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.
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.
Bontemps
Yannick
Y
Laboratory of Biochemistry, CNRS Formation de Recherche en Evolution 2534, Faculty of Medicine, 51 rue Cognacq-Jay, 51095 Reims cedex, France.
Vuillermoz
Boris
B
Antonicelli
Frank
F
Perreau
Corinne
C
Danan
Jean-Louis
JL
Maquart
François-Xavier
FX
Wegrowski
Yanusz
Y
D018943:Anthracyclines / Q000494:pharmacology; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D000860:Hypoxia; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000502:physiology*; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics*
D018943:Anthracyclines; D006025:Glycosaminoglycans; D009841:Oligonucleotides; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D016212:Transforming Growth Factor beta; D012313:RNA; D014533:Uridine Diphosphate Glucose Dehydrogenase; D008156:Luciferases; D010100:Oxygen