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All metazoans that utilize molecular oxygen (O2) for metabolic purposes have the capacity to adapt to hypoxia, the condition that arises when O2 demand exceeds supply. This is mediated through activation of the hypoxia-inducible factor (HIF) pathway. At physiological oxygen levels (normoxia), HIF-prolyl hydroxylases (PHDs) hydroxylate proline residues on HIF-α subunits leading to their destabilization by promoting ubiquitination by the von-Hippel Lindau (VHL) ubiquitin ligase and subsequent proteasomal degradation. HIF-α transactivation is also repressed in an O2-dependent way due to asparaginyl hydroxylation by the factor-inhibiting HIF (FIH). In hypoxia, the O2-dependent hydroxylation of HIF-α subunits by PHDs and FIH is reduced, resulting in HIF-α accumulation, dimerization with HIF-β and migration into the nucleus to induce an adaptive transcriptional response. Although HIFs are the canonical substrates for PHD- and FIH-mediated protein hydroxylation, increasing evidence indicates that these hydroxylases may also have alternative targets. In addition to PHD-conferred alterations in protein stability, there is now evidence that hydroxylation can affect protein activity and protein/protein interactions for alternative substrates. PHDs can be pharmacologically inhibited by a new class of drugs termed prolyl hydroxylase inhibitors which have recently been approved for the treatment of anemia associated with chronic kidney disease. The identification of alternative targets of HIF hydroxylases is important in order to fully elucidate the pharmacology of hydroxylase inhibitors (PHI). Despite significant technical advances, screening, detection and verification of alternative functional targets for PHDs and FIH remain challenging. In this review, we discuss recently proposed non-HIF targets for PHDs and FIH and provide an overview of the techniques used to identify these.

Hypoxia is a prominent feature of chronically inflamed tissues. Oxygen-sensing hydroxylases control transcriptional adaptation to hypoxia through the regulation of hypoxia-inducible factor (HIF) and nuclear factor κB (NF-κB), both of which can regulate the inflammatory response. Furthermore, pharmacologic hydroxylase inhibitors reduce inflammation in multiple animal models. However, the underlying mechanism(s) linking hydroxylase activity to inflammatory signaling remains unclear. IL-1β, a major proinflammatory cytokine that regulates NF-κB, is associated with multiple inflammatory pathologies. We demonstrate that a combination of prolyl hydroxylase 1 and factor inhibiting HIF hydroxylase isoforms regulates IL-1β–induced NF-κB at the level of (or downstream of) the tumor necrosis factor receptor-associated factor 6 complex. Multiple proteins of the distal IL-1β–signaling pathway are subject to hydroxylation and form complexes with either prolyl hydroxylase 1 or factor inhibiting HIF. Thus, we hypothesize that hydroxylases regulate IL-1β signaling and subsequent inflammatory gene expression. Furthermore, hydroxylase inhibition represents a unique approach to the inhibition of IL-1β–dependent inflammatory signaling.

Hypoxia is a feature of the microenvironment of a growing tumor. The transcription factor NFκB is activated in hypoxia, an event that has significant implications for tumor progression. Here, we demonstrate that hypoxia activates NFκB through a pathway involving activation of IκB kinase-β (IKKβ) leading to phosphorylation-dependent degradation of IκBα and liberation of NFκB. Furthermore, through increasing the pool and/or activation potential of IKKβ, hypoxia amplifies cellular sensitivity to stimulation with TNFα. Within its activation loop, IKKβ contains an evolutionarily conserved LxxLAP consensus motif for hydroxylation by prolyl hydroxylases (PHDs). Mimicking hypoxia by treatment of cells with siRNA against PHD-1 or PHD-2 or the pan-prolyl hydroxylase inhibitor DMOG results in NFκB activation. Conversely, overexpression of PHD-1 decreases cytokine-stimulated NFκB reporter activity, further suggesting a repressive role for PHD-1 in controlling the activity of NFκB. Hypoxia increases both the expression and activity of IKKβ, and site-directed mutagenesis of the proline residue (P191A) of the putative IKKβ hydroxylation site results in a loss of hypoxic inducibility. Thus, we hypothesize that hypoxia releases repression of NFκB activity through decreased PHD-dependent hydroxylation of IKKβ, an event that may contribute to tumor development and progression through amplification of tumorigenic signaling pathways. IKK

The asparagine hydroxylase, factor inhibiting HIF (FIH), confers oxygen-dependence upon the hypoxia-inducible factor (HIF), a master regulator of the cellular adaptive response to hypoxia. Studies investigating whether asparagine hydroxylation is a general regulatory oxygen-dependent modification have identified multiple non-HIF targets for FIH. However, the functional consequences of this outside of the HIF pathway remain unclear. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a substrate for hydroxylation by FIH on N22. Mutation of N22 leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. Furthermore, in cultured cells, overexpression of N22A mutant OTUB1 impairs cellular metabolic processes when compared to wild type. Based on these data, we hypothesize that OTUB1 is a target for functional hydroxylation by FIH. Additionally, we propose that our results provide new insight into the regulation of cellular energy metabolism during hypoxic stress and the potential for targeting hydroxylases for therapeutic benefit.

The emergence of generative artificial intelligence (GenAI) chatbots, such as ChatGPT, presents unique challenges and opportunities in an educational setting; however, they lack empirical evidence as teaching and learning tools. This study sought to investigate the impact of teacher-led AI-focused interventions in higher education institutions in different subject areas. Our aims were to support student engagement, explore the impact of AI tools for learning engagement and efficiency and skill development, and promote awareness of the strengths and limitations of GenAI tools in an educational context. This study was carried out with three distinct cohorts; Physiology, Initial Teacher Education, and Engineering, with year 3 and 4 undergraduate students. Each cohort received two 50 min teacher-led AI-focused interventions, including practical exercises relevant to the specific discipline. Following the interventions, students from all three cohorts received a common (optional) survey that quantitatively and qualitatively evaluated their experiences. Data from the three cohorts was pooled for analysis, with individual cohort analyses for Physiology, Initial Teacher Education, and Engineering provided. Our data indicates that teacher-led introductions to AI tools have positive effects on student engagement with peers, educators, and most notably the subject the students engage in. Students also reported very positive supportive effects with respect to learning engagement, learning efficiency, and critical thinking skills. Students found GenAI tools most useful for gathering knowledge and research purposes, while notable limitations included challenges associated with generating prompts and the accuracy of information. Students noted plagiarism as a significant ethical concern. Taken together, our data collected from diverse teaching and learning contexts support the use of teacher-led AI-focused interventions, specifically ChatGPT, in third-level education. Approaches like this are highly relevant to the university teaching of Physiology, Initial Teacher Education, and Engineering but are also more broadly applicable to third-level education in general to inform opportunities, limitations, and ethical considerations of GenAI in education.

The hypoxia-inducible factor (HIF) is a key regulator of the cellular response to hypoxia which promotes oxygen delivery and metabolic adaptation to oxygen deprivation. However, the degree and duration of HIF-1α expression in hypoxia must be carefully balanced within cells in order to avoid unwanted side effects associated with excessive activity. The expression of HIF-1α mRNA is suppressed in prolonged hypoxia, suggesting that the control of HIF1A gene transcription is tightly regulated by negative feedback mechanisms. Little is known about the resolution of the HIF-1α protein response and the suppression of HIF-1α mRNA in prolonged hypoxia. Here, we demonstrate that the Repressor Element 1-Silencing Transcription factor (REST) binds to the HIF-1α promoter in a hypoxia-dependent manner. Knockdown of REST using RNAi increases the expression of HIF-1α mRNA, protein and transcriptional activity. Furthermore REST knockdown increases glucose consumption and lactate production in a HIF-1α- (but not HIF-2α-) dependent manner. Finally, REST promotes the resolution of HIF-1α protein expression in prolonged hypoxia. In conclusion, we hypothesize that REST represses transcription of HIF-1α in prolonged hypoxia, thus contributing to the resolution of the HIF-1α response.

The nuclear receptor sub-family 4 group A (NR4A) family are early response genes that encode proteins that are activated in several tissues/cells in response to a variety of stressors. The NR4A family comprises NR4A1 , NR4A2 and NR4A3 of which NR4A2 and NR4A3 are under researched and less understood, particularly in the context of immune cells. NR4A expression is associated with multiple diseases e.g. arthritis and atherosclerosis and the development of NR4A-targetting molecules as therapeutics is a current focus in this research field. Here, we use a combination of RNA-sequencing coupled with strategic bioinformatic analysis to investigate the down-stream effects of NR4A2 and NR4A3 in monocytes and dissect their common and distinct signalling roles. Our data reveals that NR4A2 and NR4A3 depletion has a robust and broad-reaching effect on transcription in both the unstimulated state and in the presence of LPS. Interestingly, many of the genes affected were present in both the unstimulated and stimulated states revealing a previously unappreciated role for the NR4As in unstimulated cells. Strategic clustering and bioinformatic analysis identified both distinct and common transcriptional roles for NR4A2 and NR4A3 in monocytes. NR4A2 notably was linked by both bioinformatic clustering analysis and transcription factor interactome analysis to pathways associated with antigen presentation and regulation of MHC genes. NR4A3 in contrast was more closely linked to pathways associated with viral response. Functional studies further support our data analysis pointing towards preferential/selective roles for NR4A2 in the regulation of antigen processing with common roles for NR4A2 and NR4A3 evident with respect to cell migration. Taken together this study provides novel mechanistic insights into the role of the enigmatic nuclear receptors NR4A2 and NR4A3 in monocytes.

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxia-dependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.

Uncontrolled inflammation underpins a diverse range of diseases where effective therapy remains an unmet clinical need. Hypoxia is a prominent feature of the inflammatory microenvironment that regulates key transcription factors including HIF and NF-κB in both innate and adaptive immune cells. In turn, altered activity of the pathways controlled by these factors can affect the course of inflammation through the regulation of immune cell development and function. In this review, we will discuss these pathways and the oxygen sensors that confer hypoxic sensitivity in immune cells. Furthermore, we will describe how hypoxia-dependent pathways contribute to immunity and discuss their potential as therapeutic targets in inflammatory and infectious disease.

Mitochondrial biogenesis occurs in response to increased cellular ATP demand. The mitochondrial electron transport chain requires molecular oxygen to produce ATP. Thus, increased ATP generation after mitochondrial biogenesis results in increased oxygen demand that must be matched by a corresponding increase in oxygen supply. We found that overexpression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), which increases mitochondrial biogenesis in primary skeletal muscle cells, leads to increased expression of a cohort of genes known to be regulated by the dimeric hypoxia-inducible factor (HIF), a master regulator of the adaptive response to hypoxia. PGC-1α-dependent induction of HIF target genes under physiologic oxygen concentrations is not through transcriptional coactivation of HIF or up-regulation of HIF-1α mRNA but through HIF-1α protein stabilization. It occurs because of intracellular hypoxia as a result of increased oxygen consumption after mitochondrial biogenesis. Thus, we propose that at physiologic oxygen concentrations, PGC-1α is coupled to HIF signaling through the regulation of intracellular oxygen availability, allowing cells and tissues to match increased oxygen demand after mitochondrial biogenesis with increased oxygen supply.