The plasminogen-plasminogen activator (PA) system in neuroblastoma: role of PA inhibitor-1 in metastasis.
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Proteases of the plasminogen-plasminogen activator (PA) system play an important role in cancer metastasis. We have examined the expression of these proteases and their cell surface receptors and inhibitors in neuroblastoma, a tumor that originates in cells of the neural crest and is the second most common solid tumor in children. This analysis was performed in seven established human cell lines and 20 primary tumor specimens. Urokinase PA and, in particular, tissue-type PA were expressed in cell lines and in tumor tissues; however, their levels of expression did not correlate with clinical stage. There was little evidence suggesting that neuroblastoma cells concentrate PA activity at their cell surface because urokinase-type PA receptor mRNA was detected in two cell lines and in 5 of 20 tumor samples by reverse transcription-PCR only. PA inhibitor (PAI)-2 was absent in all cell lines and tumor tissue samples examined. However, PAI-1, which was not expressed by the cell lines, was expressed by stromal cells and, specifically, endothelial cells in tumor tissue. By extending the analysis of PAI-1 expression in 64 primary tumor specimens, we found that high PAI-1 expression paradoxically correlated with metastatic stage and tumor recurrence. In vitro experiments indicated that the expression of PAI-1 by human microvascular endothelial cells was stimulated in the presence of SK-N-BE(2) human neuroblastoma cells and neuroblastoma culture medium. Recombinant PAI-1 also promoted SK-N-BE(2) cell detachment from vitronectin and migration from vitronectin toward fibronectin. From these data, we conclude that the up-regulation of PAI-1 expression in endothelial cells may promote rather than inhibit metastasis in neuroblastoma. (+info)
ECM-stimulated actin bundle formation in embryonic corneal epithelia is tyrosine phosphorylation dependent.
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Previous studies demonstrated that corneal epithelial cells isolated without basal lamina respond to extracellular matrix (ECM) in an actin dependent manner; the basal cell surface flattens and the actin cortical mat reorganizes. We hypothesize that the actin reorganization is initiated by intracellular signaling mechanisms that includes tyrosine phoshporylation and activation of the Rho, MAP kinase, and PI3 kinase signal transduction pathways. Our goals were to develop a morphological assay to test this hypothesis by answering the following questions: 1) Do the actin bundle formations in the cortical mat have the same configuration in response to different ECM molecules? 2) What is the minimum time ECM molecules need to be in contact with the tissue for the actin to reorganize? 3) Will blocking tyrosine phosphorylation inhibit reorganization of the actin? 4) Are known signal transduction proteins phosphorylated in response to soluble matrix molecules? The actin cortical mat demonstrated distinct bundle configurations in the presence of different ECM molecules. Soluble fibronectin accumulated at the basal cell surfaces 75-fold over 30 min in a clustered pattern. The cells need contact with ECM for a minimum of 10 min to reform the actin bundles at 2 hr. In contrast, two substances that bind to heptahelical receptors to stimulate the Rho pathway, bombesin and lysophosphatidic acid, reorganized the actin bundles in 15-30 min. Focal adhesion kinase, p190 Rho-GAP, tensin, and paxillin were tyrosine phosphorylated in response to soluble fibronectin, type I collagen, or laminin 1. Erk-1, erk-2, and PI3 kinase were activated after 1 hr stimulation by type I collagen. Herbimycin A blocked actin reorganization induced by ECM molecules. In conclusion, we have developed two morphological assays to examine the response of corneal epithelial cells to ECM molecules. In addition, actin bundle reorganization involved tyrosine phosphorylation, MAP kinase, and PI3 kinase activation. (+info)
Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome.
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The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins. (+info)
Surface display of functional fibronectin-binding domains on Staphylococcus carnosus.
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The surface expression in Staphylococcus carnosus of three different fibronectin binding domains (FNBDs), derived from fibronectin binding proteins of Streptococcus dysgalactiae and Staphylococcus aureus, has been investigated. Surface localization of the chimeric proteins containing the FNBDs was demonstrated. All three surface-displayed FNBDs were demonstrated to bind fibronectin in whole-cell enzyme-linked binding assays. Furthermore, for one of the constructs, intranasal immunizations with the recombinant bacteria resulted in improved antibody responses to a model immunogen present within the chimeric surface proteins. The implications of the results for the design of live bacterial vaccine delivery systems are discussed. (+info)
Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc.
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Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc. (+info)
Distinct biological effects of macrophage inflammatory protein-1alpha and stroma-derived factor-1alpha on CD34+ hemopoietic cells.
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Chemokines are important regulators of both hemopoietic progenitor cell (HPC) proliferation and adhesion to extracellular matrix molecules. Here, we compared the biological effects of the CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) with those of the CXC chemokine stroma-derived factor-1alpha (SDF-1alpha) on immunomagnetically purified CD34+ cells from leukapheresis products (LP CD34+). In particular, studies on chemokine-induced alterations of LP CD34+ cell attachment to fibronectin-coated plastic surfaces, proliferation of these cells in colony-forming cell (CFC) assays and intracellular calcium mobilization were performed. MIP-1alpha but not SDF-1alpha was found to increase the adhesion of LP CD34+ cells to fibronectin in a dose-dependent manner. Both chemokines elicited growth-suppressive effects on LP CD34+ cells in CFC assays. While MIP-1alpha reduced the number of granulomonocytic (CFC-GM) and erythroid (BFU-E) colonies to the same extent, SDF-1alpha showed a significantly greater inhibitory effect on CFC-GM than BFU-E. Finally, we demonstrated that SDF-1alpha but not MIP-1alpha triggers increases in intracellular calcium in LP CD34+ cells. The SDF-1alpha-induced calcium response was rapid and concentration-dependent, with a maximal stimulation observed at > or = 15 ng/ml. In conclusion, our data suggest distinct biological properties of SDF-1alpha and MIP-1alpha in terms of modulation of LP CD34+ cell adhesion to fibronectin and intracellular calcium levels. However, comparable growth-suppressive effects on HPC proliferation were observed, indicating that this feature may be independent of chemokine-induced calcium responses. (+info)
Bone morphogenetic protein 2 inhibits platelet-derived growth factor-induced c-fos gene transcription and DNA synthesis in mesangial cells. Involvement of mitogen-activated protein kinase.
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Bone morphogenetic proteins (BMPs) play an important role in nephrogenesis. The biologic effect and mechanism of action of these proteins in the adult kidney has not yet been studied. We investigated the effect of BMP2, a member of these growth and differentiation factors, on mitogenic signal transduction pathways induced by platelet-derived growth factor (PDGF) in glomerular mesangial cells. PDGF is a growth and survival factor for these cells in vitro and in vivo. Incubation of mesangial cells with increasing concentrations of BMP2 inhibited PDGF-induced DNA synthesis in a dose-dependent manner with maximum inhibition at 250 ng/ml. Immune complex tyrosine kinase assay of PDGF receptor beta immunoprecipitates from lysates of mesangial cells treated with PDGF showed no inhibitory effect of BMP2 on PDGF receptor tyrosine phosphorylation. This indicates that the inhibition of DNA synthesis is likely due to postreceptor events. However, BMP2 significantly inhibited PDGF-stimulated mitogen-activated protein kinase (MAPK) activity that phosphorylates the Elk-1 transcription factor, a component of the ternary complex factor. Using a fusion protein-based reporter assay, we also show that BMP2 blocks PDGF-induced Elk-1-mediated transcription. Furthermore, we demonstrate that BMP2 inhibits PDGF-induced transcription of c-fos gene, a natural target of Elk-1 that normally forms a ternary complex that activates the serum response element of the c-fos gene. These data provide the first evidence that in mesangial cells, BMP2 signaling cross-talks with MAPK-based transcriptional events to inhibit PDGF-induced DNA synthesis. One target for this inhibition is the early response gene c-fos. (+info)
Integrin-mediated migration of murine B82L fibroblasts is dependent on the expression of an intact epidermal growth factor receptor.
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To evaluate the mechanisms by which epidermal growth factor (EGF) regulates actin-based cellular processes such as cell migration, we first examined the effects of EGF on cell adhesion, which is essential for cell migration. In mouse B82L fibroblasts transfected with the full-length EGF receptor, EGF promotes cell rounding and attenuates cell spreading on fibronectin, laminin, and vitronectin, and thus appears to reduce the strength of cell adhesion. Moreover, EGF synergizes with multiple extracellular matrix (ECM) components in the promotion of integrin-mediated cell migration of several different cell types, including fibroblasts and various carcinoma and osteosarcoma cell lines. Interestingly, co-presentation (co-positioning) of EGF with laminin or fibronectin is essential for EGF-stimulated migration. When EGF is mixed with the cells instead of the ECM components, it has little effect on cell migration. These results suggest that co-presentation of EGF with ECM components can enhance the polarization events required for directional cell movement. To identify the EGF receptor elements critical for the EGF stimulation of cell migration, B82L fibroblasts were transfected with either mutated or wild-type EGF receptors. Surprisingly, we found that B82L-Parental cells that lack the EGF receptor are not able to migrate to fibronectin, even though they can adhere to fibronectin. However, the introduction of wild-type EGF receptors into these fibroblasts enables them to migrate toward fibronectin even in the absence of EGF. The requirement of the EGF receptor for cell migration does not appear to result from the secretion of EGF or TGF-alpha by the cells transfected with the EGF receptor. Furthermore, cells expressing EGF receptors that are kinase-inactive, or C-terminally truncated, exhibit little migration toward fibronectin, indicating that an intact EGF receptor kinase is required for fibronectin-induced cell migration. In addition, neutralizing anti-EGF receptor antibodies attenuate cell migration in the presence of EGF, and inhibit migration to fibronectin or laminin alone. These results further suggest that the EGF receptor is downstream of integrin activation in the signal transduction pathways leading to fibroblast migration. (+info)