scholarly journals Oxygen impairs oligodendroglial development via oxidative stress and reduced expression of HIF-1α

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Christina Brill ◽  
Till Scheuer ◽  
Christoph Bührer ◽  
Stefanie Endesfelder ◽  
Thomas Schmitz
Keyword(s):  
Oxidative Stress ◽  
Hypoxia Inducible Factor ◽  
Precursor Cell ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Low Oxygen ◽  
Hypoxia Inducible Factor 1 ◽  
Reporter Assays ◽  
Induced Changes ◽  
Cells Cultured

Abstract The premature increase of oxygen tension may contribute to oligodendroglial precursor cell (OPC) damage in preterm infants. Fetal OPCs are exposed to low oxygen tissue tensions not matched when cells are cultured in room air. Maturation (A2B5, O4, O1, MBP, CNP, arborization), oxidative stress (nitrotyrosine Western blot, NRF2 and SOD2 expression), apoptosis (TUNEL), proliferation (Ki67), and expression of transcription factors regulated by Hypoxia-Inducible-Factor-1-alpha (Hif-1α) expressed in OPCs (Olig1, Olig2, Sox9, Sox10) were assessed in rat OPCs and OLN93 cells cultured at 5% O2 and 21% O2. Influences of Hif-1α were investigated by Hif-1α luciferase reporter assays and Hif-1α-knockdown experiments. At 21% O2, cell proliferation was decreased and process arborization of OPCs was reduced. Expression of MBP, CNP, Olig1, Sox9 and Sox10 was lower at 21% O2, while Nrf2, SOD2, nitrotyrosine were increased. Apoptosis was unchanged. Luciferease reporter assay in OLN93 cells indicated increased Hif-1α activity at 5% O2. In OLN93 cells at 5% O2, Hif-1α knockdown decreased the expression of MBP and CNP, similar to that observed at 21% O2. These data indicate that culturing OPCs at 21% O2 negatively affects development and maturation. Both enhanced oxidative stress and reduced expression of Hif-1α-regulated genes contribute to these hyperoxia-induced changes.

scholarly journals Sequential Activation of Hypoxia-Inducible Factor 1 and Specificity Protein 1 is Required for Hypoxia-Induced Transcriptional Stimulation of Abcc8

2011 ◽  
Vol 32 (3) ◽  
pp. 525-536 ◽  
Author(s):  
Seung Kyoon Woo ◽  
Min Seong Kwon ◽  
Zhihua Geng ◽  
Zheng Chen ◽  
Alexander Ivanov ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Binding Sites ◽  
Hypoxia Inducible Factor ◽  
Luciferase Reporter ◽  
Hypoxia Inducible Factor 1 ◽  
Specificity Protein 1 ◽  
Reporter Assays ◽  
Sequential Activation ◽  
Specificity Protein ◽  
Stimulation Of

Cerebral ischemia causes increased transcription of sulfonylurea receptor 1 (SUR1), which forms SUR1-regulated NC(Ca-ATP) channels linked to cerebral edema. We tested the hypothesis that hypoxia is an initial signal that stimulates transcription of Abcc8, the gene encoding SUR1, via activation of hypoxia-inducible factor 1 (HIF1). In the brain microvascular endothelial cells, hypoxia increased SUR1 abundance and expression of functional SUR1-regulated NC(Ca-ATP) channels. Luciferase reporter activity driven by the Abcc8 promoter was increased by hypoxia and by coexpression of HIF1α. Surprisingly, a series of luciferase reporter assays studying the Abcc8 promoter revealed that binding sites for specificity protein 1 (Sp1), but not for HIF, were required for stimulation of Abcc8 transcription by HIF1α. Luciferase reporter assays studying Sp1 promoters of three species, and chromatin immunoprecipitation analysis in rats after cerebral ischemia, indicated that HIF binds to HIF-binding sites on the Sp1 promoter to stimulate transcription of the Sp1 gene. We conclude that sequential activation of two transcription factors, HIF and Sp1, is required to stimulate transcription of Abcc8 following cerebral ischemia. Sequential gene activation in cerebral ischemia provides a plausible molecular explanation for the prolonged treatment window observed for inhibition of the end-target gene product, SUR1, by glibenclamide.

scholarly journals Induction of Gastrin Expression in Gastrointestinal Cells by Hypoxia or Cobalt Is Independent of Hypoxia-Inducible Factor (HIF)

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3006-3016 ◽  
Author(s):  
Lin Xiao ◽  
Suzana Kovac ◽  
Mike Chang ◽  
Arthur Shulkes ◽  
Graham S. Baldwin ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Hypoxia Inducible Factor ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Human Gastric Cancer ◽  
Low Oxygen ◽  
Ags Cells ◽  
Cholecystokinin Receptor ◽  
Hypoxia Inducible Factor 1

Gastrin and its precursors have been shown to promote mitogenesis and angiogenesis in gastrointestinal tumors. Hypoxia stimulates tumor growth, but its effect on gastrin gene regulation has not been examined in detail. Here we have investigated the effect of hypoxia on the transcription of the gastrin gene in human gastric cancer (AGS) cells. Gastrin mRNA was measured by real-time PCR, gastrin peptides were measured by RIA, and gastrin promoter activity was measured by dual-luciferase reporter assay. Exposure to a low oxygen concentration (1%) increased gastrin mRNA concentrations in wild-type AGS cells (AGS) and in AGS cells overexpressing the gastrin receptor (AGS-cholecystokinin receptor 2) by 2.1 ± 0.4- and 4.1 ± 0.3-fold (P < 0.05), respectively. The hypoxia mimetic, cobalt chloride (300 μM), increased gastrin promoter activity in AGS cells by 2.4 ± 0.3-fold (P < 0.05), and in AGS-cholecystokinin receptor 2 cells by 4.0 ± 0.3-fold (P < 0.05), respectively. The observations that either deletion from the gastrin promoter of the putative binding sites for the transcription factor hypoxia-inducible factor 1 (HIF-1) or knockdown of either the HIF-1α or HIF-1β subunit did not affect gastrin promoter inducibility under hypoxia indicated that the hypoxic activation of the gastrin gene is likely HIF independent. Mutational analysis of previously identified Sp1 regulatory elements in the gastrin promoter also failed to abrogate the induction of promoter activity by hypoxia. The observations that hypoxia up-regulates the gastrin gene in AGS cells by HIF-independent mechanisms, and that this effect is enhanced by the presence of gastrin receptors, provide potential targets for gastrointestinal cancer therapy.

scholarly journals Crosstalk in oxygen homeostasis networks: SKN-1/NRF inhibits the HIF-1 hypoxia-inducible factor in Caenorhabditis elegans

2021 ◽  
Author(s):  
Dingxia Feng ◽  
Zhiwei Zhai ◽  
Zhiyong Shao ◽  
Yi Zhang ◽  
Jo Anne Powell-Coffman
Keyword(s):  
Oxidative Stress ◽  
Hypoxia Inducible Factor ◽  
Regulatory Sequences ◽  
Low Oxygen ◽  
Transcriptional Responses ◽  
C Elegans ◽  
Protein Levels ◽  
Oxygen Homeostasis ◽  
Genome Wide ◽  
A Genome

AbstractDuring development, homeostasis, and disease, organisms must balance responses that allow adaptation to low oxygen (hypoxia) with those that protect cells from oxidative stress. The evolutionarily conserved hypoxia-inducible factors are central to these processes, as they orchestrate transcriptional responses to oxygen deprivation. Here, we employ genetic strategies in C. elegans to identify stress-responsive genes and pathways that modulate the HIF-1 hypoxia-inducible factor and facilitate oxygen homeostasis. Through a genome-wide RNAi screen, we show that RNAi-mediated mitochondrial or proteasomal dysfunction increases the expression of hypoxia-responsive reporter Pnhr-57:GFP in C. elegans. Interestingly, only a subset of these effects requires hif-1. Of particular importance, we found that skn-1 RNAi increases the expression of hypoxia-responsive reporter Pnhr-57:GFP and elevates HIF-1 protein levels. The SKN-1/NRF transcription factor has been shown to promote oxidative stress resistance. We present evidence that the crosstalk between HIF-1 and SKN-1 is mediated by EGL-9, the prolyl hydroxylase that targets HIF-1 for oxygen-dependent degradation. Treatment that induces SKN-1, such as heat, increases expression of a Pegl-9:GFP reporter, and this effect requires skn-1 function and a putative SKN-1 binding site in egl-9 regulatory sequences. Collectively, these data support a model in which SKN-1 promotes egl-9 transcription, thereby inhibiting HIF-1. We propose that this interaction enables animals to adapt quickly to changes in cellular oxygenation and to better survive accompanying oxidative stress.

scholarly journals MicroRNA-16, via FGF2 Regulation of the ERK/MAPK Pathway, Is Involved in the Magnesium-Promoted Osteogenic Differentiation of Mesenchymal Stem Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-14 ◽  
Author(s):  
Hong Qi ◽  
Yang Liu ◽  
Lu Wu ◽  
Su Ni ◽  
Jing Sun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Mapk Pathway ◽  
Luciferase Reporter ◽  
Erk Mapk ◽  
Marrow Mesenchymal Stem Cells ◽  
Elevated Expression ◽  
Reporter Assays ◽  
Induced Changes

microRNAs (miRNAs) participate in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). However, few reports have discussed the effect of miRNAs on the magnesium chloride (MgCl2)-induced promotion of osteogenic differentiation of BMSCs, a process involved in the healing of bone tissue. As determined in the present investigation, MgCl2 decreased miR-16 levels; increased levels of fibroblast growth factor 2 (FGF2), p-p38, and p-ERK; and promoted the osteogenic differentiation of BMSCs. Enhancement of miR-16 levels by an miR-16 mimic blocked these MgCl2-induced changes. Moreover, luciferase reporter assays confirmed that miR-16 binds to the 3′UTR region of FGF2 mRNA. Down-regulation of FGF2 blocked the MgCl2-induced increases of p-p38 and p-ERK and the promotion of the osteogenic differentiation of BMSCs. Furthermore, over-expression of miR-16 attenuated the MgCl2-induced overproduction of p-p38 and p-ERK1/2 and the high levels of osteogenic differentiation, effects that were reversed by elevated expression of FGF2. In summary, the present findings provide a mechanism by which miR-16 regulates MgCl2-induced promotion of osteogenic differentiation by targeting FGF2-mediated activation of the ERK/MAPK pathway.

scholarly journals Induction of Redox-Active Gene Expression by CoCl2 Ameliorates Oxidative Stress-Mediated Injury of Murine Auditory Cells

Antioxidants ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 399 ◽  
Author(s):  
Jhang Ho Pak ◽  
Junyeong Yi ◽  
Sujin Ryu ◽  
In Ki Kim ◽  
Jung-Woong Kim ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Ganglion Cells ◽  
Stria Vascularis ◽  
Hypoxia Inducible Factor ◽  
Spiral Ganglion ◽  
Organ Of Corti ◽  
Luciferase Reporter ◽  
Related Factor ◽  
Redox Active

Free radicals formed in the inner ear in response to high-intensity noise, are regarded as detrimental factors for noise-induced hearing loss (NIHL). We reported previously that intraperitoneal injection of cobalt chloride attenuated the loss of sensory hair cells and NIHL in mice. The present study was designed to understand the preconditioning effect of CoCl2 on oxidative stress-mediated cytotoxicity. Treatment of auditory cells with CoCl2 promoted cell proliferation, with increases in the expressions of two redox-active transcription factors (hypoxia-inducible factor 1α, HIF-1α, nuclear factor erythroid 2-related factor 2; Nrf-2) and an antioxidant enzyme (peroxiredoxin 6, Prdx6). Hydrogen peroxide treatment resulted in the induction of cell death and reduction of these protein expressions, reversed by pretreatment with CoCl2. Knockdown of HIF-1α or Nrf-2 attenuated the preconditioning effect of CoCl2. Luciferase reporter analysis with a Prdx6 promoter revealed transactivation of Prdx6 expression by HIF-1α and Nrf-2. The intense immunoreactivities of HIF-1α, Nrf-2, and Prdx6 in the organ of Corti (OC), spiral ganglion cells (SGC), and stria vascularis (SV) of the cochlea in CoCl2-injected mice suggested CoCl2-induced activation of HIF-1α, Nrf-2, and Prdx6 in vivo. Therefore, we revealed that the protective effect of CoCl2 is achieved through distinctive signaling mechanisms involving HIF-1α, Nrf-2, and Prdx6.

scholarly journals MicroRNA 648 Targets ET-1 mRNA and Is Cotranscriptionally Regulated withMICAL3by PAX5

2014 ◽  
Vol 35 (3) ◽  
pp. 514-528 ◽  
Author(s):  
Chen Li ◽  
Caryn S. Gonsalves ◽  
Marthe-Sandrine Eiymo Mwa Mpollo ◽  
Punam Malik ◽  
Stanley M. Tahara ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Posttranscriptional Regulation ◽  
Hypoxia Inducible Factor ◽  
Positive Control ◽  
Luciferase Reporter ◽  
Lim Domain ◽  
Placenta Growth Factor ◽  
Reporter Assays ◽  
Potent Vasoconstrictor ◽  
Distal Promoter

Pulmonary hypertension (PHT) is associated with high mortality in sickle cell anemia (SCA). Previously, we showed that elevated levels of placenta growth factor (PlGF) in SCA patients correlate with increased levels of the potent vasoconstrictor endothelin-1 (ET-1) and PHT. Moreover, PlGF induced the expression of ET-1 via hypoxia-inducible factor 1α. Here, we show a novel example of ET-1 posttranscriptional regulation by PlGF via action of microRNA 648 (miR-648), which is subject to transcriptional coregulation with its host gene,MICAL3(microtubule-associated monooxygenase, calponin, and LIM domain containing 3gene). PlGF repressed expression of miR-648 in endothelial cells. Luciferase reporter assays using wild-type and mutant ET-1 3′ untranslated region (UTR) constructs, and transfection of miR-648 mimics showed that miR-648 targets the 3′ UTR of ET-1 mRNA. Since miR-648 is located in a 5′-proximal intron ofMICAL3, we examined which of three potential promoters was responsible for its expression. TheMICAL3distal promoter (P1) was the predominant promoter used for transcription of pre-miR-648, and it was under positive control by PAX5 (paired box protein 5) transcription factor, as demonstrated by the loss and gain of function of PAX5 activity, and chromatin immunoprecipitation analysis. These studies provide a novel link wherein PlGF-mediated downregulation of PAX5 attenuates miR-648 expression leading to increased ET-1 levels that are known to induce PHT in SCA.

scholarly journals Propofol Attenuates Hypoxia-Induced Inflammation in BV2 Microglia by Inhibiting Oxidative Stress and NF-κB/Hif-1α Signaling

2020 ◽  
Vol 2020 ◽  
pp. 1-11 ◽  
Author(s):  
Xiaowei Peng ◽  
Chenglong Li ◽  
Wei Yu ◽  
Shuai Liu ◽  
Yushuang Cong ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hypoxia Inducible Factor ◽  
Underlying Mechanism ◽  
Beneficial Effects ◽  
Hypoxia Inducible Factor 1 ◽  
B Subunit ◽  
Bv2 Cells ◽  
Total Antioxidant ◽  
Bv2 Microglia ◽  
Interfering Rna

Hypoxia-induced neuroinflammation typically causes neurological damage and can occur during stroke, neonatal hypoxic-ischemic encephalopathy, and other diseases. Propofol is widely used as an intravenous anesthetic. Studies have shown that propofol has antineuroinflammatory effect. However, the underlying mechanism remains to be fully elucidated. Thus, we aimed to investigate the beneficial effects of propofol against hypoxia-induced neuroinflammation and elucidated its potential cellular and biochemical mechanisms of action. In this study, we chose cobalt chloride (CoCl2) to establish a hypoxic model. We found that propofol decreased hypoxia-induced proinflammatory cytokines (TNFα, IL-1β, and IL-6) in BV2 microglia, significantly suppressed the excessive production of reactive oxygen species, and increased the total antioxidant capacity and superoxide dismutase activity. Furthermore, propofol attenuated the hypoxia-induced decrease in mitochondrial membrane potential andy 2 strongly inhibited protein expression of nuclear factor-kappa B (NF-κB) subunit p65 and hypoxia inducible factor-1α (Hif-1α) in hypoxic BV2 cells. To investigate the role of NF-κB p65, specific small interfering RNA (siRNA) against NF-κB p65 were transfected into BV2 cells, followed by exposure to hypoxia for 24 h. Hypoxia-induced Hif-1α production was downregulated after NF-κB p65 silencing. Further, propofol suppressed Hif-1α expression by inhibiting the upregulation of NF-κB p65 after exposure to hypoxia in BV2 microglia. In summary, propofol attenuates hypoxia-induced neuroinflammation, at least in part by inhibiting oxidative stress and NF-κB/Hif-1α signaling.

scholarly journals Mir-141-3p Regulates Apoptosis and Mitochondrial Membrane Potential via Targeting Sirtuin1 in a 1-Methyl-4-Phenylpyridinium in vitro Model of Parkinson’s Disease

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Yumin Zheng ◽  
Li Dong ◽  
Na Liu ◽  
Xiaoguang Luo ◽  
Zhiyi He
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Western Blot ◽  
Pc12 Cells ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Luciferase Reporter ◽  
Ros Production ◽  
Reporter Assay ◽  
Induced Apoptosis

Objectives. Parkinson’s disease (PD) is a common neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons in the substantia nigra. The present study investigated miR-141-3p/sirtuin1 (SIRT1) activity in a 1-methyl-4-phenylpyridinium- (MPP+-) induced PC12-cell model of PD. Methods. PC12 cells were exposed to MMP+ following induction of differentiation by nerve growth factor (NGF). miR-141-3p and SIRT1 expressions were examined using RT-qPCR and western blot. Cell viability was evaluated using the MTT assay. Apoptosis percentage, reactive oxygen species (ROS) production, and mitochondrial membrane potential (Δψm) were evaluated using flow cytometry. Expression of Nuclear factor-kappa B- (NF-κB-) related proteins was determined by western blot. Bioinformatic analysis, RT-qPCR, and luciferase reporter assay were used to confirm the interaction between miR-141-3p and SIRT1. Results. miR-141-3p was upregulated, and SIRT1 was downregulated in MPP+-treated PC12 cells. MPP+ treatment also upregulated nitric oxide synthase 1 (Nos1) and α-synuclein. miR-141-3p induced apoptosis, oxidative stress, mitochondrial dysfunction, and downregulated the SIRT1 mRNA expression. The luciferase reporter assay showed that SIRT1 was the target of miR-141-3p. SIRT1 transfection attenuated apoptosis, ROS production and maintained Δψm. SIRT1 also downregulated Nos1, tumor necrosis factor-α (TNF-α), interleukin 1 beta (IL-1β), interleukin 6(IL-6) and upregulated B cell lymphoma 2 (Bcl-2) protein. In addition, SIRT1 activator resveratrol blocked the effects of miR-141-3p mimic on Nos1, α-synuclein, and mitochondrial membrane potential. SIRT1 inhibitor sirtinol reversed the biological effects of miR-141-3p. Conclusion. Increased miR-141-3p induced apoptosis, oxidative stress, and mitochondrial dysfunction in MPP+-treated PC12 cells by directly targeting the SIRT1 expression. Our study provided a potential therapeutic strategy for PD.

scholarly journals Mir-29b Regulates Oxidative Stress by Targeting SIRT1 in Ovarian Cancer Cells

2017 ◽  
Vol 43 (5) ◽  
pp. 1767-1776 ◽  
Author(s):  
Meng Hou ◽  
Xiaohang Zuo ◽  
Chen Li ◽  
Yan Zhang ◽  
Yue Teng
Keyword(s):  
Oxidative Stress ◽  
Ovarian Cancer ◽  
Cell Viability ◽  
Metabolic Regulation ◽  
Critical Role ◽  
Annexin V ◽  
Luciferase Reporter ◽  
Ovarian Cancer Cells ◽  
Dependent Manner ◽  
Reporter Assays

Background: Metabolic abnormalities are frequently observed in multiple malignancies including epithelial ovarian cancer (EOC), among which imbalance between generation and elimination of reactive oxygen species (ROS) plays a critical role in EOC onset and progression. Here we investigated the role of miR-29b, a well-established tumor-suppressor miRNA in metabolic regulation of EOC cells. Methods: cell viability and apoptosis in miR-29b inhibited and over-expressed EOC cells were evaluated by CCK8 and Annexin V–FITC/PI assays. Change in miR-29b was detected in EOC cells incubated in H2O2 culture by q-PCR. Relative ROS levels were also detected in different EOC cultures, including modified miR-29b and SIRT1 levels as well as H2O2 incubation. A luciferase reporter assay was employed to detect the direct binding of miR-29b to SIRT1 3’ UTR. Changes in cell viability and ROS levels were assessed in SIRT1-knocked down EOC cells. Results: miR-29b expression correlates with decreased EOC cell viability and increased apoptosis. H2O2 downregulated miR-29b in a time and dose-dependent manner. miR-29b expression negatively correlated with ROS levels, whereas SIRT1 significantly stimulated ROS formation. Luciferase reporter assays confirmed miR-29b downregulation of SIRT1by directly targeting its mRNA 3’-UTR. SIRT1 silencing rescues cell viability of H2O2 treated cells. Also, SIRT1 inhibition blocked cell apoptosis induced by H2O2 as well as reduced intracellular ROS levels. Conclusion: Together, our findings indicated that the miR-29b/SIRT1 axis has a protective effect against H2O2-induced damage of cell viability and oxidative stress and may provide novel options for miR-29b-based therapeutic approaches for EOC treatment.

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