Different cation binding to the I domains of alpha1 and alpha2 integrins: implication of the binding site structure.
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In the present work, we studied the interactions of recombinant alpha1 and alpha2 integrin I domains with cations Tb(3+), Mn(2+), Mg(2+) and Ca(2+). We observed that alpha1 and alpha2 I domains bind these cations with significantly different characteristics. The binding of Mg(2+) by the alpha1 I domain was accompanied by significant changes of tryptophan fluorescence which could be interpreted as a conformational change. Comparison of the alpha1 integrin I domain structure obtained by comparative modeling with a known structure of the alpha2 integrin I domain shows distinct differences in the metal ion binding sites which could explain the differences in cation binding. (+info)
Differential expression of alpha 1, alpha 3 and alpha 5 integrin subunits in acute and chronic stages of myocardial infarction in rats.
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OBJECTIVE: Anchoring cardiac myocytes to extracellular matrix, which is mediated mainly by integrins on their surfaces, is important for maintaining the architecture of myocardial tissues and transmitting mechanical force. We evaluated the expression of alpha integrin subunits on myocytes and the accumulation of interstitial collagen and fibronectin at acute and chronic stages after myocardial infarction. METHODS: Myocardial infarction was induced by ligation of left coronary arteries in rats. The expression of alpha 1, alpha 3 and alpha 5 integrin subunits, and accumulation of collagen and fibronectin were analyzed with immunohistochemistry or sirius-red staining. RESULTS: In hearts without infarction, moderate expression of the alpha 3 subunit and only slight expression of the alpha 5 subunit were observed on myocytes. In the first week after infarction, the alpha 1 subunit, collagen and fibronectin were increased only in the peri-infarcted area, while the alpha 5 subunit was increased both in peri-infarcted and non-infarcted areas. At day 42, the expression of the alpha 1 subunit and collagen were still increased, although the alpha 5 subunit and fibronectin were decreased. The expression of the alpha 3 subunit was not altered throughout the experimental period. CONCLUSION: These data suggest that integrin subunits play an important role in healing and remodeling processes after myocardial infarction. (+info)
Extracellular matrix composition and hypoxia regulate the expression of HLA-G and integrins in a human trophoblast cell line.
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During human placentation, extravillous cytotrophoblast cells emerge from chorionic villi contacting the decidua to invade the uterine wall. When isolated from first-trimester placentae, cytotrophoblast cells undergo step-wise differentiation in vitro that recapitulates the phenotypic heterogeneity observed in vivo. We examined a cell line, HTR-8/SVneo, that has been established from human first-trimester cytotrophoblast to determine whether these cells possess some of the unique cytotrophoblast characteristics that have been described previously. Exposure during serum-free culture to hypoxic conditions (2% oxygen concentration) increased HTR-8/SVneo cell proliferation and reduced invasion of a three-dimensional basement membrane (Matrigel). During culture on surfaces coated with individual extracellular matrix proteins, HTR-8/SVneo cells expressed cytokeratin but not the trophoblast-specific major histocompatibility protein, HLA-G. However, HLA-G expression was induced in HTR-8/SVneo cells that contacted Matrigel. Expression of the alpha5 integrin subunit was relatively unaffected by matrix composition, whereas alpha1 was up-regulated and alpha6 was down-regulated after transferring cells to Matrigel. Hypoxia increased alpha6 and decreased both alpha1 and HLA-G expression on Matrigel. HTR-8/SVneo cells retain several important characteristics associated with primary cultures of first-trimester human cytotrophoblast cells, including their altered behavior in response to a changing maternal environment. (+info)
Inhibition of distal lung morphogenesis in Nkx2.1(-/-) embryos.
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In vitro and in vivo results are consistent with a critical role for NKX2.1, an epithelial homeodomain transcription factor in lung morphogenesis. Nkx2.1 null mutant embryos die at birth due to respiratory insufficiency caused by profoundly abnormal lungs. However, the precise role of NKX2.1 in the multistep process of lung structural morphogenesis and differentiation of various pulmonary cell types remains unknown. In the current study, we tested the hypothesis that the mutant lungs do not undergo branching morphogenesis beyond the formation of the mainstem bronchi and therefore consist solely of dilated tracheobronchial structures. To test this hypothesis, we determined the spatial and temporal expression pattern of a number of extracellular matrix (ECM) proteins and their cellular receptors, including alpha-integrins, laminin, and collagen type IV. Although laminin is expressed in the mutant Nkx2.1(-/-) lungs, expression of alpha-integrins and collagen type IV is significantly reduced or absent. In addition, examination of regionally specific expression of differentially spliced Vegf (vascular endothelial growth factor) transcripts, clearly indicates that the epithelial phenotype of the Nkx2.1(-/-) lungs is similar to the tracheobronchial epithelium. In contrast to wild-type lungs in which both Vegf1 and Vegf3 are developmentally expressed, Nkx2.1(-/-) lungs are characterized by predominant expression of Vegf1 and reduced or absent Vegf3. A similar pattern of Vegf expression is also observed in isolated tracheo-bronchial tissue. The sum of these findings suggest that at least two separate pathways may exist in embryonic lung morphogenesis: proximal lung morphogenesis is Nkx2.1 independent, while distal lung morphogenesis appears to be strictly dependent on the wild-type activity of Nkx2.1. (+info)
Elevated glucocorticoid receptor transactivation and down-regulation of alpha 1 integrin are associated with loss of plasma membrane Ca2+-ATPase isoform 1.
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We have previously shown that inhibition of expression of the plasma membrane Ca(2+)-ATPase isoform 1 in PC6 cells leads to loss of nerve growth factor-mediated neurite extension (Brandt, P.C., Sisken, J.E., Neve, R.L., and Vanaman, T.C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 13843-13848). Cells lacking plasma membrane Ca(2+)-ATPase 1 did not attach to collagen-coated plates as tightly as controls, suggesting that a defect in adhesion might be underlying the inability to extend neurites. We report here that cell lines lacking plasma membrane Ca(2+)-ATPase 1 do not produce alpha(1) integrin, which is required for both collagen adherence and neurite extension. Because alpha(1) integrin gene transcription can be down-regulated by glucocorticoids, the response of cells to glucocorticoids was investigated. Cortisol-dependent transactivation from the mouse mammary tumor virus promoter in cells lacking plasma membrane Ca(2+)-ATPase 1 was stimulated 145-216-fold over untreated cells compared with 15-26-fold for controls. This increase was not due to increased binding affinity of the receptor for cortisol, an increased number of cortisol-binding sites, or increased translocation of the receptor to the nucleus. Expression of additional glucocorticoid receptor-dependent genes required for neurite extension must also be altered in cells missing the plasma membrane Ca(2+)-ATPase 1 because constitutive expression of alpha(1) integrin did not restore their nerve growth factor-mediated neurite extension capability. The impact of plasma membrane Ca(2+)-ATPase isoform 1 on other signaling systems and the resultant profound yet subtle effects on PC6 cells strongly suggests that it plays an important role in modulating signal transduction pathways downstream of Ca(2+)-mediated signals. (+info)
Potent costimulation of effector T lymphocytes by human collagen type I.
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Purified, resting peripheral blood T lymphocytes were previously reported to undergo beta(1) integrin-dependent activation when cultured with anti-CD3 mAb coimmobilized with fibronectin, but not type I collagen. However, the extravascular T cells that encounter immobilized extracellular matrix proteins and are involved in disease pathogenesis have different properties from resting peripheral blood cells. In this study, we confirm that resting CD4(+) and CD8(+) T cells from peripheral blood are costimulated by immobilized fibronectin, but not type I collagen. In contrast, Ag- or mitogen-stimulated CD4(+) and CD8(+) T cell lines, used as models of the effector cells involved in disease, are more potently costimulated by type I collagen than fibronectin. The collagen-induced effects are similar in assays with serum-free medium and in more physiological assays in which anti-CD3 mAb is replaced by a threshold concentration of Ag and irradiated autologous PBMC as APC. The responses are beta(1) integrin dependent and mediated largely by very late Ag (VLA) 1 and 2, as shown by their up-regulation on the T cell lines as compared with freshly purified resting PBL, and by the effects of blocking mAb. Reversed phase HPLC located the major costimulatory sequence(s) in the alpha1 chain of type I collagen, the structure of which was confirmed by amino acid sequencing. The results demonstrate the potential importance of type I collagen, an abundant extracellular matrix protein, in enhancing the activation of extravascular effector T cells in inflammatory disease, and point to a new immunotherapeutic target. (+info)
Integrin function and regulation in development.
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Integrins are a large family of membrane receptors, consisting of alpha and beta subunits, that play a pivotal role in the interaction of cells with the extracellular matrix. Such interaction regulates the organization of cells in organs and tissues during development as well as cell differentiation and proliferation. We have shown that unfertilized oocytes express integrins that might be important during fertilization. We also analyzed nervous system and muscle tissue development showing that integrin expression is precisely regulated during organization of these tissues. The results indicate that two distinct integrin alpha subunits mediate the outgrowth of processes in nerve and glial cells. Alpha1 integrin, a laminin receptor, is up-regulated by nerve growth factor and other differentiation stimuli and is involved in neurite extension by nerve cells. In contrast, process extension by glial cells is likely to involve the alphaV integrin. Moreover, the latter integrin subunit is also transiently expressed in muscle of the embryo body where it localizes predominantly at developing myotendinous junctions. After birth this integrin disappears and is substituted by the alpha7 subunit. At the same time, important changes also occur in the expression of the associated beta subunit. In fact, the beta1A isoform which is expressed in fetal muscles, is substituted by beta1D. These isoforms are generated by alternative splicing and differ in only a few amino acid residues at the COOH terminus of the protein. This region of the molecule is exposed at the cytoplasmic face of the plasma membrane and is connected to the actin filaments. Our results show that beta1D, which is expressed only in striated muscle tissues, binds to both cytoskeletal and extracellular matrix proteins with an affinity higher than beta1A. Thus, beta1D provides a stronger link between the cytoskeleton and extracellular matrix necessary to support mechanical tension during muscle contraction. These results indicate that cells can regulate their interactions with the extracellular matrix by changing their expression of alpha integrin subunits and thus ligand specificity, or by more subtle changes involving alternative usage of different cytoplasmic domains. The important role of both alpha and beta integrin subunit cytoplasmic domains during development is further illustrated by the analysis of targeted mutations which we have generated by homologous recombination in mice. (+info)
Differential effects of CD18, CD29, and CD49 integrin subunit inhibition on neutrophil migration in pulmonary inflammation.
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Neutrophil migration to lung alveoli is a characteristic of lung diseases and is thought to occur primarily via capillaries rather than postcapillary venules. The role of adhesion molecules CD18 and CD29 on this migration in a mouse model of lung inflammation has been investigated. The number of neutrophils present in bronchoalveolar lavage fluid was determined 4 h after intratracheal instillation of LPS (0.1-1 microg) or murine recombinant KC (CXC chemokine, 0.03-0.3 microg). Both stimuli produced a dose-related increase in neutrophil accumulation. Intravenous anti-mouse CD18 mAb, 2E6 (0.5 mg/mouse), significantly (p < 0.001) attenuated LPS (0.3 microg)- but not KC (0.3 microg)-induced neutrophil accumulation. The anti-mouse CD29 mAb, HM beta 1-1 (0.02 mg/mouse), significantly (p < 0.05) inhibited both LPS (0.3 microg)- and KC (0.3 microg)-induced neutrophil migration. A second mAb to CD18 (GAME-46) and both F(ab')(2) and Fab of HM beta 1-1 produced similar results to those above, while coadministration of mAbs did not result in greater inhibition. Electron microscopy studies showed that CD29 was involved in the movement of neutrophils from the interstitium into alveoli. The effect of mAbs to CD49 (alpha integrin) subunits of CD29 was also examined. mAbs to CD49e and CD49f inhibited both responses, while anti-CD49b and CD49d significantly inhibited responses to KC only. These data suggest that CD29 plays a critical role in neutrophil migration in pulmonary inflammation and that CD49b and CD49d mediate CD18-independent neutrophil accumulation. (+info)