Publications by authors named "Yanusz Wegrowski"

27 Publications

  • Page 1 of 1

'Click'-xylosides as initiators of the biosynthesis of glycosaminoglycans: Comparison of mono-xylosides with xylobiosides.

Chem Biol Drug Des 2017 Mar 2;89(3):319-326. Epub 2016 Nov 2.

CNRS UMR 7369, Matrice Extracellulaire et Dynamique Cellulaire, UFR de Médecine, Université de Reims Champagne Ardenne, Reims Cedex, France.

Different mono-xylosides and their corresponding xylobiosides obtained by a chemo-enzymatic approach featuring various substituents attached to a triazole ring were probed as priming agents for glycosaminoglycan (GAG) biosynthesis in the xylosyltransferase-deficient pgsA-745 Chinese hamster ovary cell line. Xylosides containing a hydrophobic aglycone moiety were the most efficient priming agents. Mono-xylosides induced higher GAG biosynthesis in comparison with their corresponding xylobiosides. The influence of the degree of polymerization of the carbohydrate part on the priming activity was investigated through different experiments. We demonstrated that in case of mono-xylosides, the cellular uptake as well as the affinity and the catalytic efficiency of β-1,4-galactosyltransferase 7 were higher than for xylobiosides. Altogether, these results indicate that hydrophobicity of the aglycone and degree of polymerization of glycone moiety were critical factors for an optimal priming activity for GAG biosynthesis.
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http://dx.doi.org/10.1111/cbdd.12865DOI Listing
March 2017

Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity.

PLoS One 2016 1;11(3):e0150226. Epub 2016 Mar 1.

CNRS UMR 7369, Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), Université de Reims Champagne Ardenne, Laboratoire de Biochimie Médicale et de Biologie Moléculaire, Reims, France.

Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150226PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773148PMC
July 2016

Lumican: a new inhibitor of matrix metalloproteinase-14 activity.

FEBS Lett 2014 Nov 7;588(23):4319-24. Epub 2014 Oct 7.

CNRS UMR 7369, Matrice Extracellulaire et Dynamique Cellulaire, Reims, France; Université de Reims Champagne Ardenne, Laboratoire de Biochimie Médicale et de Biologie Moléculaire, Reims, France. Electronic address:

We previously showed that lumican regulates MMP-14 expression. The aim of this study was to compare the effect of lumican and decorin on MMP-14 activity. In contrast to decorin, the glycosylated form of lumican was able to significantly decrease MMP-14 activity in B16F1 melanoma cells. Our results suggest that a direct interaction occurs between lumican and MMP-14. Lumican behaves as a competitive inhibitor which leads to a complete blocking of the activity of MMP-14. It binds to the catalytic domain of MMP-14 with moderate affinity (KD∼275 nM). Lumican may protect collagen against MMP-14 proteolysis, thus influencing cell-matrix interaction in tumor progression.
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http://dx.doi.org/10.1016/j.febslet.2014.09.040DOI Listing
November 2014

Glycosaminoglycan profiling in different cell types using infrared spectroscopy and imaging.

Anal Bioanal Chem 2014 Sep 15;406(24):5795-803. Epub 2014 Jul 15.

Laboratoire de Biochimie médicale et de Biologie Moléculaire, UFR de Médecine, Université de Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51095, Reims Cedex, France.

We recently identified vibrational spectroscopic markers characteristic of standard glycosaminoglycan (GAG) molecules. The aims of the present work were to further this investigation to more complex biological systems and to characterize, via their spectral profiles, cell types with different capacities for GAG synthesis. After recording spectral information from individual GAG standards (hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate) and GAG-GAG mixtures, GAG-defective mutant Chinese hamster ovary (CHO)-745 cells, wild-type CHO cells, and chondrocytes were analyzed as suspensions by high-throughput infrared spectroscopy and as single isolated cells by infrared imaging. Spectral data were processed and interpreted by exploratory unsupervised chemometric methods based on hierarchical cluster analysis and principal component analysis. Our results showed that the spectral information obtained was discriminant enough to clearly delineate between the different cell types both at the cell suspension and single-cell levels. The abilities of the technique are to perform spectral profiling and to identify single cells with different potentials to synthesize GAGs. Infrared microspectroscopy/imaging could therefore be developed for cell screening purposes and further for identifying GAG molecules in normal tissues during physiological conditions (aging, healing process) and numerous pathological states (arthritis, cancer).
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http://dx.doi.org/10.1007/s00216-014-7994-2DOI Listing
September 2014

Lumican - derived peptides inhibit melanoma cell growth and migration.

PLoS One 2013 2;8(10):e76232. Epub 2013 Oct 2.

Laboratoire de Biochimie Médicale et de Biologie Moléculaire, CNRS FRE 3481, Université de Reims-Champagne-Ardenne, Reims, France.

Lumican, a small leucine-rich proteoglycan of the extracellular matrix, presents potent anti-tumor properties. Previous works from our group showed that lumican inhibited melanoma cell migration and tumor growth in vitro and in vivo. Melanoma cells adhered to lumican, resulting in a remodeling of their actin cytoskeleton and preventing their migration. In addition, we identified a sequence of 17 amino acids within the lumican core protein, named lumcorin, which was able to inhibit cell chemotaxis and reproduce anti-migratory effect of lumican in vitro. The aim of the present study was to characterize the anti-tumor mechanism of action of lumcorin. Lumcorin significantly decreased the growth in monolayer and in soft agar of two melanoma cell lines - mice B16F1 and human SK-MEL-28 cells - in comparison to controls. Addition of lumcorin to serum free medium significantly inhibited spontaneous motility of these two melanoma cell lines. To characterize the mechanisms involved in the inhibition of cell migration by lumcorin, the status of the phosphorylation/dephosphorylation of proteins was examined. Inhibition of focal adhesion kinase phosphorylation was observed in presence of lumcorin. Since cancer cells have been shown to migrate and to invade by mechanisms that involve matrix metalloproteinases (MMPs), the expression and activity of MMPs were analyzed. Lumcorin induced an accumulation of an intermediate form of MMP-14 (~59kDa), and inhibited MMP-14 activity. Additionally, we identified a short, 10 amino acids peptide within lumcorin sequence, which was able to reproduce its anti-tumor effect on melanoma cells. This peptide may have potential pharmacological applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076232PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3788744PMC
July 2014

Quantitative analysis of type I collagen fibril regulation by lumican and decorin using AFM.

J Struct Biol 2013 Sep 6;183(3):394-403. Epub 2013 Jun 6.

DFG-Center for Functional Nanostructures, Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Strasse 1a, 76131 Karlsruhe, Germany. Electronic address:

Lumican and decorin, two members of the small leucine-rich repeat proteoglycan (SLRP) family, have been implicated as regulators of collagen I fibril structure in different tissues. Both proteoglycans consist of a core protein and a glycosaminoglycan (GAG) chain, but quantitative information regarding the precise role of the protein and GAG moieties in regulating collagen structure is still limited. In this study, we used AFM imaging and a model system of aligned collagen I nanofibrils to investigate the role of lumican and decorin on collagen I fibril structure with high resolution. When co-assembled with collagen I, recombinant lumican or decorin proteins lacking the GAG chains decreased collagen fibril width to values below <100nm and increased interfibrillar spacing in a dose-dependent manner. At lower concentrations, lumican appeared to have a stabilizing effect on newly-formed collagen fibrils, while at higher concentrations both lumican and decorin inhibited collagen fibrillogenesis. GAG-containing decorin also increased interfibrillar spacing, decreased fibril width and ultimately inhibited fibrillogenesis, but these effects required lower concentrations compared to recombinant decorin, indicating that the decorin core protein alone cannot compensate for the full regulatory and structural contribution of the GAG chain during collagen I fibrillogenesis. Using a 2D autocorrelation approach, we furthermore analyzed and compared the effects of recombinant and glycosylated decorin on collagen ultrastructure, providing a quantitative measure for the observed structural differences. AFM analysis of ordered fibrillar collagen arrays in combination with quantitative autocorrelation image analysis thus provides a useful tool for investigating SLRP-dependent nanoscale effects on collagen fibril structure.
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http://dx.doi.org/10.1016/j.jsb.2013.05.022DOI Listing
September 2013

Lumican effects in the control of tumour progression and their links with metalloproteinases and integrins.

FEBS J 2013 May 18;280(10):2369-81. Epub 2013 Mar 18.

Laboratoire de Biochimie Médicale et de Biologie Moléculaire, CNRS FRE 3481, Université de Reims-Champagne-Ardenne, Reims, France.

Lumican is a member of the small leucine-rich proteoglycan family. It is present in numerous extracellular matrices of different tissues, such as muscle, cartilage, and cornea. In skin, lumican is present as a glycoprotein. It plays a critical role in collagen fibrillogenesis, as shown by knocking out of its gene in mice. A direct link between lumican expression and melanoma progression and metastasis has been demonstrated. Lumican was shown to impede tumour cell migration and invasion by directly interacting with the α2β1 integrin. In addition, an active sequence of the lumican core protein, called lumcorin, was identified as being responsible for inhibition of melanoma cell migration. Lumican was also shown to exert angiostatic properties by downregulating the proteolytic activity associated with endothelial cell membranes, particularly matrix metalloproteinase (MMP)-14 and MMP-9. Globally, lumican appears to be a potent agent for inhibiting tumour progression rather than tumorigenesis. However, progressive changes in proteoglycans occur in the tumour environment. The complexity and diversity of proteoglycan structure might be responsible for a variety of functions that regulate cell behaviour. Through their core protein and their glycosaminoglycan chains, proteoglycans can interact with growth factors and chemokines. These interactions affect cell signalling, motility, adhesion, growth, and apoptosis. This review summarizes recent data concerning lumican control of tumour progression in different cancers, with a particular focus on its interactions with MMPs and integrins. Its potential therapeutic implications are discussed.
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http://dx.doi.org/10.1111/febs.12210DOI Listing
May 2013

Effect of lumican on the migration of human mesenchymal stem cells and endothelial progenitor cells: involvement of matrix metalloproteinase-14.

PLoS One 2012 7;7(12):e50709. Epub 2012 Dec 7.

Laboratoire de Biochimie Médicale et de Biologie Moléculaire, CNRS FRE 3481, Université de Reims Champagne-Ardenne, Reims, France.

Background: Increasing number of evidence shows that soluble factors and extracellular matrix (ECM) components provide an optimal microenvironment controlling human bone marrow mesenchymal stem cell (MSC) functions. Successful in vivo administration of stem cells lies in their ability to migrate through ECM barriers and to differentiate along tissue-specific lineages, including endothelium. Lumican, a protein of the small leucine-rich proteoglycan (SLRP) family, was shown to impede cell migration and angiogenesis. The aim of the present study was to analyze the role of lumican in the control of MSC migration and transition to functional endothelial progenitor cell (EPC).

Methodology/principal Findings: Lumican inhibited tube-like structures formation on Matrigel® by MSC, but not EPC. Since matrix metalloproteinases (MMPs), in particular MMP-14, play an important role in remodelling of ECM and enhancing cell migration, their expression and activity were investigated in the cells grown on different ECM substrata. Lumican down-regulated the MMP-14 expression and activity in MSC, but not in EPC. Lumican inhibited MSC, but not EPC migration and invasion. The inhibition of MSC migration and invasion by lumican was reversed by MMP-14 overexpression.

Conclusion/significance: Altogether, our results suggest that lumican inhibits MSC tube-like structure formation and migration via mechanisms that involve a decrease of MMP-14 expression and activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050709PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517548PMC
May 2013

Overexpression of lumican affects the migration of human colon cancer cells through up-regulation of gelsolin and filamentous actin reorganization.

Exp Cell Res 2012 Nov 16;318(18):2312-23. Epub 2012 Jul 16.

Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland.

Cell migration is a multistep process initiated by extracellular matrix components that leads to cytoskeletal changes and formation of different protrusive structures at the cell periphery. Lumican, a small extracellular matrix leucine-rich proteoglycan, has been shown to inhibit human melanoma cell migration by binding to α2β1 integrin and affecting actin cytoskeleton organization. The aim of this study was to determine the effect of lumican overexpression on the migration ability of human colon adenocarcinoma LS180 cells. The cells stably transfected with plasmid containing lumican cDNA were characterized by the increased chemotactic migration measured on Transwell filters. Lumican-overexpressing cells presented the elevated filamentous to monomeric actin ratio and gelsolin up-regulation. This was accompanied by a distinct cytoskeletal actin rearrangement and gelsolin subcellular relocation, as observed under laser scaning confocal microscope. Moreover, LS180 cells overexpressing lumican tend to form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin at the submembrane region of the cells. In conclusion, the elevated level of lumican secretion to extracellular space leads to actin cytoskeletal remodeling followed by an increase in migration capacity of human colon LS180 cells. These data suggest that lumican expression and its presence in ECM has an impact on colon cancer cells motility and may modulate invasiveness of colon cancer.
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http://dx.doi.org/10.1016/j.yexcr.2012.07.005DOI Listing
November 2012

Characterization of glycosaminoglycans by tandem vibrational microspectroscopy and multivariate data analysis.

Methods Mol Biol 2012 ;836:117-30

Laboratoire de Biochimie Médicale et de Biologie Moléculaire, CNRS UMR 6237-MEDyC, Université de Reims-Champagne-Ardenne, Reims, France.

Vibrational spectroscopies (VS), INFRARED SPECTROSCOPY and RAMAN SPECTROSCOPY, are well-established techniques for exploring the chemical composition of samples. VS are based on the molecular vibrations and give a spectral signature also called "molecular fingerprint" characteristic of the studied material. Recent advances in these techniques have rendered them faster, more sensitive, and easier to use. This chapter describes their application to characterize the main glycosaminoglycans-without any sample destruction or degradation. Nowadays, the use of multivariate statistical analysis for analyzing spectral data allows to extract rapidly the discriminant spectral information from large data sets. The combination of VS and this type of data analysis is also discussed in this chapter.
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http://dx.doi.org/10.1007/978-1-61779-498-8_8DOI Listing
May 2012

Ultraviolet-B irradiation induces epidermal up-regulation of heparanase expression and activity.

J Photochem Photobiol B 2012 Jan 31;106:107-12. Epub 2011 Oct 31.

Université de Reims Champagne-Ardenne, Laboratoire de Biochimie Médicale et Biologie Moléculaire, CNRS UMR 6237, 51 rue Cognacq Jay, 51095 Reims cedex, France.

Heparan sulfate (HS) glycosaminoglycans are abundant components of basement membranes and cell surfaces where they are present associated with specific core-proteins to form proteoglycans, mainly perlecan, glypicans and syndecans. They play many roles such as modulation of cell proliferation and differentiation, cell-matrix adhesion and assembly. It was previously shown that HS content decreases during skin aging. This decrease could be explained either by a decrease of HS synthesis or by an increased activity of its degrading enzyme, heparanase (Hpse-1). Since UV-B irradiation is one of the most important factor for skin photo-damage, we decided to study the effects of UV-B irradiation on heparanase expression and activity in human epidermal keratinocytes. Normal human keratinocytes and reconstructed epidermis were submitted to increasing doses of UV-B. HPSE1 mRNA levels were measured using real time PCR and heparanase enzymatic activity was quantified in human keratinocyte cultures using a microtiter-based assay. Expression and distribution of Hpse-1 were also studied in reconstructed epidermis by immunofluorescence. Both HPSE1 mRNA level and heparanase enzymatic activity were increased after UV-B irradiation of keratinocyte cultures in a time and dose-dependent manner. Protein expression of Hpse-1 was also up-regulated with increasing doses of UV-B in reconstructed epidermis. Increase of Hpse-1 expression and activity in the epidermis after UV-B irradiation could contribute to skin photo-aging.
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http://dx.doi.org/10.1016/j.jphotobiol.2011.10.013DOI Listing
January 2012

Lumican inhibits angiogenesis by interfering with α2β1 receptor activity and downregulating MMP-14 expression.

Thromb Res 2011 Nov 12;128(5):452-7. Epub 2011 Jul 12.

Department of Molecular and Medical Biophysics, Medical University of Lodz, Poland.

Introduction: Previous studies showed that lumican, a small leucine-rich proteoglycan that binds to α2 integrin I domain, is an efficient inhibitor of cell adhesion and migration. In this report, we tested its effect on angiogenesis in vitro and in vivo.

Materials And Methods: Effect of lumican on angiogenesis was evaluated by in vitro capillary tube formation test performed between Fibrin II Gels or in Matrigel™ and in vivo by Matrigel(™) plug assay in BALB/c mice. Changes in matrix metalloproteinases expression caused by lumican were analyzed in endothelial cells by real-time PCR, Western immunoblotting and gelatin zymography.

Results: In unchallenged endothelial cells, Matrigel™ induced robust capillary morphogenesis. In contrast, tube formation was dramatically reduced by lumican, and by siRNA to β1 integrin subunit mRNA but not by control siRNA. Similarly, lumican effectively inhibited neovascularization in vivo in assays using Matrigel™ plugs formed in BALB/c mice. Interestingly, lumican significantly reduced expression of matrix metalloproteinases, particularly MMP-14 that is known to activate other MMPs in close vicinity of endothelial cell membranes.

Conclusions: Our results provide strong evidence that lumican affects angiogenesis both by interfering with α2β1 receptor activity and downregulating proteolytic activity associated with surface membranes of endothelial cells.
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http://dx.doi.org/10.1016/j.thromres.2011.06.011DOI Listing
November 2011

Ultraviolet-B irradiation induces differential regulations of hyaluronidase expression and activity in normal human keratinocytes.

Photochem Photobiol 2011 Sep-Oct;87(5):1105-12. Epub 2011 Jul 28.

Laboratoire de Biochimie Médicale et Biologie Moléculaire, CNRS UMR 6237, Université de Reims Champagne-Ardenne, Reims, France.

Skin aging is a complex process determined by genetic factors (intrinsic aging) and environmental factors (extrinsic aging). One of the most influential environmental factor is UV-B irradiation. Hyaluronic acid (HA) is an abundant component of skin extracellular matrix where it plays many roles such as hydration and architectural support. Downregulation of HA during photoaging was reported previously. Changes in expression and function of its degrading enzymes, the hyaluronidases (Hyals) might be involved in this decrease. In the present study, normal human keratinocytes were submitted to increasing doses of UV-B. The mRNA expression of HYAL1, HYAL2 and HYAL3 and the hyaluronidase enzymatic activity were quantified using real-time PCR and a microtiter-based assay, respectively. After UV-B irradiation, HYAL1 mRNA expression was upregulated whereas HYAL2 and HYAL3 mRNAs were downregulated and hyaluronidase enzymatic activity was increased in both cell layer and culture medium. In parallel, immunohistochemical studies performed on UV-B irradiated reconstructed epidermis confirmed that Hyal-1, Hyal-2 and Hyal-3 protein expression were differently regulated by UV-B. Taken together, our results demonstrate that UV-B irradiation induces differential regulations of hyaluronidase expression and enzymatic activity in human keratinocytes. These differential modulations of hyaluronidase expression and activity by UV-B could contribute to cutaneous photoaging.
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http://dx.doi.org/10.1111/j.1751-1097.2011.00959.xDOI Listing
December 2011

Lumican inhibits cell migration through α2β1 integrin.

Exp Cell Res 2010 Oct 10;316(17):2922-31. Epub 2010 Aug 10.

CNRS UMR 6237 MEDyC, Université de Reims-Champagne-Ardenne, Reims, France.

Lumican, an extracellular matrix protein of the small leucine-rich proteoglycan family, has been shown to impede melanoma progression by inhibiting cell migration. In the present study, we show that lumican targets α2β1 integrin thereby inhibiting cell migration. A375 melanoma cells were transfected with siRNA directed against the α2 integrin subunit. Compared to A375 control cells, the anti-migratory effect of lumican was abrogated on transfected A375 cells. Moreover, lumican inhibited the chemotactic migration of Chinese hamster ovary (CHO) cells stably transfected with α2 integrin subunit (CHO-A2) but not that of wild-type CHO cells (CHO-WT) lacking this subunit. In contrast to CHO-WT cells, we observed in time-lapse microscopy a decrease of CHO-A2 cell migration speed in presence of lumican. Focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) and total FAK were analysed in CHO-WT and CHO-A2 cells. A significant decrease of the ratio pFAK/FAK was shown in presence of recombinant human lumican. Using solid phase assays, a direct binding between lumican and the α2β1 integrin was demonstrated. This interaction did not involve the glycan moiety of lumican and was cation independent. Lumican was also able to bind the activated I domain of the α2 integrin subunit with a K(d)≥200nM. In conclusion, we demonstrated for the first time that the inhibition of cell migration by lumican depends on a direct binding between the core protein of lumican and the α2β1 integrin.
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http://dx.doi.org/10.1016/j.yexcr.2010.08.002DOI Listing
October 2010

Rapid characterization of glycosaminoglycans using a combined approach by infrared and Raman microspectroscopies.

J Pharm Sci 2011 Feb;100(2):441-50

Université de Reims, Laboratoire de Biochimie Médicale et de Biologie Moléculaire, CNRS UMR 6237 MEDyC, Reims, France.

This study describes a complementary infrared (IR) and Raman spectroscopic investigations of a set of biomolecule representatives of glycosaminoglycans (GAGs) class. Both IR and Raman data exhibit characteristic spectral signatures that allow a direct molecular distinction of these compounds. Comparison of these molecular signatures clearly evidences the differences between heparan sulfate and heparin by computing the intensity ratio between the 1248 cm(-1) and 1043 cm(-1) peaks, corresponding respectively to sulfate and C-O-C linkages. Identically, chondroitin-4-sulfate and chondroitin-6-sulfate are differentiated via the 730 cm(-1) and 853 cm(-1) bands (axial orientation) and the 822 cm(-1) and 1000 cm(-1) bands (equatorial orientation). These orientations concern the sulfate groups of these molecules. Secondly, multivariate statistical methods were employed to analyze the data set. Hierarchical cluster analysis (HCA) of both IR and Raman spectra showed a very good differentiation between the different structures. In addition, the sulfatation degree could be obtained from this classification. Principal component analysis using three principal components (PCs) confirmed the findings of HCA and, furthermore, comparisons of the PC loadings highlight the main molecular differences between the members of this class of biological molecules. It is expected that these microspectroscopic methods will be useful in identifying GAG spectral signatures in complex biological systems.
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http://dx.doi.org/10.1002/jps.22288DOI Listing
February 2011

Lumcorin: a leucine-rich repeat 9-derived peptide from human lumican inhibiting melanoma cell migration.

FEBS Lett 2009 Sep 15;583(18):3027-32. Epub 2009 Aug 15.

Laboratoire de Biochimie Médicale et de Biologie Moléculaire, CNRS UMR 6237, Université Reims-Champagne-Ardenne, F-51095 Reims, France.

We previously showed that lumican decreases melanoma progression. The aim of the present study was to determine the active sequence of the lumican core protein responsible for the inhibition of melanoma cell migration. Using different recombinant and synthetic peptides derived from lumican, we localized an active site in the leucine-rich repeat 9 domain of the lumican core protein. We propose the name lumcorin (fragment of lumican core protein) for the active peptide derived from this site. Lumcorin was able to inhibit melanoma cell migration in vitro.
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http://dx.doi.org/10.1016/j.febslet.2009.08.012DOI Listing
September 2009

Lumican core protein inhibits melanoma cell migration via alterations of focal adhesion complexes.

Cancer Lett 2009 Sep 24;283(1):92-100. Epub 2009 Apr 24.

Laboratoire de Biochimie Médicale et Biologie Moléculaire, CNRS UMR 6237, Faculté de Médecine, Université de Reims-Champagne-Ardenne, 51095 Reims Cedex, France.

Lumican is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM) with anti-tumor activity. We recently demonstrated that lumican inhibits the migration of melanoma cells and identified beta1 integrin as mediator of this effect [M.F. D'Onofrio, S. Brézillon, T. Baranek, C. Perreau, P.J. Roughley, F.X. Maquart, Y. Wegrowski, Identification of beta1 integrin as mediator of melanoma cell adhesion to lumican, Biochem. Biophys. Res. Commun. 365 (2008) 266-272]. The aim of the present work was to study beta1 integrin, focal adhesion complexes, actin distribution and expression in the presence of lumican substratum in comparison to type I collagen or fibronectin substrata in A375 human melanoma cells. The protein distribution was investigated by immunocytochemistry and confocal microscopy. In parallel, their expression was evaluated by Western immunoblotting and Real-time Reverse Transcription-PCR analyses. The interaction of melanoma cells with the lumican substratum resulted in heterogeneous distribution of beta1 integrin on cell membrane after 24h of seeding. Concomitantly, a reorganization of actin stress fibers and a significant decrease in vinculin immunostaining at focal adhesion complexes were observed. No alteration of the expression was detected at protein and mRNA levels. However, a cytosolic accumulation of vinculin focal adhesion protein was observed on lumican substratum by confocal microscopy. Moreover, vinculin expression was significantly increased in cytosolic fractions in comparison to cells seeded on type I collagen or fibronectin substrata. Our results suggest that lumican induces an alteration of the link between actin filaments and beta1 integrin, characterized by a cytosolic accumulation of vinculin focal adhesion protein, which could lead to a destabilization of focal adhesion complexes. In addition, focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) was significantly decreased. Therefore, the cytoskeleton remodeling and the decreased pFAK phosphorylation induced by lumican in melanoma cells might explain, at least in part, the anti-invasive effect of this SLRP.
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http://dx.doi.org/10.1016/j.canlet.2009.03.032DOI Listing
September 2009

Lumican affects actin cytoskeletal organization in human melanoma A375 cells.

Life Sci 2008 Nov 22;83(19-20):651-60. Epub 2008 Sep 22.

Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, ul. Przybyszewskiego 63/77, 51-148 Wroclaw, Poland.

Aims: Lumican, a small leucine-rich proteoglycan (SLRP), has attracted attention as a molecule of the extracellular matrix possibly involved in signalling pathways affecting cancer cell behaviour. The remodelling of the actin cytoskeleton, induced in response to external stimuli, is crucial for cell motility and intracellular signal transduction. The main goal of this study was to examine the effects of recombinant lumican on actin organization, the state of actin polymerization, actin isoform expression, and their sub-cellular distribution in the A375 human melanoma cell line.

Main Methods: Fluorescence and confocal microscopy were used to observe actin cytoskeletal organization and the sub-cellular distribution of cytoplasmic beta- and gamma-actins. The ability of actin to inhibit DNaseI activity was used to quantify actin. Western blotting and real-time PCR were used to determine the expression levels of the actin isoforms.

Key Findings: A375 cells grown on lumican coatings changed in morphology and presented rearranged actin filament organization: from filaments evenly spread throughout the whole cell body to their condensed sub-membrane localization. In the presence of lumican, both actin isoforms were concentrated under the cellular membrane. A statistically significant increase in the total, filamentous, and monomeric actin pools was observed in A375 cells grown on lumican.

Significance: Novel biological effects of lumican, an extracellular matrix SLRP, on the actin pool and organization are identified, which may extend our understanding of the mechanism underlying the inhibitory effect of lumican on the migration of melanoma cells.
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http://dx.doi.org/10.1016/j.lfs.2008.09.008DOI Listing
November 2008

Identification of beta1 integrin as mediator of melanoma cell adhesion to lumican.

Biochem Biophys Res Commun 2008 Jan 5;365(2):266-72. Epub 2007 Nov 5.

Laboratoire de Biochimie Médicale et Biologie Moléculaire, CNRS UMR 6198, Faculté de Médecine, IFR 53, Université de Reims-Champagne-Ardenne, 51095 Reims cedex, France.

Lumican is a small leucine-rich proteoglycan (SLRP) present in the dermal extracellular matrix. Previous data from our laboratory demonstrated that lumican decreases melanoma progression in vivo. Here, we show that melanoma cell migration is decreased by lumican and that this effect is due to an enhanced cell adhesion. The adhesion of A375 human melanoma cells on lumican was dose-dependent and required Mg2+ and Mn2+ divalent cations. Using a panel of monoclonal antibodies directed against integrin subunits, we showed that A375 cells can bind to recombinant lumican through beta1 type integrins. Moreover, the use of rhodocetin, an inhibitor of alpha2 integrin, suggested that this particular subunit might also be involved in the interaction with lumican. The increased beta1 integrin-mediated adhesion of melanoma cells to lumican might explain, at least in part, the anti-invasive effect of this SLRP.
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http://dx.doi.org/10.1016/j.bbrc.2007.10.155DOI Listing
January 2008

Chondroitin sulfate proteoglycans in tumor progression.

Adv Pharmacol 2006 ;53:297-321

CNRS UMR 6198, Faculty of Medicine, IFR-53, 51095 Reims Cedex, France.

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http://dx.doi.org/10.1016/S1054-3589(05)53014-XDOI Listing
March 2007

Influence of aging on glycosaminoglycans and small leucine-rich proteoglycans production by skin fibroblasts.

Mol Cell Biochem 2005 Sep;277(1-2):63-72

Laboratory of Biochemistry and Molecular Biology, CNRS UMR 6198, IFR 53-Biomolécules, Faculty of Medicine, University of Reims Champagne-Ardenne, 51 Rue Cognacq Jay, 51095, Reims Cedex, France.

Skin aging is characterised by a progressive deterioration of its functional properties, linked to alterations of dermal connective tissue. Whereas many studies have been devoted to collagen alterations during aging, the situation is less clear concerning glycosaminoglycans and proteoglycans. Particularly, the alterations of the expression of small leucine-rich proteoglycans (SLRPs), a family of proteoglycans strongly implicated in cell regulation, have never been studied. In the present study we measured glycosaminoglycans and small leucine-rich proteoglycans synthesis by skin fibroblasts from donors of 1 month to 83 years old. [3H]-glucosamine and [35S]-sulfate incorporation did not show significant differences of sulfated GAG synthesis during aging. On the other hand, a significant positive correlation was found between hyaluronan secretion and donor's age. Northern blot analysis of SLRPs mRNAs showed a significant negative correlation of lumican mRNA with donor's age, whereas decorin and biglycan mRNAs were not significantly altered. Immunohistochemical study and quantitative image analysis confirmed a decreased lumican accumulation in aged human skin. Taken together, our results suggest that impairment of glycosaminoglycans and SLRPs synthesis might be involved in the functional alterations of aged skin.
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http://dx.doi.org/10.1007/s11010-005-5073-xDOI Listing
September 2005

c-Krox down-regulates the expression of UDP-glucose dehydrogenase in chondrocytes.

Biochem Biophys Res Commun 2005 Aug;333(4):1123-31

Laboratory of Connective Tissue Biochemistry, Faculty of Medicine, Caen, France.

Chondrocyte glycosaminoglycan (GAG) synthesis is regulated by the availability of UDP-glucuronate, the substrate of glucuronosyl transferases which form the GAG chains in proteoglycans and hyaluronan. UDP-glucose dehydrogenase (UDPGD) is therefore a key enzyme in the synthesis of UDP-glucuronate from glucose. However, the mechanisms regulating its expression in chondrocytes are not fully understood. We investigated the effect of c-Krox, a zinc-finger transcription factor previously shown to modulate several matrix genes, on the synthesis of GAG and transcriptional activity of several UDPGD gene promoter constructs, using transient transfection and decoy experiments in rabbit articular chondrocytes (RACs). We show that overexpression of c-Krox inhibits radiosulfate incorporation into neosynthesized GAG and that the effect was mediated by a cis-sequence located between +18 and +39bp of the UDPGD gene. Since that sequence can also bind Sp1/Sp3 factors, it is likely that c-Krox acts in concert with these proteins to modulate the UDPGD gene expression in articular chondrocytes.
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http://dx.doi.org/10.1016/j.bbrc.2005.06.020DOI Listing
August 2005

The small leucine-rich proteoglycan lumican inhibits melanoma progression.

Exp Cell Res 2004 Jun;296(2):294-306

Laboratory of Biochemistry, CNRS UMR 6198, Faculty of Medicine, F-51095 Reims, France.

Lumican is a member of the small leucine-rich proteoglycan (SLRP) family. It contributes to the organisation of the collagen network and plays an important role in cell migration and tissue repair. The present study aimed to determine the influence of lumican expression on adhesion, anchorage-dependent and -independent growth, migration, in vitro invasion and in vivo melanoma growth. For that purpose, B16F1 mouse melanoma cells were stably transfected with an expression plasmid containing the complete lumican cDNA. Lumican expression by tumor cells did not change the proliferative activity of mouse melanoma cells in monolayer culture and did not influence either cell adhesion to extracellular matrix gel or type I collagen or cell spreading on these substrates. In contrast, lumican-transfected cells were characterized by a strong reduction of their anchorage-independent proliferation in agarose gel and capacity to invade extracellular matrix gel. After subcutaneous injections of transfected B16F1 cells in syngenic mice, lumican expression significantly decreased subcutaneous tumor formation in vivo, with a concomitant decrease of cyclin D1 expression. Lumican induced and/or increased the apoptosis of B16F1 cells. The results suggest that lumican is involved in the control of melanoma growth and invasion and may be considered, like decorin, as an anti-tumor factor from the extracellular matrix.
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http://dx.doi.org/10.1016/j.yexcr.2004.02.005DOI Listing
June 2004

Involvement of stromal proteoglycans in tumour progression.

Crit Rev Oncol Hematol 2004 Mar;49(3):259-68

Laboratory of Biochemistry, CNRS FRE 2534, Faculty of Medicine, IFR-53, 51095 Reims Cedex, France.

Glycosaminoglycans (GAGs) and proteoglycans (PGs) belong to a class of extracellular macromolecules necessary for the growth of any multicellular structures, including tumours. Transformed cells induce stromal reaction either per se or by activation of the mesenchymal cells. Tumour stroma contains several chondroitin sulphate and heparan sulphate proteoglycans. These proteoglycans and their glycosaminoglycan chains modify cell behaviour by interacting with different molecules such as growth factors, cytokines, chemokines, proteinases and their inhibitors. This review describes the main proteoglycans of tumour stoma and discusses their implication in the regulation of the activity of extracellular proteins and peptides.
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http://dx.doi.org/10.1016/j.critrevonc.2003.10.005DOI Listing
March 2004

Specific protein-1 is a universal regulator of UDP-glucose dehydrogenase expression: its positive involvement in transforming growth factor-beta signaling and inhibition in hypoxia.

J Biol Chem 2003 Jun 7;278(24):21566-75. Epub 2003 Apr 7.

Laboratory of Biochemistry, CNRS Formation de Recherche en Evolution 2534, Faculty of Medicine, 51 rue Cognacq-Jay, 51095 Reims cedex, France.

UDP-glucose dehydrogenase (UGDH) is a key enzyme of the unique pathway for the synthesis of UDP-glucuronate, the substrate for the numerous glucuronosyl transferases, which act on the synthesis of glycosaminoglycans and glucuronidation reaction of xeno- and endobiotics. Using the bacterial artificial chromosome approach, we have cloned and characterized the human UGDH promoter. The core promoter of -644 nucleotides conferred reporter gene activity in transient transfection assay of a variety of cell types, including MRC5 fibroblasts and the HepG2 hepatoma cell line. The minimal promoter of -100 nucleotides contains a functional inverted TATA box. No consensus CAAT sequence was found up to -2133 nucleotides. The expression of UGDH was up- and down-regulated by transforming growth factor (TGF)-beta and hypoxia, respectively. TGF-beta enhanced the activity of all the deletion constructs, except the minimal promoter. Hypoxia slightly increased the activity of the short promoter-containing constructs but decreased that of the -374 nucleotides and core promoter constructs. The core promoter contained numerous GC-rich sequences for the binding of Sp1 transcription factor. Bisanthracycline, an anti-Sp1 compound, decreased UGDH mRNA expression and inhibited the core promoter constructs activity. Gel mobility shift and supershift assays after TGF-beta stimulation demonstrated an increased DNA binding of the nuclear extract proteins to the two Sp1 sequences located in the -374-bp promoter. By contrast, nuclear extract proteins from hypoxia-treated cells demonstrated a decreased binding of the consensus Sp1 sequence. These results indicate that numerous Sp1 cis-acting sequences of the UGDH core promoter are responsible for up- and down-regulation of the gene after TGF-beta stimulation and in hypoxic conditions, respectively.
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http://dx.doi.org/10.1074/jbc.M209366200DOI Listing
June 2003

Enhanced expression of plasminogen activator inhibitor-1 by dedifferentiated thyrocytes.

Biochem Biophys Res Commun 2002 Jul;295(3):737-43

Laboratoire de Biochimie, CNRS FRE 2534, Faculté de Médecine, IFR 53, 51 095 Reims Cedex, France.

Porcine thyrocytes in vitro in the presence of TSH adopt follicular-like morphology. Epidermal growth factor, phorbol esters or transforming growth factor beta-1 (TGFbeta-1) induce a rapid spreading of the cells and dedifferentiation. In addition to thyroglobulin, dedifferentiated thyrocytes secreted into the culture medium three proteins in abundant quantities. Two of them have been previously identified as thrombospondin-1 and clusterin, respectively. Using the microsequencing method we identified the third one, a M(r) 45,000 glycosylated protein, as plasminogen activator inhibitor-1 (PAI-1). EGF, phorbol esters or TGF-beta1 predominantly increased PAI-1 protein expression in TSH-treated cells. The maximal increase of PAI-1 mRNA steady-state level was observed 6 h after EGF treatment and sustained up to 48 h. Recombinant PAI-1 inhibited cell-associated plasmin activity and delayed cell spreading. Enhanced synthesis and secretion of PAI-1 upon treatment with different growth factors during dedifferentiation process and spreading may be considered a feed-back defence mechanism of the cells to harmful extracellular stimuli.
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http://dx.doi.org/10.1016/s0006-291x(02)00712-xDOI Listing
July 2002