Specific Protein-1 Is a Universal Regulator of UDP-glucose Dehydrogenase Expression

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)-β and hypoxia, respectively. TGF-β 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-β 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-β stimulation and in hypoxic conditions, respectively. 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)-β and hypoxia, respectively. TGF-β 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-β 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-β stimulation and in hypoxic conditions, respectively. UDP-glucose dehydrogenase (UGDH, 1The abbreviations used are: UGDH, UDP-glucose dehydrogenase; TGF-β, transforming growth factor-β1; GAG, glycoaminoglycan; BAC, bacterial artificial chromosome; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FCS, fetal calf serum.1The abbreviations used are: UGDH, UDP-glucose dehydrogenase; TGF-β, transforming growth factor-β1; GAG, glycoaminoglycan; BAC, bacterial artificial chromosome; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FCS, fetal calf serum. EC 1.1.1.22) is a key enzyme in the synthesis of UDP-glucuronate from glucose. In animals, it constitutes the unique pathway for glucuronate formation (1Toyoda H. Kinoshita-Toyoda A. Fox B. Selleck S.B. J. Biol. Chem. 2000; 275: 21856-21861Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Because glucuronate is a component of glycosaminoglycans, the mutation inactivation of UGDH (sugarless) abolishes glycosaminoglycan assembly and, consequently, abolishes GAG-dependent growth factor signaling. For example, such a mutation was shown to induce cardiac valve malformation in zebrafish (2Walsh E.C. Stainier D.Y. Science. 2001; 293: 1670-1673Crossref PubMed Scopus (243) Google Scholar) or the “wingless” and “no white eyes” phenotypes in Drosophila (3Binari R.C. Staveley B.E. Johnson W.A. Godavarti R. Sasisekharan R. Manoukian A.S. Development. 1997; 124: 2623Crossref PubMed Google Scholar, 4Benevolenskaya E.V. Frolov M.V. Birchler J.A. Mol. Gen. Genet. 1998; 260: 131-143Crossref PubMed Scopus (12) Google Scholar). The primary structure of the mammalian enzyme was obtained from the protein sequence of bovine UGDH (5Hempel J. Perozich J. Romovacek H. Hinich A. Kuo I. Feingold D.S. Prot. Sci. 1994; 3: 1074-1080Crossref PubMed Scopus (54) Google Scholar). The human gene, recently cloned in our laboratory (6Bontemps Y. Maquart F.X. Wegrowski Y. Biochem. Biophys. Res. Commun. 2000; 275: 981-985Crossref PubMed Scopus (7) Google Scholar), was assigned to chromosome 4p15.1 by radiation hybrid mapping (7Marcu O. Stathakis D.G. Marsh J.L. Cytogenet. Cell Genet. 1999; 86: 244-245Crossref PubMed Google Scholar). It contains 12 exons, extends over 26 kb, and has one major transcription start site (6Bontemps Y. Maquart F.X. Wegrowski Y. Biochem. Biophys. Res. Commun. 2000; 275: 981-985Crossref PubMed Scopus (7) Google Scholar). The structure of the enzyme is well conserved between the species and phyla. The cloned mammalian proteins from different species showed overall 97% identity. The human sequence has 27% identity with the Escherichia coli ortholog, with 100 and 60% identity of the NAD+ binding and the catalytic sites, respectively (8Wegrowski Y. Perreau C. Bontemps Y. Maquart F.X. Biochem. Biophys. Res. Commun. 1998; 250: 206-211Crossref PubMed Scopus (22) Google Scholar). Glycosaminoglycan chains of proteoglycans and hyaluronan are ubiquitous components of extracellular matrix and pericellular spaces. There is a growing body of information on the implication of GAGs in cell behavior, including signal transduction, cell proliferation, spreading, migration, and cancer growth and metastasis (9Iozzo R.V. San Antonio J.D. J. Clin. Invest. 2001; 108: 349-355Crossref PubMed Scopus (396) Google Scholar, 10Selva E.M. Perrimon N. Adv. Cancer Res. 2001; 83: 67-80Crossref PubMed Scopus (32) Google Scholar, 11Toole B.P. Wight T.N. Tammi M.I. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). GAG synthesis is influenced by cytokines and growth factors. TGF-β is the most potent stimulator of proteoglycan and glycosaminoglycan synthesis, including that of hyaluronan. Its action, however, depends on the cell type (12Bassols A. Massague J. J. Biol. Chem. 1988; 263: 3039-3045Abstract Full Text PDF PubMed Google Scholar). The synthesis of GAGs is also modulated by oxygen cell status. Hypoxic endothelial cells and lung fibroblasts enhanced heparan sulfate/chondroitin sulfate ratio, which led to an increase of basic fibroblast growth factor reactivity on the cell surface (13Karlinsky J.B. Rounds S. Farber H.W. Circ. Res. 1992; 71: 782-789Crossref PubMed Scopus (12) Google Scholar, 14Figueroa J.E. Tao Z. Sarphie T.G. Smart F.W. Glancy D.L. Vijayagopal P. Atherosclerosis. 1999; 143: 135-144Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). It was also shown that the level of intracellular UDP-glucuronate could influence GAG synthesis (1Toyoda H. Kinoshita-Toyoda A. Fox B. Selleck S.B. J. Biol. Chem. 2000; 275: 21856-21861Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The knowledge of the cytokine regulation of UGDH expression is scanty. The human UGDH was shown to be an early response gene after interleukin-1β treatment of ocular fibroblasts (15Spicer A.P. Kaback L.A. Smith T.J. Seldin M.F. J. Biol. Chem. 1998; 273: 25117-25124Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), as well as an early androgen response gene in breast cancer (16Lapointe J. Labrie C. Endocrinology. 1999; 140: 4486-4493Crossref PubMed Scopus (19) Google Scholar). It was also up-regulated by fetal serum (8Wegrowski Y. Perreau C. Bontemps Y. Maquart F.X. Biochem. Biophys. Res. Commun. 1998; 250: 206-211Crossref PubMed Scopus (22) Google Scholar, 17Grubb M.F. Kasofsky J. Strong J. Anderson L.W. Cysyk R.L. Biochem. Mol. Biol. Int. 1993; 30: 819-827PubMed Google Scholar). TGF-β signaling in the cells is initiated through receptor-dependent Ser/Thr kinase phosphorylation of SMAD3 (18Hartsough M.T. Mulder K.M. Pharmacol. Ther. 1997; 75: 21-41Crossref PubMed Scopus (109) Google Scholar), which forms a heterodimer complex with SMAD4 (19Lagna G. Hata A. Hemmati-Brivanlou A. Massague J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (805) Google Scholar). The complex is translocated into the nucleus where it activates its target genes (20Massague J. Nat. Rev. Mol. Cell. Biol. 2000; 1: 169-178Crossref PubMed Scopus (1627) Google Scholar). SMAD proteins, however, cooperate with other transcription factors to activate the promoters of different genes. In several systems, Sp1 is a powerful intermediate of the TGF-β signaling cascade. For example, Sp1 drives TGF-β activation of promoters such as the collagen α1(I) (21Cutroneo K.R. Mol. Biol. Rep. 2000; 27: 191-194Crossref PubMed Scopus (10) Google Scholar) and α2(I) chains (22Inagaki Y. Truter S. Ramirez F. J. Biol. Chem. 1994; 269: 14828-14834Abstract Full Text PDF PubMed Google Scholar, 23Poncelet A.C. Schnaper H.W. J. Biol. Chem. 2001; 276: 6983-6992Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), plasminogen activator inhibitor-1 (24Datta P.K. Blake M.C. Moses H.L. J. Biol. Chem. 2000; 275: 40014-40019Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), SMAD7 (25Brodin G. Ahgren A. ten Dijke P. Heldin C.H. Heuchel R. J. Biol. Chem. 2000; 275: 29023-29030Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), TGF-β receptor II, and TGF-β itself (26Kim Y. Ratziu V. Choi S.G. Lalazar A. G. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The of Sp1 in the of different genes is by its with the GC-rich the formation of a complex with The of Sp1 expression S. S. 2000; PubMed Scopus Google Scholar, S. Cancer Res. 2001; Google Scholar), or mutation of GC-rich binding sequences (24Datta P.K. Blake M.C. Moses H.L. J. Biol. Chem. 2000; 275: 40014-40019Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, G. Ahgren A. ten Dijke P. Heldin C.H. Heuchel R. J. Biol. Chem. 2000; 275: 29023-29030Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, Biophys. 2000; PubMed Scopus Google Scholar, C. A. C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), TGF-β action, of SMAD and Sp1 in signaling genes are through Sp1 after TGF-β treatment and proteins, and it was that Sp1 is a major of the expression of extracellular proteins F. J. A. J. Invest. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In Sp1 also the activity of the gene S. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). In the we characterized the regulation of UGDH The expression of its mRNA was in different cell of human cloned the human UGDH promoter and its minimal sequence and activity. showed that the core promoter of is TGF-β treatment through numerous Sp1 the other in HepG2 which a level of UGDH we demonstrated that hypoxic led to the down-regulation of enzyme by nuclear of the Sp1 that Sp1 transcription factor be a of UGDH expression and, by the enzyme the UDP-glucuronate level in different including GAG Cell cells obtained from HepG2 cell and or to from S. A. P. S. Google cell cell and MRC5 lung endothelial cells obtained from cells cell obtained from Cell in the by the with FCS, 100 and 100 TGF-β MRC5 cells for in a FCS, and the stimulation was as in the For hypoxic conditions, HepG2 cells to the was with with The cells with for and with for was with an from different cell by the and on and the with a of human UGDH (8Wegrowski Y. Perreau C. Bontemps Y. Maquart F.X. Biochem. Biophys. Res. Commun. 1998; 250: 206-211Crossref PubMed Scopus (22) Google Scholar). The and to in an for with of glyceraldehyde-3-phosphate dehydrogenase a of P. P. S. C. P. Res. PubMed Scopus Google Scholar) or with the J. Res. PubMed Scopus Google Scholar). and DNA from the the was with and the on a by a The was with a the of UGDH (6Bontemps Y. Maquart F.X. Wegrowski Y. Biochem. Biophys. Res. Commun. 2000; 275: 981-985Crossref PubMed Scopus (7) Google Scholar). which a with the was from the into a and The sequence was with the R. H. I. V. I. F. Res. 2000; PubMed Scopus Google Scholar) and H. Res. PubMed Scopus Google Scholar), the from and from the was into and the –2133 for the –644 and for the –100 respectively. constructs the with site for and for or with a the site –374 The by and into in the inverted TATA was the The with the and The was on the –100 to the and constructs to the and reporter gene constructs and for MRC5 and HepG2 into MRC5 cells or HepG2 cells with of the kinase promoter of gene the transient transfection to the of in the MRC5 and HepG2 cells and with a reporter assay in a and to activity. and from of or MRC5 or from HepG2 cells or as by J.D. Res. PubMed Scopus Google Scholar). protein by the protein assay consensus Sp1 site and an in from The to Sp1 on UGDH promoter as and The and with kinase proteins in a reaction and of For a of was as in the the was and the was by in a of on in and for The and by For supershift the nuclear with of for by as anti-Sp1 was obtained from and from was from The Sp1 the of the Sp1 transcription the with the Sp1 transcription factor by with the nuclear as and used as extract from cells used as of UGDH of TGF-β, and was shown that UGDH is in all (8Wegrowski Y. Perreau C. Bontemps Y. Maquart F.X. Biochem. Biophys. Res. Commun. 1998; 250: 206-211Crossref PubMed Scopus (22) Google Scholar, A.P. Kaback L.A. Smith T.J. Seldin M.F. J. Biol. Chem. 1998; 273: 25117-25124Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). used a of different human cells in to the expression of the the cells a of UGDH mRNA of and with a slightly different of two The mRNA expression was in endothelial cells and in HepG2 hepatoma cells the The mRNA that the of cells of or influence on UGDH fibroblasts and MRC5 fibroblasts an intermediate level of UGDH In we of the on the of and cell we to MRC5 which results as a of UGDH to the TGF-β and the HepG2 hepatoma cells as a of UGDH to the Because TGF-β is a major growth factor to influence GAG synthesis, we its on UGDH expression by TGF-β increased the of into GAG in a In TGF-β enhanced the level of UGDH The was with expression after of stimulation The expression was after stimulation and for an that the stimulation protein synthesis and that UGDH is a response gene after TGF-β The response of UGDH however, that the of stimulation be complex and the of different transcription factors. Hypoxia is factor of GAG synthesis (13Karlinsky J.B. Rounds S. Farber H.W. Circ. Res. 1992; 71: 782-789Crossref PubMed Scopus (12) Google Scholar). For that we the of oxygen on the UGDH mRNA expression in HepG2 of inhibited the UGDH mRNA level in a Hypoxia also the of into the GAG in HepG2 cells The was in MRC5 cells of UGDH the of the regulation of UGDH expression by TGF-β and hypoxia, the including the was cloned from a a of the human chromosome (6Bontemps Y. Maquart F.X. Wegrowski Y. Biochem. Biophys. Res. Commun. 2000; 275: 981-985Crossref PubMed Scopus (7) Google Scholar). a of by and was It contained of the the R. H. I. V. I. F. Res. 2000; PubMed Scopus Google Scholar) and H. Res. PubMed Scopus Google Scholar) a for the of the promoter. The inverted TATA was located from the transcription start and consensus CAAT sequence was The of contained consensus sequences for Sp1 transcription factor and one consensus sequence for R. P. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the promoter a of different of the to reporter gene was The transient transfection of the constructs the activity from the promoter activity in MRC5 and HepG2 cells The level was in HepG2 cells with MRC5 for the constructs. the of the promoter up to –644 enhanced the expression of the reporter The of the of the activity with the the of The showed but promoter activity. sequence contained one Sp1 consensus site and an inverted TATA box. of decreased the reporter gene activity to the level that –100 be as a minimal promoter for the UGDH gene and that the inverted TATA is for its activity. of TGF-β and Hypoxia on constructs different of the UGDH promoter to MRC5 cells and HepG2 cells to the of TGF-β and respectively. TGF-β on minimal and promoters but enhanced the activity of all the constructs up to –644 The stimulation was for the constructs of –374 and –644 These and Sp1 consensus respectively the into the HepG2 cells in the activity of the constructs and enhanced in to the cells in The constructs and –2133 inhibited in hypoxic that the site was located between and –374 of the promoter contains of in UGDH it was shown that as a of Sp1 transcription factor B. A. J. Res. 1999; 27: PubMed Scopus Google Scholar). used to the implication of Sp1 in the activity of UGDH promoter. a of slightly inhibited UGDH mRNA level in MRC5 fibroblasts and the of TGF-β In MRC5 influence the activity of and constructs, that the Sp1 cis-acting of sequences are in the regulation of UGDH promoter. In with the mRNA inhibited all the promoter constructs and the of TGF-β was obtained was to the of HepG2 the of inhibited UGDH mRNA level in cell inhibited all the constructs activity in a The of Sp1 was by with nuclear a consensus Sp1 sequence DNA The nuclear of MRC5 cells and HepG2 cells with showed on the Using a of Sp1 we a of the to Sp1 and that the one to the one to the binding of factor Y. H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar), and the which was also the to In to the a of the consensus sequence on the of the TGF-β enhanced the of the DNA consensus binding to Sp1 and The nuclear obtained from HepG2 cells in the of showed of Sp1 factor as with cells from showed that the TGF-β was obtained with the –374 and stimulation for the and –374-bp constructs, The of promoter contains Sp1 binding which Sp1 binding site from is in the TGF-β we used from and a Sp1 consensus as a of in of nuclear from and MRC5 cells with the different an increase of binding TGF-β treatment with Sp1 sequences located and but with the The binding was obtained with the The of binding was of type consensus Sp1 sequence or Sp1 The type but the and the of These results by supershift By the and the we demonstrated that the increased Sp1 binding activity in MRC5 cells was to the Sp1 transcription factor. the anti-Sp1 It is to of transcription factor or the formation of of the was nuclear extract from cells and with an anti-Sp1 from and supershift with the slightly with the Sp1 transcription factor and was the to a in the formation as shown by other J.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). results that Sp1 is a of UGDH promoter activity and to its and to the it increase or UGDH mRNA supershift extract from MRC5 cells with TGF-β and nuclear extract from cells with Sp1 consensus or by the with anti-Sp1 or as the The indicate the of and the indicate the of The the UDP-glucuronate is an intermediate and the substrate for numerous including in GAGs and hyaluronan The synthesis of UDP-glucuronate is by the unique pathway of UGDH in animals, and it was shown that mutation inactivation of the enzyme growth by of There is a body of that the enzyme has a in the the of UDP-glucuronate in the cells was between and Biochem. J. PubMed Scopus Google Scholar, J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), a to the of the the expression of UGDH mRNA is in the cells and with enhanced UDP-glucuronate for example, or Y. Perreau C. Bontemps Y. Maquart F.X. Biochem. Biophys. Res. Commun. 1998; 250: 206-211Crossref PubMed Scopus (22) Google and UGDH is a response gene as demonstrated by and by the response to TGF-β stimulation results with (15Spicer A.P. Kaback L.A. Smith T.J. Seldin M.F. J. Biol. Chem. 1998; 273: 25117-25124Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). the regulation of UGDH, we cloned its that the promoter activity of reporter gene in transient transfection is the of the of UGDH promoter. The minimal promoter contains an inverted TATA as it was shown for several mammalian genes J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, PubMed Scopus Google Scholar, Y. H. Biol. Chem. PubMed Scopus Google Scholar, J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (22) Google Scholar), and CAAT was in the promoter up to –2133 The core promoter contains numerous GC-rich of and promoters of genes J. Cell Biol. Int. Rep. 2001; Scopus Google Scholar). In such Sp1 consensus shown to the of TATA for assembly R. Cell. Full Text PDF PubMed Scopus Google Scholar). the inverted TATA of UGDH promoter the activity of the reporter gene a of genes that a level of expression but are by different extracellular including of UGDH promoter is from other or C. R.C. G. Mol. Cell. Biol. 1992; PubMed Google Scholar), including the of the pathway Mol. 1999; PubMed Scopus Google Scholar, B. J. Biol. Chem. 1988; 263: Full Text PDF PubMed Google Scholar). The UGDH core promoter contains Sp1 consensus The deletion of different showed a decreased activity of the reporter gene to the promoter up to –644 The cloned sequence of –2133 was the of sequence. The core promoter of was responsible for the increased activity in HepG2 cells with MRC5 that the major transcription are located in from two cell The increase of the promoter activity with its and its down-regulation by that several of the Sp1 are functional in HepG2 cell activity. The Sp1 transcription factor act as an for complex R. Cell. Full Text PDF PubMed Scopus Google Scholar, J. 1999; PubMed Scopus (10) Google Scholar), but it as an for different of B. P. Int. J. Biochem. Cell Biol. 1997; PubMed Scopus Google Scholar). of UGDH mRNA is up-regulated by serum (8Wegrowski Y. Perreau C. Bontemps Y. Maquart F.X. Biochem. Biophys. Res. Commun. 1998; 250: 206-211Crossref PubMed Scopus (22) Google Scholar, 17Grubb M.F. Kasofsky J. Strong J. Anderson L.W. Cysyk R.L. Biochem. Mol. Biol. Int. 1993; 30: 819-827PubMed Google Scholar). TGF-β is one of the major factors in the serum that GAG TGF-β increased UGDH mRNA and in a with a after an the of In the signaling of TGF-β is SMAD proteins, which to the target genes by its binding S. Heldin C.H. ten Dijke P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). binding is of and the with transcription factors to There is a body of that Sp1 in TGF-β signal we showed that the promoter of human UGDH consensus binding but Sp1 in its sequence. the other all the constructs except that of –100 showed enhanced reporter gene expression after TGF-β treatment of MRC5 cells The most the constructs –374 and –644 of the UGDH promoter. These constructs have and Sp1 binding with the the Sp1 the TGF-β stimulation of UGDH mRNA expression and the activity of all the constructs in HepG2 There was a of nuclear proteins on Sp1 consensus sequence as by and supershift we that two Sp1 located and from the transcription start site are in the enhanced binding after TGF-β the the most results the that TGF-β on the human UGDH promoter through numerous Sp1 The in the of MRC5 cells and HepG2 cells to treatment are with the in expression of UGDH The of HepG2 cells to be for enhanced and enhanced growth of cell line. the of Sp1 factor be used to gene or the be and to factor UGDH expression is In the human an hypoxic be in growing with and in the are in in The is the its Hypoxia GAG synthesis with of sulfate J.E. J. 1988; Google Scholar). In hypoxic conditions, the cell including is to which a of and the cell It was shown in hypoxic conditions, the are up-regulated J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The reaction by UGDH of NAD+ for of UDP-glucuronate In hypoxic conditions, NAD+ is for the phosphorylation It is that UGDH synthesis be down-regulated oxygen is The is and the of UGDH mRNA is after The down-regulation of the reporter gene was in transient transfection with the constructs the –374-bp and core promoters The of UGDH promoter activity in by the Sp1 factor in HepG2 treatment hypoxic conditions, and the assay showed a decreased for Sp1 on its consensus sequence in the nucleus from hypoxic results indicate that the Sp1 transcription factor a in the promoter activity of Sp1 increase the UGDH expression after TGF-β stimulation or its expression in hypoxic the enzyme could the UDP-glucuronate for glycosaminoglycan synthesis and of is for and