J. Biochem, 2004, Vol. 136, No. 3 273-277
© 2004 The Japanese Biochemical Society
MOLECULAR BIOLOGY |
Identification and Characterization of the Hypoxia-Responsive Element of the Human Placental 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase Gene
Departments of 1 Microbiology and 3 Parasitology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513; 2 Department of Molecular Medicine, Research Center for Molecular Medical Science, 35-1 Shimo, Fussa, Tokyo 197-0023; 4 Veterans Affairs Medical Center and Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 4500 South Lancaster Road, Dallas, Texas 75216, USA; and 5 Department of Nutrition, Faculty of Home Economics, Kyushu Women’s University, 1-1 Jiyugaoka, Yahatanishi-ku, Fukuoka 807-8586
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.
* To whom correspondence should be addressed. Tel/Fax: +81-93-693-3132, E-mail: rsakaki{at}kwuc.ac.jp
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