Simultaneous infection with retroviruses pseudotyped with different envelope proteins bypasses viral receptor interference associated with colocalization of gp70 and target cells on fibronectin CH-296.
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Several factors are thought to limit the efficiency of retroviral transduction in clinical gene therapy protocols that target hematopoietic stem cells. For example, the level of expression of the amphotropic receptor Pit-2, a phosphate symporter, appears to be low in human and murine hematopoietic stem cells. We have previously demonstrated that transduction of hematopoietic cells in the presence of the fibronectin (FN) fragment CH-296 is extremely efficient (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876-882, 1996). To examine functionally whether the retrovirus receptor is a limiting factor in transduction of hematopoietic cells, we performed competition experiments in the presence of FN CH-296 with retrovirus vectors pseudotyped with the same or a different envelope protein. We demonstrate in both human erythroleukemia (HEL) cells and primary human CD34(+) hematopoietic cells inhibition of efficient infection due to receptor interference when two vectors targeting the amphotropic receptor are used simultaneously. Receptor interference lasted up to 24 h. No interference was demonstrated when vectors targeting the amphotropic receptor and the gibbon ape leukemia virus (GALV) receptor Pit-1 were used concurrently. In contrast, simultaneous infection with vectors targeting both Pit-1 and Pit-2 yielded transduction efficiencies consistently higher than with either vector alone in both HEL cells and human CD34(+) hematopoietic cells. These data demonstrate that the use of FN CH-296 leads to amphotropic receptor saturation in these cells. Simultaneous infection with vectors targeting both amphotropic and GALV receptors may prove to be of additional benefit in the design of gene therapy protocols. (+info)
Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin.
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Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM. (+info)
Inhibition of factor XIIIa-mediated incorporation of fibronectin into fibrin by pulmonary surfactant.
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Intra-alveolar deposition of exudated plasma proteins is a hallmark of acute and chronic inflammatory lung diseases. In particular, fibrin and fibronectin may provide a primary matrix for fibrotic lung remodeling in the alveolar compartment. The present study was undertaken to explore the effect of two surfactant preparations on the incorporation of fibronectin into fibrin. We observed that surfactant phospholipids are associated with insoluble fibrin, factor XIIIa-cross-linked fibrin, and cross-linked fibrin with incorporated fibronectin. Factor XIIIa-mediated binding of fibronectin to fibrin was noticeably altered in the presence of surfactant. Coincubation with two different commercially available surfactants but not with dipalmitoylphosphatidylcholine alone resulted in a reduction of fibronectin incorporation into fibrin clots by approximately one-third. This effect was not dependent on the calcium concentration. We conclude that 1) factor XIIIa-cross-linked fibrin-fibronectin is able to incorporate surfactant phospholipids in amounts comparable to fibrin clots without fibronectin and 2) the binding of fibronectin to fibrin is partially inhibited in the presence of pulmonary surfactant. (+info)
Heterologous desensitization of IL-8-mediated chemotaxis in human neutrophils by a cell-binding fragment of fibronectin.
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In this study, we have explored the mechanism for the desensitization of IL-8-mediated neutrophil chemotaxis by a cell-binding fragment of fibronectin (120-kDa FN). Preincubation of neutrophil suspensions with the 120-kDa FN fragment resulted in a heterologous desensitization of IL-8-mediated chemotaxis while not affecting neutrophil chemotaxis to either fMLP or zymosan-activated serum. Preincubation of neutrophils with the beta1-integrin-activating antibody (TS2/16) mimicked the effects of the 120-kDa FN fragment while preincubating neutrophils with the beta1-integrin blocking antibody (mAb13) abrogated the inhibitory effects of the 120-kDa FN fragment on IL-8-mediated chemotaxis. Furthermore, we also demonstrated that the 120-kDa FN fragment did not inhibit chemotaxis to the CXC chemokine MGSA/GROalpha which interacts with high affinity to the IL-8 receptor B (CXCR2). By in vivo phosphorylation of neutrophils and probing lysates with an anti-CXCR1 antibody, we demonstrated that the addition of the cell-binding fragment of fibronectin resulted in a time-dependent phosphorylation of CXCR1. These findings suggest that the mechanism of heterologous desensitization of IL-8-mediated chemotaxis following ligation of FN-dependent integrins is the result of phosphorylation of the CXCR1 receptor. (+info)
Embryonic death of Mek1-deficient mice reveals a role for this kinase in angiogenesis in the labyrinthine region of the placenta.
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Mek is a dual-specificity kinase that activates the extracellular-signal-regulated (Erk) mitogen-activated protein (MAP) kinases upon agonist binding to receptors. The Erk MAP kinase cascade is involved in cell-fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single mek gene is present in Caenorhabditis elegans, Drosophila and Xenopus, two mek homologs, Mek1 and Mek2, are present in the mammalian cascade. In the present study, we describe a mutant mouse line in which the mek1 gene has been disrupted by insertional mutagenesis. The null mutation was recessive lethal, as the homozygous mutant embryos died at 10.5 days of gestation. Histopathological analyses revealed a reduction in vascularization of the placenta that was due to a marked decrease of vascular endothelial cells in the labyrinthine region. The failure to establish a functional placenta probably explains the death of the mek1-/- embryos. Cell-migration assays indicated that mek1-/- fibroblasts could not be induced to migrate by fibronectin, although the levels of Mek2 protein and Erk activation were normal. Re-expression of Mek1 in the mutant mouse embryonic fibroblasts (MEFs) restored their ability to migrate. Our findings provide genetic evidence that establishes the unique role played by Mek1 in signal transduction. They also suggest that mek1 function is required for normal response to angiogenic signals that might promote vascularization of the labyrinthine region of the placenta. (+info)
Detection of alternative splicing of fibronectin mRNA in a single cell.
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Pre-fibronectin mRNA is subject to alternative splicing at three sites, EDA, EDB and IIICS. We analyzed the alternative splicing of fibronectin mRNA in a single cell. Reverse transcription-polymerase chain reaction analyses showed cells that produced a single form of mRNA at each one of these sites as well as cells that produced multiple forms at a given site: for example, some cells produced either the EDA(+) or EDA(-) form of the mRNA and other cells produced both forms. About 80% of the cells produced both (+) and (-) forms of the mRNA at the EDA and EDB sites, and the remaining cells contained either the (+) or (-) form. Five forms of fibronectin mRNA can result from alternative splicing at the IIICS site. Complex combinations of alternative splicing products were observed among the individual cells: there were ten different combinations of mRNA isoforms with respect to the IIICS site. Statistically significant changes in alternative splicing at the IIICS site were observed during cellular senescence. (+info)
Control of type II transforming growth factor-beta receptor expression by integrin ligation.
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Ectopic expression of the alpha5 integrin subunit in cancer cells with little or no endogenous expression of this integrin often results in reduced proliferation as well as reduced malignancy. We now show that inhibition resulting from ectopic expression of alpha5 integrin is due to induction of autocrine negative transforming growth factor-beta (TGF-beta) activity. MCF-7 breast cancer cells do not express either alpha5 integrin or type II TGF-beta receptor and hence are unable to generate TGF-beta signal transduction. Ectopic expression of alpha5integrin expression enhanced cell adhesion to fibronectin, reduced proliferation, and increased the expression of type II TGF-beta receptor mRNA and cell surface protein. Receptor expression was increased to a higher level in alpha5 transfectants by growth on fibronectin-coated plates. Induction of type II TGF-beta receptor expression also resulted in the generation of autocrine negative TGF-beta activity because colony formation was increased after TGF-beta neutralizing antibody treatment. Transient transfection with a TGF-beta promoter response element in tandem with a luciferase cDNA into cells stably transfected with alpha5 integrin resulted in basal promoter activities 5-10-fold higher than those of control cells. Moreover, when alpha5 transfectants were treated with a neutralizing antibody to either TGF-beta or integrin alpha5, this increased basal promoter activity was blocked. Autocrine TGF-beta activity also induced 3-fold higher endogenous fibronectin expression in alpha5 transfectants relative to that of control cells. Re-expression of type II receptor by alpha5 transfection also restored the ability of the cells to respond to exogenous TGF-beta and led to reduced tumor growth in athymic nude mice. Taken together, these results show for the first time that TGF-beta type II receptor expression can be controlled by alpha5beta1 ligation and integrin signal transduction. Moreover, TGF-beta and integrin signal transduction appear to cooperate in their tumor-suppressive functions. (+info)
Expression of N-cadherin, N-CAM, fibronectin and tenascin is stimulated by TGF-beta1, beta2, beta3 and beta5 during the formation of precartilage condensations.
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Cell surface adhesion and extracellular matrix proteins are known to play a key role in the formation of cell condensations during skeletal development, and their formation is crucial for the expression of cartilage-specific genes. However, little is known about the relationship between adhesion molecules (N-cadherin and N-CAM), extracellular matrix proteins (fibronectin and tenascin) and TGF-beta1, TGF-beta2 and TGF-beta3 during in vitro precartilage condensations in mouse chondrogenesis. On these bases, we determined the participation of mammalian TGF-beta1, TGF-beta2 and TFG-beta3 and Xenopus TGF-beta5 on the expression of cell surface adhesion and extracellular matrix proteins during the formation of precartilage condensations. Also, we characterized the effects of TGF-betas on proteoglycan metabolism at different cellular densities in mouse embryonic limb bud mesenchymal cells. In TGF-beta1 and TGF-beta5-treated cultures, proteoglycan biosynthesis was higher than in controls, while there were no differences in proteoglycan catabolism, which caused the accumulation of cartilage extracellular matrix. When mesenchymal cells were seeded at three different cellular densities in the presence of TGF-betas, only high density cultures presented increased stimulation of proteoglycan biosynthesis, compared to low and intermediate densities. To determine whether the effect of TGF-betas on precartilage condensations is mediated through the expression of N-cadherin, N-CAM, fibronectin and tenascin, we evaluated their expression. Results showed that TGF-beta1, TGF-beta2, TGF-beta3, and TGF-beta5 differentially enhanced the expression of N-cadherin, N-CAM, fibronectin and tenascin in precartilage condensations, suggesting that TGF-beta isoforms play an important role in the establishment of cell-cell and cell-extracellular matrix interactions during precartilage condensations. (+info)