Activation Of Hypoxia-inducible Factor Research Articles

PHD2 attenuates high-glucose-induced blood retinal barrier breakdown in human retinal microvascular endothelial cells by regulating the Hif-1α/VEGF pathway.

Diabetic macular edema (DME) is one of the most frequent causes of severe vision loss. The pathogenesis of DME is still not fully understood; however, it is hypothesized to result from breakdown of the blood-retinal barrier (BRB) due to retinal inflammation by vascular endothelial growth factor (VEGF) secretion under hyperglycemic conditions. In this investigation, we discovered that Prolyl-4-hydroxylase 2 (PHD2), an upstream regulator of hypoxia-inducible factor 1 (HIF-1) modulates VEGF expression and thus preserves BRB function in the mouse retina. Primary human retinal microvascular endothelial cells (hRMECs) were cultured in human endothelial serum-free growth medium and exposed to hyperglycemia. Changes in cell viability were investigated by an MTT assay. BRB function in each group was revealed by a paracellular permeability assay and trans-endothelial electrical resistance (TEER). Morphological changes in the BRB were investigated by immunofluorescence staining of occludin and zonula occludens-1 (ZO-1). The mRNA and protein levels of the tight junction proteins, PHD2, HIF-1α, and VEGF were measured by reverse transcription-quantitative PCR (RT-qPCR), western blot analysis and ELISA. Under hyperglycemic conditions, the viability of hRMECs was decreased, and PHD2 expression was downregulated, accompanied by increased paracellular permeability and decreased trans-endothelial electrical resistance. Additionally, HIF-1α and VEGF expression levels were increased, whereas the expression levels of tight junction proteins, including occludin and ZO-1, were decreased and BRB function was compromised. The PHD2 activator R59949 (diacylglycerol kinase inhibitor II), altered these pathological changes, and the PHD2 inhibitor dimethyloxalylglycine (DMOG) resulted in the opposite effects. These results demonstrated that PHD2 inhibited HIF-1 activity by inhibiting HIF-1α expression in hRMECs under hyperglycemic conditions, which led to the downregulation of the expression of the angiogenic factor VEGF, and thus helped to maintain the functions of hRMECs. Therefore, it is reasonable to propose that PHD2 could be a potential novel target for the treatment of DME or other diseases with a similar pathogenesis.

HIF-Dependent NFATC1 Activation Upregulates ITGA5 and PLAUR in Intestinal Epithelium in Inflammatory Bowel Disease.

Intestinal epithelial cells exist in physiological hypoxia, leading to hypoxia-inducible factor (HIF) activation and supporting barrier function and cell metabolism of the intestinal epithelium. In contrast, pathological hypoxia is a common feature of some chronic disorders, including inflammatory bowel disease (IBD). This work was aimed at studying HIF-associated changes in the intestinal epithelium in IBD. In the first step, a list of genes responding to chemical activation of hypoxia was obtained in an in vitro intestinal cell model with RNA sequencing. Cobalt (II) chloride and oxyquinoline treatment of both undifferentiated and differentiated Caco-2 cells activate the HIF-signaling pathway according to gene set enrichment analysis. The core gene set responding to chemical hypoxia stimulation in the intestinal model included 115 upregulated and 69 downregulated genes. Of this set, protein product was detected for 32 genes, and fold changes in proteome and RNA sequencing significantly correlate. Analysis of publicly available RNA sequencing set of the intestinal epithelial cells of patients with IBD confirmed HIF-1 signaling pathway activation in sigmoid colon of patients with ulcerative colitis and terminal ileum of patients with Crohn’s disease. Of the core gene set from the gut hypoxia model, expression activation of ITGA5 and PLAUR genes encoding integrin α5 and urokinase-type plasminogen activator receptor (uPAR) was detected in IBD specimens. The interaction of these molecules can activate cell migration and regenerative processes in the epithelium. Transcription factor analysis with the previously developed miRGTF tool revealed the possible role of HIF1A and NFATC1 in the regulation of ITGA5 and PLAUR gene expression. Detected genes can serve as markers of IBD progression and intestinal hypoxia.

Iron Deficiency in Polycythemia Vera Increases HIF Activity and Transcription of Prothrombotic Genes

Polycythemia vera (PV) is characterized by elevated red cell mass due to gain-of-function mutations of the tyrosine kinase JAK2 gene. Major causes of morbidity and mortality in PV are venous and arterial thromboses. Risk factors of thrombosis in PV are age > 60 years, previous thrombosis history, and leukocytosis due to increased granulocytes (PMID: 31865003). High hematocrit has been considered as a causative factor of thrombosis due to high viscosity; however, this has been challenged. Phlebotomy to maintain hematocrit below 45% is performed by most hematologists to prevent thrombosis. However, in our prospective controlled study, we reported that phlebotomies, but not high hematocrit, are associated with increased risk of thrombosis in Chuvash erythrocytosis (CE). CE is due to a hypomorphic mutation of the Von Hippel-Lindau (VHL) gene, a negative regulator of hypoxia inducible factors (HIFs) (PMID 289208), leading to augmented HIFs. We hypothesize that repeated phlebotomies may increase the risk of thrombosis in PV.We reported that HIFs, and some HIFs-regulated thrombotic genes, are upregulated in PV in both granulocytes and platelets, and even more in PV with a thrombosis history (PMID: 32203583). PV patients with mutated JAK2 have lower ferritin, serum iron, transferrin saturation, and increased soluble transferrin receptor levels compared to JAK2-wild type erythrocytosis (PMID: 30042411), indicating that PV patients with JAK2 mutation are more likely to be iron deficiency (ID). Repeated phlebotomies further augment ID, which increases the level of HIF-1 and HIF-2 by inhibiting the principal negative regulator of HIFs, prolyl hydroxylase domain 2 (PHD2) enzyme. PHD2 requires iron as a co-factor (PMID: 18066546). This led us to hypothesize that further increase of HIFs due to phlebotomy-induced ID increases expression of HIF-regulated prothrombotic genes, leading to increased risk of thrombosis in PV.We studied 17 PV with ID, 32 PV without ID, and 19 healthy control and measured their prothrombotic genes' transcript levels in the granulocytes. Of 17 PVs with ID, 9 (56.3 %) had thrombosis while 14/32 PVs without ID (43.8 %) had thrombosis history. We then measured transcript levels of prothrombotic genes, such as F3 (tissue factor), SELP (P-selectin), THBS1 (thrombospondin-1), SERPINE1(plasminogen activator inhibitor-1) and HIF-target genes VEGFA and SLC2A1 (glucose transporter-1). The transcripts of these genes were upregulated in PV with and without ID compared to the controls. However, PV with ID had higher transcripts of these genes compared to PV without ID, except SERPINE1 (Figure 1). These prothrombotic gene transcripts were inversely correlated with ferritin levels and positively correlated with HIF target genes. Thus, ID in PV further augmented HIFs activity and induced expression of prothrombotic genes. In ongoing studies of PV patients serving as their own controls, PV subjects with ID are treated with hydroxyurea to prevent rebound erythrocytosis. The hydroxyurea treatment alone did not normalize high transcripts of prothrombotic and HIF target genes. However, transcripts of these prothrombotic and HIF-target genes decreased after iron correction while their JAK2V617F allele burden did not change.We then measured transcript levels of these prothrombotic genes in the patients with germline mutations augmenting HIF activity including loss of function VHL, gain-of-function EPAS1 (HIF2a), and loss-of-function EGLN1 (PHD2). These patients have high transcript levels of prothrombotic genes due to high HIF activity although they don't have ID. Transcripts of F3, SELP, VEGFA, and SLC2A1 of the patients with HIF-pathway mutations were increased compared to the controls and compared to PV without ID but similar to the levels in PV with ID. On the other hand, transcript levels of THBS1 and SERPINE1 in the patients with HIF-pathway mutations were higher than the levels seen in the patients with PV with ID. These data indicate that ID in PV augments HIF activity similar, although not identical, to the patients with germline HIF-pathway mutations and may facilitate propensity to thromboses.These data suggest that therapeutic phlebotomy to control high hematocrit in PV and other erythrocytoses has the potential to increase thrombosis risk. In our ongoing studies, transcript levels of these prothrombotic genes are being correlated with their functions and protein levels. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Regulation of Fetal Hemoglobin Expression By the VHL-HIF1α Oxygen Sensing System

Defining the mechanisms that control the perinatal switch from γ-globin (HBG1 and HBG2) to β-globin (HBB) gene expression in human red blood cells (RBCs) has informed novel approaches to reactivate fetal hemoglobin (HbF, α2γ2) therapeutically for sickle cell disease and β-thalassemia. However, one longstanding unsolved problem is to explain how HbF becomes elevated in conditions such as blood loss, hypoxia and hemolysis. These conditions are associated with accelerated RBC production, also referred to as stress erythropoiesis, driven by activation of hypoxia-inducible factor (HIF) via a canonical O 2 sensing pathway. At high O 2 levels (“normoxia”), O 2-dependent prolyl hydroxylase domain (PHD) enzymes hydroxylate HIFα, thereby targeting it for ubiquitination by the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex, followed by proteasomal degradation. At low O 2 tension (hypoxia), PHD activity is reduced, causing HIF1α to accumulate, dimerize with constitutively expressed HIF1β, and bind hypoxia response elements (HREs) to activate a broad array of genes that facilitate hypoxic adaptation. We identified VHL and HIF1α, as negative and positive regulators of HbF expression, respectively.Disruption of the VHL gene in CD34 + hematopoietic stem and progenitor cells (HSPCs) by transfection with ribonucleoprotein (RNP) consisting of Cas9 and VHL-targeting guide RNA increased HbF expression from 7.5% ±1.2% in control cells to 30.9% ± 4.8% (mean ± SD, P<0.0001) in RBC progeny generated by in vitro differentiation. Similarly, γ-globin mRNA was induced by 5 -fold after disruption of VHL in HUDEP-2 cells, an immortalized erythroid line that expresses mainly adult hemoglobin (HbA, α2/β2). Mass spectrometry and transcriptome analysis of VHL-disrupted CD34 + HSPCs revealed increased HIF1α protein expression with no change in the corresponding mRNA. Inhibition of HIF1A mRNA by RNA interference suppressed γ-globin induction in VHL-/- HUDEP-2 clones and in RBCs generated from VHL RNP-treated CD34 + cells, indicating VHL knockout induced HbF through HIF1α protein accumulation. CUT&RUN analysis of VHL-depleted erythroblasts revealed HIF1α/HIF1β heterodimer occupancy at BGLT3, a long-noncoding RNA locus approximately 2.7kb 3' of HBG1. The HIF1α-bound BGLT3 locus contains two canonical HREs (ACGTG) separated by 13 bp. Disruption of each HRE motif in VHL-/- HUDEP-2 cells by base editing caused additive reductions in BGLT3 HIF1α occupancy and γ-globin expression. Mechanistically, VHL depletion caused the accumulation of HIFα/β heterodimers at BGLT3, recruitment of the transcriptional activators GATA1 and P300, and targeted chromatin accessibility to establish the active enhancer mark H3K27ac. These changes were accompanied by altered chromosome conformation to favor long-range interactions between the γ-globin loci (HBG1 and HBG2) and the locus control region, a powerful upstream enhancer. Treatment of healthy donor CD34 + HSPCs-derived erythroblasts with the clinically approved PHD inhibitor FG-4592 (Roxadustat) caused HbF to increase from 4.76%±1.22% at baseline to 12.86 ± 2.40% (mean ± SD in 3 biological replicates, P<0.01). Treatment of the same erythroblasts with FG-4592 and hydroxyurea, a widely used SCD drug that acts partly by inducing HbF, caused HbF to increase from 4.76% ± 1.22% at baseline to 24.1% ± 5.3% (mean ± SD in 3 biological replicates, P<0.01), indicating an additive effect. Our findings link developmental globin gene regulation with O 2 sensing, provide a mechanism for HbF induction during stress erythropoiesis, and identify a novel therapeutic approach for β-hemoglobinopathies. DisclosuresBlobel: Pfizer: Consultancy; Fulcrum Therapeutics, Inc.: Consultancy. Weiss: Beam Therapeutics: Current holder of stock options in a privately-held company; Forma Therapeutics: Consultancy; Cellarity Inc.: Consultancy; Novartis: Consultancy.

Discovery of a Potent and Orally Bioavailable Hypoxia-Inducible Factor 2α (HIF-2α) Agonist and Its Synergistic Therapy with Prolyl Hydroxylase Inhibitors for the Treatment of Renal Anemia.

Activation of hypoxia-inducible factor 2 (HIF-2) has emerged as a potent renal anemia treatment strategy. Here, the benzisothiazole derivative 26 was discovered as a novel HIF-2α agonist, which first demonstrated nanomolar activity (EC50 = 490 nM, Emax = 349.2%) in the luciferase reporter gene assay. Molecular dynamics simulations indicated that 26 could allosterically enhance HIF-2 dimerization. Furthermore, compound 26 had a good pharmacokinetic profile (the oral bioavailability in rats was 41.38%) and an in vivo safety profile (the LD50 in mice was greater than 708 mg·kg-1). In the in vivo efficacy assays, the combination of 26 and the prolyl hydroxylase inhibitor, AKB-6548, was confirmed for the first time to synergistically increase the plasma erythropoietin level in mice (from 260 to 2296 pg·mL-1) and alleviate zebrafish anemia induced by doxorubicin. These results provide new insights for HIF-2α agonists and the treatment of renal anemia.

Sensing an Oxygen Sensor: Development and Application of Activity-Based Assays Directly Monitoring HIF Heterodimerization.

Conventionally, hypoxia-inducible factor (HIF) activation by prolyl hydroxylase domain enzyme (PHD) inhibition is monitored by gene reporter assays. The principle relies on the monitoring of an upstream event (HIF stabilization) by the downstream transcriptional activity. Here, we developed a novel approach to directly sense HIF activation by monitoring the heterodimerization of the HIFα/HIFβ subunits, constituting the functional HIF transcription factor. Two live cell-based biosensor assay setups were designed, utilizing functional complementation of split-nanoluciferase as a tool to measure HIFα/HIFβ protein-protein interaction resulting from the stabilization of HIF1α or HIF2α. The assay setup in a 96-well format was optimized for a duration of 2 h, and a HEK293T transfection protocol was introduced for the optimal configuration of HIFα/HIFβ-fusion proteins. These new bioassays outperformed hypoxia response element-based gene reporter assay, the current state-of-the-art assay, in terms of sensitivity. Applicability was demonstrated using a panel of PHD inhibitors, including roxadustat, molidustat, daprodustat, desidustat, vadadustat, and FG-2216, for which concentration-response curves were generated, allowing for the derivation of potency (EC50) and efficacy (Emax) data. The broad applicability of the biosensors was established via applying hypoxia mimetic CoCl2, iron chelator desferrioxamine, proteasome inhibitor MG-132, and 2-OG mimetic dimethyloxalylglycine on the assays, indicating concentration-dependent effects.

Osteoclasts adapt to physioxia perturbation through DNA demethylation

Oxygen plays an important role in diverse biological processes. However, since quantitation of the partial pressure of cellular oxygen in vivo is challenging, the extent of oxygen perturbation in situ and its cellular response remains underexplored. Using two-photon phosphorescence lifetime imaging microscopy, we determine the physiological range of oxygen tension in osteoclasts of live mice. We find that oxygen tension ranges from 17.4 to 36.4 mmHg, under hypoxic and normoxic conditions, respectively. Physiological normoxia thus corresponds to 5% and hypoxia to 2% oxygen in osteoclasts. Hypoxia in this range severely limits osteoclastogenesis, independent of energy metabolism and hypoxia-inducible factor activity. We observe that hypoxia decreases ten-eleven translocation (TET) activity. Tet2/3 cooperatively induces Prdm1 expression via oxygen-dependent DNA demethylation, which in turn activates NFATc1 required for osteoclastogenesis. Taken together, our results reveal that TET enzymes, acting as functional oxygen sensors, regulate osteoclastogenesis within the physiological range of oxygen tension, thus opening new avenues for research on in vivo response to oxygen perturbation.

HIF-1 mediated activation of antimicrobial peptide LL-37 in type 2 diabetic patients

Infections are common in patients with diabetes, but increasing antibiotic resistance hampers successful bacterial clearance and calls for alternative treatment strategies. Hypoxia-inducible factor 1 (HIF-1) is known to influence the innate immune defense and could therefore serve as a possible target. However, the impact of high glucose on HIF-1 has received little attention and merits closer investigation. Here, we show that higher levels of proinflammatory cytokines and CAMP, encoding for the antimicrobial peptide cathelicidin, LL-37, correlate with HIF-1 in type 2 diabetic patients. Chemical activation of HIF-1 further enhanced LL-37, IL-1β, and IL-8 in human uroepithelial cells exposed to high glucose. Moreover, HIF-1 activation of transurethrally infected diabetic mice resulted in lower bacterial load. Drugs activating HIF-1 could therefore in the future potentially have a therapeutic role in clearing bacteria in diabetic patients with infections where antibiotic treatment failed.Key messages• Mohanty et al. “HIF-1 mediated activation of antimicrobial peptide LL-37 in type 2 diabetic patients.”• Our study highlights induction of the antimicrobial peptide, LL-37, and strengthening of the innate immunity through hypoxia-inducible factor 1 (HIF-1) in diabetes.• Our key observations are:1. HIF-1 activation increased LL-37 expression in human urothelial cells treated with high glucose. In line with that, we demonstrated that patients with type 2 diabetes living at high altitude had increased levels of the LL-37.2. HIF-1 activation increased IL-1β and IL-8 in human uroepithelial cells treated with high glucose concentration.3. Pharmacological activation of HIF-1 decreased bacterial load in the urinary bladder of mice with hereditary diabetes.• We conclude that enhancing HIF-1 may along with antibiotics in the future contribute to the treatment in selected patient groups where traditional therapy is not possible.

Heritable disorders of oxygen sensing.

Hypoxia-inducible factors (HIFs) activate gene transcription in response to reduced O2 availability and play critical roles in development, physiology, and disease pathogenesis. Mutations that dysregulate HIF activity are the genetic basis for tumor predisposition in the von Hippel-Lindau syndrome and excess red blood cell production in hereditary erythrocytosis.

Pharmacological inhibition of Mint3 attenuates tumour growth, metastasis, and endotoxic shock

Hypoxia-inducible factor-1 (HIF-1) plays essential roles in human diseases, though its central role in oxygen homoeostasis hinders the development of direct HIF-1-targeted pharmacological approaches. Here, we surveyed small-molecule compounds that efficiently inhibit the transcriptional activity of HIF-1 without affecting body homoeostasis. We focused on Mint3, which activates HIF-1 transcriptional activity in limited types of cells, such as cancer cells and macrophages, by suppressing the factor inhibiting HIF-1 (FIH-1). We identified naphthofluorescein, which inhibited the Mint3–FIH-1 interaction in vitro and suppressed Mint3-dependent HIF-1 activity and glycolysis in cancer cells and macrophages without evidence of cytotoxicity in vitro. In vivo naphthofluorescein administration suppressed tumour growth and metastasis without adverse effects, similar to the genetic depletion of Mint3. Naphthofluorescein attenuated inflammatory cytokine production and endotoxic shock in mice. Thus, Mint3 inhibitors may present a new targeted therapeutic option for cancer and inflammatory diseases by avoiding severe adverse effects.

Modulatory role of PACAP and VIP on HIFs expression in lung adenocarcinoma

Lung adenocarcinoma is the most frequent form of non-small cell lung cancer. Inside the tumor mass, uncontrolled cell proliferation generates hypoxic areas leading to activation of hypoxia-inducible factors (HIFs) responsible for neovascularization and tumor metastasis. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two neuropeptides widely distributed in respiratory organs. Previous studies have demonstrated that these peptides interfere with hypoxic pathways in various diseases, including tumors. However, their modulatory role in HIFs expression in lung adenocarcinomas has not yet been evaluated. In the present paper, we detected the expression profile of PACAP, VIP and related receptors in healthy and adenocarcinoma human lung tissue. To characterize peptides’ modulatory effects on HIFs expression, we also exposed A549 lung adenocarcinoma cells and human normal bronchial epithelial BEAS-2B cells to microenvironmental hypoxia by treating them with deferoxamine (DFX). The results showed that PACAP and VIP significantly reduced HIF-1α and HIF-2α levels in both cell lines following hypoxic stress. The HIF-3α expression profile was related to cellular phenotype as it was lower in BEAS-2B and higher in A549 cells under low oxygen tension. In lung adenocarcinoma cells, peptide treatment restored HIF-3 α expression to control levels. These results suggest that endogenous PACAP and VIP exert controversial roles in cellular hypoxic microenvironments depending on the pathophysiological conditions of the lung tissue.

Hypoxia-Inducible Factors as Key Players in the Pathogenesis of Non-alcoholic Fatty Liver Disease and Non-alcoholic Steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) are a major public health concern with high and increasing global prevalence, and a significant disease burden owing to its progression to more severe forms of liver disease and the associated risk of cardiovascular disease. Treatment options, however, remain scarce, and a better understanding of the pathological and physiological processes involved could enable the development of new therapeutic strategies. One process implicated in the pathology of NAFLD and NASH is cellular oxygen sensing, coordinated largely by the hypoxia-inducible factor (HIF) family of transcription factors. Activation of HIFs has been demonstrated in patients and mouse models of NAFLD and NASH and studies of activation and inhibition of HIFs using pharmacological and genetic tools point toward important roles for these transcription factors in modulating central aspects of the disease. HIFs appear to act in several cell types in the liver to worsen steatosis, inflammation, and fibrosis, but may nevertheless improve insulin sensitivity. Moreover, in liver and other tissues, HIF activation alters mitochondrial respiratory function and metabolism, having an impact on energetic and redox homeostasis. This article aims to provide an overview of current understanding of the roles of HIFs in NAFLD, highlighting areas where further research is needed.

Chronic and Cycling Hypoxia: Drivers of Cancer Chronic Inflammation through HIF-1 and NF-κB Activation: A Review of the Molecular Mechanisms.

Chronic (continuous, non-interrupted) hypoxia and cycling (intermittent, transient) hypoxia are two types of hypoxia occurring in malignant tumors. They are both associated with the activation of hypoxia-inducible factor-1 (HIF-1) and nuclear factor κB (NF-κB), which induce changes in gene expression. This paper discusses in detail the mechanisms of activation of these two transcription factors in chronic and cycling hypoxia and the crosstalk between both signaling pathways. In particular, it focuses on the importance of reactive oxygen species (ROS), reactive nitrogen species (RNS) together with nitric oxide synthase, acetylation of HIF-1, and the action of MAPK cascades. The paper also discusses the importance of hypoxia in the formation of chronic low-grade inflammation in cancerous tumors. Finally, we discuss the effects of cycling hypoxia on the tumor microenvironment, in particular on the expression of VEGF-A, CCL2/MCP-1, CXCL1/GRO-α, CXCL8/IL-8, and COX-2 together with PGE2. These factors induce angiogenesis and recruit various cells into the tumor niche, including neutrophils and monocytes which, in the tumor, are transformed into tumor-associated neutrophils (TAN) and tumor-associated macrophages (TAM) that participate in tumorigenesis.

Genomic and functional evidence reveals convergent evolution in fishes on the Tibetan Plateau.

High-altitude environments are strong drivers of adaptive evolution in endemic organisms. However, little is known about the genetic mechanisms of convergent adaptation among different lineages, especially in fishes. There are three independent fish groups on the Tibetan Plateau: Tibetan Loaches, Schizothoracine fishes and Glyptosternoid fishes; all are well adapted to the harsh environmental conditions. They represent an excellent example of convergent evolution but with an unclear genetic basis. We used comparative genomic analyses between Tibetan fishes and fishes from low altitudes and detected genomic signatures of convergent evolution in fishes on the Tibetan Plateau. The Tibetan fishes exhibited genome-wide accelerated evolution in comparison with a control set of fishes from low altitudes. A total of 368 positively selected genes were identified in Tibetan fishes, which were enriched in functional categories related to energy metabolism and hypoxia response. Widespread parallel amino acid substitutions were detected among the Tibetan fishes and a subset of these substitutions occurred in positively selected genes associated with high-altitude adaptation. Functional assays suggested that von Hippel-Lindau (VHL) tumour suppressor genes from Tibetan fishes enhance hypoxia-inducible factor (HIF) activity convergently under hypoxia compared to low-altitude fishes. The results provide genomic and functional evidence supporting convergent genetic mechanisms for high-altitude adaptation in fishes on the Tibetan Plateau.

321 Deletion of hypoxia-inducible factor prolyl 4-hydroxylase 2 in FoxD1-lineage mesenchymal cells leads to congenital truncal alopecia

We aimed to investigate how the depletion of hypoxia inducible factor prolyl-4 hydroxylase 2 (HIF- P4H-2) in Forkhead Box D1 (FoxD1) lineage cells affects hair follicle (HF) and skin development. Hypoxia-inducible factors (HIFs) induce hundreds of genes regulating oxygen homeostasis in tissues. The HIF-P4Hs regulate the stability and hence the activity of HIFs in an oxygen-dependent manner. The skin is a hypoxic organ with endogenous HIF expression but the role of hypoxia signalling in skin and HFs are poorly understood. We studied a mouse line where Hif-p4h-2 is conditionally inactivated in FoxD1-expressing (FoxD1+) cells. This mouseline was studied at different developmental stages during the HF morphogenesis and cycle. To be able to pinpoint the location of FoxD1+ cells we studied the skin of a mouse line where FoxD1+ cells could be seen due to the expression of enhanced green fluorescent protein and the non-FoxD1-expressing cells due to the expression of tomato-red. Lack of Hif-p4h-2 in FoxD1-lineage mesodermal cells causes disturbed HF development during the first catagen leading to formation of large epithelial lined HF cysts filled with keratins, which eventually manifests as truncal alopecia. The cranial hair remains normal. We show that FoxD1+ cells are mesenchymal cells located in the dermis and dermal papilla of truncal skin. FoxD1+ cells are scarce in the cranial skin. The depletion of Hif-p4h-2 in FoxD1+ cells misregulates genes crucial for hypoxia response, keratin formation and differentiation, TGF-β and Notch signalling pathways, which are known to be essential for the HF development and cycling. We show here for the first time that HIF-P4H-2 function in FoxD1-lineage cells is essential for the normal development and homeostasis of HFs.

Essential role of systemic iron mobilization and redistribution for adaptive thermogenesis through HIF2-α/hepcidin axis

Iron is an essential biometal, but is toxic if it exists in excess. Therefore, iron content is tightly regulated at cellular and systemic levels to meet metabolic demands but to avoid toxicity. We have recently reported that adaptive thermogenesis, a critical metabolic pathway to maintain whole-body energy homeostasis, is an iron-demanding process for rapid biogenesis of mitochondria. However, little information is available on iron mobilization from storage sites to thermogenic fat. This study aimed to determine the iron-regulatory network that underlies beige adipogenesis. We hypothesized that thermogenic stimulus initiates the signaling interplay between adipocyte iron demands and systemic iron liberation, resulting in iron redistribution into beige fat. To test this hypothesis, we induced reversible activation of beige adipogenesis in C57BL/6 mice by administering a β3-adrenoreceptor agonist CL 316,243 (CL). Our results revealed that CL stimulation induced the iron-regulatory protein-mediated iron import into adipocytes, suppressed hepcidin transcription, and mobilized iron from the spleen. Mechanistically, CL stimulation induced an acute activation of hypoxia-inducible factor 2-α (HIF2-α), erythropoietin production, and splenic erythroid maturation, leading to hepcidin suppression. Disruption of systemic iron homeostasis by pharmacological HIF2-α inhibitor PT2385 or exogenous administration of hepcidin-25 significantly impaired beige fat development. Our findings suggest that securing iron availability via coordinated interplay between renal hypoxia and hepcidin down-regulation is a fundamental mechanism to activate adaptive thermogenesis. It also provides an insight into the effects of adaptive thermogenesis on systemic iron mobilization and redistribution.

Prolyl Hydroxylase Inhibitors: a New Opportunity in Renal and Myocardial Protection.

Hypoxia, via the activity of hypoxia-inducible factors (HIFs), plays a crucial role in fibrosis, inflammation, and oxidative injury, processes which are associated with progression of cardiovascular and kidney diseases. HIFs are key transcription heterodimers consisting of regulatory α-subunits (HIF-1α, HIF-2α, HIF-3α) and a constitutive β-subunit (HIF-β). The stability of HIFs is regulated by the prolyl hydroxylases (PHDs). Specific PHD inhibitors (PHD-i) are being investigated as a therapeutic approach to modulate the cellular signaling pathways and harness the native protective adaptive responses to hypoxia. Selective inhibition of PHD leads to the stabilization of the HIFs, which is the transcriptional gatekeeper of a multitude of genes involved in angiogenesis, energy metabolism, apoptosis, inflammation, and fibrosis. PHD-i downregulate hepcidin, improve iron absorption, and increase the endogenous production of erythropoietin. Furthermore, this pharmacological group has also been proven to ameliorate ischemic injuries in several organs, opening a new and promising field in cardiovascular research.. In this review, we present the basic and clinical potential of PHD-i treatment in different scenarios, such as ischemic heart disease, cardiac hypertrophy and heart failure, and their interplay with other pharmacological agents with proven cardiovascular benefits, such as sodium-glucose cotransporter 2 (SGLT2) inhibitors.

Hypoxia regulates overall mRNA homeostasis by inducing Met1-linked linear ubiquitination of AGO2 in cancer cells

Hypoxia is the most prominent feature in human solid tumors and induces activation of hypoxia-inducible factors and their downstream genes to promote cancer progression. However, whether and how hypoxia regulates overall mRNA homeostasis is unclear. Here we show that hypoxia inhibits global-mRNA decay in cancer cells. Mechanistically, hypoxia induces the interaction of AGO2 with LUBAC, the linear ubiquitin chain assembly complex, which co-localizes with miRNA-induced silencing complex and in turn catalyzes AGO2 occurring Met1-linked linear ubiquitination (M1-Ubi). A series of biochemical experiments reveal that M1-Ubi of AGO2 restrains miRNA-mediated gene silencing. Moreover, combination analyses of the AGO2-associated mRNA transcriptome by RIP-Seq and the mRNA transcriptome by RNA-Seq confirm that AGO2 M1-Ubi interferes miRNA-targeted mRNA recruiting to AGO2, and thereby facilitates accumulation of global mRNAs. By this mechanism, short-term hypoxia may protect overall mRNAs and enhances stress tolerance, whereas long-term hypoxia in tumor cells results in seriously changing the entire gene expression profile to drive cell malignant evolution.

MP07-05 THE MECHANISM OF KIDNEY STONE SUPPRESSION BY PROLINE HYDROXYLASE INHIBITOR ADMINISTRATION

You have accessJournal of UrologyStone Disease: Basic Research & Pathophysiology (MP07)1 Sep 2021MP07-05 THE MECHANISM OF KIDNEY STONE SUPPRESSION BY PROLINE HYDROXYLASE INHIBITOR ADMINISTRATION Kengo Kawase, Shuzo Hamamoto, Tatsuya Hattori, Tomoki Okada, Yutaro Tanaka, Takeru Sugino, Rei Unno, Kazumi Taguchi, Ryosuke Ando, Atsushi Okada, and Takahiro Yasui Kengo KawaseKengo Kawase More articles by this author , Shuzo HamamotoShuzo Hamamoto More articles by this author , Tatsuya HattoriTatsuya Hattori More articles by this author , Tomoki OkadaTomoki Okada More articles by this author , Yutaro TanakaYutaro Tanaka More articles by this author , Takeru SuginoTakeru Sugino More articles by this author , Rei UnnoRei Unno More articles by this author , Kazumi TaguchiKazumi Taguchi More articles by this author , Ryosuke AndoRyosuke Ando More articles by this author , Atsushi OkadaAtsushi Okada More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001980.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Kidney stones are associated with metabolic syndrome (MetS). MetS has been shown to create a hypoxic environment in adipose tissue, resulting in inflammation. It has been reported that the prolyl hydroxylases (PHD)-hypoxia-inducible factor (HIF) pathway is mainly involved in the hypoxic response and that PHD inhibitors suppress inflammation in adipose tissue. In this study, we investigated the inhibitory effect of a PHD inhibitor (FG) on kidney stone formation. METHODS: A group of cultured mouse renal tubular cells (M1) and a group of co-cultured M1 and 3T3L1 cells differentiated into white adipocytes were prepared. FG (0 μmol/L, 50 μmol/L) was added to all for 6 hours, and calcium oxalate crystals (20 μg/cm2) were added to M1 for a total of four groups. The extent of crystal adhesion was examined. In addition, inflammation and stress were determined by quantitative PCR using Ccl2 and Spp1, and the expression of Adiponectin in the culture medium were evaluated. RESULTS: FG treatment significantly decreased the amount of crystal adherence in both monoculture and co-culture groups (p <0.05), and FG treatment significantly decreased the expression of Ccl2 and Spp1 in M1 cells in both monoculture and co-culture groups (p <0.05). Adiponectin in culture was increased in the co-culture group by FG treatment (p <0.05). CONCLUSIONS: The activation of HIF in adipocytes by FG administration was suggested to inhibit renal stone formation by showing adiponectin-mediated anti-inflammatory effects. Source of Funding: The authors declare no conflicts of interest associated with this manuscript © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e140-e141 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kengo Kawase More articles by this author Shuzo Hamamoto More articles by this author Tatsuya Hattori More articles by this author Tomoki Okada More articles by this author Yutaro Tanaka More articles by this author Takeru Sugino More articles by this author Rei Unno More articles by this author Kazumi Taguchi More articles by this author Ryosuke Ando More articles by this author Atsushi Okada More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Loading ...

Impact of hypoxia on the pathogenesis and therapy resistance in multiple myeloma.

Multiple myeloma (MM) is a refractory plasma cell tumor. In myeloma cells, the transcription factor IRF4, the master regulator of plasma cells, is aberrantly upregulated and plays an essential role in oncogenesis. IRF4 forms a positive feedback loop with MYC, leading to additional tumorigenic properties. In recent years, molecular targeted therapies have contributed to a significant improvement in the prognosis of MM. Nevertheless, almost all patients experience disease progression, which is thought to be a result of treatment resistance induced by various elements of the bone marrow microenvironment. Among these, the hypoxic response, one of the key processes for cellular homeostasis, induces hypoxia‐adapted traits such as undifferentiation, altered metabolism, and dissemination, leading to drug resistance. These inductions are caused by ectopic gene expression changes mediated by the activation of hypoxia‐inducible factors (HIFs). By contrast, the expression levels of IRF4 and MYC are markedly reduced by hypoxic stress. Notably, an anti‐apoptotic capability is usually acquired under both normoxic and hypoxic conditions, but the mechanism is distinct. This fact strongly suggests that myeloma cells may survive by switching their dependent regulatory factors from IRF4 and MYC (normoxic bone marrow region) to HIF (hypoxic bone marrow microenvironment). Therefore, to achieve deep remission, combination therapeutic agents, which are complementarily effective against both IRF4‐MYC‐dominant and HIF‐dominated fractions, may become an important therapeutic strategy for MM.