Role of DNA methylation and methyl-DNA binding proteins in the repression of 5-lipoxygenase promoter activity

Katryniok, Careen; Schnur, Nicole; Gillis, Ad J. M.; Knethen, Andreas von; Sorg, B.; Looijenga, Leendert H. J.; Rådmark, Olof; Steinhilber, Dieter

doi:10.1016/j.bbalip.2009.09.003

Cited by 21 publications

(12 citation statements)

References 29 publications

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“…One mechanism that affects DNA accessibility is methylation of CpG dinucleotides at specific protein binding sites [32]. It has been demonstrated that epigenetic modifications, including methylation, play an important role in aberrant vav 1 expression in pancreatic cancer cell lines [22].…”

Section: Resultsmentioning

confidence: 99%

“…CpG 4 is located within a putative binding site for the transcription factors ETF and Sp1, but there are no consensus sequences predicted in the CpG 3 location. Katryniok et al reported that recruitment of Sp1 to its binding site in the human 5-lipoxygenase gene promoter is prevented by methylation [32]. However, in an earlier work, Iguchi-Ariga and Schaffner did not find an effect of CpG methylation on Sp1 binding in the cAMP promoter [52].…”

Section: Discussionmentioning

confidence: 96%

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Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation

Ilan¹,

Katzav²

2012

PLoS ONE

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Vav1 is a signal transducer protein that functions as a guanine nucleotide exchange factor for the Rho/Rac GTPases in the hematopoietic system where it is exclusively expressed. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma, lung cancer, and pancreatic ductal adenocarcinoma (PDA). Although some factors that affect vav1 expression are known, neither the physiological nor pathological regulation of vav1 expression is completely understood. We demonstrate herein that mutations in putative transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Among these sites is a consensus site for c-Myb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines. Depletion of c-Myb using siRNA led to a dramatic reduction in vav1 expression in these cells. Consistent with this, co-transfection of c-Myb activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. We also explored the methylation status of the vav1 promoter. Bisulfite sequencing revealed that the vav1 promoter was completely unmethylated in human lymphocytes, but methylated to various degrees in tissues that do not normally express vav1. The vav1 promoter does not contain CpG islands in proximity to the transcription start site; however, we demonstrated that methylation of a CpG dinucleotide at a consensus Sp1 binding site in the vav1 promoter interferes with protein binding in vitro. Our data identify two regulatory mechanisms for vav1 expression: binding of c-Myb and CpG methylation of 5′ regulatory sequences. Mutation of other putative transcription factor binding sites suggests that additional factors regulate vav1 expression as well.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 96%

Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation

Ilan¹,

Katzav²

2012

PLoS ONE

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“…Two major pathways are implicated in regulating ALOX5 expression: promoter methylation and transcription factor activation [57]. Research demonstrates that leukocyte cell lines that have methylated promoters, U-937 and HL-60TB, do not express ALOX5, while the HL-60 cell line, which is unmethylated expresses ALOX5 [58], [59]. Moreover, the treatment of the U-937 and HL-60TB cell lines with the demethylating agent, 5-aza-2'deoxycytidine resulted in the restoration of ALOX5 expression [58].…”

Section: Discussionmentioning

confidence: 99%

Chronic Hypoxia Promotes Pulmonary Artery Endothelial Cell Proliferation through H2O2-Induced 5-Lipoxygenase

et al. 2014

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Pulmonary Hypertension (PH) is a progressive disorder characterized by endothelial dysfunction and proliferation. Hypoxia induces PH by increasing vascular remodeling. A potential mediator in hypoxia-induced PH development is arachidonate 5-Lipoxygenase (ALOX5). While ALOX5 metabolites have been shown to promote pulmonary vasoconstriction and endothelial cell proliferation, the contribution of ALOX5 to hypoxia-induced proliferation remains unknown. We hypothesize that hypoxia exposure stimulates HPAEC proliferation by increasing ALOX5 expression and activity. To test this, human pulmonary artery endothelial cells (HPAEC) were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24-, 48-, or 72 hours. In a subset of cells, the ALOX5 inhibitor, zileuton, or the 5-lipoxygenase activating protein inhibitor, MK-886, was administered during hypoxia exposure. ALOX5 expression was measured by qRT-PCR and western blot and HPAEC proliferation was assessed. Our results demonstrate that 24 and 48 hours of hypoxia exposure have no effect on HPAEC proliferation or ALOX5 expression. Seventy two hours of hypoxia significantly increases HPAEC ALOX5 expression, hydrogen peroxide (H2O2) release, and HPAEC proliferation. We also demonstrate that targeted ALOX5 gene silencing or inhibition of the ALOX5 pathway by pharmacological blockade attenuates hypoxia-induced HPAEC proliferation. Furthermore, our findings indicate that hypoxia-induced increases in cell proliferation and ALOX5 expression are dependent on H2O2 production, as administration of the antioxidant PEG-catalase blocks these effects and addition of H2O2 to HPAEC promotes proliferation. Overall, these studies indicate that hypoxia exposure induces HPAEC proliferation by activating the ALOX5 pathway via the generation of H2O2.

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“…The expression of the ALOX5 gene, which encodes 5-LOX is influenced by epigenetic mechanisms such as DNA methylation (Katryniok et al, 2010; Uhl et al, 2002, 2003, Vikman et al, 2009; Zhang et al, 2004). Most current knowledge about the role of DNA methylation in regulating 5-LOX expression has been obtained from studies of the human 5-LOX gene (on chromosome 10) and its promoter which possesses a unique GC-rich region and is a target of methylation modifications (Katryniok et al, 2010).…”

mentioning

confidence: 99%

“…Most current knowledge about the role of DNA methylation in regulating 5-LOX expression has been obtained from studies of the human 5-LOX gene (on chromosome 10) and its promoter which possesses a unique GC-rich region and is a target of methylation modifications (Katryniok et al, 2010). In contrast to the human gene, the mouse 5-LOX gene (on chromosome 6) lacks the typical CpG islands found in the human sequence.…”

mentioning

confidence: 99%

5-Lipoxygenase in mouse cerebellar Purkinje cells

2010

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It has been suggested that the enzymatic pathway of 5-lipoxygenase (5-LOX) influences brain functioning and pathobiology. The mRNAs for both the enzyme 5-LOX and its activating protein FLAP have been found in the cerebellum. In this work, we investigated the cellular expression of 5-LOX in the adult mouse cerebellar cortex. We used the in situ mRNA hybridization assay, immunocytochemistry, laser capture microdissection, and our previously developed method for assaying the DNA methylation status of a putative mouse 5-LOX promoter. Since both 5-LOX mRNA in situ hybridization signal and FLAP immunoreactivity co-localize with calbindin 28kD immunoreactivity (a Purkinje cell marker) but not with S-100β immunoreactivity (a Bergmann glia marker), the suggestion is that the 5-LOX pathway is expressed in cerebellar Purkinje cells. We found that methylation in the sites targeted by methylation-sensitive restriction endonucleases AciI and HinP1I but not BstUI and HpaII was greater in DNA samples obtained from a high-5-LOXexpressing cerebellar region (Purkinje cells) vs. a low-5-LOX-expressing region (the molecular cell layer), suggesting a possible epigenetic contribution to the cell-specific 5-LOX expression in the cerebellum. We propose that Purkinje cell-localized 5-LOX and FLAP expression may be involved in the cerebellar synthesis of leukotrienes and/or could influence the Dicer-mediated microRNA formation and processes of neuroplasticity. (Chinnici et al., 2007;Chu and Praticò, 2009;Lindgren et al., 1984;Miyamoto et al., 1987;Ohtsuki et al., 1995). Information about the site and the cell types responsible for the synthesis of 5-LOX products, the inflammatory leukotrienes and the anti-inflammatory lipoxins (Radmark et al., 2007, Serhan et al., 2008 in the CNS points to brain region differences in leukotriene formation (Chinnici et al., 2007;Miyamoto et al., 1987) and indicates the possibility of transcellular biosynthesis of cysteinyl leukotrienes in neuronal and glial cells (Farias et al., 2007). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2011 December 1. 5-LOX appears to be involved in mechanisms of cell proliferation and differentiation and in neuroplasticity, such as hedgehog-dependent neurite projection (Bijlsma et al., 2008). In the adult brain, in addition to their strong hippocampal expression, the mRNAs for both 5-LOX and its activating protein FLAP (5-lipoxygenase activating protein) were found in the cerebellum (Lammers et al., 1996) but their cellular distribution has ...

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Role of DNA methylation and methyl-DNA binding proteins in the repression of 5-lipoxygenase promoter activity

Cited by 21 publications

References 29 publications

Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation

Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation

Chronic Hypoxia Promotes Pulmonary Artery Endothelial Cell Proliferation through H2O2-Induced 5-Lipoxygenase

5-Lipoxygenase in mouse cerebellar Purkinje cells

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