Tuning the Transcriptional Response to Hypoxia by Inhibiting Hypoxia-inducible Factor (HIF) Prolyl and Asparaginyl Hydroxylases

Abstract

The hypoxia-inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a tissue context-dependent manner. The major hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1–3) and an asparaginyl hydroxylase (factor-inhibiting HIF (FIH)). PHD catalysis regulates HIFα levels, and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of specific HIF target gene transcription is unknown. We report studies using small molecule HIF hydroxylase inhibitors that investigate the extent to which HIF target gene expression is induced by PHD or FIH inhibition. The results reveal substantial differences in the role of prolyl and asparaginyl hydroxylation in regulating hypoxia-responsive genes in cells. PHD inhibitors with different structural scaffolds behave similarly. Under the tested conditions, a broad-spectrum 2-oxoglutarate dioxygenase inhibitor is a better mimic of the overall transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in the transcriptional induction of a subset of genes not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.

HIF hydroxylase inhibitors decrease cellular oxygen consumption depending on their selectivity

“…Using our novel cell-based assay that measures the FIH activity-dependent stable FIH-OTUB1 interaction, we were able to qualitatively reproduce the reported HIF prolyl vs aspraginyl hydroxylase selectivities of hypoxia, DMOG, DIP, FG-4592, GSK1278863, BAY85-3934, AKB-6548, FG-2216, and IOX2. 7,25,26,52 Of note, the compounds FG-4592, GSK1278863, BAY85-3934, and AKB-6548 are the clinically most advanced HIs for the treatment of renal anemia. 18,68 Interestingly, in this assay the extracellular iron chelator DFX functions as a pan-HI, in contrast to its reported PHD-selectivity under some conditions 26,52 ; note more recent work indicates DFX and related compounds are unlikely to be selective for the PHDs.…”

“…5 FIH-dependent hydroxylation hinders the binding of the transcriptional coactivators/histone acetyltransferases p300/CBP, so attenuating HIF's transactivation activity 6 and affects the extents to which different HIF target genes are upregulated. 7,8 In addition to HIF-α, FIH also hydroxylates multiple other substrates, such as the ankyrin-repeat domain-containing proteins IκBα, tankyrase-1, TRPV3, RIPK4, and others. [9][10][11][12] FIH also hydroxylates the deubiquitinase (DUB) ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 (OTUB1), 13,14 with which it forms a covalently linked protein complex in an oxygen-dependent manner by affecting the OTUB1 DUB activity.…”

Adventures in Defining Roles of Oxygenases in the Regulation of Protein Biosynthesis

“…It is important to note, however, that the various HIF ‘isoforms’ regulate different, sometimes overlapping gene sets, with the sets that are upregulated depending on the context (e. g. cell/tissue‐type, developmental stage, environmental chemistry). Thus, obtaining ‘fully rational’ control over the sets of the many direct (>500) and indirect (1000s) of HIF affected genes is likely to be challenging . As we, and others, have found, epigenetic factors, including histone tail modifications and epigenetic enzymes likely also play important roles in regulating HIF target gene expression .…”

“…Importantly, HIF (and the PHDs) were found not only to regulate expression of the EPO gene, but also of hundreds of other genes in humans (and other animals) many of which work to ameliorate the effects of hypoxia. The precise sets of target genes expressed vary in a cell type/environment context‐dependent manner ,,. The assignment of the roles of the PHDs in the hypoxic response via transcriptional regulation was important, including because (i) it defined a new mechanism of enzyme‐mediated transcriptional regulation that was directly linked to a response of major physiological importance; (ii) it opened up new therapeutic possibilities for the treatment of hypoxia‐related diseases, including cancer and anaemia;, and (iii) it led to the proposal that the kinetic/biophysical properties of the enzymes involved are directly linked to their sensing roles.…”

Changes in the coelomic microclimate during carbon dioxide laparoscopy: morphological and functional implications

“…It then binds with constitutively expressed HIF-β to form HIF-1α/HIF-1β heterodimers [53]. Upregulation of the hypoxia response element by the HIF-1α/HIF-1β heterodimer then results, with rapid promotion of angiogenesis, glycolysis, peritoneal adhesions, cell motility and migration, epithelialmesenchymal transition (EMT) and tumour growth [52,54,55]. This can occur during laparoscopy, particularly if high insufflation pressures are used [56].…”