Reversible Inactivation of HIF-1 Prolyl Hydroxylases Allows Cell Metabolism to Control Basal HIF-1

Continuous hydroxylation of the HIF-1 transcription factor α subunit by oxygen and 2-oxoglutarate-dependent dioxygenases promotes decay of this protein and thus prevents the transcriptional activation of many genes involved in energy metabolism, angiogenesis, cell survival, and matrix modification. Hypoxia blocks HIF-1α hydroxylation and thus activates HIF-1α-mediated gene expression. Several nonhypoxic stimuli can also activate HIF-1, although the mechanisms involved are not well known. Here we show that the glucose metabolites pyruvate and oxaloacetate inactivate HIF-1α decay in a manner selectively reversible by ascorbate, cysteine, histidine, and ferrous iron but not by 2-oxoglutarate or oxygen. Pyruvate and oxaloacetate bind to the 2-oxoglutarate site of HIF-1α prolyl hydroxylases, but their effects on HIF-1 are not mimicked by other Krebs cycle intermediates, including succinate and fumarate. We show that inactivation of HIF-1 hydroxylation by glucose-derived 2-oxoacids underlies the prominent basal HIF-1 activity commonly seen in many highly glycolytic cancer cells. Since HIF-1 itself promotes glycolytic metabolism, enhancement of HIF-1 by glucose metabolites may constitute a novel feed-forward signaling mechanism involved in malignant progression. Continuous hydroxylation of the HIF-1 transcription factor α subunit by oxygen and 2-oxoglutarate-dependent dioxygenases promotes decay of this protein and thus prevents the transcriptional activation of many genes involved in energy metabolism, angiogenesis, cell survival, and matrix modification. Hypoxia blocks HIF-1α hydroxylation and thus activates HIF-1α-mediated gene expression. Several nonhypoxic stimuli can also activate HIF-1, although the mechanisms involved are not well known. Here we show that the glucose metabolites pyruvate and oxaloacetate inactivate HIF-1α decay in a manner selectively reversible by ascorbate, cysteine, histidine, and ferrous iron but not by 2-oxoglutarate or oxygen. Pyruvate and oxaloacetate bind to the 2-oxoglutarate site of HIF-1α prolyl hydroxylases, but their effects on HIF-1 are not mimicked by other Krebs cycle intermediates, including succinate and fumarate. We show that inactivation of HIF-1 hydroxylation by glucose-derived 2-oxoacids underlies the prominent basal HIF-1 activity commonly seen in many highly glycolytic cancer cells. Since HIF-1 itself promotes glycolytic metabolism, enhancement of HIF-1 by glucose metabolites may constitute a novel feed-forward signaling mechanism involved in malignant progression. Mammalian cells adapt to hypoxia through the action of the heterodimeric transcription factor HIF-1. Such adaptations can also promote carcinogenesis by inducing angiogenesis, treatment resistance, and invasiveness in hypoxic cancer cells within tumors (1Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5295) Google Scholar). In the presence of oxygen, the HIF-1α subunit undergoes rapid decay via a ubiquitin-proteasome degradation pathway involving the von Hippel-Lindau tumor suppressor gene product pVHL (2Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar, 3Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1845) Google Scholar, 4Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4118) Google Scholar). The binding of pVHL to HIF-1α requires the post-translational hydroxylation of proline residues (Pro402 and Pro564) within the HIF-1α oxygen-dependent degradation (ODD) 4The abbreviations used are: ODDoxygen-dependent degradation2-OG2-oxoglutarateDFOdesferrioxamineDMOGdimethyloxalylglycineEGFepidermal growth factorROSreactive oxygen speciesNOnitric oxideMEMminimal Eagle's mediumDMEMDulbecco's modified Eagle's mediumRTreverse transcriptionVEGFvascular endothelial growth factorHREhypoxia regulatory elementGFPgreen fluorescent proteinHPHHIF-1α prolyl hydroxylase.4The abbreviations used are: ODDoxygen-dependent degradation2-OG2-oxoglutarateDFOdesferrioxamineDMOGdimethyloxalylglycineEGFepidermal growth factorROSreactive oxygen speciesNOnitric oxideMEMminimal Eagle's mediumDMEMDulbecco's modified Eagle's mediumRTreverse transcriptionVEGFvascular endothelial growth factorHREhypoxia regulatory elementGFPgreen fluorescent proteinHPHHIF-1α prolyl hydroxylase. domain (5Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3874) Google Scholar, 6Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. von Kriegsheim A. Hebenstreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4434) Google Scholar). This modification is prevented during hypoxia, thus allowing HIF-1α to escape proteolysis, dimerize with HIF-1β, and translocate to the nucleus. A separately controlled, O2-dependent hydroxylation of asparagine 803 in the HIF-1α C-terminal transactivation domain inhibits HIF-1 interaction with the p300/CBP coactivator, thereby blocking HIF-1 transcriptional activity in the presence of oxygen (7Lando D. Peet D.J. Gorman J.J. Whelan D. A Whitelaw M.L. Bruick R.K. Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1219) Google Scholar, 8Hewitson K.S. McNeill L.A. Riordan M.V. Tian Y.M. Bullock A.N. Welford R.W. Elkins J.M. Oldham N.J. Bhattacharya S. Gleadle J.M. Ratcliffe P.J. Pugh C.W. Schofield C.J. J. Biol. Chem. 2002; 277: 26351-26355Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). Three HIF-1α prolyl hydroxylases (HPH1 to -3; also referred to as PHD3 to -1, respectively) and one O2-dependent HIF-1α asparaginyl hydroxylase (factor inhibiting HIF, or FIH) have been clearly identified so far (9Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2106) Google Scholar, 10Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke 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 (2720) Google Scholar, 11Mahon P.C. Hirota K. Semenza G.L. Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1119) Google Scholar). These enzymes are all members of the 2-oxoglutarate-dependent family of dioxygenases and have an absolute requirement for oxygen, ferrous iron, and 2-oxoglutarate (2-OG). This explains how hypoxia, iron chelators such as desferrioxamine (DFO), and artificial 2-OG analogs such as N-oxalylglycine or its cell-permeable precursor dimethyloxalylglycine (DMOG) can all prevent HIF-1α proteolysis and activate HIF-mediated gene expression. Ascorbate is also required for the sustained activity of many 2-OG-dependent dioxygenases (12Schofield C.J. Ratcliffe P.J. Nat. Rev. Mol. Cell. Biol. 2004; 5: 343-354Crossref PubMed Scopus (1611) Google Scholar, 13Hanauske-Abel H.M. Popowicz A.M. Curr. Med. Chem. 2003; 10: 1005-1019Crossref PubMed Scopus (19) Google Scholar). oxygen-dependent degradation 2-oxoglutarate desferrioxamine dimethyloxalylglycine epidermal growth factor reactive oxygen species nitric oxide minimal Eagle's medium Dulbecco's modified Eagle's medium reverse transcription vascular endothelial growth factor hypoxia regulatory element green fluorescent protein HIF-1α prolyl hydroxylase. oxygen-dependent degradation 2-oxoglutarate desferrioxamine dimethyloxalylglycine epidermal growth factor reactive oxygen species nitric oxide minimal Eagle's medium Dulbecco's modified Eagle's medium reverse transcription vascular endothelial growth factor hypoxia regulatory element green fluorescent protein HIF-1α prolyl hydroxylase. Hypoxia-independent mechanisms also regulate HIF-1 and are highly relevant to the progression of human cancers. 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Res. 2003; Google in cell lines in high glucose in or The of basal HIF-1α in cells required glucose but not by the presence or of These cells are cultured in with the energy or the cells to HIF-1 hypoxia This not only that the cells in the presence of as the energy but also clearly glycolytic and hypoxic of HIF-1 as The and human cells in used to the glucose and basal their to Krebs for cells basal HIF-1α glucose by the energy the presence of basal HIF-1α in cells by glucose many we metabolites of HIF-1α for glucose in Krebs of all all and many that not levels of cell only a of 2-oxoacids to promote HIF-1α only pyruvate and oxaloacetate to in all cancer cell lines used in this The for inducing HIF-1α by 2-oxoacids a of cell to as as HIF-1α with The 2-oxoacids and also in but not all cell and activity The and to 2-OG and to the artificial HIF-1α N-oxalylglycine and also of that and all of a thus to HIF-1α in or cells and 2-oxoacids such as and the and not HIF-1α in that the of the also for inducing and are modified on the the not to HIF-1α and of pyruvate the can HIF-1α this requires its to pyruvate via H. 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HIF-1 by by with the basal HIF-1α expression of and cells cultured in of HIF-1α by pyruvate or oxaloacetate in Krebs also by This not by and or 2-OG reverse HIF-1α by hypoxia or This of pyruvate and HIF-1α in all cells used in this not cells a hypoxia regulatory element we that pyruvate and oxaloacetate the gene as as hypoxia or effects seen cells with fluorescent protein of the HIF-1 gene by the 2-oxoacids also selectively by but not by Ascorbate not reverse activation by hypoxia or by a of HIF-1α by pyruvate and and an in we that HIF-1α expression in human cells to in Krebs in the glucose cells cultured in to Krebs or to with glucose for a of HIF-1α seen in Krebs Since Krebs the ascorbate, this to that activity that glycolytic activation of Krebs by its and of Krebs with we that the and not the basal HIF-1α The also the of HIF-1α by pyruvate in cells cultured for in Krebs with one a the in we that and the for inhibiting basal HIF-1α This of all only of are the levels of to its in also that and the of basal HIF-1α in and its and not HIF-1α by and oxaloacetate in all cell lines used in this not but not HIF-1α by hypoxia or the iron is involved in with ascorbate, cysteine, and histidine, also selectively HIF-1α by pyruvate and oxaloacetate but not by hypoxia or the levels of and histidine, but not other to the in also the basal expression of the genes and as by and also activity by pyruvate or oxaloacetate in the cells These for and of basal HIF-1 expression by cell Pyruvate and that the effects on HIF-1 we to the that 2-oxoacids such as pyruvate hypoxia in cell through enhancement of cell in activity have been to oxygen in cell K. S. W. Metzen E. PubMed Scopus Google Scholar). we used the only to reactive in C.J. J. Cancer. PubMed Scopus Google Scholar). clearly by in hypoxic with or the of HIF-1α by pyruvate or oxaloacetate reactive oxygen species can also Since ascorbate, cysteine, histidine, and all have we also to of such as or by pyruvate or the of and to HIF-1α in we not an in or levels treatment of cells with pyruvate or oxaloacetate of other signaling in HIF-1α such as protein and also to HIF-1α by pyruvate and oxaloacetate We on HIF-1α proline hydroxylation as a for we HIF-1α by the 2-oxoacids in the This the 2-oxoacids with to proline such as pVHL binding or an that in the HIF-1α C-terminal we only in HIF-1α by the HIF-1α by or the not We a cell for oxygen-dependent HIF-1α degradation cells that been with a the HIF-1α to The protein is and in an oxygen-dependent manner to with a basal as well as of been E. P. C. J. Biol. Chem. 2002; Scholar). We that basal in cultured in for selectively by ascorbate, the expression not In Krebs basal also by and oxaloacetate but not by The of succinate pyruvate or oxaloacetate in cells not to a in cell the seen in cells and also by pyruvate or These that 2-oxoacids 2-oxoglutarate selectively O2-dependent protein Pyruvate and with not activity H. A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google and pyruvate oxaloacetate to binding to 2-OG analogs such as N-oxalylglycine promote HIF-1α by for the 2-OG binding site in A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke 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. 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This by in the of metabolites in the the of pyruvate to inactivation of activity in the we cell of medium to and HIF-1α prolyl hydroxylation in cell the as in of the a in activity cell not with the of to the cell not only the but an in with in of an in but not The inactivation in cells by the of basal and as in other not reverse or HIF-1α in cells the of pyruvate to the of HIF-1α the HIF-1α we the medium with or ferrous iron or and the for We that the HIF-1α to a by ferrous iron by iron, Ascorbate also the basal expression of genes in including and matrix the basal invasiveness of cells through invasiveness These basal HIF-1 activity in cancer progression. HIF-1 by hydroxylases are dioxygenases that also oxygen, and The mechanism for activity of 2-OG dioxygenases of the by the and of of the to a as well as binding of the may also required for of oxygen to the site of the ferrous the dioxygenases oxygen to the of one oxygen the of 2-OG to succinate and with the other oxygen a that hydroxylation in C.J. Ratcliffe P.J. Nat. Rev. Mol. Cell. Biol. 2004; 5: 343-354Crossref PubMed Scopus (1611) Google and 13Hanauske-Abel H.M. Popowicz A.M. Curr. Med. Chem. 2003; 10: 1005-1019Crossref PubMed Scopus (19) Google Scholar). of the oxygen iron or 2-OG artificial 2-OG analogs such as N-oxalylglycine or is to this family of 2-OG-dependent dioxygenases can also This that as a of enzymes residues or the of the iron in sustained In this inactivation can or by in 13Hanauske-Abel H.M. Popowicz A.M. Curr. Med. Chem. 2003; 10: 1005-1019Crossref PubMed Scopus (19) Google Scholar). 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R. and used in this with