Claudin-5 Affects Endothelial Autophagy in Response to Early Hypoxia

Hypoxic injury to cerebrovascular endothelial cells (ECs) after stroke leads to blood-brain barrier (BBB) dysfunction, which is commonly associated with disruptions of endothelial tight junctions (TJs) and increased permeability. Therefore, maintaining the structural integrity and proper function of the BBB is essential for the homeostasis and physiological function of the central nervous system (CNS). Our previous study revealed that autophagy functions on protecting the BBB by regulating the dynamics of Claudin-5, the essential TJ protein, under short-term starvation or hypoxia conditions. Here, we show that in zebrafish and in vitro cells, loss of membranous Claudin-5 conversely determine the occurrence of hypoxia-induced autophagy in cerebrovascular ECs. Absence of endothelial Claudin-5 could partly attenuate endothelial cell apoptosis caused by short-term hypoxic injury. Mechanism studies revealed that under hypoxic conditions, the existence of membranous Claudin-5 affects the stimulation of hypoxia inducible factor 1 subunit alpha (HIF-1a) and the inducible nitric oxide synthase (iNOS), which are responsible for the translocation of and endocytosis of caveole-packaged Claudin-5 into cytosol. Meanwhile, loss of Claudin-5 affects the generation of reactive oxygen species (ROS) and the downstream expression of BCL2/adenovirus E1B 19kDa protein interacting protein 3 (Bnip3). These together suppress the endothelial autophagy under hypoxia. This finding provides a theoretical basis for clarifying the mechanism of hypoxia-induced BBB injury and its potential protection mechanisms.

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Hypoxia-inducible factor 1α induces osteo/odontoblast differentiation of human dental pulp stem cells via Wnt/β-catenin transcriptional cofactor BCL9

Scientific Reports ◽

10.1038/s41598-021-04453-8 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Shion Orikasa ◽

Nobuyuki Kawashima ◽

Kento Tazawa ◽

Kentaro Hashimoto ◽

Keisuke Sunada-Nara ◽

...

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Target Genes ◽

Hypoxia Inducible Factor ◽

Dental Pulp Stem Cells ◽

Pulp Tissue ◽

Odontoblast Differentiation ◽

Hypoxia Inducible Factor 1Α ◽

Hypoxic Conditions

AbstractAccelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/β-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers (RUNX2, ALP, OC), BCL9, and Wnt/β-catenin signaling target genes (AXIN2, TCF1) in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and β-catenin expression and BCL9-β-catenin co-localization. In addition, BCL9 formed a complex with β-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/β-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/β-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury.

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Possible Implication of Hypoxia-mediated Incomplete Neutrophil Extracellular Trap Formation in Pneumonia-induced Exacerbation of Lung Diseases

10.21203/rs.3.rs-388216/v1 ◽

2021 ◽

Author(s):

Sakiko Masuda ◽

Kurumi Kato ◽

Misato Ishibashi ◽

Yuka Nishibata ◽

Ayako Sugimoto ◽

...

Keyword(s):

Lung Diseases ◽

Actin Polymerization ◽

Hypoxia Inducible Factor ◽

Neutrophil Extracellular Trap ◽

Chronic Obstructive ◽

Obstructive Pulmonary Disease ◽

Hypoxic Conditions ◽

Pulmonary Arterial ◽

Underlying Diseases

Abstract When patients with preexisting lung diseases, such as chronic obstructive pulmonary disease, interstitial pneumonitis, and pulmonary arterial hypertension, develop pneumonia, the complication often exacerbates the underlying diseases. Although neutrophil extracellular traps (NETs) are important components of innate immune system, the residue of NETs in the tissue can harm the host. We examined the expression of hypoxia-inducible factor 1α (HIF-1α) and NETs in the lungs of patients with lung diseases complicated with pneumonia, and investigated the properties of NETs generated under hypoxia. This study demonstrated that the amount of NETs in pulmonary lesions was greater in patients with pneumonia than in patients without pneumonia and displayed a positive correlation between the amount of NETs and HIF-1α expression. We further demonstrated that the formation of typical lytic NETs was suppressed and round-shaped NETs were generated under hypoxic conditions in vitro. These round NETs were resistant to digestion by the principal NET regulator, DNase I. Focusing on actin rearrangement in neutrophils stimulated under hypoxic conditions, we found that G-actin polymerization and F-actin degradation—both of which are observed time-dependently under normoxic conditions—were disrupted, suggesting that hypoxia mediated the incomplete NET formation. Moreover, neutrophils stimulated under hypoxic conditions possessed cytotoxicity. Accumulation of neutrophils that form degradation-resistant NETs and possess cytotoxicity, which are generated under hypoxic circumstances, are expected to be involved in exacerbation of underlying lung diseases complicated with pneumonia.

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Activating transcription factor 2 increases transactivation and protein stability of hypoxia-inducible factor 1α in hepatocytes

Biochemical Journal ◽

10.1042/bj20090371 ◽

2009 ◽

Vol 424 (2) ◽

pp. 285-296 ◽

Cited By ~ 16

Author(s):

Jeong Hae Choi ◽

Hyun Kook Cho ◽

Yung Hyun Choi ◽

JaeHun Cheong

Keyword(s):

Transcription Factor ◽

Protein Stability ◽

Gene Transcription ◽

Hypoxia Inducible Factor ◽

Protein Stabilization ◽

Hypoxic Conditions ◽

Tumour Suppressor Protein ◽

Activating Transcription Factor

HIF-1 (hypoxia inducible factor 1) performs a crucial role in mediating the response to hypoxia. However, other transcription factors are also capable of regulating hypoxia-induced target-gene transcription. In a previous report, we demonstrated that the transcription factor ATF-2 (activating transcription factor 2) regulates hypoxia-induced gene transcription, along with HIF-1α. In the present study, we show that the protein stability of ATF-2 is induced by hypoxia and the hypoxia-mimic CoCl2 (cobalt chloride), and that ATF-2 induction enhances HIF-1α protein stability via direct protein interaction. The knockdown of ATF-2 using small interfering RNA and translation-inhibition experiments demonstrated that ATF-2 plays a key role in the maintenance of the expression level and transcriptional activity of HIF-1α. Furthermore, we determined that ATF-2 interacts directly with HIF-1α both in vivo and in vitro and competes with the tumour suppressor protein p53 for HIF-1α binding. Collectively, these results show that protein stabilization of ATF-2 under hypoxic conditions is required for the induction of the protein stability and transactivation activity of HIF-1α for efficient hypoxia-associated gene expression.

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Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis

The Journal of Cell Biology ◽

10.1083/jcb.201111063 ◽

2012 ◽

Vol 198 (1) ◽

pp. 103-114 ◽

Cited By ~ 27

Author(s):

Yanmei Qi ◽

Xiaoxiang Tian ◽

Jie Liu ◽

Yaling Han ◽

Alan M. Graham ◽

...

Keyword(s):

Protein A ◽

Hypoxia Inducible Factor ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Epithelial Morphogenesis ◽

Dependent Manner ◽

Interacting Protein ◽

The Core ◽

Adenovirus E1b ◽

Induced Apoptosis

Apoptosis is an essential step in cavitation during embryonic epithelial morphogenesis, but its mechanisms are largely unknown. In this paper, we used embryonic stem cell–differentiated embryoid bodies (EBs) as a model and found that Bnip3 (Bcl-2/adenovirus E1B 19-kD interacting protein), a BH3-only proapoptotic protein, was highly up-regulated during cavitation in a hypoxia-dependent manner. Short hairpin RNA silencing of Bnip3 inhibited apoptosis of the core cells and delayed cavitation. We show that the Bnip3 up-regulation was mediated mainly by hypoxia-inducible factor (HIF)–2. Ablation of HIF-2α or HIF-1β, the common β subunit of HIF-1 and -2, suppressed Bnip3 up-regulation and inhibited apoptosis and cavitation. We further show that apoptosis-inducing factor (AIF) cooperated with Bnip3 to promote lumen clearance. Bnip3 silencing in AIF-null EBs nearly blocked apoptosis and cavitation. Moreover, AIF also regulated Bnip3 expression through mitochondrial production of reactive oxygen species and consequent HIF-2α stabilization. These results uncover a mechanism of cavitation through hypoxia-induced apoptosis of the core cells mediated by HIFs, Bnip3, and AIF.

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The zinc-binding motif in tankyrases is required for the structural integrity and proper function of the catalytic domain

10.1101/2021.10.04.463032 ◽

2021 ◽

Author(s):

Sven T Sowa ◽

Lari Lehtiö

Keyword(s):

Structural Integrity ◽

Catalytic Domain ◽

Zinc Binding ◽

Structural Defects ◽

Proper Function ◽

Binding Motif ◽

Acceptor Site ◽

Structural Element ◽

Zinc Binding Motif

Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and they are therefore possible drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyl-transferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on activity, stability and structure of human tankyrases. We generated mutants of human TNKS1 and TNKS2 abolishing the zinc-binding capabilities and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive likely due to introduction of major structural defects, the TNKS2 mutant remained active and displayed a different activity profile compared to the wild type.

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H2S-stimulated bioenergetics in chicken erythrocytes and the underlying mechanism

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00348.2019 ◽

2020 ◽

Vol 319 (1) ◽

pp. R69-R78

Author(s):

Zhuping Jin ◽

Quanxi Zhang ◽

Eden Wondimu ◽

Richa Verma ◽

Ming Fu ◽

...

Keyword(s):

Electron Transport ◽

Mammalian Cells ◽

Electron Transport Chain ◽

Structural Integrity ◽

Underlying Mechanism ◽

Atp Content ◽

Hypoxic Conditions ◽

Transport Chain ◽

Chicken Erythrocytes

The production of H2S and its effect on bioenergetics in mammalian cells may be evolutionarily preserved. Erythrocytes of birds, but not those of mammals, have a nucleus and mitochondria. In the present study, we report the endogenous production of H2S in chicken erythrocytes, which was mainly catalyzed by 3-mercaptopyruvate sulfur transferase (MST). ATP content of erythrocytes was increased by MST-generated endogenous H2S under normoxic, but not hypoxic, conditions. NaHS, a H2S salt, increased ATP content under normoxic, but not hypoxic, conditions. ATP contents in the absence or presence of NaHS were eliminated by different inhibitors for mitochondrial electron transport chain in chicken erythrocytes. Succinate and glutamine, but not glucose, increased ATP content. NaHS treatment similarly increased ATP content in the presence of glucose, glutamine, or succinate, respectively. Furthermore, the expression and activity of sulfide:quinone oxidoreductase were enhanced by NaHS. The structural integrity of chicken erythrocytes was largely maintained during 2-wk NaHS treatment in vitro, whereas most of the erythrocytes without NaHS treatment were lysed. In conclusion, H2S may regulate cellular bioenergetics as well as cell survival of chicken erythrocytes, in which the functionality of the electron transport chain is involved. H2S may have different regulatory roles and mechanisms in bioenergetics of mammalian and bird cells.

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Hypoxia induces B-type natriuretic peptide release in cell lines derived from human cardiomyocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00250.2009 ◽

2009 ◽

Vol 297 (2) ◽

pp. H550-H555 ◽

Cited By ~ 44

Author(s):

Gregori Casals ◽

Josefa Ros ◽

Alessandro Sionis ◽

Mercy M. Davidson ◽

Manuel Morales-Ruiz ◽

...

Keyword(s):

Natriuretic Peptide ◽

Transcriptional Activity ◽

Molecular Mechanisms ◽

Ventricular Myocytes ◽

Hypoxia Inducible Factor ◽

Peptide Hormone ◽

Mrna Levels ◽

Human Cardiomyocytes ◽

Hypoxic Conditions

B-type natriuretic peptide (BNP) is a peptide hormone of myocardial origin with significant cardioprotective properties. Patients with myocardial ischemia present with high levels of BNP in plasma and elevated expression in the myocardium. However, the molecular mechanisms of BNP induction in the ischemic myocardium are not well understood. The aim of the investigation was to assess whether myocardial hypoxia induces the production of BNP in human ventricular myocytes. To test the hypothesis that reduced oxygen tension can directly stimulate BNP gene expression and release in the absence of hemodynamic or neurohormonal stimuli, we used an in vitro model system of cultured human ventricular myocytes (AC16 cells). Cells were cultured under normoxic (21% O2) or hypoxic (5% O2) conditions for up to 48 h. The accumulation of BNP, atrial natriuretic peptide (ANP), and vascular endothelial growth factor (VEGF) was then measured. Hypoxia stimulated the protein release of BNP and VEGF but not ANP. In concordance, the increased mRNA levels of BNP and VEGF but not ANP were found on culturing AC16 cells under hypoxic conditions. The analysis of the transcriptional activity of the hypoxia-inducible factor 1 (HIF-1) in nuclear extracts showed that HIF-1 activity was induced under hypoxic conditions. Finally, the treatment of AC16 cells with the HIF-1 inhibitor rotenone in hypoxia inhibited BNP and VEGF release. In conclusion, these data indicate that hypoxia induces the synthesis and secretion of BNP in human ventricular myocytes, likely through HIF-1-enhanced transcriptional activity.

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Abstract 82: Regulation Of Frataxin By HIF-1 In The Ischemic Diabetic Heart

Circulation Research ◽

10.1161/res.115.suppl_1.82 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Raj H Amin ◽

Abdullah AlAsmari ◽

Gayani Nanayakkari ◽

John Quindry ◽

Shavanthi Mouli ◽

...

Keyword(s):

Heart Failure ◽

Insulin Signalling ◽

Ischemia Reperfusion ◽

Mitochondrial Protein ◽

Hypoxia Inducible Factor ◽

Diabetic Heart ◽

Diabetic Patients ◽

Hypoxic Injury ◽

Infarction Size

Background: Diabetes is at epidemic proportions, with the major form of fatality due to congestive heart failure triggered by myocardial infarction (MI). The impaired insulin signalling in the diabetic heart leads to myocardial energy dysregulation that compromises the cardioprotective mechanism against ischemic injury. Therefore understanding how mitochondrial energetics is altered in the diabetic ischemic heart would greatly advance the knowledge base for improving outcomes from heart failure in diabetic patients. Methods/Findings: We observed that db/db mice (leptin deficient, type 2 diabetic mice) have increased infarction size (>30%) compared to wild type mice after ischemia/reperfusion (IR) injury by TTC stain. We also found that activity of Hypoxia inducible factor-1 (HIF1) is involved in the cardioprotective response to ischemia, is impaired in db/db hearts. HIF1 is known to transcriptionally regulate genes involved in myocardial energetics. We recently found that HIF1 transcriptionally regulates the mitochondrial protein frataxin (Fxn) in cardiomyocytes as determined by luciferase assays (>3 fold). In vitro studies indicate that hypoxic conditions increase Fxn protein expression in cardiomyocytes as determined by western analysis (2 fold). Fxn plays an important role in the Fe-S cluster biogenesis required for aconitase, succinate dehydrogenase and complexes in the mitochondria. Interestingly, we observed decreased expression of Fxn in the ischemic diabetic heart. Conclusion: we postulate that attenuated HIF1-Fxn signalling in ischemic diabetic heart leads to abnormally enlarged infarction size in response to IR. The decline in HIF-1 activity in response to hypoxia was further validated in cardiomyocytes cultured in high glucose media. The significance for Fxn against hypoxic injury was confirmed by utilizing overexpressed Fxn cardiomyocytes via MTT, ATP and aconitase activity assays. Current and future work: currently we are attempting to identify the HIF response element (HRE) in Fxn promoter to further validate the transcriptional activity of HIF1. In addition, we are completing the IR surgeries on HIF1 KO mice to address the cardioprotective nature of HIF1-Fxn signalling against MI.

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Monoclonal Antibody-Based Screening Assay for Factor Inhibiting Hypoxia-Inducible Factor Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108318800 ◽

2008 ◽

Vol 13 (6) ◽

pp. 494-503 ◽

Cited By ~ 23

Author(s):

Sang-Hyeup Lee ◽

Jeong Hee Moon ◽

Eun Ah Cho ◽

Seong-Eon Ryu ◽

Myung Kyu Lee

Keyword(s):

Monoclonal Antibody ◽

High Throughput Screening ◽

Transcriptional Activity ◽

Hypoxia Inducible Factor ◽

Enzyme Linked Immunosorbent Assay ◽

Sensitive Assay ◽

Screening Assay ◽

Hypoxic Conditions

The factor-inhibiting hypoxia-inducible factor (FIH) hydroxylates the asparagine 803 (Asn803) residue of the hypoxia-inducible factor 1α (HIF-1α), and the modification abrogates the transcriptional activity of HIF-1α. Because FIH is more active on HIF-1α than prolyl hydroxylase domain proteins under hypoxic conditions, its inhibitors have potential to be developed as anti-ischemic drugs targeting normal cells stressed by hypoxia. In this study, the authors developed the first monoclonal antibody, SHN-HIF1α, specifically to Asn803 hydroxylated HIF-1α and a sensitive assay system for FIH inhibitors using the monoclonal antibody (Mab). SHN-HIF1α showed 740 times higher affinity to the Asn803 hydroxylated HIF-1α peptide than the unmodified one. An enzyme-linked immunosorbent assay—based system using SHN-HIF1α displayed at least 30 times more sensitivity than previous methods for screening FIH inhibitors and was easily applicable to develop a high-throughput screening system. SHN-HIF1α also showed an Asn803 hydroxylation-dependent specificity to HIF-1α in cells. Taken together, the results suggest that it may be used to analyze the in vivo and in vitro activities of FIH inhibitors. ( Journal of Biomolecular Screening 2008:494-503)

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The MAP Kinase Phosphatase MKP-1 Modulates Neurogenesis via Effects on BNIP3 and Autophagy

Biomolecules ◽

10.3390/biom11121871 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1871

Author(s):

Yinghui Li ◽

Marc W. Halterman

Keyword(s):

Stem Cell ◽

Map Kinase ◽

Neural Stem Cell ◽

Neural Plasticity ◽

Stem Cell Differentiation ◽

Interacting Protein ◽

Critical Window ◽

Map Kinase Phosphatase ◽

Adenovirus E1b

Inherited and acquired defects in neurogenesis contribute to neurodevelopmental disorders, dysfunctional neural plasticity, and may underlie pathology in a range of neurodegenerative conditions. Mitogen-activated protein kinases (MAPKs) regulate the proliferation, survival, and differentiation of neural stem cells. While the balance between MAPKs and the family of MAPK dual-specificity phosphatases (DUSPs) regulates axon branching and synaptic plasticity, the specific role that DUSPs play in neurogenesis remains unexplored. In the current study, we asked whether the canonical DUSP, MAP Kinase Phosphatase-1 (MKP-1), influences neural stem cell differentiation and the extent to which DUSP-dependent autophagy is operational in this context. Under basal conditions, Mkp-1 knockout mice generated fewer doublecortin (DCX) positive neurons within the dentate gyrus (DG) characterized by the accumulation of LC3 puncta. Analyses of wild-type neural stem cell (NSC) differentiation in vitro revealed increased Mkp-1 mRNA expression during the initial 24-h period. Notably, Mkp-1 KO NSC differentiation produced fewer Tuj1-positive neurons and was associated with increased expression of the BCL2/adenovirus E1B 19-kD protein-interacting protein 3 (BNIP3) and levels of autophagy. Conversely, Bnip3 knockdown in differentiated Mkp-1 KO NSCs reduced levels of autophagy and increased neuronal yields. These results indicate that MKP-1 exerts a pro-neurogenic bias during a critical window in NSC differentiation by regulating BNIP3 and basal autophagy levels.

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