Savaria University Center

Szombathely, Hungary

Savaria University Center

Szombathely, Hungary
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Letoha T.,Pharmacoidea Development and Service Ltd. | Letoha T.,University of Szeged | Kolozsi C.,Pharmacoidea Development and Service Ltd. | Kolozsi C.,University of Szeged | And 7 more authors.
European Journal of Pharmaceutical Sciences | Year: 2013

The long awaited breakthrough of gene therapy significantly depends on the in vivo efficiency of targeted intracellular delivery. Hidden details of cellular uptake present a great hurdle for non-viral gene delivery with liposomes. Growing scientific evidence supports the involvement of polyanionic cell surface carbohydrates in cellular internalization of cationic liposomes. Syndecans, a highly conserved family of transmembrane heparan sulfate proteoglycans serve attachment sites for great variety of cationic ligands including growth factors, cytokines and even parasites. In the present study we quantitatively measured the contribution of various syndecan isoforms to liposome-mediated gene transfer. The obtained data show the superiority of syndecan-4, the ubiquitously expressed isoform of the syndecan family, in cellular uptake of liposomes. Applied mutational analysis demonstrated that gene delivery could be abolished by mutating the glycosaminoglycan attachment site of syndecans, highlighting the importance of polyanionic heparan sulfate side chains in the attachment of cationic liposomes. Blocking sulfation of syndecans also diminished gene delivery, a finding that confirms the essential role of polyanionic charges in binding cationic liposomes. Mutating other parts of the syndecan extracellular domain, including the cell-binding domain, had clearly smaller effect on liposome internalization. Mutational analyses also revealed that superiority of syndecan-4 in liposome-mediated gene delivery is significantly influenced by its cytoplasmic domain that orchestrates signaling pathways leading to macropinocytosis. In summary our study present a mechanistic insight into syndecan-mediated macropinocytic uptake of lipoplexes and highlights syndecan-4 as a superior target for cationic liposomes. © 2013 Elsevier B.V.

Keller-Pinter A.,Semmelweis University | Keller-Pinter A.,University of Szeged | Bottka S.,Hungarian Academy of Sciences | Timar J.,Semmelweis University | And 7 more authors.
Cellular and Molecular Life Sciences | Year: 2010

During mitosis, cells detach, and the cell-matrix interactions become restricted. At the completion of cytokinesis, the two daughter cells are still connected transiently by an intercellular bridge (ICB), which is subjected to abscission, as the terminal step of cytokinesis. Cell adhesion to the matrix is mediated by syndecan-4 (SDC4) transmembrane heparan sulfate proteoglycan. Our present work demonstrated that SDC4 promotes cytokinesis in a phosphorylation-dependent manner in MCF-7 breast adenocarcinoma cells. The serine179-phosphorylation and the ectodomain shedding of SDC4 changed periodically in a cell cycle-dependent way reaching the maximum at G2/M phases. On the contrary, the phospho-resistant Ser179Ala mutant abrogated the shedding. The phosphorylated full-length and shed remnants enriched along the mitotic spindles, and subsequently in the ICBs, however, proper membrane insertion was necessary for midbody localization. Expression of phosphomimicking Ser179Glu SDC4 resulted in incomplete abscission, whereas expression of the phospho-resistant SDC4 led to giant, multinucleated cells. © 2010 Springer Basel AG.

Letoha T.,Hungarian Academy of Sciences | Letoha T.,Pharmacoidea Ltd. | Keller-Pinter A.,University of Szeged | Kusz E.,Hungarian Academy of Sciences | And 8 more authors.
Biochimica et Biophysica Acta - Biomembranes | Year: 2010

Cell-penetrating peptides (CPPs) are short peptides capable of translocating across the plasma membrane of live cells and transporting conjugated compounds intracellularly. Fifteen years after discovering the first model cationic CPPs, penetratin and TAT, CPP internalization is still challenging many questions. Particularly it has been unknown whether CPPs enter the cells with or without mediation of a specific surface receptor. Here we report that syndecan-4, the universally expressed isoform of the syndecan family of transmembrane proteoglycans, binds and mediates transport of the three most frequently utilized cationic CPPs (penetratin, octaarginine and TAT) into the cells. Quantitative uptake studies and mutational analyses demonstrate that attachment of the cationic CPPs is mediated by specific interactions between the heparan sulfate chains of syndecan-4 and the CPPs. Protein kinase C alpha is also heavily involved in the uptake mechanism. The collected data give the first direct evidence on the receptor-mediated uptake of cationic CPPs and may replace the long-thought, but already contradicted membrane penetration hypothesis. Thus our study might give an answer for a decade long debate and foster the development of rationalized, syndecan-4 targeted novel delivery technologies. © 2010 Elsevier B.V.

Zong F.,Karolinska Institutet | Fthenou E.,University of Crete | Castro J.,Karolinska Institutet | Peterfia B.,Semmelweis University | And 4 more authors.
Cell Proliferation | Year: 2010

Objectives: Syndecan-1 is a transmembrane proteoglycan involved in various biological processes. Its extracellular, transmembrane and cytoplasmic domains may all participate in signal transduction. The aim of this study was to investigate the biological roles of these domains of syndecan-1. Materials and methods: We transfected cells of two mesenchymal tumour cell lines with a full-length syndecan-1 construct and three truncated variants, namely 78 construct lacking the EC domain with exception of DRKE sequence; 77 construct lacking extracellular the whole domain and RMKKK corresponding to a short cytoplasmic motif. Subcellular distribution was revealed using confocal laser microscopy. Overexpression of the constructs was verified using real-time RT-PCR and by FACS analysis and effects of syndecan-1 on cell behaviour were explored. Cell cycle analysis allowed for dissection of mechanisms regulating cell proliferation. Results: Overexpression of syndecan-1 influenced expression profile of the other syndecan members, and decreased tumour cell proliferation significantly by two mechanisms, as follows: increased length of G0/G1 phase was the most evident change in RMKKK and 77 transfectants, whereas prolonged S phase was more obvious in full-length transfectants. Overexpression of syndecan-1 changed the tumour cell morphology in an epithelioid direction. Conclusions: Both full-length and truncated syndecan-1 inhibited proliferation of the mesenchymal tumour cells, providing new insights into the importance for cancer growth of different functional domains of this proteoglycan. © 2009 Blackwell Publishing Ltd.

Zong F.,Karolinska Institutet | Fthenou E.,University of Crete | Mundt F.,Karolinska Institutet | Szatmari T.,Karolinska Institutet | And 6 more authors.
PLoS ONE | Year: 2011

Background: Syndecans are proteoglycans whose core proteins have a short cytoplasmic domain, a transmembrane domain and a large N-terminal extracellular domain possessing glycosaminoglycan chains. Syndecans are involved in many important cellular processes. Our recent publications have demonstrated that syndecan-1 translocates into the nucleus and hampers tumor cell proliferation. In the present study, we aimed to investigate the role of syndecan-1 in tumor cell adhesion and migration, with special focus on the importance of its distinct protein domains, to better understand the structure-function relationship of syndecan-1 in tumor progression. Methodology/Principal Findings: We utilized two mesenchymal tumor cell lines which were transfected to stably overexpress full-length syndecan-1 or truncated variants: the 78 which lacks the extracellular domain except the DRKE sequence proposed to be essential for oligomerization, the 77 which lacks the whole extracellular domain, and the RMKKK which serves as a nuclear localization signal. The deletion of the RMKKK motif from full-length syndecan-1 abolished the nuclear translocation of this proteoglycan. Various bioassays for cell adhesion, chemotaxis, random movement and wound healing were studied. Furthermore, we performed gene microarray to analyze the global gene expression pattern influenced by syndecan-1. Both full-length and truncated syndecan-1 constructs decrease tumor cell migration and motility, and affect cell adhesion. Distinct protein domains have differential effects, the extracellular domain is more important for promoting cell adhesion, while the transmembrane and cytoplasmic domains are sufficient for inhibition of cell migration. Cell behavior seems to depend also on the nuclear translocation of syndecan-1. Many genes are differentially regulated by syndecan-1 and a number of genes are actually involved in cell adhesion and migration. Conclusions/Significance: Our results demonstrate that syndecan-1 regulates mesenchymal tumor cell adhesion and migration, and different domains have differential effects. Our study provides new insights into better understanding of the role of syndecans in tumor progression. © 2011 Zong et al.

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