Identification and characterization of the hypoxia-responsive element of the human placental 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.
The placenta-type isozyme of human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K, identical to PFKFB3) is expressed in a variety of cells and tissues such as placenta, brain, testis, liver, kidney, skeletal muscle, primary blood mononuclear cells and cancer cells. We observed previously that the enhancer region of the HP2K gene, which has been identified in the 5'-flanking region between -1265 and -1329, could respond to serum stimulation following the transfection of human choriocarcinoma BeWo cells with HP2K promoter-luciferase constructs. The HP2K enhancer region also contains two copies of the hypoxia-inducible factor-1 (HIF-1) binding motif (5'-ACGTG-3'). In this study we performed characterization of the HP2K gene expression in response to hypoxic conditions. Both electrophoretic mobility shift and co-transfection assays of the HP2K promoter-luciferase reporter with HIF-1 expression vectors indicated that HIF-1 binds to the hypoxia-responsive element (HRE) of HP2K, thereby upregulating its gene expression. In addition, we demonstrated using site-directed mutagenesis that a complete tandem repeat of the HIF-1 binding motif with a 4-bp interruption is required for full induction of HP2K expression (up to 22-fold) under hypoxic conditions, and that this response is much stronger than that of the erythropoietin (EPO) gene. These results suggest that the sequence 5'-ACGTGNNNNACGTG-3' in the HP2K enhancer is the authentic HRE consensus motif that mediates increased transcription, under hypoxic conditions, via HIF-1.
Fukasawa
Masashi
M
Department of Microbiology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan.
Tsuchiya
Terumasa
T
Takayama
Eiji
E
Shinomiya
Nariyoshi
N
Uyeda
Kosaku
K
Sakakibara
Ryuzo
R
Seki
Shuhji
S
D020816:Amino Acid Motifs; D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006019:Glycolysis; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D025481:Phosphofructokinase-2 / Q000737:chemistry* / Q000502:physiology; D010920:Placenta / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014018:Tissue Distribution; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009841:Oligonucleotides; D020033:Protein Isoforms; D014157:Transcription Factors; D004921:Erythropoietin; D008156:Luciferases; D025481:Phosphofructokinase-2