The PFKFB4 gene encodes
isoenzyme of 6-phosphofructo-2-
kinase/
fructose-2,6-biphosphatase (PFKFB or PFK-2/FBPase-2) which originally was found in the testes. We have studied hypoxic regulation of PFKFB4 gene in
prostate cancer cell line, PC-3, and several other
cancer cell lines. It was shown that
hypoxia significantly induced PFKFB4
mRNA levels in PC-3 as well as in HeLa, Hep3B and HepG2 cell lines.
Hypoxia increased PFKFB4
protein levels also. Moreover,
desferrioxamine and
cobalt chloride, which are known to mimic
hypoxia, also had a stimulatory effect on the expression of PFKFB4
mRNA. In order to investigate the mechanisms of hypoxic regulation of PFKFB4 gene expression, we used
dimethyloxalylglycine, which has the ability to mimic effect of
hypoxia by significant induction of
hypoxia-inducible factor (HIF-1alpha)
protein levels. Our studies showed that PFKFB4
mRNA expression in PC-3, HeLa, Hep3B and HepG2 cell lines was highly responsive to
dimethyloxalylglycine, an inhibitor of HIF-1alpha
hydroxylase enzymes, suggesting that the
hypoxia responsiveness of this gene is regulated by HIF
proteins. To better understand the hypoxic regulation of PFKFB4 gene expression, we isolated genomic
DNA, which includes the promoter region of PFKFB4. Cell transfection, deletion and site-specific mutagenesis of the PFKFB4 promoter region indicates that hypoxic induction of PFKFB4 gene expression is mediated by the
hypoxia-responsive
element (HRE). These experiments identified a HRE 422-429 bp upstream from the translation start site. Thus, our results indicate that testis-specific form of PFKFB or PFK-2/FBPase-2 is also expressed in several
cancer cell lines and that
hypoxia induces transcription of PFKFB4 gene in these cell lines by HIF-1alpha dependent mechanism. HRE in 5'-promoter region of PFKFB4 gene mediates hypoxic induction of PFKFB4 gene transcription.