Hypoxia-inducible Factor-1-mediated Expression of the 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) Gene

One of the key mediators of the hypoxic response in animal cells is the hypoxia-inducible transcription factor-1 (HIF-1) complex, in which the α-subunit is highly susceptible to oxygen-dependent degradation. The hypoxic response is manifested in many pathophysiological processes such as tumor growth and metastasis. During hypoxia, cells shift to a primarily glycolytic metabolic mode for their energetic needs. This is also manifested in the HIF-1-dependent up-regulation of many glycolytic genes. Paradoxically, tumor cells growing under conditions of normal oxygen tension also show elevated glycolytic rates that correlate with the increased expression of glycolytic enzymes and glucose transporters (the Warburg effect). A key regulator of glycolytic flux is the relatively recently discovered fructose-2,6-bisphosphate (F-2,6-P2), an allosteric activator of 6-phosphofructo-1-kinase (PFK-1). Steady state levels of F-2,6-P2 are maintained by the bifunctional enzyme PFK-2/F2,6-Bpase, which has both kinase and phosphatase activities. Herein, we show that one isozyme, PFKFB3, is highly induced by hypoxia and the hypoxia mimics cobalt and desferrioxamine. This induction could be replicated by the use of an inhibitor of the prolyl hydroxylase enzymes responsible for the von Hippel Lindau (VHL)-dependent destabilization and tagging of HIF-1α. The absolute dependence of the PFKFB3 gene on HIF-1 was confirmed by its overexpression in VHL-deficient cells and by the lack of hypoxic induction in mouse embryonic fibroblasts conditionally nullizygous for HIF-1α. One of the key mediators of the hypoxic response in animal cells is the hypoxia-inducible transcription factor-1 (HIF-1) complex, in which the α-subunit is highly susceptible to oxygen-dependent degradation. The hypoxic response is manifested in many pathophysiological processes such as tumor growth and metastasis. During hypoxia, cells shift to a primarily glycolytic metabolic mode for their energetic needs. This is also manifested in the HIF-1-dependent up-regulation of many glycolytic genes. Paradoxically, tumor cells growing under conditions of normal oxygen tension also show elevated glycolytic rates that correlate with the increased expression of glycolytic enzymes and glucose transporters (the Warburg effect). A key regulator of glycolytic flux is the relatively recently discovered fructose-2,6-bisphosphate (F-2,6-P2), an allosteric activator of 6-phosphofructo-1-kinase (PFK-1). Steady state levels of F-2,6-P2 are maintained by the bifunctional enzyme PFK-2/F2,6-Bpase, which has both kinase and phosphatase activities. Herein, we show that one isozyme, PFKFB3, is highly induced by hypoxia and the hypoxia mimics cobalt and desferrioxamine. This induction could be replicated by the use of an inhibitor of the prolyl hydroxylase enzymes responsible for the von Hippel Lindau (VHL)-dependent destabilization and tagging of HIF-1α. The absolute dependence of the PFKFB3 gene on HIF-1 was confirmed by its overexpression in VHL-deficient cells and by the lack of hypoxic induction in mouse embryonic fibroblasts conditionally nullizygous for HIF-1α. The rate of glucose utilization via the glycolytic pathway is highly regulated and depends upon the energetic and metabolic needs of the cell. It is coordinated with other pathways of energy generation and utilization, notably gluconeogenesis, the pentose phosphate pathway, and the citric acid cycle. Fructose-2,6-bisphosphate (F-2,6-P2) 1F-26-P2, fructose-2,6-bisphosphatePFK-16-phosphofructo-1-kinasePFKFB36-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3HIF-1αhypoxia-inducible factor-1αVHLvon Hippel LindauGlut-1glucose transporter-1VEGFvascular endothelial growth factorHREhypoxia-responsive element 1F-26-P2, fructose-2,6-bisphosphatePFK-16-phosphofructo-1-kinasePFKFB36-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3HIF-1αhypoxia-inducible factor-1αVHLvon Hippel LindauGlut-1glucose transporter-1VEGFvascular endothelial growth factorHREhypoxia-responsive element is considered to be the major regulator controlling carbon flux through glycolysis. F-2,6-P2 is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), the key regulatory enzyme in glycolysis as well as an inhibitor of frucrose-1,6-bisphosphatase (1Kawaguchi T. Veech R.L. Uyeda K. J. Biol. Chem. 2001; 276: 28554-28561Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 2Hue L. Rousseau G.G. Adv. Enzyme Regul. 1993; 33: 97-110Crossref PubMed Scopus (74) Google Scholar, 3Okar D.A. Lange A.J. Biofactors. 1999; 10: 1-14Crossref PubMed Scopus (119) Google Scholar). The synthesis and degradation of F-2,6-P2 depends upon a single enzyme, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFK-2/F-2,6-BPase), which has both kinase and phosphatase activities. This bifunctional enzyme is regulated by phosphorylation and dephosphorylation that are dependent upon intracellular cAMP levels (4Pilkis S.J. Claus T.H. Kurland I.J. Lange A.J. Annu. Rev. Biochem. 1995; 64: 799-835Crossref PubMed Scopus (226) Google Scholar). Furthermore, PFK-2/F-2,6-BPase synthesis can be induced by mitogens, growth factors, and inflammatory cytokines, implicating its role in setting the glycolytic rate under multiple physiologic and pathologic conditions (5Chesney J. Mitchell R. Benigni F. Bacher M. Spiegel L., Al- Abed Y. Han J.H. Metz C. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3047-3052Crossref PubMed Scopus (244) Google Scholar). 6-P2, fructose-2,6-bisphosphate 6-phosphofructo-1-kinase 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 hypoxia-inducible factor-1α von Hippel Lindau glucose transporter-1 vascular endothelial growth factor hypoxia-responsive element 6-P2, fructose-2,6-bisphosphate 6-phosphofructo-1-kinase 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 hypoxia-inducible factor-1α von Hippel Lindau glucose transporter-1 vascular endothelial growth factor hypoxia-responsive element Four different genes coding different isozymes (PFKFB1–4) have been identified to date (6Algaier J. Uyeda K. Biochem. Biophys. Res. Commun. 1988; 153: 328-333Crossref PubMed Scopus (38) Google Scholar, 7Heine-Suner D. Diaz-Guillen M.A. Lange A.J. Rodriguez de Cordoba S. Eur. J. Biochem. 1998; 254: 103-110Crossref PubMed Scopus (31) Google Scholar, 8Sakai A. Kato M. Fukasawa M. Ishiguro M. Furuya E. Sakakibara R. J. Biochem. 1996; 119: 506-511Crossref PubMed Scopus (54) Google Scholar, 9Manzano A. Rosa J.L. Ventura F. Perez J.X. Nadal M. Estivill X. Ambrosio S. Gil J. Bartrons R. Cytogenet. Cell Genet. 1998; 83: 214-217Crossref PubMed Scopus (65) Google Scholar). These isoenzymes differ not only in their tissue distribution but also in their kinetic and regulatory properties. The PFKFB3 isozyme has the highest kinase:phosphatase activity ratio and thus maintains elevated F-2,6-P2 levels, which in turn sustains high glycolytic rates (5Chesney J. Mitchell R. Benigni F. Bacher M. Spiegel L., Al- Abed Y. Han J.H. Metz C. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3047-3052Crossref PubMed Scopus (244) Google Scholar, 10Sakakibara R. Kato M. Okamura N. Nakagawa T. Komada Y. Tominaga N. Shimojo M Fukasawa M. J. Biochem. 1997; 122: 122-128Crossref PubMed Scopus (102) Google Scholar). Significantly, this isoform is constitutively expressed in several human cancer cell lines having high proliferative rates that require the elevated activity of the enzyme for the synthesis of 5-phosphoribosyl-1-pyrophosphate, a precursor for purines and pyrimidines (5Chesney J. Mitchell R. Benigni F. Bacher M. Spiegel L., Al- Abed Y. Han J.H. Metz C. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3047-3052Crossref PubMed Scopus (244) Google Scholar, 11Boada J. Roig T. Perez X. Gamez A. Bartrons R. Cascante M. Bermudez J. FEBS Lett. 2000; 480: 261-264Crossref PubMed Scopus (48) Google Scholar, 12Hirata T. Watanabe M. Miura S. Ijichi K. Fukasawa M. Sakakibara R. Biosci. Biotechnol. Biochem. 2000; 64: 2047-2052Crossref PubMed Scopus (17) Google Scholar). Thus, this could serve as an explanation for the high glycolytic rates present in transformed cells even under normal oxygen tension (the Warburg effect). Hypoxia is a potent inducer of gene expression. It is also an important component of many pathophysiological processes including tumor growth and metastasis (13Maxwell P.H. Dachs G.U. Gleadle J.M. Nicholls L.G. Harris A.L. Stratford I.J. Hankinson O. Pugh C.W. Ratcliffe PJ Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8104-8109Crossref PubMed Scopus (942) Google Scholar, 14Ryan H.E. Poloni M. McNulty W. Elson D. Gassmann M. Arbeit J.M. Johnson R.S. Cancer Res. 2000; 60: 4010-4015PubMed Google Scholar). In hypoxic conditions, as oxidative phosphorylation is impaired, cells turn to glycolysis to meet their energetic demands (the Pasteur effect). During the adaptive response to hypoxia, the expression of genes encoding several of the glycolytic enzymes and glucose transporters is increased (15Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Crossref PubMed Scopus (443) Google Scholar, 16Semenza G.L. Jiang B.-H. Leung S.W. Passantino R. Concordet J.-P. Maire P. Giallongo A. J. Biol. Chem. 1996; 271: 32529-32537Abstract Full Text Full Text PDF PubMed Scopus (1339) Google Scholar). In animals, the hypoxia-inducible transcription factor-1 (HIF-1) complex mediates the activation of these genes. HIF-1 is a heterodimeric protein complex composed of two subunits: a constitutively expressed β-subunit, and an α-subunit for which expression and activity are controlled by intracellular oxygen concentration (reviewed in Refs. 17Wenger R.H. Gassmann M. Storey K.B. Environmental Stress and Gene Regulation. BIOS Scientific Publishers Ltd., Oxford1999: 25-45Google Scholar and 18Semenza G.L. Genes Dev. 2000; 14: 1983-1991PubMed Google Scholar). During normoxia, HIF-1α is rapidly degraded by the ubiquitin proteasome system, whereas exposure to hypoxic conditions prevents its degradation (19Huang L.E., Gu, J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1835) Google Scholar, 20Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1393) Google Scholar, 21Kallio P.J. Wilson W.J. O'Brien S. Makino Y. Poellinger L. J. Biol. Chem. 1999; 274: 6519-6525Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). The enzymatic hydroxylation of proline 564 of HIF-1α controls the turnover of the protein by tagging it for interaction with the von Hippel Lindau (VHL) protein (22Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin W.G., Jr. Science. 2001; 292: 464-468Crossref PubMed Scopus (3842) Google Scholar, 23Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A.V. Hebestreti H.F. Mukherju M. Schofield C.J. Maxwell Ph.H. Pugh C, W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4391) Google Scholar, 24Yu F. White S.B. Zhao Q. Lee F.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9630-9636Crossref PubMed Scopus (638) Google Scholar). The VHL protein forms a multiprotein complex that contains, at a minimum, elongins B and C, Cul2, and Rbx, and acts as the ubiquitin ligase that targets HIF-1α for degradation. The effect of hypoxia on Pro-564 hydroxylation can be mimicked by transition metals like cobalt, iron chelators and by inhibitors of the prolyl hydroxylase enzymes (22Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin W.G., Jr. Science. 2001; 292: 464-468Crossref PubMed Scopus (3842) Google Scholar,23Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A.V. Hebestreti H.F. Mukherju M. Schofield C.J. Maxwell Ph.H. Pugh C, W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4391) Google Scholar). Despite its importance in regulating glycolysis and gluconeogenesis, the role of PFK-2/F-2,6-BPase enzyme in the hypoxic response pathway in mammals has not been characterized. We report here that hypoxia, cobalt, and iron chelators produce a significant induction of PFKFB3 mRNA in several human and mouse cell lines. Furthermore, by utilizing conditional knock-out cell lines of the HIF-1αgene, we demonstrate that the hypoxia inducibility of this gene is dependent on the presence of an active HIF-1 complex. Cobalt chloride and desferrioxamine were purchased from Sigma. Dimethyloxalylglycine was a gift of Peter Ratcliffe (Oxford, UK). Fetal calf serum was obtained from HyClone (Logan, UT). 32PUTP (800 Cu/mmol) was from PerkinElmer Life Sciences. T3 and T7 RNA polymerases, RNase inhibitor, and DNase I (Rnase free) where from Roche cells and cells were in with and embryonic fibroblasts and cell were obtained from R. S. Johnson of H.E. Poloni M. McNulty W. Elson D. Gassmann M. Arbeit J.M. Johnson R.S. Cancer Res. 2000; 60: 4010-4015PubMed Google and in high glucose with of this conditionally nullizygous fibroblasts for HIF-1α has the of HIF-1α mRNA and protein H.E. Poloni M. McNulty W. Elson D. Gassmann M. Arbeit J.M. Johnson R.S. Cancer Res. 2000; 60: 4010-4015PubMed Google Scholar). cells and their cell were N. M. A. Caro J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The cells and a cell were by hypoxic the were for in a and with a carbon and This oxygen concentration was to HIF-1α hypoxic as by Jiang G.L. C. J. 1996; 271: PubMed Google Scholar). RNA was from cell lines the acid by and P. N. Biochem. PubMed Scopus Google Scholar). were with of and in the of of and of a were to cell with the of RNA was with an of RNA were with and in The for synthesis of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase for was by synthesis of a RNA from cells and by PFKFB3 was and These to and of the human PFKFB3 A. Rosa J.L. Ventura F. Perez J.X. Nadal M. Estivill X. Ambrosio S. Gil J. Bartrons R. Cytogenet. Cell Genet. 1998; 83: 214-217Crossref PubMed Scopus (65) Google Scholar). The was from this to of the human PFKFB3 was and with this was to a for This a expressed in both the and PFKFB3 A was for the synthesis of a mouse mouse RNA as The and to and of the mouse PFKFB3 The was and with was for the mouse PFKFB3 The human was by synthesis of a RNA from cells and by was and These to and of the human and the was with to a The for synthesis of mouse for was by synthesis of a RNA from mouse and by was and These to and of the mouse of was This was with to a The for the human has been A. T. S. Caro J. 1994; Google Scholar). The for synthesis of mouse was by synthesis of a mouse RNA and by of the as The to and of mouse A was and with this was to a that was to mouse and human were by the and to be to the of was Roche T7 T3 RNA and of RNA were under and in of were for at and for at as A. T. S. Caro J. 1994; Google Scholar). The were on a in for at The was and to at of mRNA was of mRNA was for RNA shift where as N. M. A. Caro J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google from and hypoxic The for the shift the from the cell as N. M. A. Caro J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). was from and was from from was the of hypoxia, cobalt and desferrioxamine on PFKFB3 gene mRNA levels were by RNase in hypoxia, cobalt, and desferrioxamine PFKFB3 and mRNA expression in Furthermore, as in mRNA is by with desferrioxamine and is at A of induction is for The effect of hypoxia and desferrioxamine was also in cells that lack and have N. M. A. Caro J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). These cells on glycolysis for their energetic and have been on their response to hypoxia as both normal and N. M. A. Caro J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Rodriguez J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, E. Ratcliffe P.J. 2001; 98: PubMed Scopus Google Scholar). in as well as their have a normal induction of the PFKFB3 gene in response to hypoxia and desferrioxamine. induction of PFKFB3 was in cells the role of HIF-1 in the hypoxic response of the PFKFB3 we a mouse cell with a conditional of the HIF-1α gene H.E. Poloni M. McNulty W. Elson D. Gassmann M. Arbeit J.M. Johnson R.S. Cancer Res. 2000; 60: 4010-4015PubMed Google Scholar). cells and HIF-1α cells were to hypoxia for and PFKFB3 was the RNase in HIF-1α cells have a induction of PFKFB3 mRNA in hypoxia, were in the HIF-1α were for the and for which the response to hypoxia is to be dependent on The shift in the hypoxic induction of the HIF-1 complex in the HIF-1α whereas such complex is in the The presence of HIF-1α in the hypoxic HIF-1α cells was confirmed by is by an oxygen-dependent hydroxylation of Pro-564 in the of HIF-1α This is by prolyl which as a Gleadle J.M. J. Mole D.R. M. E. Wilson M.I. A. Tian N. P. R. J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Science. 2001; PubMed Scopus Google Scholar). of these enzymes can HIF-1α under conditions P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A.V. Hebestreti H.F. Mukherju M. Schofield C.J. Maxwell Ph.H. Pugh C, W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4391) Google Scholar). We a inhibitor of to the of enzyme on mRNA expression. in for in the of PFKFB3 and mRNA to a to that by hypoxic in two different cell lines. HIF-1α expression as by in HIF-1α hydroxylation at and at N. C. Maxwell C.W. Pugh C.W. Ratcliffe P.J. J. 2001; PubMed Scopus Google its interaction with which targets it for and degradation by the in have been to and HIF-1-dependent genes such as and P.H. Pugh C.W. Ratcliffe P.J. 1999; PubMed Scopus Google Scholar). the role of on PFKFB3 mRNA we the cell These cells a VHL and to produce a VHL in VHL-deficient cells PFKFB3, and The expression of these genes is in cells with of a the of the increased levels of in the VHL cells to the of The hypoxic response of an is manifested both at the and at the oxygen cells from oxidative phosphorylation to glycolysis. is the potent activator of glycolysis and the rate of glucose utilization (1Kawaguchi T. Veech R.L. Uyeda K. J. Biol. Chem. 2001; 276: 28554-28561Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 2Hue L. Rousseau G.G. Adv. Enzyme Regul. 1993; 33: 97-110Crossref PubMed Scopus (74) Google Scholar, 3Okar D.A. Lange A.J. Biofactors. 1999; 10: 1-14Crossref PubMed Scopus (119) Google Scholar, S.J. Claus T.H. Kurland I.J. Lange A.J. Annu. Rev. Biochem. 1995; 64: 799-835Crossref PubMed Scopus (226) Google Scholar). It and the enzyme PFK-2/F-2,6-BPase controls the levels of by its synthesis and degradation. the PFK-2/F-2,6-BPase of PFKFB3 has the highest kinase:phosphatase It is also the isoform highly expressed in transformed that it to the high glycolytic rate in (5Chesney J. Mitchell R. Benigni F. Bacher M. Spiegel L., Al- Abed Y. Han J.H. Metz C. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3047-3052Crossref PubMed Scopus (244) Google Scholar, 10Sakakibara R. Kato M. Okamura N. Nakagawa T. Komada Y. Tominaga N. Shimojo M Fukasawa M. J. Biochem. 1997; 122: 122-128Crossref PubMed Scopus (102) Google Scholar). several have that the genes coding for of the enzymes of the glycolytic pathway are hypoxia, the response of the PFK-2/F-2,6-BPase gene has not been characterized. with the PFKFB3 isoform demonstrate that exposure to hypoxia a significant in its mRNA in several cell lines. which have a high of not show to The response to hypoxia was mimicked by exposure to the iron desferrioxamine and the transition Furthermore, the induction of gene was not by the of oxidative as by the normal response in These which lack and on have with to their to hypoxia N. M. A. Caro J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Rodriguez J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, E. Ratcliffe P.J. 2001; 98: PubMed Scopus Google Scholar). The here that gene activation in response to hypoxia is of the presence of an active The role of HIF-1 was cells in the HIF-1α It has been that the HIF-1α cells have growth rate under hypoxic conditions as well as glycolytic response to hypoxia manifested by levels, acid and H. M. P. K. Johnson R.S. Biol. 2001; PubMed Scopus Google Scholar). The response to hypoxia of the PFKFB3 and the genes was in an absolute of a HIF-1 complex. conditions, is and rapidly degraded by the proteasome (19Huang L.E., Gu, J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1835) Google Scholar, 20Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1393) Google Scholar, 21Kallio P.J. Wilson W.J. O'Brien S. Makino Y. Poellinger L. J. Biol. Chem. 1999; 274: 6519-6525Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). The by which oxygen the of the hydroxylation of other by prolyl hydroxylase enzymes (22Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin W.G., Jr. Science. 2001; 292: 464-468Crossref PubMed Scopus (3842) Google Scholar, 23Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A.V. Hebestreti H.F. Mukherju M. Schofield C.J. Maxwell Ph.H. Pugh C, W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4391) Google Scholar). HIF-1α with which acts as a ubiquitin thus it for degradation. We the role of VHL and prolyl hydroxylation in the up-regulation of PFKFB3 cells and inhibitors of the prolyl hydroxylase VHL-deficient cells several of the HIF-1-dependent genes as a of their to P.H. Pugh C.W. Ratcliffe P.J. 1999; PubMed Scopus Google Scholar). The VHL-deficient cells show elevated levels of PFKFB3, and that could be but by the expression of a VHL are a of recently enzymes that HIF-1α in an oxygen Gleadle J.M. J. Mole D.R. M. E. Wilson M.I. A. Tian N. P. R. J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Science. 2001; PubMed Scopus Google Scholar). The and of of of this have the for iron and for their and P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A.V. Hebestreti H.F. Mukherju M. Schofield C.J. Maxwell Ph.H. Pugh C, W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4391) Google that the enzymatic activity of the could be by The present demonstrate that in a significant up-regulation of PFKFB3 and mRNA expression. The that the complex mediates the activation of the PFKFB3 The of gene has been well characterized. The gene at A. A. L. Rosa J.L. Ventura F. Bartrons R. 2001; PubMed Scopus Google Scholar). This for the and forms by of which from could not these two of the of the PFKFB3 gene a and for several transcription present in the are with for the of and M. E. N. A. M. N. Sakakibara R. Biochem. Biophys. Res. Commun. 2000; PubMed Scopus Google Scholar, A. A. L. Rosa J.L. Ventura F. Bartrons R. 2001; PubMed Scopus Google Scholar). are at several in the of the One of at to from the transcription to the in of the glycolytic enzymes and G.L. Genes Dev. 2000; 14: 1983-1991PubMed Google Scholar). the HIF-1 in the hypoxic of the PFKFB3 gene has not been In a effect of hypoxia on the of PFKFB3 mRNA has not been is the key enzyme that glycolysis in It is by and and by It is that is the potent activator of the Furthermore, the levels of are controlled by a of the kinase and phosphatase of which in different cell and are under metabolic conditions D.A. Lange A.J. 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In HIF-1α is expressed even under conditions as is the in VHL as a of other H. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Harris A.L. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, D. H. J.M. E. G.L. 2000; PubMed Scopus Google Scholar, M.A. Ohh M. Yang H. J.M. M. Kaelin W.G., Jr. Genet. 2001; 10: PubMed Scopus Google Scholar). It is to that in PFKFB3 be thus an explanation for the Warburg which is in tumor cells O. Science. PubMed Scopus Google Scholar). We the the cells and for this Peter Ratcliffe of for the R. S. Johnson of for the mouse embryonic N. for the We also the of D. with and R. with