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ABSTRACT: 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 evidenced by knocking out of its gene in mouse. A direct link between lumican expression and melanoma progression and metastasis was 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 responsible for the inhibition of melanoma cell migration. Lumican was also shown to exert angiostatic properties by down-regulating proteolytic activity associated with endothelial cell membranes, particularly MMP-14 and MMP-9. Globally, lumican appears as a potent agent for inhibiting tumour progression rather than tumourigenesis. However, progressive changes in proteoglycans occur in 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 signaling, motility, adhesion, growth and apoptosis. This review summarizes recent data concerning lumican control of tumour progression in different cancers with particular interest for its interactions with MMPs and integrins. Its potential therapeutic implications will be discussed. © 2013 The Authors Journal compilation © 2013 FEBS.
FEBS Journal 02/2013; · 3.79 Impact Factor
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ABSTRACT: 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.
Experimental Cell Research 07/2012; 318(18):2312-23. · 3.58 Impact Factor
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ABSTRACT: 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.
Methods in molecular biology (Clifton, N.J.) 01/2012; 836:117-30.
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Mariusz Malinowski,
Katarzyna Pietraszek,
Corinne Perreau,
Mateusz Boguslawski,
Véronique Decot,
Jean-François Stoltz,
Laurent Vallar,
Jolanta Niewiarowska,
Czeslaw Cierniewski,
François-Xavier Maquart, Yanusz Wegrowski,
Stéphane Brézillon
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ABSTRACT: 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).
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.
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.
PLoS ONE 01/2012; 7(12):e50709. · 4.09 Impact Factor
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ABSTRACT: 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.
Journal of photochemistry and photobiology. B, Biology 10/2011; 106:107-12. · 1.87 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
Thrombosis Research 07/2011; 128(5):452-7. · 2.44 Impact Factor
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ABSTRACT: 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.
Photochemistry and Photobiology 06/2011; 87(5):1105-12. · 2.41 Impact Factor
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ABSTRACT: 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.
Journal of Pharmaceutical Sciences 02/2011; 100(2):441-50. · 3.06 Impact Factor
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Cédric Zeltz,
Stéphane Brézillon,
Jarmo Käpylä,
Johannes A Eble,
Hélène Bobichon,
Christine Terryn,
Corinne Perreau,
Clemens M Franz,
Jyrki Heino,
François-Xavier Maquart, Yanusz Wegrowski
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ABSTRACT: 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.
Experimental Cell Research 10/2010; 316(17):2922-31. · 3.58 Impact Factor
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ABSTRACT: 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.
FEBS letters 09/2009; 583(18):3027-32. · 3.54 Impact Factor
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ABSTRACT: 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.
Cancer letters 05/2009; 283(1):92-100. · 4.86 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
Life Sciences 10/2008; 83(19-20):651-60. · 2.53 Impact Factor
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ABSTRACT: 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.
Biochemical and Biophysical Research Communications 02/2008; 365(2):266-72. · 2.48 Impact Factor
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Marie-France D'Onofrio,
Stéphane Brézillon,
Thomas Baranek,
Corinne Perreau,
Agata Radwanska,
Bertrand Brassart,
Peter J Roughley,
Maria Malicka-Blaszkiewicz,
Sylvie Ricard-Blum,
François-Xavier Maquart, Yanusz Wegrowski
Journal of Biological Chemistry 12/2007; · 4.77 Impact Factor
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Advances in pharmacology (San Diego, Calif.) 02/2006; 53:297-321.
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ABSTRACT: 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.
Molecular and Cellular Biochemistry 10/2005; 277(1-2):63-72. · 2.06 Impact Factor
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Gallic Beauchef,
Magdalini Kypriotou,
Christos Chadjichristos,
Russell L Widom,
Benoît Porée,
Emmanuelle Renard,
Safa Moslemi, Yanusz Wegrowski,
François-Xavier Maquart,
Jean-Pierre Pujol,
Philippe Galéra
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ABSTRACT: 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.
Biochemical and Biophysical Research Communications 09/2005; 333(4):1123-31. · 2.48 Impact Factor
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ABSTRACT: 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.
Experimental Cell Research 07/2004; 296(2):294-306. · 3.58 Impact Factor
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ABSTRACT: 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.
Critical Reviews in Oncology/Hematology 04/2004; 49(3):259-68. · 4.41 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 07/2003; 278(24):21566-75. · 4.77 Impact Factor
