Inhibition of Hypoxia-inducible Factor (HIF) Hydroxylases by Citric Acid Cycle Intermediates: POSSIBLE LINKS BETWEEN CELL METABOLISM AND STABILIZATION OF HIF

• Published in: The Journal of biological chemistry Vol. 282; no. 7; pp. 4524 - 4532
• Main Authors: Koivunen, Peppi, Hirsilä, Maija, Remes, Anne M, Hassinen, Ilmo E, Kivirikko, Kari I, Myllyharju, Johanna
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 16.02.2007
• Subjects: Cell Line; Citric Acid Cycle - drug effects; Citric Acid Cycle - genetics; Dose-Response Relationship, Drug; Enzyme Inhibitors - metabolism; Erythropoietin - biosynthesis; Fibroblasts - chemistry; Fibroblasts - enzymology; Fibroblasts - pathology; Fumarate Hydratase - chemistry; Fumarate Hydratase - deficiency; Fumarate Hydratase - metabolism; Gene Expression Regulation, Enzymologic - drug effects; Gene Expression Regulation, Enzymologic - genetics; Hypoxia-Inducible Factor 1, alpha Subunit - chemistry; Hypoxia-Inducible Factor 1, alpha Subunit - genetics; Hypoxia-Inducible Factor 1, alpha Subunit - metabolism; Mixed Function Oxygenases - antagonists & inhibitors; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Neoplasms - chemistry; Neoplasms - enzymology; Neoplasms - genetics; Neoplasms - pathology; p300-CBP Transcription Factors - metabolism; Procollagen-Proline Dioxygenase - antagonists & inhibitors; Procollagen-Proline Dioxygenase - chemistry; Procollagen-Proline Dioxygenase - genetics; Procollagen-Proline Dioxygenase - metabolism; Succinate Dehydrogenase - chemistry; Succinate Dehydrogenase - deficiency; Succinate Dehydrogenase - metabolism; Transcription, Genetic - drug effects; Vascular Endothelial Growth Factor A - biosynthesis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M610415200

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 Ki values of 50-80 μM and succinate 350-460 μM, whereas neither inhibited FIH. Oxaloacetate was an additional inhibitor of all three HIF-P4Hs with Ki values of 400-1000 μM and citrate of HIF-P4H-3, citrate being the most effective inhibitor of FIH with a Ki of 110 μM. Culturing of cells with fumarate diethyl or dimethyl ester, or a high concentration of monoethyl ester, stabilized HIF-1α 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-αs and effects on tumor pathology.