title: Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF.
abstract: The stability and transcriptional activity of the hypoxia-inducible factors (HIFs) are regulated by two oxygen-dependent events that are catalyzed by three HIF prolyl 4-hydroxylases (HIF-P4Hs) and one HIF asparaginyl hydroxylase (FIH). We have studied possible links between metabolic pathways and HIF hydroxylases by analyzing the abilities of citric acid cycle intermediates to inhibit purified human HIF-P4Hs and FIH. Fumarate and succinate were identified as in vitro inhibitors of all three HIF-P4Hs, fumarate having K(i) values of 50-80 microM and succinate 350-460 microM, whereas neither inhibited FIH. Oxaloacetate was an additional inhibitor of all three HIF-P4Hs with K(i) values of 400-1000 microM and citrate of HIF-P4H-3, citrate being the most effective inhibitor of FIH with a K(i) of 110 microM. Culturing of cells with fumarate diethyl or dimethyl ester, or a high concentration of monoethyl ester, stabilized HIF-1alpha and increased production of vascular endothelial growth factor and erythropoietin. Similar, although much smaller, changes were found in cultured fibroblasts from a patient with fumarate hydratase (FH) deficiency and upon silencing FH using small interfering RNA. No such effects were seen upon culturing of cells with succinate diethyl or dimethyl ester. As FIH was not inhibited by fumarate, our data indicate that the transcriptional activity of HIF is quite high even when binding of the coactivator p300 is prevented. Our data also support recent suggestions that the increased fumarate and succinate levels present in the FH and succinate dehydrogenase-deficient tumors, respectively, can inhibit the HIF-P4Hs with consequent stabilization of HIF-alphas and effects on tumor pathology.
authors.affiliation: Collagen Research Unit, University of Oulu, FIN-90014 Oulu, Finland; Biocenter Oulu, Departments of University of Oulu, FIN-90014 Oulu, Finland; Medical Biochemistry and Molecular Biology and, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Peppi
authors.initials: P
authors.lastname: Koivunen
authors.affiliation: Collagen Research Unit, University of Oulu, FIN-90014 Oulu, Finland; Biocenter Oulu, Departments of University of Oulu, FIN-90014 Oulu, Finland; Medical Biochemistry and Molecular Biology and, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Maija
authors.initials: M
authors.lastname: Hirsilä
authors.affiliation: Neurology, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Remes
authors.affiliation: Medical Biochemistry and Molecular Biology and, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Ilmo E
authors.initials: IE
authors.lastname: Hassinen
authors.affiliation: Collagen Research Unit, University of Oulu, FIN-90014 Oulu, Finland; Biocenter Oulu, Departments of University of Oulu, FIN-90014 Oulu, Finland; Medical Biochemistry and Molecular Biology and, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Kari I
authors.initials: KI
authors.lastname: Kivirikko
authors.affiliation: Collagen Research Unit, University of Oulu, FIN-90014 Oulu, Finland; Biocenter Oulu, Departments of University of Oulu, FIN-90014 Oulu, Finland; Medical Biochemistry and Molecular Biology and, University of Oulu, FIN-90014 Oulu, Finland. Electronic address: johanna.myllyharju@oulu.fi.
authors.forename: Johanna
authors.initials: J
authors.lastname: Myllyharju
mesh_terms: D002460:Cell Line; D002952:Citric Acid Cycle / Q000187:drug effects / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism; D004921:Erythropoietin / Q000096:biosynthesis; D005347:Fibroblasts / Q000737:chemistry / Q000201:enzymology* / Q000473:pathology; D005649:Fumarate Hydratase / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009369:Neoplasms / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis; D050880:p300-CBP Transcription Factors / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D042461:Vascular Endothelial Growth Factor A; D004921:Erythropoietin; D006899:Mixed Function Oxygenases; C058291:aspartic acid 2-oxoglutarate-dependent dioxygenase; D011393:Procollagen-Proline Dioxygenase; D013385:Succinate Dehydrogenase; D050880:p300-CBP Transcription Factors; D005649:Fumarate Hydratase