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

Abstract

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