Hypoxia induces transcription of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 gene via hypoxia-inducible factor-1alpha activation.
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.
Minchenko
Oleksandr
O
Department of Molecular Biology, Palladin Institute of Biochemistry, National Academy of Science of Ukraine, Kyiv 01601, Ukraine. o_minchenko@hotmail.co
Opentanova
Iryna
I
Minchenko
Dmytro
D
Ogura
Tsutomu
T
Esumi
Hiroyasu
H
D006528:Carcinoma, Hepatocellular / Q000201:enzymology; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D003035:Cobalt / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D025481:Phosphofructokinase-2 / Q000096:biosynthesis* / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000201:enzymology; D012333:RNA, Messenger / Q000096:biosynthesis; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes; D012333:RNA, Messenger; D014157:Transcription Factors; D003035:Cobalt; D025481:Phosphofructokinase-2; D003676:Deferoxamine; D005998:Glycine