Targeting hypoxia signalling for the treatment of ischaemic and inflammatory diseases.
ABSTRACT: Hypoxia-inducible factors (HIFs) are stabilized during adverse inflammatory processes associated with disorders such as inflammatory bowel disease, pathogen infection and acute lung injury, as well as during ischaemia-reperfusion injury. HIF stabilization and hypoxia-induced changes in gene expression have a profound impact on the inflamed tissue microenvironment and on disease outcomes. Although the mechanism that initiates HIF stabilization may vary, the final molecular steps that control HIF stabilization converge on a set of oxygen-sensing prolyl hydroxylases (PHDs) that mark HIFs for proteasomal degradation. PHDs are therefore promising therapeutic targets. In this Review, we discuss the emerging potential and associated challenges of targeting the PHD-HIF pathway for the treatment of inflammatory and ischaemic diseases.
Project description:Hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs) are ?-ketoglutarate (?KG)-dependent dioxygenases that function as cellular oxygen sensors. However, PHD activity also depends on factors other than oxygen, especially ?KG, a key metabolic compound closely linked to amino-acid metabolism. We examined the connection between amino-acid availability and PHD activity. We found that amino-acid starvation leads to ?KG depletion and to PHD inactivation but not to HIF stabilization. Furthermore, pharmacologic or genetic inhibition of PHDs induced autophagy and prevented mammalian target of rapamycin complex 1 (mTORC1) activation by amino acids in a HIF-independent manner. Therefore, PHDs sense not only oxygen but also respond to amino acids, constituting a broad intracellular nutrient-sensing network.
Project description:Hypoxia is a major driving force in vascularization and vascular remodeling. Pharmacological inhibition of prolyl hydroxylases (PHDs) leads to an oxygen-independent and long-lasting activation of hypoxia-inducible factors (HIFs). Whereas effects of HIF-stabilization on transcriptional responses have been thoroughly investigated in endothelial cells, the molecular details of cytoskeletal changes elicited by PHD-inhibition remain largely unknown. To investigate this important aspect of PHD-inhibition, we used a spheroid-on-matrix cell culture model.Microvascular endothelial cells (glEND.2) were organized into spheroids. Migration of cells from the spheroids was quantified and analyzed by immunocytochemistry. The PHD inhibitor dimethyloxalyl glycine (DMOG) induced F-actin stress fiber formation in migrating cells, but only weakly affected microvascular endothelial cells firmly attached in a monolayer. Compared to control spheroids, the residual spheroids were larger upon PHD inhibition and contained more cells with tight VE-cadherin positive cell-cell contacts. Morphological alterations were dependent on stabilization of HIF-1? and not HIF-2? as shown in cells with stable knockdown of HIF-? isoforms. DMOG-treated endothelial cells exhibited a reduction of immunoreactive Rac-1 at the migrating front, concomitant with a diminished Rac-1 activity, whereas total Rac-1 protein remained unchanged. Two chemically distinct Rac-1 inhibitors mimicked the effects of DMOG in terms of F-actin fiber formation and orientation, as well as stabilization of residual spheroids. Furthermore, phosphorylation of p21-activated kinase PAK downstream of Rac-1 was reduced by DMOG in a HIF-1?-dependent manner. Stabilization of cell-cell contacts associated with decreased Rac-1 activity was also confirmed in human umbilical vein endothelial cells.Our data demonstrates that PHD inhibition induces HIF-1?-dependent cytoskeletal remodeling in endothelial cells, which is mediated essentially by a reduction in Rac-1 signaling.
Project description:The response to hypoxia in animals involves the expression of multiple genes regulated by the ??-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIF? isoforms, as catalysed by prolyl hydroxylases (PHD 1-3). Prolyl hydroxylation promotes binding of HIF? to the von Hippel-Lindau protein (VHL)-elongin B/C complex, thus signalling for proteosomal degradation of HIF?. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.
Project description:As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIF? in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.
Project description:Long-term survival of renal allografts depends on the chronic immune response and is probably influenced by the initial injury caused by ischemia and reperfusion. Hypoxia-inducible transcription factors (HIFs) are essential for adaptation to low oxygen. Normoxic inactivation of HIFs is regulated by oxygen-dependent hydroxylation of specific prolyl-residues by prolyl-hydroxylases (PHDs). Pharmacological inhibition of PHDs results in HIF accumulation with subsequent activation of tissue-protective genes. We examined the effect of donor treatment with a specific PHD inhibitor (FG-4497) on graft function in the Fisher-Lewis rat model of allogenic kidney transplantation (KTx). Orthotopic transplantation of the left donor kidney was performed after 24 h of cold storage. The right kidney was removed at the time of KTx (acute model) or at day 10 (chronic model). Donor animals received a single dose of FG-4497 (40 mg/kg i.v.) or vehicle 6 h before donor nephrectomy. Recipients were followed up for 10 days (acute model) or 24 weeks (chronic model). Donor preconditioning with FG-4497 resulted in HIF accumulation and induction of HIF target genes, which persisted beyond cold storage. It reduced acute renal injury (serum creatinine at day 10: 0.66 +/- 0.20 vs. 1.49 +/- 1.36 mg/dL; P < 0.05) and early mortality in the acute model and improved long-term survival of recipient animals in the chronic model (mortality at 24 weeks: 3 of 16 vs. 7 of 13 vehicle-treated animals; P < 0.05). In conclusion, pretreatment of organ donors with FG-4497 improves short- and long-term outcomes after allogenic KTx. Inhibition of PHDs appears to be an attractive strategy for organ preservation that deserves clinical evaluation.
Project description:Background:In humans and other animals, the chronic hypoxic response is mediated by hypoxia inducible transcription factors (HIFs) which regulate the expression of genes that counteract the effects of limiting oxygen. Prolyl hydroxylases (PHDs) act as hypoxia sensors for the HIF system in organisms ranging from humans to the simplest animal Trichoplax adhaerens. Methods:We report structural and biochemical studies on the T. adhaerens HIF prolyl hydroxylase (TaPHD) that inform about the evolution of hypoxia sensing in animals. Results:High resolution crystal structures (?1.3 Å) of TaPHD, with and without its HIF? substrate, reveal remarkable conservation of key active site elements between T. adhaerens and human PHDs, which also manifest in kinetic comparisons. Conclusion:Conserved structural features of TaPHD and human PHDs include those apparently enabling the slow binding/reaction of oxygen with the active site Fe(II), the formation of a stable 2-oxoglutarate complex, and a stereoelectronically promoted change in conformation of the hydroxylated proline-residue. Comparison of substrate selectivity between the human PHDs and TaPHD provides insights into the selectivity determinants of HIF binding by the PHDs, and into the evolution of the multiple HIFs and PHDs present in higher animals.
Project description:Studies of many cell types show that levels of hypoxia inducible factor (HIF)-1? and HIF-2? are primarily controlled by oxygen-dependent proteasomal degradation, catalyzed by HIF prolyl-hydroxylases (PHDs). However, in the hypoxic niche of the intervertebral disc, the mechanism of HIF-? turnover in nucleus pulposus cells is not yet known. We show that in nucleus pulposus cells HIF-1? and HIF-2?, degradation was mediated through 26S proteasome irrespective of oxygen tension. It is noteworthy that HIF-2? degradation through 26S proteasome was more pronounced in hypoxia. Surprisingly, treatment with DMOG, a PHD inhibitor, shows the accumulation of only HIF-1? and induction in activity of its target genes, but not of HIF-2?. Loss and gain of function analyses using lentiviral knockdown of PHDs and overexpression of individual PHDs show that in nucleus pulposus cells only PHD2 played a limited role in HIF-1? degradation; again HIF-2? degradation was unaffected. We also show that the treatment with inhibitors of lysosomal proteolysis results in a strong accumulation of HIF-1? and to a much smaller extent of HIF-2? levels. It is thus evident that in addition to PHD2 catalyzed degradation, the HIF-1? turnover in nucleus pulposus cells is primarily regulated by oxygen-independent pathways. Importantly, our data clearly suggests that proteasomal degradation of HIF-2? is not mediated by a classical oxygen-dependent PHD pathway. These results for the first time provide a rationale for the normoxic stabilization as well as the maintenance of steady-state levels of HIF-1? and HIF-2? in nucleus pulposus cells.
Project description:Hypoxia-inducible transcription factors (HIFs) control adaptation to low oxygen environments by activating genes involved in metabolism, angiogenesis, and redox homeostasis. The finding that HIFs are also regulated by small molecule metabolites highlights the need to understand the complexity of their cellular regulation. Here we use a forward genetic screen in near-haploid human cells to identify genes that stabilize HIFs under aerobic conditions. We identify two mitochondrial genes, oxoglutarate dehydrogenase (OGDH) and lipoic acid synthase (LIAS), which when mutated stabilize HIF1? in a non-hydroxylated form. Disruption of OGDH complex activity in OGDH or LIAS mutants promotes L-2-hydroxyglutarate formation, which inhibits the activity of the HIF? prolyl hydroxylases (PHDs) and TET 2-oxoglutarate dependent dioxygenases. We also find that PHD activity is decreased in patients with homozygous germline mutations in lipoic acid synthesis, leading to HIF1 activation. Thus, mutations affecting OGDHC activity may have broad implications for epigenetic regulation and tumorigenesis.
Project description:Cell adaptation to changes in oxygen (O(2)) availability is controlled by two subfamilies of O(2)-dependent enzymes: the hypoxia inducible factor (HIF)-prolyl and asparaginyl hydroxylases [prolyl hydroxylases domain (PHDs) and factor inhibiting HIF (FIH)]. These oxygen sensors regulate the activity of the HIF, a transcriptional complex central in O(2) homeostasis. In well oxygenated cells, PHDs hydroxylate the HIFalpha subunits, thereby targeting them for proteasomal degradation. In contrast, acute hypoxia inhibits PHDs, leading to HIFalpha stabilisation. However, here we show that chronic hypoxia induces HIF1/2alpha"desensitization" in cellulo and in mice. At the basis of this general adaptative mechanism, we demonstrate that chronic hypoxia not only increases the pool of PHDs but also overactivates the three PHD isoforms. This overactivation appears to be mediated by an increase in intracellular O(2) availability consequent to the inhibition of mitochondrial respiration. By using in cellulo and in vivo siRNA, we found that the PHDs are the key enzymes triggering HIFalpha desensitization, a feedback mechanism required to protect cells against necrotic cell death and thus to adapt them across a chronic hypoxia. Hence, PHDs serve as dual enzymes, for which inactivation and later overactivation is necessary for cell survival in acute or chronic hypoxia, respectively.
Project description:Oxygen-dependent regulation of the transcription factor HIF-1? relies on a family of prolyl hydroxylases (PHDs) that hydroxylate hypoxia-inducible factor 1? (HIF-1?) protein at two prolines during normal oxygen conditions, resulting in degradation by the proteasome. During low-oxygen conditions, these prolines are no longer hydroxylated and HIF-1? degradation is blocked. Hypoxia-induced miRNA-210 (miR-210) is a direct transcriptional target of HIF-1?, but its complete role and targets during hypoxia are not well understood. Here, we identify the enzyme glycerol-3-phosphate dehydrogenase 1-like (GPD1L) as a novel regulator of HIF-1? stability and a direct target of miR-210. Expression of miR-210 results in stabilization of HIF-1? due to decreased levels of GPD1L resulting in an increase in HIF-1? target genes. Altering GPD1L levels by overexpression or knockdown results in a decrease or increase in HIF-1? stability, respectively. GPD1L-mediated decreases in HIF-1? stability can be reversed by pharmacological inhibition of the proteasome or PHD activity. When rescued from degradation by proteasome inhibition, elevated amounts of GPD1L cause hyperhydroxylation of HIF-1?, suggesting increases in PHD activity. Importantly, expression of GPD1L attenuates the hypoxic response, preventing complete HIF-1? induction. We propose a model in which hypoxia-induced miR-210 represses GPD1L, contributing to suppression of PHD activity, and increases of HIF-1? protein levels.