Search

While it is well-established that distal hypoxia response elements (HREs) regulate hypoxia-inducible factor (HIF) target genes such as erythropoietin (Epo), an interplay between multiple distal and proximal (promoter) HREs has not been described so far. Hepatic Epo expression is regulated by a HRE located downstream of the EPO gene, but this 3' HRE is dispensable for renal EPO gene expression. We previously identified a 5' HRE and could show that both HREs direct exogenous reporter gene expression. Here, we show that whereas in hepatic cells the 3' but not the 5' HRE is required, in neuronal cells both the 5' and 3' HREs contribute to endogenous Epo induction. Moreover, two novel putative HREs were identified in the EPO promoter. In hepatoma cells HIF interacted mainly with the distal 3' HRE, but in neuronal cells HIF most strongly bound the promoter, to a lesser extent the 3' HRE, and not at all the 5' HRE. Interestingly, mutation of either of the two distal HREs abrogated HIF binding to the 3' and promoter HREs. These results suggest that a canonical functional HRE can recruit multiple, not necessarily HIF, transcription factors to mediate HIF binding to different distant HREs in an organ-specific manner.

Hypoxia regulates autophagy and nucleotide-binding oligomerization domain receptor, pyrin domain containing (NLRP)3, two innate immune mechanisms linked by mutual regulation and associated to IBD. Here we show that hypoxia ameliorates inflammation during the development of colitis by modulating autophagy and mammalian target of rapamycin (mTOR)/NLRP3 pathway. Hypoxia significantly reduces tumor necrosis factor α, interleukin (IL)-6 and NLRP3 expression, and increases the turnover of the autophagy protein p62 in colon biopsies of Crohn’s disease patients, and in samples from dextran sulfate sodium-treated mice and Il-10 −/− mice. In vitro, NF-κB signaling and NLRP3 expression are reduced through hypoxia-induced autophagy. We also identify NLRP3 as a novel binding partner of mTOR. Dimethyloxalylglycine-mediated hydroxylase inhibition ameliorates colitis in mice, downregulates NLRP3 and promotes autophagy. We suggest that hypoxia counteracts inflammation through the downregulation of the binding of mTOR and NLRP3 and activation of autophagy., Hypoxia and HIF-1α activation are protective in mouse models of colitis, and the latter regulates autophagy. Here Cosin-Roger et al. show that hypoxia ameliorates intestinal inflammation in Crohn’s patients and murine colitis models by inhibiting mTOR/NLRP3 pathway and promoting autophagy.

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.

Two main features common to all solid tumors are tissue hypoxia and inflammation, both of which cause tumor progression, metastasis, therapy resistance and increased mortality. Chronic inflammation is associated with increased cancer risk, as demonstrated for inflammatory bowel disease patients developing colon cancer. However, the interplay between hypoxia and inflammation on the molecular level remains to be elucidated. We found that MC-38 mouse colon cancer cells contain functional hypoxic (HIF-1α) and inflammatory (p65/RelA) signaling pathways. In contrast to cells of the myeloid lineage, HIF-1α levels remained unaffected in MC-38 cells treated with LPS, and hypoxia failed to induce NF-κB. A similar regulation of canonical HIF and NF-κB target genes confirmed these results. RNA deep sequencing of HIF-1α and p65/RelA knock-down cells revealed that a surprisingly large fraction of HIF target genes required p65/RelA for hypoxic regulation and a number of p65/RelA target genes required HIF-1α for proinflammatory regulation, respectively. Hypoxia attenuated the inflammatory response to LPS by inhibiting nuclear translocation of p65/RelA independently of HIF-1α, which was associated with enhanced IκBα levels and decreased IKKβ phosphorylation. These data demonstrate that the interaction between hypoxic and inflammatory signaling pathways needs to be considered when designing cancer therapies targeting HIF or NF-κB. ISSN:1949-2553

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.

Prolyl-4-hydroxylation is necessary for proper structural assembly of collagens and oxygen-dependent protein stability of hypoxia-inducible transcription factors (HIFs). In vitro function of HIF prolyl-4-hydroxylase domain (PHD) enzymes requires oxygen and 2-oxoglutarate as cosubstrates with iron(II) and vitamin C serving as cofactors. Although vitamin C deficiency is known to cause the collagen-disassembly disease scurvy, it is unclear whether cellular oxygen sensing is similarly affected. Here, we report that vitamin C–deprived Gulo−/− knockout mice show normal HIF-dependent gene expression. The systemic response of Gulo−/− animals to inspiratory hypoxia, as measured by plasma erythropoietin levels, was similar to that of animals supplemented with vitamin C. Hypoxic HIF induction was also essentially normal under serum- and vitamin C–free cell-culture conditions, suggesting that vitamin C is not required for oxygen sensing in vivo. Glutathione was found to fully substitute for vitamin C requirement of all 3 PHD isoforms in vitro. Consistently, glutathione also reduced HIF-1α protein levels, transactivation activity, and endogenous target gene expression in cells exposed to CoCl2. A Cys201Ser mutation in PHD2 increased basal hydroxylation rates and conferred resistance to oxidative damage in vitro, suggesting that this surface-accessible PHD2 cysteine residue is a target of antioxidative protection by vitamin C and glutathione.

PASKIN links energy flux and protein synthesis in yeast, regulates glycogen synthesis in mammals, and has been implicated in glucose-stimulated insulin production in pancreatic beta-cells. Using newly generated monoclonal antibodies, PASKIN was localized in the nuclei of human testis germ cells and in the midpiece of human sperm tails. A speckle-like nuclear pattern was observed for endogenous PASKIN in HeLa cells in addition to its cytoplasmic localization. By yeast two-hybrid screening, we identified the multifunctional eukaryotic translation elongation factor eEF1A1 as a novel interaction partner of PASKIN. This interaction was mapped to the PAS A and kinase domains of PASKIN and to the C-terminus of eEF1A1 using mammalian two-hybrid and GST pull-down assays. Kinase assays, mass spectrometry and site-directed mutagenesis revealed PASKIN auto-phosphorylation as well as eEF1A1 target phosphorylation mainly but not exclusively at Thr432. Wild-type but not kinase-inactive PASKIN increased the in vitro translation of a reporter cRNA. Whereas eEF1A1 did not localize to the nucleus, it co-localizes with PASKIN to the cytoplasm of HeLa cells. The two proteins also showed a remarkably similar localization in the midpiece of the sperm tail. These data suggest regulation of eEF1A1 by PASKIN-dependent phosphorylation in somatic as well as in sperm cells.

The Per-ARNT-Sim (PAS) domain serine/threonine kinase PASKIN, or PAS kinase, links energy flux and protein synthesis in yeast and regulates glycogen synthase in mammals. A recent report suggested that PASKIN mRNA, protein, and kinase activity are increased in pancreatic islet beta-cells under hyperglycemic conditions and that PASKIN is necessary for insulin gene expression. We previously generated Paskin knockout mice by targeted replacement of the kinase domain with the beta-geo fusion gene encoding beta-galactosidase reporter activity. Here we show that no 5-bromo-4-chloro-3-indolyl-ss-d-galactopyranoside (X-gal) staining was observed in islet beta-cells derived from Paskin knockout mice, irrespective of the ambient glucose concentration, whereas adenoviral expression of the lacZ gene in beta-cells showed strong X-gal staining. No induction of PASKIN mRNA could be detected in insulinoma cell lines or in islet beta-cells. Increasing glucose concentrations resulted in PASKIN-independent induction of insulin mRNA levels and insulin release. PASKIN mRNA levels were high in testes but undetectable in pancreas and in islet beta-cells. Finally, blood glucose levels and glucose tolerance after intraperitoneal glucose injection were indistinguishable between Paskin wild-type and knockout mice. These results suggest that Paskin gene expression is not induced by glucose in pancreatic beta-cells and that glucose-stimulated insulin production is independent of PASKIN.

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.

Prolyl 4-hydroxylase domain (PHD) proteins are oxygen-dependent enzymes that hydroxylate hypoxia-inducible transcription factor (HIF) alpha-subunits, leading to their subsequent ubiquitination and degradation. Paradoxically, the expression of two family members (PHD2 and PHD3) is induced in hypoxic cell culture despite the reduced availability of the oxygen co-substrate, and it has been suggested that they become functionally relevant following re-oxygenation to rapidly terminate the HIF response. Here we show that PHDs are also induced in hypoxic mice in vivo, albeit in a tissue-specific manner. As demonstrated under chronically hypoxic conditions in vitro, PHD2 and PHD3 show a transient maximum but remain up-regulated over more than 10 days, suggesting a feedback down-regulation of HIF-1alpha which then levels off at a novel set point. Indeed, hypoxic induction of PHD2 and PHD3 is paralleled by the attenuation of endogenous HIF-1alpha. Using an engineered oxygen-sensitive reporter gene in a cellular background lacking endogenous HIF-1alpha and hence inducible PHD expression, we could show that increased exogenous PHD levels can compensate for a wide range of hypoxic conditions. Similar data were obtained in a reconstituted cell-free system in vitro. In summary, these results suggest that due to their high O2 Km values, PHDs have optimal oxygen-sensing properties under all physiologically relevant oxygen concentrations; increased PHDs play a functional role even under oxygen-deprived conditions, allowing the HIF system to adapt to a novel oxygen threshold and to respond to another hypoxic insult. Furthermore, such an autoregulatory oxygen-sensing system would explain how a single mechanism works in a wide variety of differently oxygenated tissues.

The prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins hydroxylate hypoxia-inducible transcription factor (HIF)-alpha (alpha) subunits, leading to their subsequent ubiquitinylation and degradation. Since oxygen is a necessary cosubstrate, a reduction in oxygen availability (hypoxia) decreases PHD activity and, subsequently, HIF-alpha hydroxylation. Non-hydroxylated HIF-alpha cannot be bound by the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL), and HIF-alpha proteins thus become stabilized. HIF-alpha then heterodimerizes with HIF-beta (beta) to form the functionally active HIF transcription factor complex, which targets approximately 200 genes involved in adaptation to hypoxia. The three HIF-alpha PHDs are of a different nature compared with the prototype collagen prolyl-4-hydroxylase, which hydroxylates a mass protein rather than a rare transcription factor. Thus, novel assays had to be developed to express and purify functionally active PHDs and to measure PHD activity in vitro. A need also exists for such assays to functionally distinguish the three different PHDs in terms of substrate specificity and drug function. We provide a detailed description of the expression and purification of the PHDs as well as of an HIF-alpha-dependent and a HIF-alpha-independent PHD assay.

Aryl hydrocarbon receptor (AhR) and hypoxiainducible factor-1α (HIF-1α) are conditionally regulated transcription factor subunits that form heterodimeric complexes with their common partner, AhR nuclear translocator (ARNT/HIF-1β). Whereas the environmentally toxic compound 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) initiates the trans-activation activity of AhR:ARNT/HIF-1β, hypoxic exposure stabilizes HIF-1α and functionally activates the HIF-1α:ARNT/HIF-1β complex. To analyze a possible crosstalk between these two pathwaysin vivo, rats were given dioxin orally and/or were exposed to carbon monoxide (CO), causing functional anemia. We found that exposure to CO inhibited the xenobiotic response while dioxin application had no significant negative impact on hypoxia-mediated gene transcription.

Objectives The core of advanced atherosclerotic plaques turns hypoxic as the arterial wall thickens and oxygen diffusion capacity becomes impaired. Macrophage-derived foam cells play a pivotal role in atherosclerotic plaque formation by expressing scavenger receptors that regulate lipid uptake. However, the role of hypoxia in scavenger receptor regulation remains incompletely understood. Methods and results Using RT-qPCR, flow cytometry and immunoblotting, we found that mRNA and protein expression levels of the scavenger receptor A (SRA) and the cluster of differentiation 36 (CD36) were upregulated by oxidized low-density lipoprotein (oxLDL), but decreased following exposure of macrophages to hypoxia. In contrast, lectin-like oxLDL receptor (Lox-1) mRNA and protein levels were upregulated under hypoxic conditions. Flow cytometry confirmed the increased lipid content in macrophages after exposure to 0.2% oxygen and the hypoxia-mimetic dimethyloxalylglycine (DMOG). Antibody-mediated blocking of Lox-1 receptor decreased the hypoxic induction of oxLDL uptake and lipid content. RNAi-mediated knock-down of hypoxia-inducible factor (HIF)-1α in macrophages attenuated the hypoxic induction of Lox-1. Conclusions Hypoxia increases lipid uptake into macrophages and differentially regulates the expression of oxLDL receptors. Lox-1 plays a major role in hypoxia-induced foam cell formation which is, at least in part, mediated by HIF-1α.