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Hypoxic regulation of gene expression is dominated by the HIF system and can be mimicked by DMOG

ABSTRACT: The response of cells to hypoxia is characterised by co-ordinated regulation of many genes. Studies of the regulation of the expression of many of these genes by oxygen has implicated a role for the heterodimeric transcription factor hypoxia inducible factor (HIF). The mechanism of oxygen sensing which controls this heterodimeric factor is via oxygen dependent prolyl and asparaginyl hydroxylation by specific 2-oxoglutarate dependent dioxygenases (PHD1, PHD2, PHD3 and FIH-1). Whilst HIF appears to have a major role in hypoxic regulation of gene expression, it is unclear to what extent other transcriptional mechanisms are also involved in the response to hypoxia. The extent to which 2-oxoglutarate dependent dioxygenases are responsible for the oxygen sensing mechanism in HIF-independent hypoxic gene regulation is also unclear. Both the prolyl and asparaginyl hydroxylases can be inhibited by dimethyloxalylglycine (DMOG). Such inhibition can produce activation of the HIF system with enhanced transcription of target genes and might have a role in the therapy of ischaemic disease. We have examined the extent to which the HIF system contributes to the regulation of gene expression by hypoxia, to what extent 2-oxoglutarate dependent dioxygenase inhibitor can mimic the hypoxic response and the nature of the global transcriptional response to hypoxia. We have utilised microarray assays of mRNA abundance to examine the gene expression changes in response to hypoxia and to DMOG. We demonstrate a large number of hypoxically regulated genes, both known and novel, and find a surprisingly high level of mimicry of the hypoxic response by use of the 2-oxoglutarate dependent dioxygenase inhibitor, dimethyloxalylglycine. We have also used microarray analysis of cells treated with small interfering RNA (siRNA) targeting HIF-1alpha and HIF-2alpha to demonstrate the differing contributions of each transcription factor to the transcriptional response to hypoxia. Candidate transcripts were confirmed using an independent microarray platform and real-time PCR. The results emphasise the critical role of the HIF system in the hypoxic response, whilst indicating the dominance of HIF-1alpha and defining genes that only respond to HIF-2alpha. Keywords: Hypoxia response, gene knockdown, chemical treatment

ORGANISM(S): Homo sapiens

PROVIDER: GSE3188 | GEO | 2005/12/01

SECONDARY ACCESSION(S): PRJNA92841

REPOSITORIES: GEO

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Project description:This a model from the article: Hypoxia-dependent sequestration of an oxygen sensor by a widespread structural motif can shape the hypoxic response - a predictive kinetic model Bernhard Schmierer, Béla Novák1 and Christopher J Schofield BMC Systems Biology2010, 4:139 20955552, Abstract: Background The activity of the heterodimeric transcription factor hypoxia inducible factor (HIF) is regulated by the post-translational, oxygen-dependent hydroxylation of its α-subunit by members of the prolyl hydroxylase domain (PHD or EGLN)-family and by factor inhibiting HIF (FIH). PHD-dependent hydroxylation targets HIFα for rapid proteasomal degradation; FIH-catalysed asparaginyl-hydroxylation of the C-terminal transactivation domain (CAD) of HIFα suppresses the CAD-dependent subset of the extensive transcriptional responses induced by HIF. FIH can also hydroxylate ankyrin-repeat domain (ARD) proteins, a large group of proteins which are functionally unrelated but share common structural features. Competition by ARD proteins for FIH is hypothesised to affect FIH activity towards HIFα; however the extent of this competition and its effect on the HIF-dependent hypoxic response are unknown. Results To analyse if and in which way the FIH/ARD protein interaction affects HIF-activity, we created a rate equation model. Our model predicts that an oxygen-regulated sequestration of FIH by ARD proteins significantly shapes the input/output characteristics of the HIF system. The FIH/ARD protein interaction is predicted to create an oxygen threshold for HIFα CAD-hydroxylation and to significantly sharpen the signal/response curves, which not only focuses HIFα CAD-hydroxylation into a defined range of oxygen tensions, but also makes the response ultrasensitive to varying oxygen tensions. Our model further suggests that the hydroxylation status of the ARD protein pool can encode the strength and the duration of a hypoxic episode, which may allow cells to memorise these features for a certain time period after reoxygenation. Conclusions The FIH/ARD protein interaction has the potential to contribute to oxygen-range finding, can sensitise the response to changes in oxygen levels, and can provide a memory of the strength and the duration of a hypoxic episode. These emergent properties are predicted to significantly shape the characteristics of HIF activity in animal cells. We argue that the FIH/ARD interaction should be taken into account in studies of the effect of pharmacological inhibition of the HIF-hydroxylases and propose that the interaction of a signalling sensor with a large group of proteins might be a general mechanism for the regulation of signalling pathways. There are there models described in the paper. 1) Skeleton Model 1 (SKM1) - HIFα CAD-hydroxylation in the absence of the FIH/AR-interaction. 2) Skeleton Model 2 (SKM2) - FIG sequestration by ARD proteins and oxygen-dependent FIH-release. 3) Full Model (Fusion of SKM1 and SKM2) - the effects of the FIH/ARD proteins interaction on HIFα CAD-hydroxylation. This model corresponds to the "Full Model" described in the paper. The model reproduces figure 5 of the publication. This model originates from BioModels Database: A Database of Annotated Published Models (http://www.ebi.ac.uk/biomodels/). It is copyright (c) 2005-2011 The BioModels.net Team. For more information see the terms of use. To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.

Project description:Retinopathy of prematurity (ROP) is the most common cause of childhood blindness worldwide and is caused by oxygen therapy necessary to prevent mortality after premature birth. We have previously demonstrated the efficacy of systemic hypoxia inducible factor (HIF) stabilization through HIF prolyl hydroxylase inhibition (HIF PHi) in protecting retinal vasculature from oxygen toxicity in a mouse model of ROP or oxygen induced retinopathy (OIR). We definitively demonstrated that hepatic HIF-1 can be activated to confer this protection using systemic dimethyloxalylglycine (DMOG) to prevent HIF-1α degradation. In this study we compare Roxadustat, a small molecule stabilizer of HIF-1 currently in phase 3 clinical trials for increasing erythropoiesis in adult patients with chronic kidney disease, to DMOG. We demonstrate that Roxadustat induces vascular protection during hyperoxia to induce the coordinated sequential growth of retinal vasculature with a 3-fold reduction in oxygen induced capillary loss (p-=0.001). In order to define the molecular mechanism of protection, we further compared the transcriptome of both liver and retina after systemic treatment with Roxadustat or DMOG. Similar gene expression profiles were identified in liver but very different effects on transcription were found in retinal tissues because Roxadustat, in contrast to DMOG, directly targets retina, confirmed by western blot and by rescue of the hepatic HIF-1 KO, two criteria that DMOG treatment is unable to fulfill. Systems pharmacologic analysis demonstrates that Roxadustat induces typical HIF regulated genes critical to aerobic glycolysis in liver and retinal tissues whereas DMOG, acting through either secreted hepatokines or by influence of systemic DMOG, downregulates cell adhesion/extracellular matrix interaction pathways while increasing expression of histone cluster genes. Stratification of liver transcriptomes to secreted gene products again shows close consensus of hepatic genes induced by both small molecules, and includes upregulation of a plethora of angiogenic proteins such as plasminogen activator inhibitor (PAI-1), erythropoietin (EPO), and orosomucosoid 2 (ORM2). Secondary validation of these transcripts by serum ELISA confirms secretion of EPO and PAI-1 into blood from liver. These findings definitively demonstrate that HIF stabilization can prevent OIR by two pathways: direct retinal HIF stabilization and induction of aerobic glycolysis or indirect, hepatic HIF-1 stabilization and increased serum angiokines. Systems pharmacology analysis therefore explains why intermittent, low dosage of small molecule HIF stabilizers creates a profound protective phenotype, because both pathways can take advantage of cytoprotection induced by the liver and by retina synergistically. These data provide a rationale for considering low dose, intermittent systemic administration of Roxadustat, currently in phase 3 trials in adults with chronic kidney disease, to eradicate ROP in children.

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Hypoxic regulation of gene expression is dominated by the HIF system and can be mimicked by DMOG

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