The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. Hydroxylation of one or two critical prolines by specific hydroxylases (P4Hs) targets their HIF-␣ subunits for proteasomal degradation. By studying the three human HIF-P4Hs, we found that the longest and shortest isoenzymes have major transcripts encoding inactive polypeptides, which suggest novel regulation by alternative splicing. Recombinant HIFP4Hs expressed in insect cells required peptides of more than 8 residues, distinct differences being found between isoenzymes.

The product of the von Hippel-Lindau gene, pVHL, targets the ␣ subunits of the heterodimeric transcription factor hypoxiainducible factor (HIF) for polyubiquitination in the presence of oxygen. The binding of pVHL to HIF is governed by the enzymatic hydroxylation of conserved prolyl residues within peptidic motifs present in the HIF␣ family members. By using a biochemical purification strategy, we have identified a human homolog of Caenorhabditis elegans Egl9 as a HIF prolyl hydroxylase. In addition, we studied the activity of a structurally diverse collection of low molecular weight inhibitors of procollagen prolyl 4-hydroxylase as potential inhibitors of the HIF hydroxylase. A model compound of this series stabilized HIF in a variety of cells, leading to the increased production of its downstream target, vascular endothelial growth factor.

The activity of hypoxia-inducible transcription factor HIF, an ␣␤ heterodimer that has an essential role in adaptation to low oxygen availability, is regulated by two oxygen-dependent hydroxylation events. Hydroxylation of specific proline residues by HIF prolyl 4-hydroxylases targets the HIF-␣ subunit for proteasomal destruction, whereas hydroxylation of an asparagine in the C-terminal transactivation domain prevents its interaction with the transcriptional coactivator p300. The HIF asparaginyl hydroxylase is identical to a previously known factor inhibiting HIF (FIH). We report here that recombinant FIH has unique catalytic and inhibitory properties when compared with those of the HIF prolyl 4-hydroxylases. FIH was found to require particularly long peptide substrates so that omission of only a few residues from the N or C terminus of a 35-residue HIF-1␣ sequence markedly reduced its substrate activity. Hydroxylation of two HIF-2␣ peptides was far less efficient than that of the corresponding HIF-1␣ peptides. The K m of FIH for O 2 was about 40% of its atmospheric concentration, being about one-third of those of the HIF prolyl 4-hydroxylases but 2.5 times that of the type I collagen prolyl 4-hydroxylase. Several 2-oxoglutarate analogs were found to inhibit FIH but with distinctly different potencies from the HIF prolyl 4-hydroxylases. For example, the two most potent HIF prolyl 4-hydroxylase inhibitors among the compounds studied were the least effective ones for FIH. It should therefore be possible to develop specific small molecule inhibitors for the two enzyme classes involved in the hypoxia response.The hypoxia-inducible transcription factor HIF 1 has a major role in the conserved oxygen-sensitive response pathway that is activated in hypoxic cells. HIF-regulated hypoxia-inducible genes are involved in angiogenesis, vascularization, and anaerobic energy production, for instance. HIFs are ␣␤ heterodimers in which the stability of the ␣ subunit is regulated in an oxygen-dependent manner (for reviews, see Refs. 1-4). Hydroxylation of at least one of two proline residues, Pro 402 and Pro 564 , in the oxygen-dependent degradation domain of human HIF-1␣ mediates the interaction of HIF-␣ with the von Hippel Lindau E3 (ubiquitin-protein isopeptide ligase) ubiquitin ligase complex that targets it for rapid proteasomal degradation under normoxic conditions (5-8). This hydroxylation is catalyzed in humans by three novel cytoplasmic and nuclear HIF prolyl 4-hydroxylases (9 -11) that are distinct from the well characterized collagen prolyl 4-hydroxylases, which reside in the lumen of the endoplasmic reticulum (12-16). Under hypoxic conditions, the oxygen-requiring process of hydroxylation is prevented, and HIF-␣ escapes degradation and dimerizes with HIF-␤, the dimer then recognizing a specific element in the promoters of hypoxia-responsive target genes (1-4).Transcriptional activation in an oxygen-dependent manner is another key step that regulates HIF-␣ activity. Hydroxylation of a specific asparagine residue, Asn 80...

Many human diseases are characterized by the development of tissue hypoxia. Inadequate oxygenation can cause cellular dysfunction and death. Tissues use many strategies, including induction of angiogenesis and alterations in metabolism, to survive under hypoxic conditions. The heterodimeric transcription factor hypoxia-inducible factor (HIF) is a master regulator of genes that promote adaptation to hypoxia. HIF activity is linked to oxygen availability because members of the EGLN family hydroxylate HIF␣ subunits on specific prolyl residues when oxygen is present, which marks them for ubiquitination and proteasomal degradation. We created a mouse that ubiquitously expresses a bioluminescent reporter consisting of firefly luciferase fused to a region of HIF that is sufficient for oxygen-dependent degradation. Our validation studies suggest that this mouse will be useful for monitoring hypoxic tissues and evaluating therapeutic agents that stabilize HIF. One such agent, the HIF prolyl hydroxylase inhibitor FG-4383, was active in the liver and kidney after systemic administration as determined by bioluminescence imaging, transcription profiling, and production of erythropoietin, indicating that the HIF transcriptional program can be manipulated in vivo with orally active organic small molecules.bioluminescence ͉ imaging ͉ von Hippel-Lindau

The reasons for inadequate production of erythropoietin (EPO) in patients with ESRD are poorly understood. A better understanding of EPO regulation, namely oxygen-dependent hydroxylation of the hypoxia-inducible transcription factor (HIF), may enable targeted pharmacological intervention. Here, we tested the ability of fibrotic kidneys and extrarenal tissues to produce EPO. In this phase 1 study, we used an orally active prolyl-hydroxylase inhibitor, FG-2216, to stabilize HIF independent of oxygen availability in 12 hemodialysis (HD) patients, six of whom were anephric, and in six healthy volunteers. FG-2216 increased plasma EPO levels 30.8-fold in HD patients with kidneys, 14.5-fold in anephric HD patients, and 12.7-fold in healthy volunteers. These data demonstrate that pharmacologic manipulation of the HIF system can stimulate endogenous EPO production. Furthermore, the data indicate that deranged oxygen sensing-not a loss of EPO production capacity-causes renal anemia.

No abstract

Activation of hypoxia-inducible transcription factor (HIF) has been identified as an important mechanism of cellular adaptation to low oxygen. Normoxic degradation of HIF is mediated by oxygen-dependent hydroxylation of specific prolyl residues of the regulative ␣-subunits by HIF prolyl hydroxylases (PHD). It was hypothesized that inhibition of HIF degradation by either hypoxia or pharmacologic inhibition of PHD would confer protection against subsequent ischemic injury. For testing this hypothesis ischemic acute renal failure was induced in rats by 40 min of clamping of the left renal artery after right-sided nephrectomy. Before surgery, pretreatment with either carbon monoxide, leading to tissue hypoxia, or the novel PHD inhibitor FG-4487 was applied. No toxic effects of FG-4487 were observed. Both pretreatments strongly induced the accumulation of HIF-1␣ and HIF-2␣ in tubular and peritubular cells, respectively, as well as HIF target gene expression. The course of subsequent ischemic injury was significantly ameliorated by both strategies of preconditioning, as evident from a significant improvement of serum creatinine and serum urea after 24 and 72 h. Furthermore, tissue injury and apoptosis were less severe, which were quantified by application of a standardized histologic scoring system in a blinded manner. In conclusion, the data provide proof of principle that preconditional activation of the HIF system protects against ischemic injury. Inhibiting the activity of HIF hydroxylases therefore seems to have considerable clinical perspectives.

The structure and function of the 2-oxoglutarate binding site of prolyl4-hydroxylase was studied by assaying the inhibitory potential of 24 selected aliphatic or aromatic compounds. All except one of them inhibited the enzyme competitively with respect to 2-oxoglutarate and noncompetitively with respect to Fez+, the Ki values ranging from 0.8 pM to over 15 mM. The Ki values for the two most effective inhibitors, pyridine 2,5-dicarboxylate and 2,4-dicarboxylate, were about 0.8 pM and 2 pM, these compounds being the most potent inhibitors of prolyl 4-hydroxylase with respect to 2-oxoglutarate known so far. Only one of the compounds tested, 2-oxoadipinate, was able to support hydroxylation by replacing 2-oxoglutarate as a cosubstrate. The data suggest that the 2-oxoglutarate binding site can be divided into three distinct subsites. Subsite I is probably a positively charged side chain of the enzyme that ionically binds the C5 carboxyl group of the 2-oxoglutarate, subsite I1 consists of two cis-positioned equatorial coordination sites of the enzyme-bound ferrous ion and is chelated by the C1-C2 moiety, while subsite I11 involves a hydrophobic binding site in the C3 -C4 region of the cosubstrate. The sp3 rehybridization of C2 within the chelating moiety of the cosubstrate appears to be a crucial event during decarboxylation that proceeds in the form of a ligand reaction inside the Fe2+ coordination sphere.Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens and other proteins with collagen-like amino acid sequences by the hydroxylation of certain proline residues in peptide linkages (for reviews, see [l-31). The minimum sequence requirement for hydroxylation is fulfilled by an -Xaa-Pro-Gly-triplet, and the reaction requires ferrous ions, 2-oxoglutarate, molecular oxygen and ascorbate. The 2-oxoglutarate is stoichiometrically decarboxylated, one atom of the O2 molecule being incorporated into the succinate while the other is incorporated into the hydroxyl group [l -31. The results of extensive kinetic studies [4-61 and other data [7, 81 are consistent with an ordered binding of Fe2 +, 2-oxoglutarate, O2 and the peptide substrate to the enzyme in this order, and an ordered release of the hydroxylated peptide, C02, succinate, and Fez+, in which Fe2 + does not leave the enzyme between the majority of the catalytic cycles and in which the order of release of the hydroxylated peptide and CO, is uncertain. Ascorbate is not consumed stoichiometrically [5, 91, and pure enzyme preparations can catalyze hydroxylation for a number of catalytic cycles in the complete absence of this vitamin [6, 81. These findings, together with the kinetic [6,10) and other data [S], suggest that ascorbate is required to prevent oxidation of the enzyme-bound iron and possibly some other groups on the enzyme molecule between some catalytic cycles, but not the majority.A detailed stereochemical mechanism has recently been suggested for prolyl4-hydroxylase [ll], in which it is proposed that binding of 2-oxoglutarate (compound...