Selective Hypoxia-Sensitive Oxomer Formation by FIH Prevents Binding of the NF-κB Inhibitor IκBß to NF-κB Subunits.

Pharmacologic inhibitors of cellular hydroxylase oxygen sensors are protective in multiple preclinical in vivo models of inflammation. However, the molecular mechanisms underlying this regulation are only partly understood, preventing clinical translation. We previously proposed a new mechanism for cellular oxygen sensing: oxygen-dependent, (likely) covalent protein oligomer (oxomer) formation. Here, we report that the oxygen sensor factor inhibiting HIF (FIH) forms an oxomer with the NF-κB inhibitor ß (IκBß). The formation of this protein complex required FIH enzymatic activity and was prevented by pharmacologic inhibitors. Oxomer formation was highly hypoxia-sensitive and very stable. No other member of the IκB protein family formed an oxomer with FIH, demonstrating that FIH-IκBß oxomer formation was highly selective. In contrast to the known FIH-dependent oxomer formation with the deubiquitinase OTUB1, FIH-IκBß oxomer formation did not occur via an IκBß asparagine residue, but depended on the amino acid sequence VAERR contained within a loop between IκBß ankyrin repeat domains 2 and 3. Oxomer formation prevented IκBß from binding to its primary interaction partners p65 and c-Rel, subunits of NF-κB, the master regulator of the cellular transcriptional response to pro-inflammatory stimuli. We therefore propose that FIH-mediated oxomer formation with IκBß contributes to the hypoxia-dependent regulation of inflammation.

PHD1-3 oxygen sensors in vivo-lessons learned from gene deletions.

Oxygen sensors enable cells to adapt to limited oxygen availability (hypoxia), affecting various cellular and tissue responses. Prolyl-4-hydroxylase domain 1-3 (PHD1-3; also called Egln1-3, HIF-P4H 1-3, HIF-PH 1-3) proteins belong to the Fe2+- and 2-oxoglutarate-dependent dioxygenase superfamily and utilise molecular oxygen (O2) alongside 2-oxoglutarate as co-substrate to hydroxylate two proline residues of α subunits of the dimeric hypoxia inducible factor (HIF) transcription factor. PHD1-3-mediated hydroxylation of HIF-α leads to its degradation and inactivation. Recently, various PHD inhibitors (PHI) have entered the clinics for treatment of renal anaemia. Pre-clinical analyses indicate that PHI treatment may also be beneficial in numerous other hypoxia-associated diseases. Nonetheless, the underlying molecular mechanisms of the observed protective effects of PHIs are only partly understood, currently hindering their translation into the clinics. Moreover, the PHI-mediated increase of Epo levels is not beneficial in all hypoxia-associated diseases and PHD-selective inhibition may be advantageous. Here, we summarise the current knowledge about the relevance and function of each of the three PHD isoforms in vivo, based on the deletion or RNA interference-mediated knockdown of each single corresponding gene in rodents. This information is crucial for our understanding of the physiological relevance and function of the PHDs as well as for elucidating their individual impact on hypoxia-associated diseases. Furthermore, this knowledge highlights which diseases may best be targeted by PHD isoform-selective inhibitors in case such pharmacologic substances become available.

Oxomer- and Reporter Gene-Based Analysis of FIH Activity in Cells.

Cellular and tissue adaptations to oxygen deprivation (hypoxia) are necessary for both normal physiology and disease. Responses to hypoxia are initiated by the cellular oxygen sensors prolyl-4-hydroxylase domain (PHD) proteins 1-3 and factor inhibiting HIF (FIH). These enzymes regulate the transcription factor hypoxia-inducible factor (HIF) in a hypoxia-sensitive manner. FIH also regulates proteins outside the HIF pathway, including the deubiquitinase OTUB1. Numerous preclinical analyses have demonstrated that treatment with HIF hydroxylase inhibitors is beneficial and protective in many hypoxia-associated diseases. However, clinically available HIF hydroxylase inhibitors increase erythropoietin (EPO) gene expression and red blood cell production, which can be detrimental in hypoxia-associated conditions, such as ischemia/reperfusion injury of the heart or chronic inflammation. Our understanding of the relevance of FIH in (patho)physiology is only in its infancy, but FIH activity does not govern erythropoietin expression. Therefore, it is of prime interest to assess the relevance of FIH activity in (patho)physiology in detail, as it may contribute to developing novel therapeutic options for treating hypoxia-associated diseases that do not affect Epo regulation. Here, we describe specific protocols for two different methods to assess FIH enzymatic activity within cells, using a HIF-dependent firefly luciferase-reporter gene and an oxomer-dependent assay. Oxomers are oxygen-dependent stable protein oligomers formed by FIH, for example, with the deubiquitinase OTUB1. Oxomer formation directly depends on FIH activity, providing a suitable cellular readout for an easy-to-use analysis of FIH enzymatic activity in cellulo. These techniques permit an analysis of FIH activity toward HIF and outside the HIF pathway, allowing the investigation of FIH activity under different (patho)physiological conditions and assessment of novel (putative) inhibitors.

Evolution of hypoxia and hypoxia-inducible factor asparaginyl hydroxylase regulation in chronic kidney disease.

BACKGROUND: The roles of hypoxia and hypoxia inducible factor (HIF) during chronic kidney disease (CKD) are much debated. Interventional studies with HIF-α activation in rodents have yielded contradictory results. The HIF pathway is regulated by prolyl and asparaginyl hydroxylases. While prolyl hydroxylase inhibition is a well-known method to stabilize HIF-α, little is known about the effect asparaginyl hydroxylase factor inhibiting HIF (FIH). METHODS: We used a model of progressive proteinuric CKD and a model of obstructive nephropathy with unilateral fibrosis. In these models we assessed hypoxia with pimonidazole and vascularization with three-dimensional micro-computed tomography imaging. We analysed a database of 217 CKD biopsies from stage 1 to 5 and we randomly collected 15 CKD biopsies of various severity degrees to assess FIH expression. Finally, we modulated FIH activity in vitro and in vivo using a pharmacologic approach to assess its relevance in CKD. RESULTS: In our model of proteinuric CKD, we show that early CKD stages are not characterized by hypoxia or HIF activation. At late CKD stages, some areas of hypoxia are observed, but these are not colocalizing with fibrosis. In mice and in humans, we observed a downregulation of the HIF pathway, together with an increased FIH expression in CKD, according to its severity. Modulating FIH in vitro affects cellular metabolism, as described previously. In vivo, pharmacologic FIH inhibition increases the glomerular filtration rate of control and CKD animals and is associated with decreased development of fibrosis. CONCLUSIONS: The causative role of hypoxia and HIF activation in CKD progression is questioned. A pharmacological approach of FIH downregulation seems promising in proteinuric kidney disease.

Fount, fate, features, and function of renal erythropoietin-producing cells.

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.

The effect of HIF on metabolism and immunity.

Cellular hypoxia occurs when the demand for sufficient molecular oxygen needed to produce the levels of ATP required to perform physiological functions exceeds the vascular supply, thereby leading to a state of oxygen depletion with the associated risk of bioenergetic crisis. To protect against the threat of hypoxia, eukaryotic cells have evolved the capacity to elicit oxygen-sensitive adaptive transcriptional responses driven primarily (although not exclusively) by the hypoxia-inducible factor (HIF) pathway. In addition to the canonical regulation of HIF by oxygen-dependent hydroxylases, multiple other input signals, including gasotransmitters, non-coding RNAs, histone modifiers and post-translational modifications, modulate the nature of the HIF response in discreet cell types and contexts. Activation of HIF induces various effector pathways that mitigate the effects of hypoxia, including metabolic reprogramming and the production of erythropoietin. Drugs that target the HIF pathway to induce erythropoietin production are now approved for the treatment of chronic kidney disease-related anaemia. However, HIF-dependent changes in cell metabolism also have profound implications for functional responses in innate and adaptive immune cells, and thereby heavily influence immunity and the inflammatory response. Preclinical studies indicate a potential use of HIF therapeutics to treat inflammatory diseases, such as inflammatory bowel disease. Understanding the links between HIF, cellular metabolism and immunity is key to unlocking the full therapeutic potential of drugs that target the HIF pathway.

The Asparagine Hydroxylase FIH: A Unique Oxygen Sensor.

Significance: Limited oxygen availability (hypoxia) commonly occurs in a range of physiological and pathophysiological conditions, including embryonic development, physical exercise, inflammation, and ischemia. It is thus vital for cells and tissues to monitor their local oxygen availability to be able to adjust in case the oxygen supply is decreased. The cellular oxygen sensor factor inhibiting hypoxia-inducible factor (FIH) is the only known asparagine hydroxylase with hypoxia sensitivity. FIH uniquely combines oxygen and peroxide sensitivity, serving as an oxygen and oxidant sensor. Recent Advances: FIH was first discovered in the hypoxia-inducible factor (HIF) pathway as a modulator of HIF transactivation activity. Several other FIH substrates have now been identified outside the HIF pathway. Moreover, FIH enzymatic activity is highly promiscuous and not limited to asparagine hydroxylation. This includes the FIH-mediated catalysis of an oxygen-dependent stable (likely covalent) bond formation between FIH and selected substrate proteins (called oxomers [oxygen-dependent stable protein oligomers]). Critical Issues: The (patho-)physiological function of FIH is only beginning to be understood and appears to be complex. Selective pharmacologic inhibition of FIH over other oxygen sensors is possible, opening new avenues for therapeutic targeting of hypoxia-associated diseases, increasing the interest in its (patho-)physiological relevance. Future Directions: The contribution of FIH enzymatic activity to disease development and progression should be analyzed in more detail, including the assessment of underlying molecular mechanisms and relevant FIH substrate proteins. Also, the molecular mechanism(s) involved in the physiological functions of FIH remain(s) to be determined. Furthermore, the therapeutic potential of recently developed FIH-selective pharmacologic inhibitors will need detailed assessment. Antioxid. Redox Signal. 37, 913-935.

The Deubiquitinase OTUB1 Is a Key Regulator of Energy Metabolism.

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.

Fate-mapping of erythropoietin-producing cells in mouse models of hypoxaemia and renal tissue remodelling reveals repeated recruitment and persistent functionality.

AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

OTUB1 regulates lung development, adult lung tissue homeostasis, and respiratory control.

OTUB1 is one of the most highly expressed deubiquitinases, counter-regulating the two most abundant ubiquitin chain types. OTUB1 expression is linked to the development and progression of lung cancer and idiopathic pulmonary fibrosis in humans. However, the physiological function of OTUB1 is unknown. Here, we show that constitutive whole-body Otub1 deletion in mice leads to perinatal lethality by asphyxiation. Analysis of (single-cell) RNA sequencing and proteome data demonstrated that OTUB1 is expressed in all lung cell types with a particularly high expression during late-stage lung development (E16.5, E18.5). At E18.5, the lungs of animals with Otub1 deletion presented with increased cell proliferation that decreased saccular air space and prevented inhalation. Flow cytometry-based analysis of E18.5 lung tissue revealed that Otub1 deletion increased proliferation of major lung parenchymal and mesenchymal/other non-hematopoietic cell types. Adult mice with conditional whole-body Otub1 deletion (wbOtub1del/del ) also displayed increased lung cell proliferation in addition to hyperventilation and failure to adapt the respiratory pattern to hypoxia. On the molecular level, Otub1 deletion enhanced mTOR signaling in embryonic and adult lung tissues. Based on these results, we propose that OTUB1 is a negative regulator of mTOR signaling with essential functions for lung cell proliferation, lung development, adult lung tissue homeostasis, and respiratory regulation.

Hypoxia in chronic kidney disease: towards a paradigm shift?

Chronic kidney disease (CKD) is defined as an alteration of kidney structure and/or function lasting for >3 months [1]. CKD affects 10% of the general adult population and is responsible for large healthcare costs [2]. Since the end of the last century, the role of hypoxia in CKD progression has controversially been discussed. To date, there is evidence of the presence of hypoxia in late-stage renal disease, but we lack time-course evidence, stage correlation and also spatial co-localization with fibrotic lesions to ensure its causative role. The classical view of hypoxia in CKD progression is that it is caused by peritubular capillary alterations, renal anaemia and increased oxygen consumption regardless of the primary injury. In this classical view, hypoxia is assumed to further induce pro-fibrotic and pro-inflammatory responses, as well as oxidative stress, leading to CKD worsening as part of a vicious circle. However, recent investigations tend to question this paradigm, and both the presence of hypoxia and its role in CKD progression are still not clearly demonstrated. Hypoxia-inducible factor (HIF) is the main transcriptional regulator of the hypoxia response. Genetic HIF modulation leads to variable effects on CKD progression in different murine models. In contrast, pharmacological modulation of the HIF pathway [i.e. by HIF hydroxylase inhibitors (HIs)] appears to be generally protective against fibrosis progression experimentally. We here review the existing literature on the role of hypoxia, the HIF pathway and HIF HIs in CKD progression and summarize the evidence that supports or rejects the hypoxia hypothesis, respectively.

Inhibition of firefly luciferase activity by a HIF prolyl hydroxylase inhibitor.

The three hypoxia-inducible factor (HIF) prolyl-4-hydroxylase domain (PHD) 1-3 enzymes confer oxygen sensitivity to the HIF pathway and are novel therapeutic targets for treatment of renal anemia. Inhibition of the PHDs may further be beneficial in other hypoxia-associated diseases, including ischemia and chronic inflammation. Several pharmacologic PHD inhibitors (PHIs) are available, but our understanding of their selectivity and its chemical basis is limited. We here report that the PHI JNJ-42041935 (JNJ-1935) is structurally similar to the firefly luciferase substrate D-luciferin. Our results demonstrate that JNJ-1935 is a novel inhibitor of firefly luciferase enzymatic activity. In contrast, the PHIs FG-4592 (roxadustat) and FG-2216 (ICA, BIQ, IOX3, YM 311) did not affect firefly luciferase. The JNJ-1935 mode of inhibition is competitive with a Ki of 1.36 µM. D-luciferin did not inhibit the PHDs, despite its structural similarity to JNJ-1935. This study provides insights into a previously unknown JNJ-1935 off-target effect as well as into the chemical requirements for firefly luciferase and PHD inhibitors and may inform the development of novel compounds targeting these enzymes.

HIF hydroxylase inhibitors decrease cellular oxygen consumption depending on their selectivity.

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.

Oxygen-dependent bond formation with FIH regulates the activity of the client protein OTUB1.

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.

Hydroxylase Inhibition Selectively Induces Cell Death in Monocytes.

Hypoxia is a common and prominent feature of the microenvironment at sites of bacteria-associated inflammation in inflammatory bowel disease. The prolyl-hydroxylases (PHD1/2/3) and the asparaginyl-hydroxylase factor-inhibiting HIF are oxygen-sensing enzymes that regulate adaptive responses to hypoxia through controlling the activity of HIF and NF-κB-dependent transcriptional pathways. Previous studies have demonstrated that the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) is effective in the alleviation of inflammation in preclinical models of inflammatory bowel disease, at least in part, through suppression of IL-1ß-induced NF-κB activity. TLR-dependent signaling in immune cells, such as monocytes, which is important in bacteria-driven inflammation, shares a signaling pathway with IL-1ß. In studies into the effect of pharmacologic hydroxylase inhibition on TLR-induced inflammation in monocytes, we found that DMOG selectively triggers cell death in cultured THP-1 cells and primary human monocytes at concentrations well tolerated in other cell types. DMOG-induced apoptosis was independent of increased caspase-3/7 activity but was accompanied by reduced expression of the inhibitor of apoptosis protein 1 (cIAP1). Based on these data, we hypothesize that pharmacologic inhibition of the HIF-hydroxylases selectively targets monocytes for cell death and that this may contribute to the anti-inflammatory activity of HIF-hydroxylase inhibitors.

Generation of renal Epo-producing cell lines by conditional gene tagging reveals rapid HIF-2 driven Epo kinetics, cell autonomous feedback regulation, and a telocyte phenotype.

Erythropoietin (Epo) is essential for erythropoiesis and is mainly produced by the fetal liver and the adult kidney following hypoxic stimulation. Epo regulation is commonly studied in hepatoma cell lines, but differences in Epo regulation between kidney and liver limit the understanding of Epo dysregulation in polycythaemia and anaemia. To overcome this limitation, we have generated a novel transgenic mouse model expressing Cre recombinase specifically in the active fraction of renal Epo-producing (REP) cells. Crossing with reporter mice confirmed the inducible and highly specific tagging of REP cells, located in the corticomedullary border region where there is a steep drop in oxygen bioavailability. A novel method was developed to selectively grow primary REP cells in culture and to generate immortalized clonal cell lines, called fibroblastoid atypical interstitial kidney (FAIK) cells. FAIK cells show very early hypoxia-inducible factor (HIF)-2α induction, which precedes Epo transcription. Epo induction in FAIK cells reverses rapidly despite ongoing hypoxia, suggesting a cell autonomous feedback mechanism. In contrast, HIF stabilizing drugs resulted in chronic Epo induction in FAIK cells. RNA sequencing of three FAIK cell lines derived from independent kidneys revealed a high degree of overlap and suggests that REP cells represent a unique cell type with properties of pericytes, fibroblasts, and neurons, known as telocytes. These novel cell lines may be helpful to investigate myofibroblast differentiation in chronic kidney disease and to elucidate the molecular mechanisms of HIF stabilizing drugs currently in phase III studies to treat anemia in end-stage kidney disease.

Genetic Knockdown and Pharmacologic Inhibition of Hypoxia-Inducible Factor (HIF) Hydroxylases.

Reduced oxygen supply that does not satisfy tissue and cellular demand (hypoxia) regularly occurs both in health and disease. Hence, the capacity for cellular oxygen sensing is of vital importance for each cell to be able to alter its energy metabolism and promote adaptation to hypoxia. The hypoxia-inducible factor (HIF) prolyl hydroxylases 1-3 (PHD1-3) and the asparagine hydroxylase factor-inhibiting HIF (FIH) are the primary cellular oxygen sensors, which confer cellular oxygen-dependent sensitivity upon HIF as well as other hypoxia-sensitive pathways, such as nuclear factor κB (NF-κB). Studying these enzymes allows us to understand the oxygen-dependent regulation of cellular processes and has led to the development of several putative novel therapeutics, which are currently in clinical trials for the treatment of anemia associated with kidney disease. Pharmacologic inhibition and genetic knockdown are commonly established techniques in protein biochemistry and are used to investigate the activity and function of proteins. Here, we describe specific protocols for the knockdown and inhibition of the HIF prolyl hydroxylases 1-3 (PHD1-3) and the asparagine hydroxylase factor-inhibiting HIF (FIH) using RNA interference (RNAi) and hydroxylase inhibitors, respectively. These techniques are essential tools for the analysis of the function of the HIF hydroxylases, allowing the investigation and discovery of novel functions and substrates of these enzymes.

Carbon dioxide-dependent regulation of NF-κB family members RelB and p100 gives molecular insight into CO2-dependent immune regulation.

CO2 is a physiological gas normally produced in the body during aerobic respiration. Hypercapnia (elevated blood pCO2 >≈50 mm Hg) is a feature of several lung pathologies, e.g. chronic obstructive pulmonary disease. Hypercapnia is associated with increased susceptibility to bacterial infections and suppression of inflammatory signaling. The NF-κB pathway has been implicated in these effects; however, the molecular mechanisms underpinning cellular sensitivity of the NF-κB pathway to CO2 are not fully elucidated. Here, we identify several novel CO2-dependent changes in the NF-κB pathway. NF-κB family members p100 and RelB translocate to the nucleus in response to CO2 A cohort of RelB protein-protein interactions (e.g. with Raf-1 and IκBα) are altered by CO2 exposure, although others are maintained (e.g. with p100). RelB is processed by CO2 in a manner dependent on a key C-terminal domain located in its transactivation domain. Loss of the RelB transactivation domain alters NF-κB-dependent transcriptional activity, and loss of p100 alters sensitivity of RelB to CO2 Thus, we provide molecular insight into the CO2 sensitivity of the NF-κB pathway and implicate altered RelB/p100-dependent signaling in the CO2-dependent regulation of inflammatory signaling.

The functional interplay between the HIF pathway and the ubiquitin system - more than a one-way road.

The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue functions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIFα subunits leads to the recruitment of an E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIFα subunits, demonstrating the regulation of the HIF pathway by the ubiquitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the best-studied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway. Understanding these mechanisms and their relevance for the activity of each other is of major importance for the comprehensive elucidation of the oxygen-dependent regulation of cellular processes. This review describes the current knowledge of the functional bidirectional interplay between the HIF pathway and the ubiquitin system on the protein level.

Intermittent hypoxia confers pro-metastatic gene expression selectively through NF-κB in inflammatory breast cancer cells.

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Treatment options are limited and the mechanisms underlying its aggressiveness are poorly understood. Intermittent hypoxia (IH) causes oxidative stress and is emerging as important regulator of tumor metastasis. Vessels in IBC tumors have been shown to be immature, which is a primary cause of IH. We therefore investigated the relevance of IH for the modulation of gene expression in IBC cells in order to assess IH as potential regulator of IBC aggressiveness. Gene array analysis of IBC cells following chronic IH (45-60 days) demonstrated increased expression of pro-metastatic genes of the extracellular matrix, such as tenascin-C (TNC; an essential factor of the metastatic niche) and matrix metalloproteinase 9 (MMP9), and of pro-inflammatory processes, such as cyclooxygenase-2 (COX-2). Investigating the oxidative stress-dependent regulation of TNC, we found a gradual sensitivity on mRNA and protein levels. Oxidative stress activated NF-E2-related factor 2 (Nrf2), c-Jun N-terminal kinase (JNK), c-Jun and nuclear factor κB (NF-κB), but TNC upregulation was only dependent on NF-κB activation. Pharmacological inhibition of inhibitor of NF-κB α (IκBα) phosphorylation as well as overexpression of IκBα prevented TNC, MMP9 and COX-2 induction, whereas the pro-inflammatory cytokine interleukin-1ß (IL-1ß) increased their expression levels. Analysis of the gene array data showed NF-κB binding sites for 64% of all upregulated genes, linking NF-κB with IH-dependent regulation of pro-metastatic gene expression in IBC cells. Our results provide a first link between intermittent hypoxia and pro-metastatic gene expression in IBC cells, revealing a putative novel mechanism for the high metastatic potential of IBC.