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 Characterization of the human topoisomerase IIβ (TOP2B) promoter activity: essential roles of the nuclear factor-Y (NF-Y)- and specificity protein-1 (Sp1)-binding sites

2002 ◽  
Vol 368 (3) ◽  
pp. 741-751 ◽  
Author(s):  
Chun-Nam LOK ◽  
Alexander J. LANG ◽  
Shelagh E.L. MIRSKI ◽  
Susan P.C. COLE
Keyword(s):  
Binding Sites ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Cancer Drug ◽  
Resting Cells ◽  
Nuclear Factor ◽  
Nuclear Factor Y ◽  
Specificity Protein 1 ◽  
Topo Ii ◽  
Specificity Protein

Eukaryotic topoisomerase II (topo II) catalyses topological genomic changes essential for chromosome segregation, chromatin reorganization, DNA replication and transcription. Mammalian topo II exists as two isoforms, designated α and β. Human topo IIα is an important cancer drug target, and an established determinant of drug sensitivity and resistance. Human topo IIβ is also the target of anticancer drugs but its role in drug resistance is less clear. The two human topo II proteins are encoded by the TOP2A and TOP2B genes, respectively, which despite their highly conserved structural organization, are subject to distinctly different modes of regulation. In the present study, we have cloned and characterized the human TOP2B promoter containing a 1.3kb fragment of the 5′-flanking and untranslated region (-1067 to +193). We found that the promoter activity of this TOP2B fragment was constant throughout the cell cycle, in contrast to the activity of the proximal promoter of TOP2A which was low in resting cells and enhanced during proliferation. Analyses of 5′-serially and internally deleted luciferase reporter constructs revealed that 80% of the TOP2B promoter activity could be attributed to the region between −533 and −481. Mutational analyses of putative regulatory elements indicated that two inverted CCAAT boxes (ICBs) within this region were essential for TOP2B promoter activity and gel mobility-shift assays indicated these sites bound the transcription factor nuclear factor-Y (NF-Y). Co-transfection experiments using a dominant-negative form of subunit A of NF-Y suggested that TOP2B promoter activity required direct interaction of NF-Y with the ICBs. In addition, a specificity protein-1 (Sp1)-binding GC box located just upstream of the ICBs was shown to contribute to TOP2B promoter activity in a synergistic manner with the ICBs. Our results suggest that the binding sites for NF-Y and Sp1 are critical for TOP2B transcription.

scholarly journals Inherited/Genetically-Associated Pheochromocytoma/ Paraganglioma Syndromes and COVID-19

Medicina ◽  
2021 ◽  
Vol 57 (10) ◽  
pp. 1033
Author(s):  
Ioannis Ilias ◽  
Gregory Kaltsas ◽  
Konstantinos Barkas ◽  
George P. Chrousos
Keyword(s):  
Protective Effect ◽  
Viral Entry ◽  
Binding Sites ◽  
Hypoxia Inducible Factor ◽  
Therapeutic Interventions ◽  
Spike Protein ◽  
Hypoxia Inducible Factor 1 ◽  
Stimulation Of ◽  
Angiotensin Converting Enzymes

In some subjects with inherited pheochromocytoma/paraganglioma (PPG) syndromes, hypoxia-inducible factor 1 alpha (HIF1α) stabilization/activation could lead to an increase in angiotensin converting enzymes (ACE). This would result in the stimulation of angiotensin (AT) II production and, hence, reduce the availability of ACE 2. The latter would provide decreased numbers of binding sites for the spike protein of SARS-CoV-2 and, therefore, result in less points of viral entry into cells. Thus, subjects with HIF1α-associated PPG syndromes may benefit from an inherent protective effect against COVID-19. Such an implication of HIF1α vis-à-vis COVID-19 could open ways of therapeutic interventions.

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 Neuronal insulin receptor substrate 2 (IRS2) expression is regulated by ZBP89 and SP1 binding to the IRS2 promoter

2009 ◽  
Vol 204 (2) ◽  
pp. 199-208 ◽  
Author(s):  
Michael Udelhoven ◽  
Mareike Pasieka ◽  
Uschi Leeser ◽  
Wilhelm Krone ◽  
Markus Schubert
Keyword(s):  
Insulin Receptor ◽  
Binding Sites ◽  
Luciferase Reporter ◽  
Serum Starvation ◽  
Insulin Receptor Substrate ◽  
Dependent Manner ◽  
Reporter Assays ◽  
Phosphoinositide 3 Kinase ◽  
Translational Start ◽  
Specificity Protein

Since neuronal insulin receptor substrate 2 (IRS2)-mediated signals coordinate key processes in rodent physiology such as food intake, fertility, longevity, and aging-related behavior, we analyzed the mechanisms of neuronal IRS2 expression in neuroblastoma (SHSY5Y) and hypothalamic (GT1-7) cell lines. Using dual luciferase reporter assays and IRS2 promoter deletion constructs, we identified a regulatory cassette within the IRS2 promoter between −779 and −679 bp from the translational start which is responsible for ∼50% of neuronal IRS2 promoter activity. Chromatin immunoprecipitation assays and electromobility shift assay revealed four overlapping ZBP89/specificity protein 1 (SP1) binding sites which alternatively bind to ZBP89 (ZNF148 as listed in the HUGO Database) or SP1. Activation of this cassette is inhibited by phosphoinositide-3-kinase (PI3K) via increased ZBP89 binding to the promoter. Serum starvation caused increased SP1 binding at one specific SP1 site and decreased binding to another, proving a regulatory interaction between the different binding sites within this promoter cassette to tightly control IRS2 expression. Mutants containing all the possible combinations of one, two, three, or all the four SP1 binding sites of the IRS2 promoter revealed that SP1 binding to one particular site is most important for promoter activation. Stable downregulation of ZBP89 using siRNA substantially increased IRS2 mRNA and protein expression. Thus, alternative binding of ZBP89 or SP1 to the described region in the IRS2 promoter regulates neuronal IRS2 expression in a PI3K-dependent manner.

scholarly journals Krüppel-like factor 4 plays a role in the luteal transition in steroidogenesis by downregulating Cyp19A1 expression

2019 ◽  
Vol 316 (6) ◽  
pp. E1071-E1080 ◽  
Author(s):  
Hyeonhae Choi ◽  
Ki-Young Ryu ◽  
Jaesook Roh
Keyword(s):  
Luciferase Reporter ◽  
Luteal Cell ◽  
Rapid Decline ◽  
Binding Motif ◽  
Steroidogenic Factor 1 ◽  
Estrogen Production ◽  
Reporter Assays ◽  
Direct Binding ◽  
Serum Gonadotropin ◽  
Specificity Protein

The transition from granulosa cell (GC) to luteal cell involves a change from estrogen production to predominantly progesterone production. We analyzed the role of Krüppel-like factor 4 ( Klf4), a transcriptional repressor used to generate pluripotent cells, in that transition. After luteinizing hormone (LH)/human chorionic gonadotropin treatment of preovulatory follicles, a major but transient increase in Klf4 transcript levels was detected. Therefore, we enquired whether Klf4 is involved in the rapid decline of aromatase, the key estrogen-producing enzyme, using preovulatory GCs obtained from pregnant mare serum gonadotropin-primed immature rat ovaries. Cyp19A1 expression in GCs transfected with FLAG- Klf4 or Klf4-specific siRNA was analyzed by real-time PCR and immunofluorescence staining. Cyp19A1 decreased when Klf4 was overexpressed, and Cyp19A1 and estradiol biosynthesis increased when Klf4 was knocked down. The mechanism by which Klf4 regulates Cyp19A1 expression was investigated using Cyp19A1 promoter-luciferase reporter assays and chromatin immunoprecipitation assays. The results revealed that the steroidogenic factor-1 (SF1)-binding motif, but not the specificity protein 1 (Sp1) binding element or the CACCC motif, was required for Klf4-mediated repression of Cyp19A1 promoter activity. Here we showed that Klf4 suppressed endogenous Cyp19A1 transcript and protein production, and this resulted from direct binding of Klf4 to the SF1 recognition motif in the Cyp19A1 promoter. These findings suggest that Klf4 is a physiologic regulator of Cyp19A1 expression in response to the LH surge in preovulatory GCs and that it has an essential role in the luteal transition in steroidogenesis.

scholarly journals LINC00511 accelerated the process of gastric cancer by targeting miR-625-5p/NFIX axis

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Zhaosheng Chen ◽  
Honglei Wu ◽  
Zhen Zhang ◽  
Guangchun Li ◽  
Bin Liu
Keyword(s):  
Gastric Cancer ◽  
Binding Sites ◽  
Regulatory System ◽  
Tumor Promoter ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Ki67 Expression ◽  
Downstream Target ◽  
Reporter Assays ◽  
Non Coding Rnas

Abstract Background Gastric cancer (GC) is a common-sighted cancer which is hard to cure over the world. Substantial researches revealed that long non-coding RNAs (lncRNAs) were fundamental regulators in the process of cancers. Nevertheless, the biological function of LINC00511 and how LINC00511 was involved in the regulatory system in GC remained unclear. Methods RIP assays and luciferase reporter assays were performed to illustrate combination between LINC00511 and miR-625-5p. Loss-of-function assays were applied for identifying LINC00511 function in GC. Results In our study, LINC00511 was discovered significantly high in expression in GC tissues and cell lines. Moreover, LINC00511 showed a strong expression in I/II and III/IV stage. Knockdown of LINC00511 could inhibit the cell proliferation while enhanced cell apoptosis rate in GC. We used nuclear–cytoplasmic fractionation to judge the subcellular localization of LINC00511. Furthermore, miR-625-5p was found to have binding sites for LINC00511 and negatively regulated by LINC00511. Overexpression of miR-625-5p repressed the course of GC. And knockdown of miR-625-5p could recover the effects of LINC00511 silence. Besides, NFIX was discovered as a downstream target of miR-625-5p and overexpression of NFIX could offset the influence of LINC00511 silence. The results of vivo studies manifested that down-regulation of LINC00511 could reduce the Ki67 expression and NFIX while lifted the expression of miR-625-5p. Conclusion Overall, the results from our study demonstrated that LINC00511 could function as a tumor promoter by targeting miR-625-5p NFIX axis, suggesting LINC00511 could be considered as a target for GC treatment.

scholarly journals Carboxyl-Terminal Transactivation Activity of Hypoxia-Inducible Factor 1α Is Governed by a von Hippel-Lindau Protein-Independent, Hydroxylation-Regulated Association with p300/CBP

2002 ◽  
Vol 22 (9) ◽  
pp. 2984-2992 ◽  
Author(s):  
Nianli Sang ◽  
Jie Fang ◽  
Vickram Srinivas ◽  
Irene Leshchinsky ◽  
Jaime Caro
Keyword(s):  
Hypoxia Inducible Factor ◽  
Adaptation To Hypoxia ◽  
Physical Interaction ◽  
Prolyl Hydroxylases ◽  
Von Hippel Lindau ◽  
Hypoxia Inducible Factor 1 ◽  
Transactivation Activity ◽  
Oxygen Homeostasis ◽  
Stimulation Of

ABSTRACT Hypoxia-inducible factor 1 complex (HIF-1) plays a pivotal role in oxygen homeostasis and adaptation to hypoxia. Its function is controlled by both the protein stability and the transactivation activity of its alpha subunit, HIF-1α. Hydroxylation of at least two prolyl residues in the oxygen-dependent degradation domain of HIF-1α regulates its interaction with the von Hippel-Lindau protein (VHL) that targets HIF-1α for ubiquitination and proteasomal degradation. Several prolyl hydroxylases have been found to specifically hydroxylate HIF-1α. In this report, we investigated possible roles of VHL and hydroxylases in the regulation of the transactivation activity of the C-terminal activating domain (CAD) of HIF-1α. We demonstrate that regulation of the transactivation activity of HIF-1α CAD also involves hydroxylase activity but does not require functional VHL. In addition, stimulation of the CAD activity by a hydoxylase inhibitor, hypoxia, and desferrioxamine was severely blocked by the adenoviral oncoprotein E1A but not by an E1A mutant defective in targeting p300/CBP. We further demonstrate that a hydroxylase inhibitor, hypoxia, and desferrioxamine promote the functional and physical interaction between HIF-1α CAD and p300/CBP in vivo. Taken together, our data provide evidence that hypoxia-regulated stabilization and transcriptional stimulation of HIF-1α function are regulated through partially overlapping but distinguishable pathways.

Ethyl pyruvate stabilizes hypoxia-inducible factor 1 alpha via stimulation of the TCA cycle

2010 ◽  
Vol 295 (2) ◽  
pp. 236-241 ◽  
Author(s):  
Seon-Ye Kim ◽  
Jae-Sun Choi ◽  
Chan Park ◽  
Joo-Won Jeong
Keyword(s):  
Ethyl Pyruvate ◽  
Hypoxia Inducible Factor ◽  
Tca Cycle ◽  
Hypoxia Inducible Factor 1 ◽  
The Tca Cycle ◽  
Stimulation Of

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.

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