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.

Terabyte-scale supervised 3D training and benchmarking dataset of the mouse kidney.

The performance of machine learning algorithms, when used for segmenting 3D biomedical images, does not reach the level expected based on results achieved with 2D photos. This may be explained by the comparative lack of high-volume, high-quality training datasets, which require state-of-the-art imaging facilities, domain experts for annotation and large computational and personal resources. The HR-Kidney dataset presented in this work bridges this gap by providing 1.7 TB of artefact-corrected synchrotron radiation-based X-ray phase-contrast microtomography images of whole mouse kidneys and validated segmentations of 33 729 glomeruli, which corresponds to a one to two orders of magnitude increase over currently available biomedical datasets. The image sets also contain the underlying raw data, threshold- and morphology-based semi-automatic segmentations of renal vasculature and uriniferous tubules, as well as true 3D manual annotations. We therewith provide a broad basis for the scientific community to build upon and expand in the fields of image processing, data augmentation and machine learning, in particular unsupervised and semi-supervised learning investigations, as well as transfer learning and generative adversarial networks.

Intrinsic TGF-ß signaling attenuates proximal tubule mitochondrial injury and inflammation in chronic kidney disease.

Excessive TGF-ß signaling and mitochondrial dysfunction fuel chronic kidney disease (CKD) progression. However, inhibiting TGF-ß failed to impede CKD in humans. The proximal tubule (PT), the most vulnerable renal segment, is packed with giant mitochondria and injured PT is pivotal in CKD progression. How TGF-ß signaling affects PT mitochondria in CKD remained unknown. Here, we combine spatial transcriptomics and bulk RNAseq with biochemical analyses to depict the role of TGF-ß signaling on PT mitochondrial homeostasis and tubulo-interstitial interactions in CKD. Male mice carrying specific deletion of Tgfbr2 in the PT have increased mitochondrial injury and exacerbated Th1 immune response in the aristolochic acid model of CKD, partly, through impaired complex I expression and mitochondrial quality control associated with a metabolic rewiring toward aerobic glycolysis in the PT cells. Injured S3T2 PT cells are identified as the main mediators of the maladaptive macrophage/dendritic cell activation in the absence of Tgfbr2. snRNAseq database analyses confirm decreased TGF-ß receptors and a metabolic deregulation in the PT of CKD patients. This study describes the role of TGF-ß signaling in PT mitochondrial homeostasis and inflammation in CKD, suggesting potential therapeutic targets that might be used to mitigate CKD progression.

The transcriptional and regulatory identity of erythropoietin producing cells.

Erythropoietin (Epo) is the master regulator of erythropoiesis and oxygen homeostasis. Despite its physiological importance, the molecular and genomic contexts of the cells responsible for renal Epo production remain unclear, limiting more-effective therapies for anemia. Here, we performed single-cell RNA and transposase-accessible chromatin (ATAC) sequencing of an Epo reporter mouse to molecularly identify Epo-producing cells under hypoxic conditions. Our data indicate that a distinct population of kidney stroma, which we term Norn cells, is the major source of endocrine Epo production in mice. We use these datasets to identify the markers, signaling pathways and transcriptional circuits characteristic of Norn cells. Using single-cell RNA sequencing and RNA in situ hybridization in human kidney tissues, we further provide evidence that this cell population is conserved in humans. These preliminary findings open new avenues to functionally dissect EPO gene regulation in health and disease and may serve as groundwork to improve erythropoiesis-stimulating therapies.

Low-Dose Near-Infrared Light-Activated Mitochondria-Targeting Photosensitizers for PDT Cancer Therapy.

Phthalocyanines (Pcs) are promising candidates for photodynamic therapy (PDT) due to their absorption in the phototherapeutic window. However, the highly aromatic Pc core leads to undesired aggregation and decreased reactive oxygen species (ROS) production. Therefore, short PEG chain functionalized A3B type asymmetric Pc photosensitizers (PSs) were designed in order to decrease aggregation and increase the aqueous solubility. Here we report the synthesis, characterization, optical properties, cellular localization, and cytotoxicity of three novel Pc-based agents (LC31, MLC31, and DMLC31Pt). The stepwise functionalization of the peripheral moieties has a strong effect on the distribution coefficient (logP), cellular uptake, and localization, as well as photocytotoxicity. Additional experiments have revealed that the presence of the malonic ester moiety in the reported agent series is indispensable in order to induce photocytotoxicity. The best-performing agent, MLC31, showed mitochondrial targeting and an impressive phototoxic index (p.i.) of 748 in the cisplatin-resistant A2780/CP70 cell line, after a low-dose irradiation of 6.95 J/cm2. This is the result of a high photocytotoxicity (IC50 = 157 nM) upon irradiation with near-infrared (NIR) light, and virtually no toxicity in the dark (IC50 = 117 µM). Photocytotoxicity was subsequently determined under hypoxic conditions. Additionally, a preliminarily pathway investigation of the mitochondrial membrane potential (MMP) disruption and induction of apoptosis by MLC31 was carried out. Our results underline how agent design involving both hydrophilic and lipophilic peripheral groups may serve as an effective way to improve the PDT efficiency of highly aromatic PSs for NIR light-mediated cancer therapy.

Sphk1 and Sphk2 Differentially Regulate Erythropoietin Synthesis in Mouse Renal Interstitial Fibroblast-like Cells.

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 S1P1 and S1P3 receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1-/-, or Sphk2-/- mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2-/- renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2-/- cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P1 and S1P3 antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P1 and S1P3. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P1+3 to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic 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.

Androglobin, a chimeric mammalian globin, is required for male fertility.

Spermatogenesis is a highly specialized differentiation process driven by a dynamic gene expression program and ending with the production of mature spermatozoa. Whereas hundreds of genes are known to be essential for male germline proliferation and differentiation, the contribution of several genes remains uncharacterized. The predominant expression of the latest globin family member, androglobin (Adgb), in mammalian testis tissue prompted us to assess its physiological function in spermatogenesis. Adgb knockout mice display male infertility, reduced testis weight, impaired maturation of elongating spermatids, abnormal sperm shape, and ultrastructural defects in microtubule and mitochondrial organization. Epididymal sperm from Adgb knockout animals display multiple flagellar malformations including coiled, bifid or shortened flagella, and erratic acrosomal development. Following immunoprecipitation and mass spectrometry, we could identify septin 10 (Sept10) as interactor of Adgb. The Sept10-Adgb interaction was confirmed both in vivo using testis lysates and in vitro by reciprocal co-immunoprecipitation experiments. Furthermore, the absence of Adgb leads to mislocalization of Sept10 in sperm, indicating defective manchette and sperm annulus formation. Finally, in vitro data suggest that Adgb contributes to Sept10 proteolysis in a calmodulin-dependent manner. Collectively, our results provide evidence that Adgb is essential for murine spermatogenesis and further suggest that Adgb is required for sperm head shaping via the manchette and proper flagellum formation.

The HIFα-Stabilizing Drug Roxadustat Increases the Number of Renal Epo-Producing Sca-1+ Cells.

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.

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.

Neurogenic and pericytic plasticity of conditionally immortalized cells derived from renal erythropoietin-producing cells.

In adult mammals, the kidney is the main source of circulating erythropoietin (Epo), the master regulator of erythropoiesis. In vivo data in mice demonstrated multiple subtypes of interstitial renal Epo-producing (REP) cells. To analyze the differentiation plasticity of fibroblastoid REP cells, we used a transgenic REP cell reporter mouse model to generate conditionally immortalized REP-derived (REPD) cell lines. Under nonpermissive conditions, REPD cells ceased from proliferation and acquired a stem cell-like state, with strongly enhanced hypoxia-inducible factor 2 (HIF-2α), stem cell antigen 1 (SCA-1), and CD133 expression, but also enhanced alpha-smooth muscle actin (αSMA) expression, indicating myofibroblastic signaling. These cells maintained the "on-off" nature of Epo expression observed in REP cells in vivo, whereas other HIF target genes showed a more permanent regulation. Like REP cells in vivo, REPD cells cultured in vitro generated long tunneling nanotubes (TNTs) that aligned with endothelial vascular structures, were densely packed with mitochondria and became more numerous under hypoxic conditions. Although inhibition of mitochondrial oxygen consumption blunted HIF signaling, removal of the TNTs did not affect or even enhance the expression of HIF target genes. Apart from pericytes, REPD cells readily differentiated into neuroglia but not adipogenic, chondrogenic, or osteogenic lineages, consistent with a neuronal origin of at least a subpopulation of REP cells. In summary, these results suggest an unprecedented combination of differentiation features of this unique cell type.

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.

Activation of the Hypoxia-Inducible Factor Pathway Inhibits Epithelial Sodium Channel-Mediated Sodium Transport in Collecting Duct Principal Cells.

BACKGROUND: Active sodium reabsorption is the major factor influencing renal oxygen consumption and production of reactive oxygen species (ROS). Increased sodium reabsorption uses more oxygen, which may worsen medullary hypoxia and produce more ROS via enhanced mitochondrial ATP synthesis. Both mechanisms may activate the hypoxia-inducible factor (HIF) pathway. Because the collecting duct is exposed to low oxygen pressure and variations of active sodium transport, we assessed whether the HIF pathway controls epithelial sodium channel (ENaC)-dependent sodium transport. METHODS: We investigated HIF's effect on ENaC expression in mpkCCD cl4 cells (a model of collecting duct principal cells) using real-time PCR and western blot and ENaC activity by measuring amiloride-sensitive current. We also assessed the effect of hypoxia and sodium intake on abundance of kidney sodium transporters in wild-type and inducible kidney tubule-specific Hif1α knockout mice. RESULTS: In cultured cells, activation of the HIF pathway by dimethyloxalylglycine or hypoxia inhibited sodium transport and decreased expression of ß ENaC and γ ENaC, as well as of Na,K-ATPase. HIF1 α silencing increased ß ENaC and γ ENaC expression and stimulated sodium transport. A constitutively active mutant of HIF1 α produced the opposite effect. Aldosterone and inhibition of the mitochondrial respiratory chain slowly activated the HIF pathway, suggesting that ROS may also activate HIF. Decreased γ ENaC abundance induced by hypoxia in normal mice was abolished in Hif1α knockout mice. Similarly, Hif1α knockout led to increased γ ENaC abundance under high sodium intake. CONCLUSIONS: This study reveals that γ ENaC expression and activity are physiologically controlled by the HIF pathway, which may represent a negative feedback mechanism to preserve oxygenation and/or prevent excessive ROS generation under increased sodium transport.

S1P Stimulates Erythropoietin Production in Mouse Renal Interstitial Fibroblasts by S1P1 and S1P3 Receptor Activation and HIF-2α Stabilization.

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.

Hypoxia Reduces the Transcription of Fibrotic Markers in the Intestinal Mucosa.

INTRODUCTION: Intestinal fibrosis, characterized by excessive deposition of extracellular matrix proteins, is a common and severe clinical complication of inflammatory bowel disease (IBD). However, the mechanisms underlying fibrosis remain elusive, and currently, there are limited effective pharmacologic treatments that target the development of fibrosis. Hypoxia is one of the key microenvironmental factors influencing intestinal inflammation and has been linked to fibrosis. OBJECTIVE: In the present study, we sought to elucidate the impact of hypoxia on fibrotic gene expression in the intestinal mucosa. METHODS: Human volunteers, IBD patients, and dextran sulphate sodium-treated mice were exposed to hypoxia, and colonic biopsies were collected. The human intestinal epithelial cell line Caco-2, human THP-1 macrophages, and primary human gut fibroblasts were subjected to hypoxia, and changes in fibrotic gene expression were assessed. RESULTS: Human volunteers subjected to hypoxia presented reduced transcriptional levels of fibrotic and epithelial-mesenchymal transition markers in the intestinal mucosa. IBD patients showed a trend towards a decrease in tissue inhibitor of metalloproteinase 1 protein expression. In mice, hypoxic conditions reduced the colonic expression of several collagens and matrix metalloproteinases. Hypoxic Caco-2 cells, THP-1 cells, and primary gut fibroblasts showed a significant downregulation in the expression of fibrotic and tissue remodelling factors. CONCLUSIONS: Stabilization of hypoxia-inducible factors might represent a novel therapeutic approach for the treatment of IBD-associated fibrosis.

Interfering with Tumor Hypoxia for Radiotherapy Optimization.

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.

Distal and proximal hypoxia response elements cooperate to regulate organ-specific erythropoietin gene expression.

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.