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The recent bestowal of the Nobel Prize 2019 in Physiology or Medicine to Gregg L. Semenza, Sir Peter J. Ratcliffe, and William G. Kaelin Jr. celebrates a series of remarkable discoveries that span from the physiological research question on how oxygen deficiency (hypoxia) induces the red blood cell forming hormone erythropoietin (Epo) to the first clinical application of a novel family of Epo-inducing drugs to treat patients suffering from renal anemia. This review looks back at the most important findings made by the three Nobel laureates, highlights current research trends, and sheds an eye on future perspectives of hypoxia research, including emerging and potential clinical applications.

Fibroblast-like renal erythropoietin (Epo) producing (REP) cells of the corticomedullary border region "sense" a decrease in blood oxygen content following anaemia or hypoxaemia. Burst-like transcription of Epo during tissue hypoxia is transient and is lost during fibrotic tissue remodelling, as observed in chronic kidney disease. The reason for this loss of Epo expression is under debate. Therefore, we tested the hypothesis that REP cell migration, loss and/or differentiation may cause Epo inhibition.Using a reporter mouse that allows permanent labelling of active REP cells at any given time point, we analysed the spatiotemporal fate of REP cells following their initial hypoxic recruitment in models of hypoxaemia and renal tissue remodelling.In long-term tracing experiments, tagged REP reporter cells neither died, proliferated, migrated nor transdifferentiated into myofibroblasts. Approximately 60% of tagged cells re-expressed Epo upon a second hypoxic stimulus. In an unilateral model of tissue remodelling, tagged cells proliferated and ceased to produce Epo before a detectable increase in myofibroblast markers. Treatment with a hypoxia-inducible factor (HIF) stabilizing agent (FG-4592/roxadustat) re-induced Epo expression in the previously active REP cells of the damaged kidney to a similar extent as in the contralateral healthy kidney.Rather than cell death or differentiation, these results suggest cell-intrinsic transient inhibition of Epo transcription: following long-term dormancy, REP cells can repeatedly be recruited by tissue hypoxia, and during myofibrotic tissue remodelling, dormant REP cells are efficiently rescued by a pharmaceutic HIF stabilizer, demonstrating persistent REP cell functionality even during phases of Epo suppression.

Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P

Genetic induction of hypoxia signaling by deletion of the von Hippel-Lindau (Vhl) protein in mesenchymal PDGFR-β+ cells leads to abundant HIF-2 dependent erythropoietin (EPO) expression in the cortex and outer medulla of the kidney. This rather unique feature of kidney PDGFR-β+ cells promote questions about their special characteristics and general functional response to hypoxia. To address these issues, we characterized kidney PDGFR-β+ EPO expressing cells based on additional cell markers and their gene expression profile in response to hypoxia signaling induced by targeted deletion of Vhl or exposure to low oxygen and carbon monoxide respectively, and after unilateral ureteral obstruction. CD73+, Gli1+, tenascin C+ and interstitial SMMHC+ cells were identified as zonally distributed subpopulations of PDGFR-β+ cells. EPO expression could be induced by Vhl deletion in all PDGFR-β+ subpopulations. Under hypoxemic conditions, recruited EPO+ cells were mostly part of the CD73+ subpopulation. Besides EPO production, expression of adrenomedullin and regulator of G-protein signaling 4 was upregulated in PDGFR-β+ subpopulations in response to the different hypoxic stimuli. Thus, different kidney interstitial PDGFR-β+ subpopulations exist, capable of producing EPO in response to different stimuli. Activation of hypoxia signaling in these cells also induces factors likely contributing to improved kidney interstitial tissue oxygenation.

Renal erythropoietin (Epo)-producing (REP) cells represent a rare and incompletely understood cell type. REP cells are fibroblast-like cells located in close proximity to blood vessels and tubules of the corticomedullary border region. Epo mRNA in REP cells is produced in a pronounced “on–off” mode, showing transient transcriptional bursts upon exposure to hypoxia. In contrast to “ordinary” fibroblasts, REP cells do not proliferate ex vivo, cease to produce Epo, and lose their identity following immortalization and prolonged in vitro culture, consistent with the loss of Epo production following REP cell proliferation during tissue remodelling in chronic kidney disease. Because Epo protein is usually not detectable in kidney tissue, and Epo mRNA is only transiently induced under hypoxic conditions, transgenic mouse models have been developed to permanently label REP cell precursors, active Epo producers, and inactive descendants. Future single-cell analyses of the renal stromal compartment will identify novel characteristic markers of tagged REP cells, which will provide novel insights into the regulation of Epo expression in this unique cell type.

Inhibition of the prolyl-4-hydroxylase domain (PHD) enzymes, leading to the stabilization of hypoxia-inducible factor (HIF) α as well as to the stimulation of erythropoietin (Epo) synthesis, is the functional mechanism of the new anti-anemia drug roxadustat. Little is known about the effects of roxadustat on the Epo-producing cell pool. To gain further insights into the function of PHD inhibitors, we characterized the abundance of mesenchymal stem cell (MSC)-like cells after roxadustat treatment of mice. The number of Sca-1+ mesenchymal cells following roxadustat treatment increased exclusively in the kidneys. Isolated Sca-1+ cells demonstrated typical features of MSC-like cells, including adherence to tissue culture plates, trilineage differentiation potential, and expression of MSC markers. Kidney-derived Sca-1+ MSC-like cells were cultured for up to 21 days. Within the first few days in culture, cells stabilized HIF-1α and HIF-2α and temporarily increased Epo production upon incubation in hypoxia. In summary, we have identified a Sca-1+ MSC-like cell population that is involved in renal Epo production and might contribute to the strong anti-anemic effect of the PHD inhibitor roxadustat.

Hypoxia in solid tumors is an important predictor of treatment resistance and poor clinical outcome. The significance of hypoxia in the development of resistance to radiotherapy has been recognized for decades and the search for hypoxia-targeting, radiosensitizing agents continues. This review summarizes the main hypoxia-related processes relevant for radiotherapy on the subcellular, cellular and tissue level and discusses the significance of hypoxia in radiation oncology, especially with regard to the current shift towards hypofractionated treatment regimens. Furthermore, we discuss the strategies to interfere with hypoxia for radiotherapy optimization, and we highlight novel insights into the molecular pathways involved in hypoxia that might be utilized to increase the efficacy of radiotherapy.

Erythropoietin (Epo) is the critical hormone for erythropoiesis. In adults, Epo is mainly produced by a subset of interstitial fibroblasts in the kidney, with minor amounts being produced in the liver and the brain. In this study, we used the immortalized renal interstitial fibroblast cell line FAIK F3-5 to investigate the ability of the bioactive sphingolipid sphingosine 1-phosphate (S1P) to stimulate Epo production and to reveal the mechanism involved. Stimulation of cells with exogenous S1P under normoxic conditions (21% O2) led to a dose-dependent increase in Epo mRNA and protein levels and subsequent release of Epo into the medium. S1P also enhanced the stabilization of HIF-2α, a key transcription factor for Epo expression. S1P-stimulated Epo mRNA and protein expression was abolished by HIF-2α mRNA knockdown or by the HIF-2 inhibitor compound 2. Furthermore, the approved S1P receptor modulator FTY720, and its active form FTY720-phosphate, both exerted a similar effect on Epo expression as S1P. The effect of S1P on Epo was antagonized by the selective S1P1 and S1P3 antagonists NIBR-0213 and TY-52156, but not by the S1P2 antagonist JTE-013. Moreover, inhibitors of the classical MAPK/ERK, the p38-MAPK, and inhibitors of protein kinase (PK) C and D all blocked the effect of S1P on Epo expression. Finally, the S1P and FTY720 effects were recapitulated in the Epo-producing human neuroblastoma cell line Kelly, suggesting that S1P receptor-dependent Epo synthesis is of general relevance and not species-specific. In summary, these data suggest that, in renal interstitial fibroblasts, which are the primary source of plasma Epo, S1P1 and 3 receptor activation upregulates Epo under normoxic conditions. This may have a therapeutic impact on disease situations such as chronic kidney disease, where Epo production is impaired, causing anemia, but it may also have therapeutic value as Epo can mediate additional tissue-protective effects in various organs.

Dysregulated energy metabolism is a major contributor to a multitude of pathologies, including obesity and diabetes. Understanding the regulation of metabolic homeostasis is of utmost importance for the identification of therapeutic targets for the treatment of metabolically driven diseases. We previously identified the deubiquitinase OTUB1 as substrate for the cellular oxygen sensor factor-inhibiting HIF (FIH) with regulatory effects on cellular energy metabolism, but the physiological relevance of OTUB1 is unclear. Here, we report that the induced global deletion of OTUB1 in adult mice (Otub1 iKO) elevated energy expenditure, reduced age-dependent body weight gain, facilitated blood glucose clearance and lowered basal plasma insulin levels. The respiratory exchange ratio was maintained, indicating an unaltered nutrient oxidation. In addition, Otub1 deletion in cells enhanced AKT activity, leading to a larger cell size, higher ATP levels and reduced AMPK phosphorylation. AKT is an integral part of insulin-mediated signaling and Otub1 iKO mice presented with increased AKT phosphorylation following acute insulin administration combined with insulin hypersensitivity. We conclude that OTUB1 is an important regulator of metabolic homeostasis.

Protein:protein interactions are the basis of molecular communication and are usually of transient non-covalent nature, while covalent interactions other than ubiquitination are rare. For cellular adaptations, the cellular oxygen and peroxide sensor factor inhibiting HIF (FIH) confers oxygen and oxidant stress sensitivity to the hypoxia inducible factor (HIF) by asparagine hydroxylation. We investigated whether FIH contributes to hypoxia adaptation also through other mechanisms and identified a hypoxia sensitive, likely covalent, bond formation by FIH with several client proteins, including the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1). Biochemical analyses were consistent with a co-translational amide bond formation between FIH and OTUB1, occurring within mammalian and bacterial cells but not between separately purified proteins. Bond formation is catalysed by FIH and highly dependent on oxygen availability in the cellular microenvironment. Within cells, a heterotrimeric complex is formed, consisting of two FIH and one covalently linked OTUB1. Complexation of OTUB1 by FIH regulates OTUB1 deubiquitinase activity. Our findings reveal an alternative mechanism for hypoxia adaptation with remarkably high oxygen sensitivity, mediated through covalent protein-protein interactions catalysed by an asparagine modifying dioxygenase., Graphical abstract Image 1, Highlights • FIH forms a (likely amide) bond with client proteins. • Bond formation is highly hypoxia sensitive and occurs co-translationally. • FIH forms a heterotrimer with the client protein OTUB1 (FIH2OTUB11). • Complex formation between OTUB1 and FIH regulates OTUB1 deubiquitinase activity. • Bond formation by hydroxylases is an alternative mechanism for hypoxia adaptation.

Portal vein ligation (PVL) induces liver growth prior to resection. Associating liver partition and portal vein ligation (PVL plus transection=ALPPS) or the addition of the prolyl-hydroxylase inhibitor dimethyloxalylglycine (DMOG) to PVL both accelerate growth via stabilization of HIF-α subunits. This study aims at clarifying the crosstalk of hepatocytes (HC), hepatic stellate cells (HSC) and liver sinusoidal endothelial cells (LSEC) in accelerated liver growth. In vivo, liver volume, HC proliferation, vascular density and HSC activation were assessed in PVL, ALPPS, PVL+DMOG and DMOG alone. Proliferation of HC, HSC and LSEC was determined under DMOG in vitro. Conditioned media experiments of DMOG-exposed cells were performed. ALPPS and PVL+DMOG accelerated liver growth and HC proliferation in comparison to PVL. DMOG alone did not induce HC proliferation, but led to increased vascular density, which was also observed in ALPPS and PVL+DMOG. Activated HSC were detected in ALPPS, PVL+DMOG and DMOG, again not in PVL. In vitro, DMOG had no proliferative effect on HC, but conditioned supernatant of DMOG-treated HSC induced VEGF-dependent proliferation of LSEC. Transcriptome analysis confirmed activation of proangiogenic factors in hypoxic HSC. Hypoxia signaling in HSC induces VEGF-dependent angiogenesis. HSC play a crucial role in the cellular crosstalk of rapid liver regeneration.

Pharmacologic HIF hydroxylase inhibitors (HIs) are effective for the treatment of anemia in chronic kidney disease patients and may also be beneficial for the treatment of diseases such as chronic inflammation and ischemia-reperfusion injury. The selectivities of many HIs for HIF hydroxylases and possible off-target effects in cellulo are unclear, delaying the translation from preclinical studies to clinical trials. We developed a novel assay that discriminates between the inhibition of HIF-α prolyl-4-hydroxylase domain (PHD) enzymes and HIF-α asparagine hydroxylase factor inhibiting HIF (FIH). We characterized 15 clinical and preclinical HIs, categorizing them into pan-HIF-α hydroxylase (broad spectrum), PHD-selective, and FIH-selective inhibitors, and investigated their effects on HIF-dependent transcriptional regulation, erythropoietin production, and cellular energy metabolism. While energy homeostasis was generally maintained following HI treatment, the pan-HIs led to a stronger increase in pericellular pO2 than the PHD/FIH-selective HIs. Combined knockdown of FIH and PHD-selective inhibition did not further increase pericellular pO2 . Hence, the additional increase in pericellular pO2 by pan- over PHD-selective HIs likely reflects HIF hydroxylase independent off-target effects. Overall, these analyses demonstrate that HIs can lead to oxygen redistribution within the cellular microenvironment, which should be considered as a possible contributor to HI effects in the treatment of hypoxia-associated diseases.

Aurelia Lelli,1,2,* Karen A Nolan,1,2,* Sara Santambrogio,1,2 Ana Filipa Gonçalves,1,2 Miriam J Schönenberger,1,2 Anna Guinot,1,2 Ian J Frew,1,2 Hugo H Marti,3 David Hoogewijs,1,2,4 Roland H Wenger1,2 1Institute of Physiology and Zurich Center for Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland; 2National Center of Competence in Research "Kidney.CH", Zurich, Switzerland; 3Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany; 4Institute of Physiology, University of Duisburg-Essen, Essen, Germany *These authors contributed equally to this work Abstract: Long thought to be “junk DNA”, in recent years it has become clear that a substantial fraction of intergenic genomic DNA is actually transcribed, forming long noncoding RNA (lncRNA). Like mRNA, lncRNA can also be spliced, capped, and polyadenylated, affecting a multitude of biological processes. While the molecular mechanisms underlying the function of lncRNAs have just begun to be elucidated, the conditional regulation of lncRNAs remains largely unexplored. In genome-wide studies our group and others recently found hypoxic transcriptional induction of a subset of lncRNAs, whereof nuclear-enriched abundant/autosomal transcript 1 (NEAT1) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) appear to be the lncRNAs most ubiquitously and most strongly induced by hypoxia in cultured cells. Hypoxia-inducible factor (HIF)-2 rather than HIF-1 seems to be the preferred transcriptional activator of these lncRNAs. For the first time, we also found strong induction primarily of MALAT1 in organs of mice exposed to inspiratory hypoxia. Most abundant hypoxic levels of MALAT1 lncRNA were found in kidney and testis. In situ hybridization revealed that the hypoxic induction in the kidney was confined to proximal rather than distal tubular epithelial cells. Direct oxygen-dependent regulation of MALAT1 lncRNA was confirmed using isolated primary kidney epithelial cells. In summary, high expression levels and acute, profound hypoxic induction of MALAT1 suggest a hitherto unrecognized role of this lncRNA in renal proximal tubular function. Keywords: hypoxia-inducible factor, kidney, oxygen, proximal tubule, testis

Introduction Inflammatory bowel disease (IBD) is a chronic intestinal disorder, often leading to an impaired quality of life in affected patients. The importance of environmental factors in the pathogenesis of IBD, including their disease-modifying potential, is increasingly recognised. Hypoxia seems to be an important driver of inflammation, as has been reported by our group and others. The aim of the study is to evaluate if hypoxia can alter disease activity of IBD measured by Harvey-Bradshaw Activity Index in Crohn9s disease (increase to ≥5 points) and the partial Mayo Score for ulcerative colitis (increase to ≥2 points). To test the effects of hypoxia under standardised conditions, we designed a prospective and controlled investigation in healthy controls and patients with IBD in stable remission. Methods and analysis This is a prospective, controlled and observational study. Participants undergo a 3-hour exposure to hypoxic conditions simulating an altitude of 4000 metres above sea level (m.a.s.l.) in a hypobaric pressure chamber. Clinical parameters, as well as blood and stool samples and biopsies from the sigmoid colon are collected at subsequent time points. Ethics and dissemination The study protocol was approved by the Ethics Committee of the Kanton Zurich (reference KEK-ZH-number 2013-0284). The results will be published in a peer-reviewed journal and shared with the worldwide medical community. Trials registration number NCT02849821; Pre-results.

Erythropoietin (Epo) is the main humoral regulator of erythropoiesis. During development, Epo production switches in a species-specific manner from the liver to the kidneys which account for approximately 90% of total Epo synthesis in the adult. Renal Epo is produced by peritubular interstitial

Jerry H Fuady,1,* Mattia R Bordoli,1,* Irene Abreu-Rodríguez,1,* Glen Kristiansen,2 David Hoogewijs,1,** Daniel P Stiehl,1,** Roland H Wenger1,**1Institute of Physiology and Zurich Center for Human Physiology, University of Zurich, Zurich, Switzerland; 2University Hospital Bonn, Institute of Pathology, Bonn, Germany*,**These authors contributed equally to this workAbstract: Hypoxia and the hypoxia-inducible factor (HIF) signaling pathway trigger the expression of several genes involved in cancer progression and resistance to therapy. Transcriptionally active HIF-1 and HIF-2 regulate overlapping sets of target genes, and only few HIF-2 specific target genes are known so far. Here we investigated oxygen-regulated expression of Wnt-1 induced signaling protein 2 (WISP-2), which has been reported to attenuate the progression of breast cancer. WISP-2 was hypoxically induced in low-invasive luminal-like breast cancer cell lines at both the messenger RNA and protein levels, mainly in a HIF-2α-dependent manner. HIF-2-driven regulation of the WISP2 promoter in breast cancer cells is almost entirely mediated by two phylogenetically and only partially conserved functional hypoxia response elements located in a microsatellite region upstream of the transcriptional start site. High WISP-2 tumor levels were associated with increased HIF-2α, decreased tumor macrophage density, and a better prognosis. Silencing WISP-2 increased anchorage-independent colony formation and recovery from scratches in confluent cell layers of normally low-invasive MCF-7 cancer cells. Interestingly, these changes in cancer cell aggressiveness could be phenocopied by HIF-2α silencing, suggesting that direct HIF-2-mediated transcriptional induction of WISP-2 gene expression might at least partially explain the association of high HIF-2α tumor levels with prolonged overall survival of patients with breast cancer.Keywords: invasion, metastasis, motility, oxygen, tumor, transcriptional regulation

Presenilin (PSEN) 1 and 2 are the catalytic components of the γ-secretase complex, which cleaves a variety of proteins, including the amyloid precursor protein (APP). Proteolysis of APP leads to the formation of the APP intracellular domain (AICD) and amyloid β that is crucially involved in the pathogenesis of Alzheimer's disease. Prolyl-4-hydroxylase-domain (PHD) proteins regulate the hypoxia-inducible factors (HIFs), the master regulators of the hypoxic response. We previously identified the FK506 binding protein 38 (FKBP38) as a negative regulator of PHD2. Genetic ablation of PSEN1/2 has been shown to increase FKBP38 protein levels. Therefore, we investigated the role of PSEN1/2 in the oxygen sensing pathway using a variety of genetically modified cell and mouse lines. Increased FKBP38 protein levels and decreased PHD2 protein levels were found in PSEN1/2-deficient mouse embryonic fibroblasts and in the cortex of forebrain-specific PSEN1/2 conditional double knock-out mice. Hypoxic HIF-1α protein accumulation and transcriptional activity were decreased, despite reduced PHD2 protein levels. Proteolytic γ-secretase function of PSEN1/2 was needed for proper HIF activation. Intriguingly, PSEN1/2 mutations identified in Alzheimer patients differentially affected the hypoxic response, involving the generation of AICD. Together, our results suggest a direct role for PSEN in the regulation of the oxygen sensing pathway via the APP/AICD cleavage cascade.

Roland H Wenger,1 Dörthe M Katschinski21Institute of Physiology, University of Zurich, Zurich, Switzerland; 2Institute of Cardiovascular Physiology, Georg-August University Göttingen, Göttingen, GermanyWho does not know them? One bearded like a pirate, one knighted by the Queen, and one of noble spirit. United in the fight for truth and honor! No, this is not about Alexandre Dumas’ characters Porthos, Athos, and Aramis, well-known from the famous novel The Three Musketeers.1 This is about the most recent awardees of the Albert Lasker Basic Medical Research Award, the three protagonists of hypoxia research Gregg Semenza, Sir Peter Ratcliffe, and Bill Kaelin. The recent bestowal of the Lasker Prize is just another hallmark in a long list of recognitions of the major achievements by these three outstanding colleagues. What major victories in the battle against the unknowns in hypoxia research did our three heroes achieve?

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 oxygen-dependent asparagine hydroxylase FIH regulates the transcription factor HIF during the cellular response to hypoxia. This study suggests that FIH may also contribute to the hypoxia response by affecting cellular metabolism via altered deubiquitinase targeting., Author Summary Hypoxia is a commonly encountered physiologic and pathophysiologic stress to which mammalian cells have evolved an effective adaptive response. This response is governed by a transcription factor termed the hypoxia-inducible factor (HIF). The mechanisms linking the cellular sensing of oxygen levels to HIF activation have been elucidated and involve oxygen-dependent hydroxylation of HIF on proline and asparagine residues by a family of hydroxylases. A key question that remains unclear is the extent to which oxygen-dependent hydroxylation occurs as a functional post-translational modification outside of the HIF pathway. This is key to developing our understanding of whether hydroxylation is a general regulatory modification or one which has specifically evolved for the regulation of HIF. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a target for functional hydroxylation by the FIH hydroxylase. Hydroxylation of OTUB1 by FIH on asparagine residue N22 results in a restriction in its interactome, leading us to hypothesize a possible role for hydroxylation in substrate targeting. Of interest, interactions of OTUB1 with a number of proteins involved in metabolism are altered upon removal of the hydroxylation site—implicating OTUB1 as a possible link between oxygen sensing and the regulation of metabolism.

Cellular oxygen partial pressure is sensed by a family of prolyl-4-hydroxylase domain (PHD) enzymes that modify hypoxia-inducible factor (HIF)α subunits. Upon hydroxylation under normoxic conditions, HIFα is bound by the von Hippel-Lindau tumor suppressor protein and targeted for proteasomal destruction. Since PHD activity is dependent on oxygen and ferrous iron, HIF-1 mediates not only oxygen- but also iron-regulated transcriptional gene expression. Here we show that copper (CuCl2) stabilizes nuclear HIF-1α under normoxic conditions, resulting in hypoxia-response element (HRE)-dependent reporter gene expression. In in vitro hydroxylation assays CuCl2 inhibited prolyl-4-hydroxylation independently of the iron concentration. Ceruloplasmin, the main copper transport protein in the plasma and a known HIF-1 target in vitro, was also induced in vivo in the liver of hypoxic mice. Both hypoxia and CuCl2 increased ceruloplasmin (as well as vascular endothelial growth factor [VEGF] and glucose transporter 1 [Glut-1]) mRNA levels in hepatoma cells, which was due to transcriptional induction of the ceruloplasmin gene (CP) promoter. In conclusion, our data suggest that PHD/HIF/HRE-dependent gene regulation can serve as a sensory system not only for oxygen and iron but also for copper metabolism, regulating the oxygen-, iron- and copper-binding transport proteins hemoglobin, transferrin, and ceruloplasmin, respectively. (Blood. 2005;105:4613-4619)

Hypoxia has a profound influence on progression and metastasis of malignant tumors. In the present report, we used the oligonucleotide microarray technique to identify new hypoxia-inducible genes in malignant melanoma with a special emphasis on angiogenesis factors. A commercially available Affymetrix gene chip system was used to analyze five melanoma cell lines of different aggressiveness. A total of 160 hypoxia-inducible genes were identified, clustering in four different functional clusters. In search of putative angiogenesis and tumor progression factors within these clusters, Cyr61, a recently discovered angiogenesis factor, was identified. Cyr61 was hypoxia-inducible in low aggressive melanoma cells; however, it showed constitutive high expression in highly aggressive melanoma cells. Further analyses of transcriptional mechanisms underlying Cyr61 gene expression under hypoxia demonstrated that an AP-1 binding motif within the Cyr61 promoter plays a central role in the hypoxic regulation of Cyr61. It could be shown by use of in vitro luciferase assays, electrophoretic mobility shift assays, and immunoprecipitation that hypoxia-inducible factor-1alpha interacts with c-Jun/AP-1 and may thereby contribute to Cyr61 transcriptional regulation under hypoxia. Taken together, the presented data show that Cyr61 is a hypoxia-inducible angiogenesis factor in malignant melanoma with tumor stage-dependent expression. This may argue for a hypoxia-induced selection process during tumor progression toward melanoma cells with constitutive high Cyr61 expression.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homeostasis, including erythropoietin, vascular endothelial growth factor, and factors involved in glucose transport and metabolism. The mouse Hif1a gene is expressed from two distinct promoter/first exon combinations resulting in tissue-specific (mHIF-1alphaI.1) and ubiquitous (mHIF-1alphaI.2) mRNA isoforms. By in situ hybridization, we detected mHIF-1alphaI.1 mRNA exclusively in the elongated spermatids of the testis. In vitro studies indicated that the switch from mHIF-1alphaI.2 to mHIF-1alphaI.1 mRNA expression does not occur at the premeiotic stages of mouse spermatogenesis. Exposure of mice to hypoxic conditions induced mHIF-1alphaI.2 protein in spermatocytes and probably in Sertoli cells but not in spermatogonia. In contrast, expression of the putative mHIF-1alphaI.1 protein in spermatozoa of the testis and epididymis was oxygen independent and located to the midpiece of the spermatozoal flagellum. Both the switch in transcript expression during spermiogenesis and the unexpected protein localization in mature sperm cells suggest a so far unrecognized function of HIF-1alpha.

Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated alpha subunit and a constitutively expressed beta subunit. Although HIF-1 is regulated mainly by oxygen tension through the oxygen-dependent degradation of its alpha subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF-1 activation in vivo, we determined the spatial and temporal distribution of HIF-1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohistochemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF-1alpha is present in mice kept under normoxic conditions and is further increased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF-1alpha expression varies among different organs. In liver and kidney, HIF-1alpha reaches maximal levels after 1 h and gradually decreases to baseline levels after 4 h of continuous hypoxia. In the brain, however, HIF-1alpha is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF-1beta is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF-1alpha. Overall, HIF-1alpha expression in normoxic mice suggests that HIF-1 has an important role in tissue homeostasis.

Background Hypoxia-inducible factors (HIFs) are well-established mediators of tumor growth, the epithelial to mesenchymal transition (EMT) and metastasis. In several types of solid tumors, including breast cancers, the HIFs play a critical role in maintaining cancer stem cell (CSC) activity. Thus, we hypothesized that HIFs may also regulate transcription of markers of breast CSC activity. One approach to enrich for breast cells with stem-like phenotypes is FACS sorting, in which sub-populations of live cells are gated based on the expression of cell surface antigens, including various integrin subunits. Integrin alpha 6 (ITGA6; CD49f) is routinely used in combination with other integrin subunits to enrich for breast stem cells by FACS. Integrins not only mediate interactions with the extracellular matrix (ECM), but also drive intracellular signaling events that communicate from the tumor microenvironment to inside of the tumor cell to alter phenotypes including migration and invasion. Methods We used two models of metastatic breast cancer (MBC), polyoma middle T (MMTV-PyMT) and MDA-MB-231 cells, to compare the expression of ITGA6 in wild type and knockout (KO) or knockdown cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays verified that ITGA6 is a direct HIF transcriptional target. We also used FACS sorting to enrich for CD49f + cells to compare tumorsphere formation, tumor initiating cell activity, invasion and HIF activity relative to CD49fneg or low cells. Knockdown of ITGA6 significantly reduced invasion, whereas re-expression of ITGA6 in the context of HIF knockdown partially rescued invasion. A search of public databases also revealed that ITGA6 expression is an independent prognostic factor of survival in breast cancer patients. Results We report that ITGA6 is a HIF-dependent target gene and that high ITGA6 expression enhances invasion and tumor-initiating cell activities in models of MBC. Moreover, cells that express high levels of ITGA6 are enriched for HIF-1α expression and the expression of HIF-dependent target genes. Conclusions Our data suggest that HIF-dependent regulation of ITGA6 is one mechanism by which sorting for CD49f + cells enhances CSC and metastatic phenotypes in breast cancers. Our results are particularly relevant to basal-like breast cancers which express higher levels of the HIFα subunits, core HIF-dependent target genes and ITGA6 relative to other molecular subtypes. Electronic supplementary material The online version of this article (doi:10.1186/s12943-016-0510-x) contains supplementary material, which is available to authorized users.

The erythroid-specific isoform of 5-aminolevulinate synthase (ALAS2) catalyzes the rate-limiting step in heme biosynthesis. The hypoxia-inducible factor–1 (HIF-1) transcriptionally up-regulates erythropoietin, transferrin, and transferrin receptor, leading to increased erythropoiesis and hematopoietic iron supply. To test the hypothesis that ALAS2 expression might be regulated by a similar mechanism, we exposed murine erythroleukemia cells to hypoxia (1% O2) and found an up to 3-fold up-regulation of ALAS2 mRNA levels and an increase in cellular heme content. A fragment of the ALAS2 promoter ranging from −716 to +1 conveyed hypoxia responsiveness to a heterologous luciferase reporter gene construct in transiently transfected HeLa cells. In contrast, iron depletion, known to induce HIF-1 activity but inhibit ALAS2 translation, did not increase ALAS2 promoter activity. Mutation of a previously predicted HIF-1–binding site (−323/−318) within this promoter fragment and DNA-binding assays revealed that hypoxic up-regulation is independent of this putative HIF-1 DNA-binding site.

Solid tumors often have an inadequate blood supply, which results in large regions that are subjected to hypoxic or anoxic stress. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates much of the transcriptional response of cells to hypoxia. Activating transcription factor 3 (ATF3) is another transcription factor that responds to a variety of stresses and is often upregulated in cancer. We investigated the regulation of ATF3 by oxygen deprivation. ATF3 induction occurred most robustly under anoxia, is common, and it is not dependent on presence of HIF-1 or p53, but is sensitive to the inhibition of c-Jun NH2-terminal kinase activation and the antioxidant N-acetylcystein. ATF3 could also be induced by desferrioxamine but not by the mitochondrial poison cyanide or the nonspecific 2-oxoglutarate dioxygenase inhibitor dimethyloxalylglycine. We also show that anoxic ATF3 mRNA is more stable than normoxic mRNA providing a mechanism for this induction. Thus, this study demonstrates that the regulation of ATF3 under anoxia is independent of 2-oxoglutarate dioxygenase, HIF-1 and p53, presumably involving multiple regulatory pathways.

Roland H Wenger,1,2 Vartan Kurtcuoglu,1,2 Carsten C Scholz,1,2 Hugo H Marti,3 David Hoogewijs1,2,4 1Institute of Physiology and Zurich Center for Human Physiology (ZIHP), University of Zurich, 2National Center of Competence in Research “Kidney.CH”, Zurich, Switzerland; 3Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, 4Institute of Physiology, University of Duisburg-Essen, Essen, Germany Abstract: “What is the O2 concentration in a normoxic cell culture incubator?” This and other frequently asked questions in hypoxia research will be answered in this review. Our intention is to give a simple introduction to the physics of gases that would be helpful for newcomers to the field of hypoxia research. We will provide background knowledge about questions often asked, but without straightforward answers. What is O2 concentration, and what is O2 partial pressure? What is normoxia, and what is hypoxia? How much O2 is experienced by a cell residing in a culture dish in vitro vs in a tissue in vivo? By the way, the O2 concentration in a normoxic incubator is 18.6%, rather than 20.9% or 20%, as commonly stated in research publications. And this is strictly only valid for incubators at sea level. Keywords: gas laws, hypoxia-inducible factor, Krogh tissue cylinder, oxygen diffusion, partial pressure, tissue oxygen levels