Podoplanin is a small membrane mucin expressed in tumors associated with malignant progression. It is enriched at cell-surface protrusions where it colocalizes with members of the ERM (ezrin, ...radixin, moesin) protein family. Here, we found that human podoplanin directly interacts with ezrin (and moesin) in vitro and in vivo through a cluster of basic amino acids within its cytoplasmic tail, mainly through a juxtamembrane dipeptide RK. Podoplanin induced an epithelial-mesenchymal transition in MDCK cells linked to the activation of RhoA and increased cell migration and invasiveness. Fluorescence time-lapse video observations in migrating cells indicate that podoplanin might be involved in ruffling activity as well as in retractive processes. By using mutant podoplanin constructs fused to green fluorescent protein we show that association of the cytoplasmic tail with ERM proteins is required for upregulation of RhoA activity and epithelial-mesenchymal transition. Furthermore, expression of either a dominant-negative truncated variant of ezrin or a dominant-negative mutant form of RhoA blocked podoplanin-induced RhoA activation and epithelial-mesenchymal transition. These results provide a mechanistic basis to understand the role of podoplanin in cell migration or invasiveness.
The formation of new blood vessels, or angiogenesis, is a necessary process during development but also for tumour growth and other pathologies. It is promoted by different growth factors that ...stimulate endothelial cells to proliferate, migrate, and generate new tubular structures. Syndecans, transmembrane heparan sulphate proteoglycans, bind such growth factors through their glycosaminoglycan chains and could transduce the signal to the cytoskeleton, thus regulating cell behaviour. We demonstrated that syndecan-2, the major syndecan expressed by human microvascular endothelial cells, is regulated by growth factors and extracellular matrix proteins, in both bidimensional and tridimensional culture conditions. The role of syndecan-2 in “
in vitro” tumour angiogenesis was also examined by inhibiting its core protein expression with antisense phosphorothioate oligonucleotides. Downregulation of syndecan-2 reduces spreading and adhesion of endothelial cells, enhances their migration, but also impairs the formation of capillary-like structures. These results suggest that syndecan-2 has an important function in some of the necessary steps that make up the angiogenic process. We therefore propose a pivotal role of this heparan sulphate proteoglycan in the formation of new blood vessels.
Claudin-1 is an integral membrane protein component of tight junctions. The Snail family of transcription factors are repressors that play a central role in the epithelial-mesenchymal transition, a ...process that occurs during cancer progression. Snail and Slug members are direct repressors of E-cadherin and act by binding to the specific E-boxes of its proximal promoter. In the present study, we demonstrate that overexpression of Slug or Snail causes a decrease in transepithelial electrical resistance. Overexpression of Slug and Snail in MDCK (Madin-Darby canine kidney) cells down-regulated Claudin-1 at protein and mRNA levels. In addition, Snail and Slug are able to effectively repress human Claudin-1-driven reporter gene constructs containing the wild-type promoter sequence, but not those with mutations in two proximal E-box elements. We also demonstrate by band-shift assay that Snail and Slug bind to the E-box motifs present in the human Claudin-1 promoter. Moreover, an inverse correlation in the levels of Claudin-1 and Slug transcripts were observed in breast cancer cell lines. E-box elements in the Claudin-1 promoter were found to play a critical negative regulatory role in breast cancer cell lines that expressed low levels of Claudin-1 transcript. Significantly, in invasive human breast tumours, high levels of Snail and Slug correlated with low levels of Claudin-1 expression. Taken together, these results support the hypothesis that Claudin-1 is a direct downstream target gene of Snail family factors in epithelial cells.
Patients with antibody deficiency disorders are highly susceptible to microbial infections. Intravenous (i.v.) immunoglobulin concentrates were originally developed as replacement therapy for such ...patients. The present study assesses the measles virus neutralizing antibody titers and the antibody- dependent cell-mediated cytotoxicity (ADCC) capacities against Epstein-Barr virus (EBV)-infected cells of immunoglobulin G (IgG) preparations produced for i.v. use (i.v. IgG). The level of neutralizing antibodies against measles virus was determined by a syncytium neutralization test with Vero cells as targets. The measles virus neutralizing antibody titers of the i.v. IgG preparations were >3 x 10 and were an average of 1.0 log higher than the titers in pooled plasma from healthy subjects. The two IgG preparations tested showed similar ADCC activities against EBV-infected Raji cells, being active at concentrations of 3 mg/ml or higher. i.v. IgG bound to Raji cells but not to the EBV-negative Ramos cells, as evaluated by flow cytometry. Our in vitro findings may provide further support for the use of i.v. IgG for the prevention and treatment of infections caused by specific viral pathogens.
To improve our understanding of how retinal pigment epithelial (RPE) cells behave in vivo and to establish similarities with dedifferentiation and adaptive events observed in RPE cells cultured under ...simulated intraocular pathologic conditions. At the same time, to examine the origin of epithelioid-shaped and fibroblast/fusiform-shaped cells in epiretinal membranes (ERM) from proliferative vitreoretinopathy (PVR).
Cells of ERM were studied by electron-immunocytochemical techniques, using simple, double, and triple immunostaining for cytokeratins (CK), vimentin (Vim), and glial fibrillary acidic protein (GFAP). Ultrastructural morphology analysis was also carried out. Adult human RPE cells were obtained and cultured with normal and pathologic vitreous from proliferative vitreoretinal disorders, subretinal fluid aspirates from retinal detachment, and normal human serum. Their cytoskeleton was fractionated at 7 (early cultures) and 24 (late cultures) days of culture, electrophoresed, immunoblotted for intermediate filament proteins, and quantified by densitometric analysis for each condition. Changes in phenotype characteristics were also evaluated.
Epithelioid-shaped and fibroblast/fusiform-shaped cells, resembling RPE cells, expressed CK-Vim-GFAP simultaneously as intermediate filament proteins in their cytoskeleton. RPE cells in culture also expressed CK-Vim-GFAP and changed from an epithelial shape to a migratory fibroblast/fusiform-shaped phenotype in the presence of subretinal fluid aspirates and pathologic vitreous from proliferative intraocular disorders. In simulated cultures of proliferative intraocular disorders, cells decreased or retained their CK7, CK8, and CK18, retained Vim, and increased CK19 and GFAP, while their mesenchymal morphology became clearer over time.
Studies of intermediate filament proteins in vivo suggest that dedifferentiation occurs in RPE cells in ERM. Dedifferentiated RPE cells may be responsible for epithelioid-like and fibroblast/fusiform-like cells. Furthermore, changes in intermediate filament protein levels were observed in RPE cells in simulated cultures of proliferative intraocular disorders. These changes were linked to cells acquiring a mesenchymal migratory, phenotype. Results indicate that the dedifferentiation of RPE cells occurs both in vivo and in vitro and that it can be explained as an epithelial-mesenchymal transition.
Lipoprotein lipase (LPL) is produced by cells in the artery wall and can mediate binding of lipoproteins to cell surface heparan sulfate proteoglycans (HSPG), resulting in endocytosis (the bridging ...function). Active, dimeric LPL may dissociate to inactive monomers, the main form found in plasma. We have studied binding/internalization of human low density lipoprotein (LDL), mediated by bovine LPL, using THP-1 monocytes and macrophages. Uptake of
125I-LDL was similar in monocytes and macrophages and was not affected by the LDL-receptor family antagonist receptor-associated protein (RAP) or by the phagocytosis inhibitor cytochalasin D. In contrast, uptake depended on HSPG and on membrane cholesterol. Incubation in the presence of dexamethasone increased the endogenous production of LPL by the cells and also increased LPL-mediated binding of LDL to the cell surfaces. Monomeric LPL was bound to the cells mostly in a heparin-resistant fashion. We conclude that the uptake of LDL mediated by LPL dimers is receptor-independent and involves cholesterol-enriched membrane areas (lipid rafts). Dimeric and monomeric LPL differ in their ability to mediate binding/uptake of LDL, probably due to different mechanisms for binding/internalization.
Platelet-derived growth factor (PDGF) has been implicated in vascular smooth muscle cell proliferation and migration, a key process in vascular disease. PDGF is a family of dimeric isoforms of ...structurally related A-, B-, C- and D-chains that bind to PDGF receptors. PDGF A- and B-chains occur with and without basic C-terminal amino acid extensions as long (A(L) and B(L)) and short (A(S) and B(S)) isoforms. This basic sequence has been implicated as a cell retention signal through binding to glycosaminoglycans, especially to heparan sulfate. The aim of this study was to evaluate the biological relevance of PDGF interaction with glycosaminoglycans on the PDGF function in human arterial smooth muscle cells (hASMC). Here, we show that long PDGF isoforms showed greater affinity for hASMC cell surface and that they also presented more colocalization with heparan and chondroitin sulfates present on hASMC cell membrane than did short isoforms. Furthermore, all PDGF isoforms colocalized more with heparan sulfate than with chondroitin sulfate and there was little colocalization between heparan and chondroitin sulfate. PDGF-stimulated hASMC activation of DNA synthesis and directed migration (chemotaxis) was also examined. The isoform PDGF-BB(S) induced maximal proliferation and migration of hASMC. Collagen-I coating significantly increased hASMC motility towards PDGF isoforms, and particularly toward PDGF-BB(S). These results strongly support the notion that cell surface glycosaminoglycans are not essential for receptor-mediated activity of PDGF and may contribute basically to the retention and accumulation of long PDGF isoforms.
ERM (Ezrin/Radixin/Moesin) proteins are crosslinkers between plasma membrane proteins and the actin cytoskeleton, thereby involved in the formation of cell adhesion sites. Earlier work showed that ...Ezrin links syndecan-2 to the actin cytoskeleton. Here we provide evidence that the Ezrin N-terminal domain binds to the syndecan-2 cytoplasmic domain with an estimated
K
D of 0.71 μM and without the requirement of other proteins. We also studied the regions in the syndecan-2 cytoplasmic domain implicated in the binding to Ezrin. By truncating the syndecan-2 cytoplasmic domain and by oligopeptide competition assays we show that the Ezrin-binding sequence is not located in the positively charged juxtamembrane region (RMRKK), but in the neighboring sequence DEGSYD. We therefore conclude that the consensus sequence for Ezrin binding is unique among membrane proteins, suggesting a distinct regulation.
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