoplasmic tail is not necessary for the tested developmental functions. (4) Four highly conserved amino acid residues found in the N-terminal portion of the cytoplasmic tail are important but not critical for the developmental functions of beta PS; furthermore, the efficiencies with which these mutant proteins function during different morphogenetic processes vary greatly, strongly suggesting that the cytoplasmic interactions involving PS integrins are developmentally modulated.
Amino Acid Sequence↗
Null mutations in the alpha PS2 and beta PS integrin subunit genes have distinct phenotypes.
The two Drosophila position-specific (PS) integrins are expressed on complementary sides of sites where different cell layers adhere to each other, such as the attachments of the embryonic muscles to the epidermis. While there is suggestive evidence that the PS integrin-mediated adhesion is via the extracellular matrix, it is also possible that it occurs through the direct interaction of the two integrins, alpha PS1 beta PS and alpha PS2 beta PS. To help distinguish between these possibilities a comparison between the phenotypes caused by the absence of the beta PS subunit and the absence of one of the PS alpha subunits, alpha PS2, has been made. Two pieces of evidence are provided that prove that the alpha PS2 subunit is encoded by the locus inflated (if). Firstly, three new if alleles have been isolated, each of which is associated with a molecular lesion in the alpha PS2 gene, and each of which results in the complete loss of if activity. Secondly, a 39 kb fragment of genomic DNA that encompasses the alpha PS2 gene completely rescues if mutations when introduced into the germline by P-element-mediated transformation. A comparison of the null inflated phenotype with that of the locus that encodes the beta PS subunit, myospheroid (mys), reveals that while the beta PS subunit is required for the adhesion of the epidermis along the dorsal midline, the alpha PS2 subunit is not. In if mutant embryos, the muscles remain attached to the other cell layers significantly longer than in a mys mutant embryo. This shows that the alpha PS2 beta PS integrin only contributes part of the adhesive activity at the sites of PS integrin adhesion, and rules out a model where PS integrin function occurs solely by the direct interaction of the two PS integrins.
Animals↗
Tiggrin, a novel Drosophila extracellular matrix protein that functions as a ligand for Drosophila alpha PS2 beta PS integrins.
Genetic and other studies of Drosophila integrins have implicated these extracellular matrix receptors in various morphogenetic events, but identification of their endogenous ligands has been elusive. We report the biochemical purification and cloning of tiggrin, a novel extracellular matrix protein from Drosophila. This 255 x 10(3) M(r) polypeptide contains the potential integrin recognition sequence Arg-Gly-Asp (RGD) and 16 repeats of a novel 73-77 amino acid motif. The tiggrin gene is at chromosome locus 26D1-2 and is expressed by embryonic hemocytes and fat body cells. Tiggrin protein is detected in matrices, especially at muscle attachment sites that also strongly express integrins. Tiggrin-coated surfaces support primary embryo cell culture and provide excellent substrates for alpha PS2 beta PS integrin-mediated cell spreading. Soluble RGD-peptides inhibit this cell spreading.
Amino Acid Sequence↗
The role of apterous in the control of dorsoventral compartmentalization and PS integrin gene expression in the developing wing of Drosophila.
During the development of Drosophila appendages from imaginal discs lineage restrictions appear that prevent dividing cells from crossing between regionally distinct compartments. These compartments correspond not only to regions of cell lineage restrictions but also to regions of specific gene expression. When compartments were first discovered, it was proposed that their formation relied on compartment-specific 'selector' gene activity; engrailed is thought to play such a role for the early-arising anterior-posterior restriction. Recent results suggest that the dorsally expressed transcription factor encoded by apterous may control dorsoventral identity in the wing. In this study we use mosaic analysis to show that apterous maintains the late-arising dorsoventral lineage restriction in a manner that strongly supports the selector gene hypothesis: loss of apterous function from dorsal cells after the formation of the boundary causes them to cross into the ventral compartment. Moreover, we show that apterous plays a role controlling patterns of gene expression in the developing wing disc. The PS1 and PS2 integrins are normally expressed in primarily dorsal-specific and ventral-specific patterns, respectively. We show that ectopic expression of apterous induces ectopic ventral expression of PS1 integrin and alpha PS1 mRNA, while loss of apterous can induce the ectopic dorsal expression of PS2 integrin. Thus, apterous plays a selector-like role both in terms of the control of lineage restrictions and the regulation of downstream gene expression.
Animals↗