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

Abstract

Continuous hydroxylation of the HIF-1 transcription factor ␣ 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-1␣ hydroxylation and thus activates HIF-1␣-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-1␣ 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-1␣ 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 (1). In the presence of oxygen, the HIF-1␣ subunit undergoes rapid decay via a ubiquitin-proteasome degradation pathway involving the von HippelLindau tumor suppressor gene product pVHL (2-4). The binding of pVHL to HIF-1␣ requires the post-translational hydroxylation of proline residues (Pro 402 and Pro 564 ) within the HIF-1␣ oxygen-dependent degradation (ODD) 4 domain (5, 6). This modification is prevented during hypoxia, thus allowing HIF-1␣ to escape proteolysis, dimerize with HIF-1␤, and translocate to the nucleus. A separately controlled, O 2 -dependent hydroxylation of asparagine 803 in the HIF-1␣ C-terminal transactivation domain inhibits HIF-1 interaction with the p300/CBP coactivator, thereby blocking HIF-1 transcriptional activity in the presence of oxygen (7,8). Three HIF-1␣ prolyl hydroxylases (HPH1 to -3; also referred to as PHD3 to -1, respectively) and one O 2 -dependent HIF-1␣ asparaginyl hydroxylase (factor inhibiting HIF, or FIH) have been clearly identified so far (9 -11). These enzymes are all members of the 2-oxoglutarate-dependent family of dioxygenases and have an absolute requirement for oxygen, ferrous iron, and 2-oxoglutarate (2-OG). This explains how hypoxia, iron chelators such as desferrioxamine (DFO), and artificial 2-OG analogs such as N-oxalylglycine or its cellpermeable precursor dimethyloxalylglycine (DMOG) can all prevent HIF-1␣ proteolysis and activate HIF-mediated gene expression. Ascorbate is also required for the sustained activity of many 2-OG-dependent dioxygenases (12, 1...

Reactivating HIF prolyl hydroxylases under hypoxia results in metabolic catastrophe and cell death

“…Indeed, in the in vitro hydroxylation assay, increasing a-ketoglutarate alone resulted in increased hydroxylation activity in the extract. Interestingly, there are data indicating that increasing levels of ascorbate can reactivate PHDs in hypoxia (Lu et al, 2005;Gao et al, 2007). The data from our in vitro hydroxylation assay suggest that under these conditions the concentration of ascorbate has a limited role in hypoxia-mediated PHD inhibition.…”

“…There are data to suggest that levels of ascorbate and Fe(II) are also important for PHD activity. It has been shown that increasing ascorbate levels under hypoxia can increase PHD activity and therefore lead to HIF1a hydroxylation and degradation (Knowles et al, 2003;Lu et al, 2005;Gao et al, 2007). This effect appears not to be ascorbate specific, as another antioxidant, N-acetylcysteine (NAC) can also decrease hypoxic HIF1a levels in a PHD2-dependent manner (Gao et al, 2007).…”

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).…”

“…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).…”

Mitochondrial metabolites extend lifespan

“…These new findings are in line with earlier Western analyses using human cancer cell lines that report stabilization of endogenous HIF‐1α protein upon treatment with these same compounds (Lu et al ., 2005). Our studies extend these findings by revealing that HIF‐1α is not only stabilized, but indeed the holo HIF‐1 transcriptional complex is functionally activated.…”