Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that activates the cellular response to hypoxia. The HIF1␣ subunit is constantly synthesized and degraded under normoxia, but degradation is rapidly inhibited when oxygen levels drop. Oxygen-dependent hydroxylation by prolyl-4-hydroxylases (PHD) mediates HIF1␣ proteasome degradation. Brain ischemia limits the availability not only of oxygen but also of glucose. We hypothesized that this circumstance could have a modulating effect on HIF. We assessed the separate involvement of oxygen and glucose in HIF1␣ regulation in differentiated neuroblastoma cells subjected to ischemia. We report higher transcriptional activity and HIF1␣ expression under oxygen deprivation in the presence of glucose (OD), than in its absence (oxygen and glucose deprivation, OGD). Unexpectedly, HIF1␣ was not degraded at reoxygenation after an episode of OGD. This was not due to impairment of proteasome function, but was associated with lower HIF1␣ hydroxylation. Krebs cycle metabolites fumarate and succinate are known inhibitors of PHD, while ␣-ketoglutarate is a co-substrate of the reaction. Lack of HIF1␣ degradation in the presence of oxygen was accompanied by a very low ␣-ketoglutarate/fumarate ratio. Furthermore, treatment with a fumarate analogue prevented HIF1␣ degradation under normoxia. In all, our data suggest that postischemic metabolic alterations in Krebs cycle metabolites impair HIF1␣ degradation in the presence of oxygen by decreasing its hydroxylation, and highlight the involvement of metabolic pathways in HIF1␣ regulation besides the well known effects of oxygen.The hypoxia-inducible transcription factor (HIF) 5 is expressed at very low levels in cells under normal oxygen tension, but is rapidly induced upon exposure to hypoxia (1), triggering the activation of a genetic program that enables the metabolic adaptation of cells (2). HIF is a heterodimeric factor composed of a hypoxia-regulated ␣-subunit (HIF1␣ or HIF2␣) and constitutively expressed HIF1␤ (also known as aryl hydrocarbon receptor nuclear translocator, ARNT) (2). Although the ␣-subunit is constantly transcribed and translated, it is also degraded in an oxygen-dependent mechanism. It is only with dwindling oxygen levels that HIF1␣ or HF2␣ expression is readily detected (3). In the presence of oxygen, HIF prolyl-hydroxylases (PHD) hydroxylate two proline residues (positions 402 and 564 in human HIF1␣), in a reaction that requires molecular oxygen and ␣-ketoglutarate as co-substrates (4). These hydroxyproline residues are recognized by the Von Hippel-Lindau tumor suppressor protein (pVHL), one of the components of a E3 ubiquitin-ligase complex that also contains elongins B and C, cullin2, and Rbx, which conjugates ubiquitin to HIF␣ (4, 5). This results in the oxygen-dependent targeting of HIF␣ to the proteasome. Decreased oxygen concentration results in impaired prolyl-hydroxylation, reduced targeting of HIF␣ to the proteasome and the accumulation of HIF in the nucleus, where it activates a pleth...

By driving the primary transcriptional response, the h ypoxia i nducible f actor (HIF) is a master player of the hypoxia-signaling cascade, activation of which is essential to maintain oxygen homeostasis. HIF is formed by the interaction of a constitutive HIF-1 β subunit with a HIF-α subunit tightly regulated through the concerted action of the p rolyl h ydroxylase d omain containing proteins (PHDs) and factor inhibiting HIF. In welloxygenated cells, HIF-α prolyl-hydroxylation by PHDs is the recognition signal for the binding of the ubiquitin E3 ligase pVHL, allowing protein poly-ubiquitination and degradation by the proteasome. Factor inhibiting HIFmediated asparaginyl hydroxylation prevents interaction with the CBP/p300 coactivator and hence reduces HIF-dependent transcriptional activity. Upon low oxygen availability, HIF-α hydroxylation is blocked, resulting in protein stabilization and HIF complex activation. Post-translational modifications other than hydroxylation appear to be important in the cellular response to hypoxia. S mall ubiquitin-like mo difier (SUMO) is a 10 kDa protein readily conjugated to the lysine (K) residues of numerous cellular substrates in a sequential process termed SUMOylation. Recent data support the idea that a fine balance in SUMOylation/deSUMOylation is required for the adequate activation of the hypoxiasignaling cascade. In the present review, we will concentrate on the mechanisms of SUMOylation and its consequences in the cellular response to hypoxia.

By controlling HIFa hydroxylation and stability, the prolyl hydroxylase domain (PHD)-containing proteins are essential to the maintenance of oxygen homeostasis; therefore these enzymes are tightly regulated. Small ubiquitin-like modifier (SUMO) is a 10-kDa protein readily conjugated to lysine residues of the targeted proteins in a process termed SUMOylation. In this study, we introduce SUMO conjugation as a novel regulator of PHD3 (also known as EGLN3). PHD3 SUMOylation occurs at a cluster of four lysines at the C-terminal end of the protein. Furthermore, PHD3 SUMOylation by SUMO2 or SUMO3 contributes to PHD3-mediated repression of HIF1-dependent transcriptional activity. Interestingly, PHD3-SUMO conjugation does not affect PHD3 hydroxylase activity or HIF1a stability, providing new evidence for a dual role of PHD3 in HIF1 regulation. Moreover, we show that hypoxia modulates PHD3-SUMO conjugation and that this modification inversely correlates with HIF1 activation. PHD3 SUMOylation highlights a new and additional layer of regulation that is likely required to fine-tune HIF function.

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