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.
Highlights
The hypoxia-inducible transcription factors (HIFs)2 play a central role in the regulation of oxygen homeostasis
The Ki values determined here for fumarate and succinate under initial velocity conditions and using four inhibitor concentrations are in reasonable agreement with the IC50 of about 0.5 mM reported for succinate in assays of nonspecified total hypoxia-inducible factors (HIFs)-P4H activity in crude cell extracts in vitro [18] but the Ki for fumarate is about 20-fold and succinate 50-fold when compared with the apparent Ki values reported for these two compounds using purified HIF-P4H-2 and an in vitro von Hippel-Lindau (VHL) capture assay performed under nonsaturating substrate concentrations and in the presence of only one inhibitor concentration [19]
Membrane-permeable diethyl and dimethyl ester derivatives of fumarate produced stabilization of HIF-1␣ in all the cell types studied, and a very low extent of HIF-1␣ stabilization was demonstrated in cultured fibroblasts from a patient with autosomal recessive fumarate hydratase (FH) deficiency and in cells transfected with FH siRNA
Summary
The hypoxia-inducible transcription factors (HIFs) play a central role in the regulation of oxygen homeostasis. HIF-1␣ and HIF-2␣ are synthesized constitutively, and hydroxylation of at least one of two critical proline residues mediates their interaction with the von Hippel-Lindau (VHL) E3 ubiquitin-ligase complex, which targets them for rapid proteasomal degradation under normoxic conditions (4 – 6) This hydroxylation is catalyzed in humans by three cytoplasmic and nuclear HIF prolyl 4-hydroxylases (HIFP4Hs, known as PHDs 1–3, HPHs 3-1, and EGLNs 2, 1, and 3, respectively) [7,8,9], which are distinct from the well characterized endoplasmic reticulum lumenal collagen P4Hs (C-P4Hs) [10, 11]. Oxaloacetate and pyruvate have likewise been reported to stabilize HIF-1␣ in cultured cancer cell lines and inactivate HIF-P4Hs in a manner reversible by ascorbate [20, 21]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
