6-Phosphofructo-2-kinase (pfkfb3) Gene Promoter Contains Hypoxia-inducible Factor-1 Binding Sites Necessary for Transactivation in Response to Hypoxia
The up-regulation of glycolysis to enhance the production of energy under reduced pO2 is a hallmark of the hypoxic response. A key regulator of glycolytic flux is fructose-2,6-bisphosphate, and its steady state concentration is regulated by the action of different isozymes product of four genes (pfkfb1–4). pfkfb3 has been found in proliferating cells and tumors, being induced by hypoxia. To understand the organization of cis-acting sequences that are responsible for the oxygen-regulated pfkfb3 gene, we have studied its 5′-flanking region. Extensive analysis of the 5′ pfkfb3 promoter sequence revealed the presence of putative consensus binding sites for various transcription factors that could play an important role in pfkfb3 gene regulation. These DNA consensus sequences included estrogen receptor, hypoxia response element (HRE), early growth response, and specific protein 1 putative binding sites. Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit constructs demonstrate that the sequence located from -1269 to -1297 relative to the start site is required for hypoxia-inducible factor 1 (HIF-1) induction. The effective binding of HIF-1 transcription factor to the HREs at -1279 and -1288 was corroborated by electrophoretic mobility shift assay and biotinylated oligonucleotide pull-down. In addition, HIF-1α null mouse embryo fibroblasts transfected with a full-length pfkfb3 promoter-luciferase reporter construct further demonstrated that HIF-1 protein was critically involved for hypoxia transactivation of this gene. Altogether, these results demonstrate that pfkfb3 is a hypoxia-inducible gene that is stimulated through HIF interaction with the consensus HRE site in its promoter region. The up-regulation of glycolysis to enhance the production of energy under reduced pO2 is a hallmark of the hypoxic response. A key regulator of glycolytic flux is fructose-2,6-bisphosphate, and its steady state concentration is regulated by the action of different isozymes product of four genes (pfkfb1–4). pfkfb3 has been found in proliferating cells and tumors, being induced by hypoxia. To understand the organization of cis-acting sequences that are responsible for the oxygen-regulated pfkfb3 gene, we have studied its 5′-flanking region. Extensive analysis of the 5′ pfkfb3 promoter sequence revealed the presence of putative consensus binding sites for various transcription factors that could play an important role in pfkfb3 gene regulation. These DNA consensus sequences included estrogen receptor, hypoxia response element (HRE), early growth response, and specific protein 1 putative binding sites. Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit constructs demonstrate that the sequence located from -1269 to -1297 relative to the start site is required for hypoxia-inducible factor 1 (HIF-1) induction. The effective binding of HIF-1 transcription factor to the HREs at -1279 and -1288 was corroborated by electrophoretic mobility shift assay and biotinylated oligonucleotide pull-down. In addition, HIF-1α null mouse embryo fibroblasts transfected with a full-length pfkfb3 promoter-luciferase reporter construct further demonstrated that HIF-1 protein was critically involved for hypoxia transactivation of this gene. Altogether, these results demonstrate that pfkfb3 is a hypoxia-inducible gene that is stimulated through HIF interaction with the consensus HRE site in its promoter region. In eukaryotic cells exposure to a low oxygen environment induces a hypoxic response pathway through a hypoxia-inducible transcription factor (HIF) 1The abbreviations used are: HIF-1, hypoxia-inducible factor 1; Glut-1, glucose transporter-1; DFO, deferoxamine; HRE, hypoxia response element; E3, ubiquitin-protein isopeptide ligase; Fru-2,6-P2, fructose-2,6-bisphosphate; PFK-2, 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase; mEF, mouse embryo fibroblast; DMEM, Dulbecco's modified Eagle's medium. (1Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5416) Google Scholar). The active transcription factor is a heterodimeric protein complex composed of two subunits HIF-1α and HIF-1β. This dimer recognizes the hypoxia response element (HRE; 5′-ACGTG-3′) present in hypoxia-inducible promoters. The HIF-1β is a constitutively expressed protein, whereas the α subunit is rapidly degraded in normoxic conditions through the ubiquitin-proteasome system (2Semenza G.L. Trends Mol. Med. 2001; 7: 345-350Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar). The protein that initiates this degradation process is the tumor suppressor VHL (Von Hippel-Lindau), which is the recognition component of an E3 ubiquitin-protein ligase complex that targets HIF-1α for proteasomal degradation when HIF-1α prolines -564 and -402 are hydroxylated (3Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2141) Google Scholar, 4Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourk J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2761) Google Scholar, 5Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1860) Google Scholar). This hydroxylation process is controlled by specific Fe2+, oxoglutarate, and oxygen-dependent hydroxylase enzymes. Thus, stabilization of HIF-1α is induced by oxygen deficiency, allowing its nuclear translocation and dimerization with HIF-1β (6Jiang B.H. Semenza G.L. Bauer C. Marti H.H. Am. J. Physiol. 1996; 271: C1172-C1180Crossref PubMed Google Scholar). Chelating or substituting Fe2+ with deferoxamine (7Wang G.L. Semenza G.L. Blood. 1993; 82: 3610-3615Crossref PubMed Google Scholar) and cobalt chloride (8Ebert B.L. Bunn H.F. Blood. 1999; 94: 1864-1877Crossref PubMed Google Scholar), respectively, or inhibiting oxoglutarate with dimethyloxalylglycine reduces the hydroxylase activity and mimics the hypoxia effects. There is now substantial evidence in support of the hypothesis that HIF-1 functions as a mediator of the adaptive response to hypoxia. Among all of the adaptations, transcriptional activation of genes associated to metabolism is of special interest. Many of these target genes promote cellular adaptation to reduced oxygen availability by increasing glucose uptake and glycolysis. Several genes encoding enzymes of the glycolytic pathway and glucose transport are activated by low pO2: aldolase-A, phosphoglycerate kinase-1, pyruvate kinase M, lactate dehydrogenase A, phosphofructokinase L, and glucose transporter-1 (Glut-1) (1Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5416) Google Scholar, 9Chen C. Pore N. Behrooz A. Ismail-Beigi F. Maity A. J. Biol. Chem. 2001; 276: 9519-9525Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 10Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar, 11Minchenko A. Leshchinsky I. Opentanova I. Sang N. Srinivas V. Armstead V. Caro J. J. Biol. Chem. 2002; 277: 6183-6187Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). High glycolytic flux is essential for tumor growth in hypoxic conditions, and many transformed cells display a high rate of glycolysis that is maintained even under aerobic conditions (Warburg effect) (12Warburg O. Science. 1956; 123: 309-314Crossref PubMed Scopus (9940) Google Scholar). Glycolytic flux is mainly controlled by 6-phosphofructo-1-kinase (13Krebs H.A. Essays Biochem. 1972; 8: 1-34PubMed Google Scholar), with fructose-2,6-bisphosphate (Fru-2,6-P2) being its most powerful allosteric activator (14Van Schaftingen E. Adv. Enzymol. Relat. Areas Mol. Biol. 1987; 59: 315-395PubMed Google Scholar, 15Pilkis S.J. Claus T.H. Kurland I.J. Lange A.J. Annu. Rev. Biochem. 1995; 64: 799-835Crossref PubMed Scopus (230) Google Scholar). These properties confer to this metabolite a key role in the control of the glycolytic pathway. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) is the bifunctional enzyme that catalyzes the synthesis and degradation of Fru-2,6-P2 and hence critically regulates carbohydrate metabolism (14Van Schaftingen E. Adv. Enzymol. Relat. Areas Mol. Biol. 1987; 59: 315-395PubMed Google Scholar, 15Pilkis S.J. Claus T.H. Kurland I.J. Lange A.J. Annu. Rev. Biochem. 1995; 64: 799-835Crossref PubMed Scopus (230) Google Scholar, 16Hue L. Rousseau G.G. Adv. Enzyme Regul. 1993; 33: 97-110Crossref PubMed Scopus (74) Google Scholar). Four independent genes, pfkfb1–4, code for the different isoforms of the PFK-2 family. These isoforms show differences in their tissue distribution and kinetic properties in response to allosteric effector, hormonal, and growth factor signals (17Okar D.A. Manzano A. Navarro-Sabate A. Riera L. Bartrons R. Lange A.J. Trends Biochem. Sci. 2001; 26: 30-35Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). The pfkfb3 gene product has the highest kinase/phosphatase activity ratio (18Sakakibara R. Kato M. Okamura N. Nakagawa T. Komada Y. Tominaga N. Shimojo M. Fukasawa M. J. Biochem. (Tokyo). 1997; 122: 122-128Crossref PubMed Scopus (109) Google Scholar). This that in is Fru-2,6-P2 are and high glycolytic are is a gene constitutively expressed in proliferating J. R. F. M. L. Y. C. R. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, Mol. 1997; PubMed Scopus Google Scholar, F. A. E. J. 1997; PubMed Scopus Google Scholar, A. F. M. S. J. Bartrons R. 1998; PubMed Scopus Google Scholar, N. Manzano A. Riera L. S. F. Bartrons R. Mol. PubMed Scopus Google Scholar, A. Kato M. Fukasawa M. M. E. R. J. Biochem. (Tokyo). 1996; PubMed Scopus Google Scholar), in transformed J. R. F. M. L. Y. C. R. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, T. Kato M. Okamura N. Fukasawa M. R. Biochem. 1998; PubMed Scopus Google Scholar, L. Manzano A. Navarro-Sabate A. Bartrons R. 2002; PubMed Scopus Google Scholar), and in various T. J. C. L. S. R. R. 2002; Google Scholar). The present the 5′-flanking of the pfkfb3 gene and its transcriptional by evidence that the consensus binding site located at -1279 and -1288 in the pfkfb3 promoter is for of this gene by hypoxia. and through the embryo fibroblasts type or and HIF-1α or by R. S. of at M. P. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). The cells in Dulbecco's modified Eagle's with and and in a of at conditions by cells in with deferoxamine or 1 dimethyloxalylglycine by hypoxic in a and with a from cells as was from was from cells under hypoxia for for and was to the The concentration and of all the ratio and of was a from pfkfb3 and by the for pfkfb3 and for The was and the relative of the specific in was the with the The relative of gene was to that of the protein gene and gene in was with in conditions cells under hypoxia for HIF-1α HIF-1α in an HIF-1α was transfected cells by in medium. The was by the promoter with a the promoter and sequences to to a the HIF-1α cells by a to the promoter in we used a from the and the reporter construct A. Manzano A. Riera L. F. Bartrons R. 2001; PubMed Scopus Google Scholar). to and to The was used as a for a the to and the to sites in the was at for and of at at at and at The was in a A from this construct to from to of the pfkfb3 was in The of the was from The was was by of the construct and at the the The a DNA and constructs of the pfkfb3 promoter the with the gene as a reporter have been A. Manzano A. Riera L. F. Bartrons R. 2001; PubMed Scopus Google Scholar). A with the c-fos minimal promoter unit as the for reporter constructs and The of from pfkfb3 promoter -1297 to with the the two putative HRE in was to the The with a in two in of the sites was used for the The of was by sequence A protein that for the protein was used to and or and the The different and of the control Four the cells with and maintained in hypoxia transfected cells maintained in with or cobalt chloride or hypoxia for the the cells with and in activity was in with DNA was to in different The at in and the to the of The protein of was a protein activity was in a activity was in of the The are as the with DNA of HIF-1α and HIF-1β protein pfkfb3 promoter constructs these and constructs transfected various of that all cells a of 1 and protein an oxygen-dependent degradation HIF-1α and HIF-1β respectively, of a constructs by F. Bunn and L. E. cellular protein was from cells of with and for 5′ at of cellular protein under conditions and to The was with a specific the of L. Manzano A. Navarro-Sabate A. Bartrons R. 2002; PubMed Scopus Google Scholar) at and with and HIF-1α and was with The by of for from or with and HIF-1β protein cells with and by of and by at for The was with of an and The cells by the through a at The was at for at The was used as for The at protein by of in to for and at the sequence from -1297 to -1269 of the pfkfb3 gene which two consensus HRE sequence of and a consensus The sequence of the control oligonucleotide The with in the presence of and in a of protein in the presence of binding and with 1 of for at DNA for at In HIF-1β protein was and for at with the The low to for A was to the to an of the The at or cells with 1 and of and of The with 1 of biotinylated the sequence from -1297 to -1269 of pfkfb3 gene and of for with for 1 with and a was used to the by for in the pfkfb3 nuclear sites found the of binding sites and the consensus sequences early growth response, estrogen receptor, HRE, specific protein and of from different promoter constructs an analysis of of their the different constructs was a α was as A was used to from and of and in the of hypoxia pfkfb3 gene we used hypoxia and the cobalt chloride a DFO, an and a of These HIF-1 activity (7Wang G.L. Semenza G.L. Blood. 1993; 82: 3610-3615Crossref PubMed Google Scholar). and of gene for pfkfb3 A. F. M. S. J. Bartrons R. 1998; PubMed Scopus Google Scholar). results from show the of HIF-1α subunit and of with cobalt or hypoxia. in the To in hypoxia conditions, was in hypoxia pfkfb3 at results Glut-1, HIF as a control of hypoxia response. These results that hypoxia and hypoxia mimics an of in this as was for cells A. Leshchinsky I. Opentanova I. Sang N. Srinivas V. Armstead V. Caro J. J. Biol. Chem. 2002; 277: 6183-6187Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). to the of pfkfb3 gene as an HIF of pfkfb3 was in HIF-1α analysis from HIF-1α cells to hypoxia or these a the of HIF-1α and a was whereas of in the transfected these results demonstrate the of HIF-1α subunit in the transcription of pfkfb3 gene in of for from the pfkfb3 pfkfb3 promoter being of the for putative this we various from the promoter and to of the 5′-flanking of the pfkfb3 gene. The of the the pfkfb3 promoter in an analysis of 5′ pfkfb3 promoter the revealed the presence of putative consensus binding sites for various transcription factors in pfkfb3 gene regulation. These DNA consensus sequences included estrogen receptor, HRE, early growth response, and specific protein sites. Among all the putative response sequences found in the pfkfb3 promoter estrogen and HRE are of special of the of these factors in the transcriptional of the gene. the presence of sites the consensus sequence for transcription factors that have been in the of genes in response to hypoxia. are sequences for specific protein and In the of hypoxia response element sequences located in the pfkfb3 promoter are A of four HREs that are to the consensus HIF binding site are The analysis of pfkfb3 promoter included a that a putative HRE at the of to revealed hypoxia binding sites at and that to to to the pfkfb3 by hypoxia. of of the of pfkfb3 Promoter to and the promoter activation by or DFO, different of the pfkfb3 promoter and of a reporter cells transfected with these reporter constructs and of to the in activity by or was activity from promoter in from cells transfected with constructs and and with and are and transfected with constructs and Thus, and for and respectively, and for the respectively, differences found the two construct and in and of the and and respectively, of the activity in its results with and in analysis revealed that and Thus, when constructs the putative HRE located at -1279 and -1288 with the differences results with and that the of the pfkfb3 promoter are essential in the hypoxic response. In that and are the putative HRE located at for hypoxic response in the pfkfb3 gene. of of HIF the pfkfb3 could the the pfkfb3 promoter as with or HIF subunits promoter construct was with and of The construct has a deletion of the oxygen-dependent degradation of the This deletion stabilization of HIF-1α under normoxic an of the is of with the HIF-1α a in activity in to the of with the was when and HIF-1β with that the of HIF-1β are for when cells are constitutively active the HRE in the pfkfb3 from -1269 to -1297 to a to the HIF element of the of the two HRE To the of this we used a of the pfkfb3 in of a c-fos minimal promoter unit in a reporter This relative to the transcription start was used to reporter constructs and and are that the a in two in of the sites. To the we relative of cells transfected with or constructs and of to in the presence or of or in type and constructs display reporter a or was in the construct or as the type construct was induced by hypoxia the To the of sites located at -1279 and we a type or with a deletion of the HIF-1α gene M. P. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). of construct a with in the whereas was in of construct in show in activity the response these results demonstrate the of HIF to these HREs for pfkfb3 hypoxia response. HIF-1 to the HRE at -1279 and -1288 in the pfkfb3 demonstrate the binding of HIF-1 to these HRE two different a of from the pfkfb3 promoter that the HREs was used in an electrophoretic mobility shift assay with HIF-1α and HIF-1β. in a in the presence of HIF-1α and HIF-1β in the presence of the of specific that present in in the presence of an HIF-1β that HIF-1 to the pfkfb3 promoter sequence a by electrophoretic mobility shift assay a the type site from the gene L.E. Bunn H.F. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). a was and HIF-1 binding was in the presence of of an oligonucleotide assay and in the presence of HIF-1 in the with hypoxic and whereas in the normoxic these results show that HIF-1α to the HRE consensus sequence from -1297 to -1269 of pfkfb3 promoter under hypoxia. pfkfb3 an HIF of HIF-1 in the hypoxic response of pfkfb3 gene promoter was studied in the with a deletion of the HIF-1α gene M. P. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). In hypoxic conditions an of pfkfb3 was in the HIF whereas in the HIF-1 cells A. Leshchinsky I. Opentanova I. Sang N. Srinivas V. Armstead V. Caro J. J. Biol. Chem. 2002; 277: 6183-6187Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). results by when the reporter construct was transfected and and the cells to hypoxia or maintained in a substantial in hypoxic whereas was in hypoxic cells Thus, HIF-1 is to the transcription of the pfkfb3 gene in response to hypoxia. The to to of oxygen is important to all The most adaptation to hypoxia is the which and an in glycolysis (13Krebs H.A. Essays Biochem. 1972; 8: 1-34PubMed Google Scholar). of the that of different glycolytic isozymes is through HIF-1α stabilization G.L. B.H. R. P. A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). HIF-1 is a of cellular adaptation to and HIF-1α null cells show in energy metabolism M. P. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). is that HIF-1, of its role in is a mediator of the in which tumor cells show glycolytic activity under oxygen conditions (12Warburg O. Science. 1956; 123: 309-314Crossref PubMed Scopus (9940) Google Scholar). have evidence for the of glycolytic enzyme gene cis-acting DNA sequences putative sites G.L. B.H. R. P. A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). analysis revealed the presence of putative HREs the of the pfkfb3 which could the of hypoxia the of pfkfb3 gene A. Leshchinsky I. Opentanova I. Sang N. Srinivas V. Armstead V. Caro J. J. Biol. Chem. 2002; 277: 6183-6187Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, O. Opentanova I. Caro J. 2003; PubMed Scopus Google Scholar). that the two constructs and the hypoxic response, the of sites of glycolytic genes as lactate dehydrogenase A, and phosphoglycerate revealed that the hypoxia response a of HIF-1 binding sites by G.L. B.H. R. P. A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). results that putative HRE at is for hypoxic response and of in these we have the -1269 to -1297 two HRE sequences and by This could to the cis-acting DNA sequences required for HIF-1 binding and transcriptional response to hypoxia. of the of HIF consensus binding sites from -1269 to -1297 from and constructs of the pfkfb3 promoter in a high hypoxia response in the type whereas the of construct in a type mouse embryo or with a deletion of the HIF-1α gene, M. P. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar), in of in that this sequence is essential for the pfkfb3 hypoxic response. The of the HRE sequence in the binding to HIF-1 complex was corroborated by electrophoretic mobility shift assay and biotinylated oligonucleotide pull-down. in the and HIF-1β and a was the of the with an HIF-1 binding to the sequence was by with Altogether, these results demonstrate that pfkfb3 is a hypoxia-inducible gene that is stimulated in transformed through HIF factor interaction with the consensus HRE sites located at -1279 and -1288 of the promoter region. To the of HIF-1 pfkfb3 gene was induced with the of the factors and HIF-1β. differences in when or with HIF-1β. Thus, cells demonstrate HIF-1β to HIF-1α with results G.L. B.H. R. P. A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). cells for HIF-1α with to The of cells demonstrate the of pfkfb3 protein in the of HIF-1α subunit the of an active factor to the pfkfb3 promoter regulation. The differences found in hypoxia pfkfb3 of the 5′-flanking and in transfected cells with promoter could the of binding to sequences of transcription which could to a high of that the of glycolytic enzymes in hypoxic and is at the transcriptional by This could an in the flux of glycolytic pathway that is The specific role of of pfkfb3 by hypoxia with its key of the enzymes A. Regul. PubMed Scopus Google Scholar). is mainly in the in the of allosteric and the role of Fru-2,6-P2 is to its allowing glycolysis to F. S. J. Bartrons R. Biochem. J. 276: PubMed Scopus Google Scholar). The enzyme responsible of its synthesis and PFK-2, is in to by through protein kinase at increasing its the C. L. L. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). a Fru-2,6-P2 putative sites for protein kinase A and have been (18Sakakibara R. Kato M. Okamura N. Nakagawa T. Komada Y. Tominaga N. Shimojo M. Fukasawa M. J. Biochem. 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