Hematopoietic reconstitution of SLP-76 corrects hemostasis and platelet signaling through alpha IIb beta 3 and collagen receptors.
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Mice deficient in the hematopoietic cell-specific adapter protein SLP-76 demonstrate a failure of T cell development and fetal hemorrhage. Although SLP-76-deficient platelets manifest defective collagen receptor signaling, this alone may not explain the observed bleeding diathesis. Because alpha IIb beta 3, the platelet fibrinogen receptor, is required for normal hemostasis, we explored a potential role for SLP-76 in alpha IIb beta 3 signaling. Interaction of soluble or immobilized fibrinogen with normal human or murine platelets triggers rapid tyrosine phosphorylation of SLP-76. Moreover, platelet adhesion to fibrinogen stimulates actin rearrangements, filopodial and lamellipodial extension, and localization of tyrosine phosphorylated proteins to the cell periphery. In contrast, SLP-76-deficient murine platelets bind fibrinogen normally, but spread poorly and exhibit reduced levels of phosphotyrosine. The in vivo bleeding diathesis as well as the defects in platelet responses to fibrinogen and collagen are reversed by retroviral transduction of SLP-76 into bone marrow derived from SLP-76-deficient mice. These studies establish that SLP-76 functions downstream of alpha IIb beta 3 and collagen receptors in platelets. Furthermore, expression of SLP-76 in hematopoietic cells, including platelets, plays a necessary role in hemostasis. (+info)
Human renal fibroblast contraction of collagen I lattices is an integrin-mediated process.
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BACKGROUND: Expression of the beta1 family of integrins allows dermal fibroblasts in wounds to contribute to the healing process through migration, adhesion, synthesis, and rearrangement of extracellular matrix. To date the ability of human renal fibroblasts to reorganize collagens and the role of cell surface receptors in this process remain unknown. METHODS: Renal fibroblasts were grown from the cortical tissue of surgically removed human kidneys. The ability of human renal fibroblasts to reorganize interstitial collagen I was examined in vitro using solidified collagen I lattices. Integrin function was blocked by incubating fibroblasts with isotype-specific antibodies prior to addition to collagen I lattices. RESULTS: Human renal fibroblasts embedded in collagen I lattices progressively decreased lattice diameter to 60.6+/-11.4% of initial diameter at 48 h post-release (P:<0.01). Fibroblasts incubated in the presence of antibody to beta1 integrin failed to contract collagen I lattices, whilst fibroblasts incubated with non-specific antibody reduced lattice diameter to 60.1+/-12.4% of initial diameter at 48 h post-release (P:<0.01). Further characterization of integrin alpha subunits showed that blocking alpha2beta1 integrin prevented lattice contraction (P:<0.05, alpha2beta1 integrin antibody vs non-specific antibody), whilst blocking of alpha5beta1, alpha3beta1 and alpha1beta1 integrins did not influence this process. CONCLUSIONS: We postulate that collagen I fibril rearrangement by human renal fibroblasts in vitro appears to be an integrin-mediated process involving the alpha2beta1 integrin. (+info)
Expression of cbsA encoding the collagen-binding S-protein of Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC 393(T).
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The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed in L. casei ATCC 393(T). The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting signal of the proteinase, PrtP, of L. casei, CbsA was presented at the surface, rendering the transformants able to bind to immobilized collagens. (+info)
Roles of SLP-76, phosphoinositide 3-kinase, and gelsolin in the platelet shape changes initiated by the collagen receptor GPVI/FcR gamma-chain complex.
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How platelet shape change initiated by a collagen-related peptide (CRP) specific for the GPVI/FcR gamma-chain complex (GPVI/FcR gamma-chain) is coupled to SLP-76, phosphoinositide (PI) 3-kinase, and gelsolin is reported. As shown by video microscopy, platelets rapidly round and grow dynamic filopodial projections that rotate around the periphery of the cell after they contact a CRP-coated surface. Lamellae subsequently spread between the projections. All the actin-driven shape changes require SLP-76 expression. SLP-76 is essential for the Ca(++) mobilization induced by CRP, whereas PI 3-kinase only modulates it. The extension of lamellae requires net actin assembly and an exposure of actin filament barbed ends downstream of PI 3-kinase. Gelsolin expression is also required for the extension of lamellae, but not for the formation of filopodia. Altogether, the data describe the role of SLP-76 in the platelet activation initiated by GPVI/FcR gamma-chain and the roles of PI 3-kinase and gelsolin in lamellae spreading. (Blood. 2000;96:3786-3792) (+info)
A mechanism for modulation of cellular responses to VEGF: activation of the integrins.
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Many similarities exist in the cellular responses elicited by VEGF and governed by integrins. Here, we identify a basis for these interrelationships: VEGF activates integrins. VEGF enhanced cell adhesion, migration, soluble ligand binding, and adenovirus gene transfer mediated by alphavbeta3 and also activated other integrins, alphavbeta5, alpha5beta1, and alpha2beta1, involved in angiogenesis. Certain tumor cells exhibited high spontaneous adhesion and migration, which were attributable to a VEGF-dependent autocrine/paracrine activation of integrins. This activation was mediated by the VEGFR2 receptor and regulated via phosphatidylinositol-3-kinase, Akt, and the PTEN signaling axis. Thus, integrin activation provides a mechanism for VEGF to induce a broad spectrum of cellular responses. (+info)
Promotion of adhesion and migration of RPE cells to provisional extracellular matrices by TNF-alpha.
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PURPOSE: Adhesion and migration of retinal pigment epithelial (RPE) cells to provisional extracellular matrices (ECM) is important in the development of epiretinal membranes found in proliferative vitreoretinopathy (PVR). Tumor necrosis factor alpha (TNF-alpha) is found in PVR membranes and regulates many functions of RPE cells. In this study, the effects of TNF-alpha on adhesion and migration of RPE cells to various components of ECM were examined and elucidation of the mechanism of the response was attempted. METHODS: Mitogen activated protein kinase (ERK1/2; MAPK) activation was measured by immunoblot. RPE cells pretreated with TNF-alpha (10 ng/ml) or TNF-alpha + PD98059 (a specific inhibitor of MAPK, 30 microM) for 24 hours were compared with control RPE. Attachment was measured by modified MTT assay on fibronectin and collagen types I and IV. Spreading was measured by staining with fluo3-AM and confocal laser scanning microscopy. Migration of RPE cells on substrates was determined by Boyden chamber assay using PDGF-BB (20 ng/ml) as a chemotactic factor. Integrin expression was determined by flow cytometry and RT-PCR. RESULTS: TNF-alpha rapidly activated MAPK and increased the extent of attachment, spreading and migration on fibronectin and collagen type I (P < 0.01) but not on collagen type IV. TNF-stimulated RPE cells showed increased mRNA and surface protein expression for alpha1 and alpha5 integrin (P < 0.01) but not alpha3 integrin subunit. Neutralizing the anti-alpha1 antibody inhibited migration on collagen type I, whereas alpha5 antibody inhibited fibronectin-induced migration. Treatment with both TNF and PD98095 reduced attachment and migration on provisional ECM and reduced the upregulated integrin expression to control levels. CONCLUSIONS: After treatment with TNF-alpha, there is increased expression of specific integrins associated with increased adhesion and migration on provisional ECM (fibronectin and collagen type I). This effect is mediated, at least in part, by activation of MAPK signaling pathway. (+info)
Integrin alpha2beta1 mediates the cell attachment of the rotavirus neuraminidase-resistant variant nar3.
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It was previously reported that integrins alpha2beta1, alpha4beta1, and alphaXbeta2 are involved in rotavirus cell infection. In this work we studied the role of integrin subunits alpha2, alpha4, and beta2 on the attachment of rotaviruses RRV and nar3 to MA104 cells. Integrin alpha2beta1 was found to serve as the binding receptor for the neuraminidase-resistant virus nar3, whereas the neuraminidase-sensitive strain RRV interacted with this integrin at a postattachment step. It was shown that nar3 binds alpha2beta1 through the DGE integrin-recognition motif located in the virus surface protein VP5. Integrin subunits alpha4 and beta2 do not seem to be involved in the initial cell binding of either virus. (+info)
Involvement of actin filaments and integrins in the binding step in collagen phagocytosis by human fibroblasts.
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In physiological conditions, collagen degradation by fibroblasts occurs primarily via phagocytosis, an intracellular pathway that is thought to require collagen receptors and actin assembly for fibril internalization and degradation. Currently it is unclear which specific steps of collagen phagocytosis in fibroblasts involve actin filament assembly. As studies of phagocytosis in fibroblasts are complicated by the relatively slow rate of particle internalization compared to professional phagocytes, we have examined the role of collagen receptors and actin only in the initial collagen binding step. Prior to the binding of collagen-coated fluorescent beads by human gingival fibroblasts, a cell type that is avidly phagocytic in vitro, cells were treated with cytochalasin D (actin filament barbed-end capping) or swinholide A (actin dimer sequestering and severing) or latrunculin B (actin monomer sequestering). Bead binding and immunostaining of (alpha)(2)(beta)(1) and (alpha)(3)(beta)(1) integrin collagen receptors were measured by flow cytometry. After 1-3 hours of coincubation with beads, cytochalasin D or swinholide A eliminated actin filaments stained by rhodamine-phalloidin and inhibited collagen bead binding (reductions of 25% and 50%, respectively), possibly because of cell rounding and restricted interactions with beads. In contrast, latrunculin enhanced binding dose-dependently over controls (twofold at 1 microM) and induced the formation of brightly staining aggregates of actin and the retention of long cytoplasmic extensions. Latrunculin also reduced surface (beta)(1), (alpha)(2) and (alpha)(3) integrin staining up to 40% in bead-free and bead-loaded cells, indicating that latrunculin enhanced collagen receptor internalization. As determined by fluorescence recovery after photobleaching, latrunculin increased the mobility of surface-bound (beta)(1) integrin. The stimulatory effect of latrunculin on collagen bead binding was reduced to control levels by treatment with a (beta)(1) integrin inactivating antibody while a (beta)(1) integrin blocking antibody abrogated both bead binding and the latrunculin-induced stimulation. Immunoblotting of bead-associated proteins showed that latrunculin completely eliminated binding of (beta)-actin to collagen beads but did not affect (beta)(1) integrin binding. These data indicate that latrunculin-induced sequestration of actin monomers facilitates the disengagement of actin from (beta)(1) integrin receptors, increases collagen bead binding and enhances collagen receptor mobility. We suggest that these alterations increase the probability of adhesive bead-to-cell interactions. (+info)