Endogenous 2-oxoacids differentially regulate expression of oxygen sensors

Abstract

Adaptations to change in oxygen availability are crucial for survival of multi-cellular organisms and are also implicated in several disease states. Such adaptations rely upon gene expression regulated by the heterodimeric transcription factors HIFs (hypoxia-inducible factors). Enzymes that link changes in oxygen tensions with the stability and transcriptional activity of HIFs are considered as oxygen sensors. These enzymes are oxygen-, iron- and 2-oxoglutarate-dependent dioxygenases that hydroxylate key proline and asparagine residues in HIFalpha subunits. The constitutive inhibitory action of these enzymes on HIFs is relieved by hypoxia and by agents that displace iron or 2-oxoglutarate. Two of the enzymes, HPH (HIF prolyl hydroxylase)-1 and HPH-2, are known to be inducible by hypoxia in a HIF-dependent manner. This suggests the existence of a novel feedback loop for adjusting hypoxia-regulated gene expression. We have recently shown that HIF-1alpha stability, HIF-1 nuclear translocation and HIF-mediated gene expression in human glioma cell lines can be stimulated by pyruvate independently of hypoxia. In the present study we show that the endogenous 2-oxoacid oxaloacetate can also activate HIF-mediated gene expression. Pyruvate and oxaloacetate treatment of cells also up-regulates HPH-1 and HPH-2, but not HPH-3 or the HIF asparaginyl hydroxylase FIH-1 (factor inhibiting HIF). Regulation of HIF-1 and the expression of HPH homologue genes can thus be influenced by specific glycolytic and tricarboxylic acid cycle metabolites. These findings may underlie important interactions between oxygen homoeostasis, glycolysis, the tricarboxylic acid cycle and gluconeogenesis.

Inhibition of Hypoxia-inducible Factor (HIF) Hydroxylases by Citric Acid Cycle Intermediates

“…In agreement with these data, cultur-ing of cancer cells with 20 mM succinate dimethyl ester for 48 h (18) or with 2.5 mM succinate diethyl ester or 0.5 mM fumarate monoethyl ester together with 3-nitropropionic acid (19), a dual inhibitor of succinate dehydrogenase (SDH) and fumarate hydratase (FH), led to stabilization of HIF-1␣, and a similar effect was seen upon silencing of SDH or FH by the corresponding siRNA (18,19). 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).…”

“…The K i values determined here for fumarate and succinate under initial velocity conditions and using four inhibitor concentrations are in reasonable agreement with the IC 50 of about 0.5 mM reported for succinate in assays of nonspecified total HIF-P4H activity in crude cell extracts in vitro (18) but the K i for fumarate is about 20-fold and succinate 50-fold when compared with the apparent K i values reported for these two compounds using purified HIF-P4H-2 and an in vitro VHL capture assay performed under nonsaturating substrate concentrations and in the presence of only one inhibitor concentration (19). Although oxaloacetate and pyruvate have been reported to stabilize HIF-1␣ in cultured cancer cells and to inhibit the HIF- P4Hs in an in vitro VHL capture assay in the presence of nonsaturating ascorbate levels (20,21), we found only a weak inhibition by oxaloacetate, especially in the case of HIF-P4H-2, and no inhibition by pyruvate in the presence of 2 mM ascorbate and no increase in the level of inhibition in the presence of lower ascorbate concentrations. 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 additionally demonstrated in cultured fibroblasts from a patient with autosomal recessive FH deficiency and in cells transfected with FH siRNA.…”

“…As oxaloacetate and pyruvate have been reported to inhibit the HIF-P4Hs in an in vitro VHL capture assay in the presence of nonsaturating ascorbate levels (30 -100 M) (20,21), we also studied inhibition of the HIF-P4Hs by these two compounds in the presence of varying ascorbate concentrations. However, the IC 50 values obtained under these conditions were identical to those determined in the presence of 2 mM ascorbate (details not shown).…”

Mitochondria: Oxygen sinks rather than sensors?

“…Remarkably, succinate and fumarate are efficient inhibitors of PHD activity and the Krebs cycle enzymes involved in their production are known as tumor suppressors, consistent with a constitutive HIFα activation in cancer cells with inactivation mutations of these tumor suppressors [38][39][40]. 6 • ROS The discovery of the PHD oxygen sensors provided a plausible site of ROS action.…”

HIF Prolyl-4-hydroxylase Interacting Proteins: Consequences for Drug Targeting

“…Indeed, it has been shown that ascorbate [47], transition metals [48,49], and reactive oxygen species (ROS) including NO [50][51][52] influence or completely block the activity of the PHDs, establishing a molecular cross-talk between oxygen homeostasis and redox-active substances. Of major physiological importance, however, is the fact that Krebs cycle intermediates interfere with PHD function [53][54][55]. Germline mutations of the genes encoding fumarate hydratase (FH) or succinate dehydrogenase (SDH) result in the accumulation of fumarate and succinate, respectively, two potent inhibitors of PHD activity [56,57].…”